MSCZO-602

M.Sc. III Semester

DEVELOPMENTAL BIOLOGY

DEPARTMENT OF ZOOLOGY
SCHOOL OF SCIENCES
UTTARAKHAND OPEN UNIVERSITY
 Developmental Biology
(MSCZO-602)

DEPARTMENT OF ZOOLOGY
SCHOOL OF SCIENCES
UTTARAKHAND OPEN UNIVERSITY
Phone No. 05946-261122, 261123
Toll free No. 18001804025
Fax No. 05946-264232, E. mail [email protected]
htpp://uou.ac.in
MEMBERS OF THE BOARD OF STUDIES AND PROGRAMME COORDINATOR

Dr. Neera Kapoor Dr. A. K. Dobriyal

Professor & Head Professor & Head

Department of Zoology, Department of Zoology

School of Sciences BGR Campus Pauri

IGNOU Maidan Garhi, New Delhi HNB Srinagar Garhwal

Dr. S. P. S. Bisht Dr. S. S. Kunjwal

Professor & Head Assistant Professor

Department of Zoology Department of Zoology

DSB Campus School of Sciences, Uttarakhand Open

Kumaon University Nainital University Haldwani, Nainital

Dr. Mukta Joshi

Assistant Professor

Department of Zoology

School of Sciences, UOU, Haldwani, Nainital

PROGRAMME COORDINATOR

Dr. Pravesh Kumar Sehgal

Associate Professor

Department of Zoology

School of Sciences, UOU, Haldwani, Nainital

 UNIT EDITOR AND WRITERS

EDITOR
Dr. Beena Joshi Bhatt
Dolphin (P.G) Institute
Biomedical & Natural Science
Deharadun, Uttarakhand Dr. Anuharika Chuchan ( Unit No. 4 & 6)
Assistant Professor
Department of Zoology
UNIT WRITER Govt. P.G. College
Dr. Shyam S. Kunjwal (Unit No.1) Khtima
Assistant Professor
Department of Zoology, Dr. Mamtesh Kumari (Unit No: 5)
Uttarakhand Open University Assistant Professor
Haldwani, Nainital. Department of Zoology
Govt. P.G. College
Dr. Shruti Saxena (Unit No. 2 & 3) Kashipur
Assistant Professor
Department of Zoology Dr. Mukta Joshi (Unit No:7 & 8)
S.G.R.R. Dehradun Assistant Professor
Department of Zoology
Uttarakhand Open University
Nainital, Uttarakhand

Course Title and Code : Developmental Biology (MSCZO-602)

ISBN :
Copyright : Uttarakhand Open University
Edition : 2022
Published II
COURSE By: DEVELOPMENTAL
: Uttarakhand Open University, Haldwani, Nainital- 263139
BIOLOGY
Course code: MSCZO- 602 Credit:
COURSE II: DEVELOPMENTAL BIOLOGY Credit: 3
Course code: MSCZO- 602
Unit Page
Block and Unit title
Number Number
Block I: Developmental Biology 1- 177

Unit : 1 Concept of Developmental Biology: Introduction, History of development, Coelom, 1- 14

Segmentation, Somites, Diploblast, Protostomes and Deuterostomes, Development in
Eukaryotes, Summary
Unit : 2 Gamete and Fertilization: Introduction, Ultra structure of Gamete, Sperms, Egg, 15- 33
Mechanism of Fertilization, Pre Fertilization, Post Fertilization, Biochemistry of
Fertilization, Summary
Unit : 3 Cleavage, Blastulation and Gastrulation: Introduction, Patterns of Cleavage 34-58
Determinate and Indeterminate Cleavage, Influence of Yolk on Cleavage, Metabolic
Changes during Cleavage, Morulation and Blastulation in Frog, Chick and Rabbit
Types of Blastulae, Major events of Gastrulation and Fate maps, Morphogenetic
movements in Frog, Chick and Rabbit, Significance of Gastrulation and
Exogastrulation, Summary

Unit : 4 Early Development: Introduction, Neurulation and Ectoderm origin and fate of 59-84
Neural Crest cells, Neurulation, Primary Neurulation, Secondary Neurulation,
D Development of Mesoderm, Development of Endoderm, Summary

Block II: BIOTECHNOLOGY

Unit : 5 Organogenesis and Organizer Concept: Introduction, Development of organs in 85-127

Chick, Brain, Eye and Heart, Embryonic induction, Primary organiser and its
Morphological Differentiation, Origin of primary Organiser, Inductive Interactions,
Nature of Inductive Signal (Possible mechanism of neural induction), Competences,
Summary
Unit : 6 Regeneration and Metaplasia: Introduction, Distribution of Regenerative 128-146
Ability, Polarity in Regeneration, Mechanism of regeneration of Amphibian limb and
Lens, Metaplasia, Super-regeneration and heteromorphosis, Summary

Unit : 7 Metamorphosis: Introduction, Kinds of Metamorphosis, Metamorphosis in 147-164

Amphibia
Physiological and Biochemical changes during Metamorphosis, Hormonal control of
Metamorphosis, Summary
Unit:8 Teratogenesis: Introduction, Types of teratogenesis, Mechanisms of genetic and 165-177
Evironmental teratogenesis , Phenocopies, Developmental mechanisms teratogenesis
Summary
DEVELOPMENTAL BIOLOGY MSCZO- 602

UNIT 1: CONCEPT OF DEVELOPMENTAL BIOLOGY

CONTENTS
1.1 Objectives
1.2 Introduction
1.3 History of Development
1.4 Principal features and patterns of development
1.4.1 Coelom
1.4.2 Segmentation
1.4.3 Somites
1.4.4 Diploblast
1.4.5 Protostomes and Deuterostomes
1.5 Terminal Questions and Answers
1.6 References

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1.1 OBJECTIVES

After studying this module, you shall be able to learn and understand about:
i. Principal features and patterns of development
ii. Coelom,
iii. Segmentation,
iv. Somites
v. Diploblast
vi. Protostomes and deuterostomes

1.2 INTRODUCTION

From the patterns in development, developmental biology evolved and became one of the old and
young disciplines in biology; it originated in the 1950 and formally formed an independent
discipline in the 1970.
A new discipline gradually was formed in the process of learning molecular embryology which
also comprehensively strengthened and further developed this discipline
Since the 1980s, due to the development of disciplines such as genetics, cell biology, and
molecular biology, a large number of new research methods were also applied, and developmental
biology made great progress.
The research content of this subject includes the occurrence and formation of gametes, fertilization
process, cell differentiation, and morphogenesis. It also includes how different cell groups in the
development process are reconfigured and specialized.
The emergence of various cell types, the appearance of the final organ phenotypic characteristics,
the establishment of special functions, the expression, control, and regulation of genes at different
developmental stages, the causal relationship between genotype and phenotypic expression, the
relationship between nucleus and cytoplasm during development, interrelationships between cells,
and the effects of external factors on embryonic development were predominantly seen. Among
them, cell differentiation became a core problem in the process of developmental biology.

1.3 HISTORY OF DEVELOPMENT

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It originated in the 1950 and formally formed an independent discipline in the 1970. A new
discipline gradually formed in the process of learning molecular embryology which is also a
comprehensive and further developed discipline.
The multidisciplinary approach to the study of development first arose before the turn of the
Twentieth Century as integration of embryology (initially the descriptive study of embryonic
development) with cytology (the study of cellular structure and function) and later with genetics
(the study of inheritance).
The leading cytologists of that time (primarily E.B. Wilson at Columbia University in New York
City) recognized that the development of the embryo is a manifestation of changes in individual
cells and that an understanding of the fundamental principles of development would come from
studying cellular structure and function.
Wilson recognized that the characteristics of an organism gradually emerge by utilization of the
inherited information that is located on the chromosomes. Therefore, it was important to
comprehend the nature of that information and how it is utilized during development. However, in
the absence of concrete evidence, there was a great deal of rampant speculation as to how the
chromosomes participate in development. The German embryologist Wilhelm Roux was the source
of much of this speculation.
Roux believed that the fertilized egg receives substances that represent different characteristics of
the organism, which - as cell division occurs - become linearly aligned on the chromosomes and
are subsequently distributed unequally to daughter cells.
This "qualitative division" fixes the fate of the cells and their descendants because some of the
determinants are lost to a cell at each division.
Roux (1888) appeared to have confirmed his theories through an experiment he conducted on frog
eggs.
Another German embryologist, Hans Driesch (1892), approached the problem differently with sea
urchin embryos. Instead of destroying one of the cells of the two-celled embryo, he separated the
cells from one another and found that isolated cells at the four-cell stage also develop normally.
Thus, Driesch concluded that each cell retains all the developmental potential of the zygote.
The conflict between these two opposing views of development has been settled in favor of
Driesch's interpretation by numerous cell separation experiments.
The Role of the Hereditary Material in Development

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Although the equal distribution of hereditary information to all cells had been established in the
late 1800s, its role in development remained an enigma. Two key contributions at the dawn of the
Twentieth Century provided the impetus for additional progress:
• In 1900, the significance of Gregor Mendel's work on heredity was finally appreciated.
• The other contribution was made by Theodor Boveri, who in a paper published in 1902
demonstrated that normal development is dependent upon the normal combination of
chromosomes. Each chromosome must have qualitatively unique effects on development.
Developmental biology is one of the important basic branches of biological sciences. The research
content is infiltrated by many other disciplines, especially genetics, cell biology, and molecular
biology.
It uses modern science and technology to study and analyze the processes and mechanisms of
organisms from spermatogenesis and egg development, fertilization, growth, aging, and death from
the molecular level, sub-microscopic level, and cellular level.
Although there are many species of animals, the development of embryos still has a similar pr
ocess, which can be divided into stages of fertilization, cleavage, morula, blastocyst, gastrula, and
organ formation.
In addition, during the embryonic development of vertebrates, the characteristics common to
various animals will appear first (such as the skin), and then specialized structures (such as fish
scales) will be developed.
In general, the ectoderm forms the epidermis and nerve tissue. The endoderm forms the intestinal
epithelium and the digestive gland epithelium, which forms bone, muscle, blood, lymph, and other
connective tissues. Others are derived from the mesoderm. But there are exceptions: the sphincter
of the eye iridescence does not come from the mesoderm, nor from the mesenchyme, but from a
part of the retina, that is, from the ectoderm.
The smooth muscle of the sweat gland is not from the mesoderm, but from the ectoderm; the
mesenchyme itself is unclear, as it may come from the ectoderm or the mesoderm, or even from the
endoderm. Research on developmental biology needs further advancement, which will help to
understand the developmental mechanisms of organisms.

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Fig. 1.1: Developmental stages

1.4 PRINCIPLE FEATURES AND PATTERNS OF DEVELOPMENT

Developmental biology is the study of the process by which animals and plants grow and develop.
Developmental biology also encompasses the biology of regeneration, asexual reproduction,
metamorphosis, and the growth and differentiation of stem cells in the adult organism.
Developmental biology includes the production of gametes, fertilization, and development of the
embryo, emergence of the adult organism, senescence, and death. Developmental biologists in the
department attempt to understand the molecular, genetic, cellular, and integrative aspects of
building an organism.
The main processes involved in the embryonic development of animals are tissue patterning (via
regional specification and patterned cell differentiation); tissue growth; and tissue morphogenesis.
The regional specification refers to the processes that create a spatial pattern in a ball or sheet of
initially similar cells. This generally involves the action of cytoplasmic determinants, located
within parts of the fertilized egg, and of inductive signals emitted from signaling centers in the
embryo.

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The early stages of the regional specification do not generate functional differentiated cells, but cell
populations committed to developing to a specific region or part of the organism. These are defined
by the expression of specific combinations of transcription factors.
Cell differentiation relates specifically to the formation of functional cell types such as nerve,
muscle, secretory epithelia, etc. Differentiated cells contain large amounts of specific proteins
associated with cell function.
Morphogenesis relates to the formation of three-dimensional shapes. It mainly involves the
orchestrated movements of cell sheets and individual cells. Morphogenesis is important for creating
the three germ layers of the early embryo (ectoderm, mesoderm, and endoderm) and for building
up complex structures during organ development.
Tissue growth involves both an overall increase in tissue size, and also the differential growth of
parts (allometry) which contributes to morphogenesis. Growth mostly occurs through cell
proliferation but also through changes in cell size or the deposition of extracellular materials.
The development of plants involves similar processes to that of animals. However, plant cells are
mostly immotile so morphogenesis is achieved by differential growth, without cell movements.
Also, the inductive signals and the genes involved are different from those that control animal
development.
1.4.1 COELOM
All animal has a cavity. Different animals use these cavities for different purposes. A vast fluid-
filled area between the body wall and the internal organs is typically referred to as a body cavity.
The alimentary canal is located between the body wall and the coelom, a perivisceral cavity. The
coelom develops during embryonic development as a split in the mesoderm, which divides into two
layers: a somatic layer that surrounds the endoderm and a splanchnic layer that lies close to the
epidermis.
The coelomic epithelium, which secretes coelomic fluid, surrounds the coelom. On one end, the
excretory organs open into the coelom, and on the other, they open to the outside. Coelomoducts,
which transport sperms or eggs from the coelom to the outside, are produced by the coelom wall,
which also gives rise to reproductive cells. The per visceral cavity, also known as the
splanchnocoel, is formed by the majority of the coelom and houses the visceral organs. The
gonocoel and nephrocoel, whose coelomic character can only be appreciated if their developmental
histories are followed, are examples of confined cavities that are formed when specific areas of the
perivisceral cavity are cut off from it. The first animals with a real coelom are annelids.

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Coeloms evolved from acoelomates, then pseudocoelomates, and finally coelomates. The absence
or presence of peritoneum or epithelial lining distinguishes a pseudocoelomate from a coelomate
animal. It exists in coelomate animals but not in pseudocoelomate ones. A real coelom might have
different embryological origins. It is referred to as schizocoelous if it arises from a split in
mesoderm cells. It is referred to as enterocoelous if it arises from out pocketing from the embryonic
gut.
According to the mode of coelom formation, there are generally two types of animals:
(A) Schizocoelomate: When coelom arises by the splitting of mesodermal bands or masses
during embryonic development, it is called schizocoel, and animals are called schizocoelomates
(Fig. 1.2). The animals belonging to the phylum Mollusca, Annelida, Arthropoda, and
Onychophora are schizocoelomates.

Fig.1.2 Coelomformation by the splitting of mesoderm

(B) Enterocoelomate: When coelom is formed by the evagination from the embryonic
archenteron and the pouch-like structures are detached from the archenteron and gradually occupy
the whole body by enlargement, called enterocoel (Fig. 1.2). The animals having enterocoel are
called enterocoelomate. The animals belonging to phylum Echinochordata, Hemichordata, and
Chordata are enterocoelomates.

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Fig 1.3 Coelom formation by out pocketing of the primitive gut

1.4.2 SEGMENTATION
Segmentation is a difficult process to satisfactorily define. Many taxa (for example the mollusks)
have some form of serial repetition in their units but are not conventionally thought of as
segmented. Segmented animals are those considered to have organs that were repeated, or to have a
body composed of self-similar units, but usually, it is the parts of an organism that are referred to
as being segmented.
Segmentation in animals typically falls into three types, characteristic of different arthropods,
vertebrates, and annelids. Arthropods such as the fruit fly form segments from a field of equivalent
cells based on transcription factor gradients. Vertebrates like the zebra fish use oscillating gene
expression to define segments known as somites. Annelids such as the leech use smaller blast cells
budded off from large teloblast cells to define segments.

Arthropods: Although Drosophila segmentation is not representative of the arthropod phylum in

general, it is the most highly studied. Early screens to identify genes involved in cuticle
development led to the discovery of a class of genes that were necessary for proper segmentation of
the Drosophila embryo.
To properly segment the Drosophila embryo, the anterior-posterior axis is defined by maternally
supplied transcripts giving rise to gradients of these proteins. This gradient then defines the
expression pattern for gap genes, which set up the boundaries between the different segments. The
gradients produced from gap gene expression then define the expression pattern for the pair-rule
genes. The pair-rule genes are mostly transcription factors, expressed in regular stripes down the

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length of the embryo. These transcription factors then regulate the expression of segment polarity
genes, which define the polarity of each segment. Boundaries and identities of each segment are
later defined.
Within the arthropods, the body wall, nervous system, kidneys, muscles, and body cavity are
segmented, as are the appendages (when they are present). Some of these elements (e.g.
musculature) are not segmented in their sister taxon, the Onychophora.
1.4.3 SOMITES
The somites (outdated term: primitive segments) are a set of bilaterally paired blocks of paraxial
mesoderm that form in the embryonic stage of somitogenesis, along the head-to-tail axis
in segmented animals. In vertebrates, somites subdivide into the sclerotomes, myotomes,
syndetomes, and dermatomes that give rise to the vertebrae of the vertebral column, rib cage, part
of the occipital bone, skeletal muscle, cartilage, tendons, and skin (of the back).
The word somite is sometimes also used in place of the word metamere. In this definition, the
somite is a homologously-paired structure in an animal body plan, such as is visible
in annelids and arthropods.
The mesoderm forms at the same time as the other two germ layers, the ectoderm and endoderm.
The mesoderm at either side of the neural tube is called the paraxial mesoderm. It is distinct from
the mesoderm underneath the neural tube which is called the chordamesoderm that becomes the
notochord. The paraxial mesoderm is initially called the “segmental plate” in the chick embryo or
the “unregimented mesoderm” in other vertebrates. As the primitive streak regresses and neural
folds gather (to eventually become the neural tube), the paraxial mesoderm separates into blocks
called somites.
Formation: The pre-somitic mesoderm commits to the somitic fate before the mesoderm becomes
capable of forming somites. The cells within each somite are specified based on their location
within the somite. Additionally, they retain the ability to become any kind of somite-derived
structure until relatively late in the process of somitogenesis.
The development of the somites depends on a clock mechanism as described by the clock and
wavefront model. In one description of the model, oscillating Notch and Wnt signals provide the
clock. The wave is a gradient of the FGF protein that is rostral to caudal (nose to tail gradient).
Somites form one after the other down the length of the embryo from the head to the tail, with each
new somite forming on the caudal (tail) side of the previous one.

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The timing of the interval is not universal. Different species have different interval timing. In
the chick, embryo somites are formed every 90 minutes. In the mouse, the interval is 2 hours.
For some species, the number of somites may be used to determine the stage of embryonic
development more reliably than the number of hours post-fertilization because the rate of
development can be affected by temperature or other environmental factors. The somites appear on
both sides of the neural tube simultaneously. Experimental manipulation of the developing somites
will not alter the rostral/caudal orientation of the somites, as the cell fates have been determined
before somitogenesis. Somite formation can be induced by Noggin-secreting cells. The number of
somites is species-dependent and independent of embryo size (for example, if modified via surgery
or genetic engineering). Chicken embryos have 50 somites; mice have 65, while snakes have 500.
As cells within the paraxial mesoderm begin to come together, they are termed somitomeres,
indicating a lack of complete separation between segments. The outer cells undergo
a mesenchymal-epithelial transition to form an epithelium around each somite. The inner cells
remain as mesenchyme.
1.4.4 DIPLOBLAST
Diploblasty is a condition of the blastula in which there are two primary germ layers:
the ectoderm and endoderm. Diploblastic organisms are organisms that develop from such a
blastula and include cnidaria and ctenophore, formerly grouped in the phylum Coelenterate, but
later understanding of their differences resulted in their being placed in separate phyla. The
endoderm allows them to develop true tissue. This includes tissue associated with the gut and
associated glands. The ectoderm, on the other hand, gives rise to the epidermis, the nervous tissue,
and if present, nephridia.
Simpler animals, such as sea sponges, have one germ layer and lack true tissue organization.
All the more complex animals (from flatworms to humans) are triploblastic with three germ layers
(a mesoderm as well as ectoderm and endoderm). The mesoderm allows them to develop
true organs. Groups of diploblastic animals alive today include jellyfish, corals, sea anemones, and
comb jellies.
Features of the diploblastic animal
a) They consist of jelly-like noncellular mesenchyma or coagulated mesoglea in the middle
among ectoderm and endoderm.
b) They show radial symmetry, biradial, or rotational symmetry.
c) A lesser degree of specialization.

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d) No proper transport system.

e) Absence of coelom.
f) Sac-like digestive system and gastrovascular cavity.
g) Diploblastic creatures might have cell types that serve different capabilities, for example,
epitheliomuscular cells, which act as a covering as well as contractile cells.
h) The endoderm of diploblastic animals has true tissues and intestines. A non-living layer
named mesoglea is present between the ectoderm and endoderm.
i) Mesoglea helps in protecting the gut lining and body.
j) These animals do not develop organs.
Examples: Phylum Porifera and Cnidaria.

1.4.5 PROTOSTOMES AND DEUTEROSTOMES

The creation of the mouth first or the anus can be used to separate the bilateral metazoans into two
major assemblages. Protostomes are metazoans in which the animal's anus and mouth are formed
largely by the blastopore, and Deutrostomes in which the animal's anus and mouth are formed
mostly by the blastopore. The next subsections will provide your study material on these two
groupings.
Protostomia: The metazoans in which the mouth is derived from blastopore on the anterior end
and anus that appears later to complete the alimentary canal are included in Protostomia. As the
mouth forms first, their animals are included in the ‘Protostomia” (Mouth first) division of the
animal kingdom. The nerve cord is ventral in protostomes.

.
Fig. 1.4 Diagram showing the development of mouth from the blastopore

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The developmental characteristics of protostomes are as follows.

1. The pattern of embryonic cleavage: Cleavage is spiral in protostomes, i.e., the axis of the
cleavage plane is oblique, so blastomeres have a spiral arrangement in which one tier of cells
alternates with the next tier of cells. The spiral cleavage is masked at the 6th cleavage 64-cell
stage.
2. The fate of embryonic blastomeres: The fate of blastomeres is determined very early
during holoblastic cleavage. This is called determinate or mosaic cleavage, which means
blastomeres are destined to form a particular organ in the very early stage of cleavage. In
Figure 1.4 just after the first cleavage ablation of one of the cells takes place it leads to the
loss of head structure in the embryo that drives from it. Such a type of development is said to
be mosaic.
3. Fate of blastopore: The blastopore either becomes mouth (e.g., Mollusca) or gives rise to
both mouth and anus (e.g., some mollusks, polychaetes, and onychophorans) in adults.
4. Formation of mesoderm: Mesoderm originates from the fourth cell, named mesentoblast
(also called as ‘4d’ cell) which increases in number by proliferation.
5. Formation of coelom: Coelom originates from the splitting of the mesodermal cell mass.
This process of coelom formation is known as schizocoely and coelom are called
schizocoelom (‘schizo’ means split). Examples: Coelomate protostomes include Sipuncula,
Echiura, Annelida, Pogonophora, Mollusca, Onychophora, Tardigrada, Pentastomida, and
some groups of arthropods.
Deuterostomia: The metazoans in which anal opening are derived from blastopore during
embryonic development and represents the posterior end of the body and mouth are formed later
are included in deuterostomia. As the anus forms first and the mouth is formed secondarily, these
animals are grouped in deuterostomia (Mouth second). The nerve cord is dorsal in deuterostomes.

Fig. 1.5 Diagram showing the development of the anus from the blastopore

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The developmental characteristics of deuterostomes are as follows.

1. Pattern of embryonic cleavage: The radial pattern of embryonic cleavage occurs in which the
cleavage plane is either parallel or at a right angle to the polar axis. Blastomeres are arranged
directly above or below one another.
2. Fate of embryonic blastomeres:
Cleavage is indeterminate and if blastomeres are separated at 4 cell stages, each one will develop
into a complete individual. Cleavage is regulative because each of the blastomeres if separated can
regulate its development (Fig 1.6). In figure 1.6, if ablation of one cell takes place, then the
descendants of the remaining cell can give rise to the structure in the embryo that would have
developed from the lost cell. In this case, the green cell can regenerate the head structure as well as
the trunk region. Such development is said to be regulative.
3. Fate of blastopore: Blastopore becomes the adult anus and then the formation of the mouth
takes place from a second opening on the dorsal surface of the embryo.
4. Formation of mesoderm: Mesodermal tissue is formed by the outgrowth of the endodermal
wall of the archenteron.
5. Formation of coelom: Coelom is formed by evagination of pouches from the wall of the arch-
enteron and each diverticulum becomes separated from the archenteron and develops an inde-
pendent coelomic pouch. This process of formation of the coelom is called enterocoely and the
coelom is called enterocoelom.
Examples: Deuterostomes include Echinoderms, Chordates, Pogonophores, Hemichordates, and
some minor phyla.

1.5 TERMINAL QUESTIONS AND ANSWERS

Q.1 Explain in detail about Coelom.

Q.2 what do you understand by Segmentation? Explain in detail.
Q.3 Write a short note on Somites.
Q.5 Define the Diploblastic Animal.
Q.6.Explain in detail the Protostomes and Deuterostomes ?

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1.6 REFERENCES

 Mauch T J, Schoenwolf G C. Developmental Biology. Sixth Edition. By Scott F.

Gilbert. American Journal of Medical Genetics Part A. 2001, 99(2):170–171.
 Slack J M. Developmental biology of the pancreas. Cell Biochemistry & Biophysics. 2004,
40(3):127-142.
 Hirsch A M. Developmental biology of legume nodulation. New Phytologist. 2010,
122(2):211-237.
 Browder, L.W., C. A. Erickson and W.R. Jeffery. 1991. Developmental Biology. Third
Edition. Saunders College Publishing. Philadelphia.
 Driesch H. 1892. Entwick lungs mechanism Studien. I. Der Werth der beiden ersten
Furchungszellen in der Echinodermentwicklung. Experimentelle Erzeugen von Theil und
Doppelbildung. Zeitschrift für wissenschaftliche Zoologie, 53: 160-178; 183-184.
 Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer. Sunderland, MA.
 Roux, W. 1888. Beiträge zur Entwicklungsmechanik des Embryo. Ueber die künstliche
Hervorbringung halber Embryonen durch Zerstörung einer der beiden ersten
Furchungskugelin, sowie über die Nachentwicklung (Postgeneration) der fehlenden
Köperhälfte. Virchows Arch. Pathol. Anat. Physiol., 114: 113-153; 289-291.
 Shostak, S. 1991. Embryology. An Introduction to Developmental Biology. HarperCollins.
New York.
 Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and Meyerowitz, E.
1998. Principles of Development. Current Biology. London.
 Brusca, R. C., and G. J. Brusca. Invertebrates, 2nd ed. New York: Sinauer Associates, 2003.
 Nielsen, C. Animal Evolution: Interrelationships of the Living Phyla, 2nd ed. New
York: Oxford University Press, 2001.

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UNIT 2: GAMETE AND FERTILIZATION

CONTENTS
2.1 Objectives
2.2 Introduction
2.3 Ultrastructure of gamete
2.3.1 Sperms
2.3.2 Egg
2.4 Mechanism of fertilization
2.4.1 Pre-fertilization
2.4.2 Post- fertilization
2.4.3 Biochemistry of fertilization
2.5 Summary
2.6 Terminal questions and answers

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2.1 OBJECTIVES

The study of this unit will let the students understand the following:
 The structure of Gametes
 Difference between egg and sperm
 The concept of Fertilization.
 Preparation of cell for the fertilization
 Significance of fertilization.
 Elucidate the type of Fertilization.
 Elucidate the various types of organization in insects.
 Various factors involved in Fertilization.
 The Bio-Chemistry involved during fertilization.
 Difference between prefertilization and postfertilization events.

2.2 INTRODUCTION

A cell is the fundamental Unit of Life. Every Living Being is a combination of several Cells
combined as tissue, muscles, and organs. These cells undergo drastic changes during their entire
life, generating, regenerating, dying, etc. Evolotion in the cells apart from the rest of the world was
quiet evident and remarkable. This course of evolution leads to certain cells becoming specialized
for reproduction that came to be known as Gamete.
Gametes have evolved according to their sex named spermatozoa and Ova for Male and females
respectively. These gametes carry a single copy of the genetic material which fuses with
themselves (ova and sperm). They have unique structures that evolved to facilitate their fusion.
The entire process of fusing is known as fertilization. This fertilization undergoes two main
processes namely pre-fertilization and post-fertilization. Fertilization had a huge significance in
maintaining diploidy in the race and genetic variation. The fertilization is subjected to a lot of
factors that influence its timing, structure, etc. A set of events happens before fertilization and a lot
of changes happen in the cytoplasm of the egg after the sperm penetrates its membrane. The
process of fertilization has been studied extensively in marine invertebrates like sea urchins,
echinoids, amphibians, mammals, and vertebrates also.

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2.3 ULTRA STRUCTURE OF GAMETE

Gamete means the reproductive cell or sex cells. These gametes are Female gametes and male gametes.
Female gametes and Male gametes are known as ova or egg cells and sperm respectively. All Gamete
cells are haploid cells which mean that they carry only one copy of a chromosome. The Male and
Female gamete fuse them to form a new cell known as a zygote. Sperm cells or spermatozoa are small
and motile whereas egg cell or ovum is relatively large and non-motile. Ova mature in the ovaries of
females and sperm in the testes of males. Spermatozoon and ovum fuse during fertilization to form a
new diploid organism. Gametes carry half the genetic information of an individual, one ploidy of each
type. They are created through the process of meiosis. A germ cell undergoes two fissions, resulting in
the production of four gametes.
2.3.1 SPERMS
Sperms are male gametes developed in the testis through a process of spermatogenesis.
Spermatogenesis forms spermatids. These spermatids produced a specialized structure called sperm.
2.3.1.1 STRUCTURE OF SPERM
The sperm's body is covered by the plasma membrane. Their body is divided into head, neck,
Middle-piece, and tail.
The Head contains the nucleus which has the genetic code, which is the main contribution to the
new offspring. Heads have the acrosome which contains digestive enzymes needed to penetrate the
ovum.
Mid-piece is posterior to the head. It contains mitochondria to provide energy to the sperm. These
mitochondria convert fructose and other energy substrates into high-energy compounds.
The tail joins the head at the proximal centriole and is called the implantation region. The head and
tail get separated at this point during fertilization.
A tail is a flagellum that causes the sperm to swim. A major feature of the tail is the axial filament.
The axial filament starts at the proximal centriole and runs through the entire body of the tail and is
made up of a small bundle of tiny fibrils.

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Fig 2.1 Diagram of Sperm

2.3.2 EGG
The egg is the Female gametes developed in the ovary through a process of oogenesis. Eggs are
single cells that are capable to produce a new organism when fused with sperms. These Eggs are
released from an ovary and move to the uterus waiting for the sperm to fuse and produce a new
organism.

Fig 2.2 Diagram of Egg

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In shape, the eggs are spherical or oval and are non–motile. Their sizes vary in different animals
with the smallest size found in mammals and the largest in birds. These cell sizes are greater than
other body cells. Inside the Egg spherical boundary, a large amount of cytoplasm exists which is
called ooplasm, a big nucleus exists within the cytoplasm known as a germinal vesicle. The side of
the egg containing the nucleus is called the animal pole and another side is known as the vegetal
pole. The nucleus is surrounded by a plasma membrane known as oolemma. This oolemma leads to
the rise of microvilli which is responsible for absorbing the food material to facilitate the growth of
the cell. Three egg membranes namely outer corona radiata, middle zona pellucida, and inner
plasma membrane cover the cell. Follicle cell forms corona radiata and is the outer layer of the
cell-attached with zona pellucida. Zona pellucida forms the vitelline membrane of the cell that
surrounds the egg. This membrane is thick and transparent. The plasma membrane is the innermost
layer. There is some space between zona pellucida and plasma membrane known as perivitelline
space.

2.4 MECHANISM OF FERTILIZATION

Fertilization involves the fusion of two haploid cells namely sperm and ova to produce a new diploid
organism.
Types of Fertilization: There are two different types of fertilization:
1. External Fertilization: The fusion happens outside the body. It occurs in the environment. Various
physical conditions like temperature, water, soil, humidity, etc., affect the fertilization process.
2. Internal Fertilization: The fusion happens inside the female body. This fertilization is free from
changes in physical conditions like temperature, water, soil, humidity, etc. of the environment.
FACTORS INVOLVED IN FERTILIZATION:
1. Chemotaxis: Some animal eggs attract the sperms by the release of certain chemicals. These
chemicals are found in the chorion lining the micropyle. Once the chorion is removed the activities
of sperm are ceased. This is found in coelenterates, fishes, insects, etc.
2. Life Span of Gametes: In External Fertilization life span is short whereas in internal
Fertilization is long which can vary from 17 hrs in Rats to 4 years in Turtles inside the female
genital tract.
3. Production of an enormous number of sperms: A large number of sperms are produced to
fertilize the lesser number of the egg.

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4. Mechanical Juxtaposition of Gametes: This refers to the mechanical position by which sperm
can reach the ovum. It is different from animal to animal like Sepia uses an arm to transfer their
sperm, copulation in mammals, etc.
5. Synchrony in the Production and Release of Gametes: The synchronization of male and
female maturity and their release to facilitate fertilization.
The fertilazation process involves two different phases namely Pre-Fertilization and Post Fertilization
Step 1: Pre –Fertilization: This refers to the process which occurs before the fusion of gametes.
Step 2: Post- Fertilization: This refers to the process after the fusion of gametes Fusion of male and
female gametes produces a new organism in the fallopian tube and its attachment to the uterus of the
female body.

Fig 2.3 Steps of Pre- Fertilization

2.4.1 PRE- FERTILIZATION

Pre Fertilization comprises the five stages:
STEP 1: Bringing of sperm and ova to the same place: Most Invertebrates and vertebrates
maintain a close distance between spermatozoa and ovum by the act of some behavior.
Spermatozoa and ovum need to be in a liquid medium at the same time for fertilization. There are
two types of fertilization based on nature and place. In External Fertilization, eggs are released on a
liquid medium followed by the release of sperm near them. In Internal Fertilization they are
brought together by an act of copulation. In some animals especially where the fertilization is
external Sperm are attracted to eggs by the release of chemicals called Chemotaxis. These
Chemotaxes are released by the egg. These chemicals are present in the chorion lining the
micropyle.
STEP 2: Capacitation and contact: Capacitation refers to the act where the sperm fertilizes the
egg of the same species. There exists a set of chemicals i.e fertilizins and antifertilizins which

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ensure that fertilization will occur between the gametes of the same species. These chemicals also
ensure that only one sperm can fertilize the egg.

Fig. 2.4: Mechanism of Fertilizin and Anti fertilizin Reaction

Fertillizin – antifertilizin reaction: F.R. Lillie postulated the Fertilizin and Anti Fertilizin Theory.
This reaction between Fertilizin and Anti Fertilizin ensures the sperm and ova of a certain species
can only mate. Fertilizin is the chemical found on the surface of the egg. These Fertilizin molecules
have many receptors or binding sites to bind with the sperm. These receptors are species-specific to
fuse with the sperms of the same species. These glycoproteins molecules are embedded with the
jelly coat or plasma membrane of the ovum.
Anti-Fertilizin is the chemical found on the surface of sperm. They are acid proteins with a smaller
molecular weight than fertilizin. This anti-fertilizin is also species-specific. The reaction between
Fertilizin and Anti Fertilizin is quite analogous to the lock and key mechanism of enzymes.
Mechanisms of Fertilizin and Anti fertilizin Reaction:
1. Sperms identify the eggs and react by identifying with Fertilizin molecules.
2. The first attachment between egg and sperm is due to the link between fertilizin and anti-
fertilizin particles.
3. Eggs are released in a liquid medium. Due to the liquid medium, few molecules of fertilizin
combine with the medium. These start to attract all the sperms currently in the same
medium.

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4. This aggregation of sperms leads to agglutination. Only a few sperms reach the surface of
the egg. This mechanism helps to reduce polyspermy.
5. This action ensures that fertilization will be done within species.
6. Fertilizin activates the sperm to initiate the acrosome reaction to dissolve the egg
membrane.
STEP 3: Acrosome reaction and penetration: Eggs (Ovum) are covered with one or more layers
of membrane or gelatinous or follicle cell to prevent the egg from getting fertilized except in
Porifera and Coelenterates. Spermatozoa need to break these layers to make the process of
fertilization. Spermatozoa get attached to these layers and become motionless. The acrosome found
in the head of the sperm performs physiochemical activity to break these layers. Acrosome releases
a certain enzymatic protein called sperm lysins. In mammals corona radiata acts as a barrier that
prevents spermatozoa to reach the ovum. To facilitate the sperm acrosome has an enzyme called
hyaluronidase which aids in dissolving the adhesive layer and cell disbursement. The acrosome
undergoes morphological change and transforms into a filament to help the release of sperm inside
the egg.

Fig. 2.5: Stages of the sperm-egg association during fertilization

Acrosome reaction and penetration in Saccoglossus: Colwin and Colwin, 1967 described the
acrosome reaction and penetration in Saccoglossus. In Saccoglosus spermatozoon have a spherical
nucleus, a tail, and an acrosomal vesicle at the front. These acrosomal vesicles are surrounded by
acrosomal granules except at the apex. Apex has a space filled with a material called peri
acrosomal material.
The following events occur when sperm comes in contact with an egg. They are
1. Bursting of acrosome: Acrosomal apex bursts and expose acrosomal vesicles.

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2. Lytic enzymes are released: Acrosomal vesicles touch the egg layer and release Lytic
enzymes to make passage through the layer.
3. Acrosomal tubules are formed: Acrosomal membrane starts to move towards the nucleus
and form a long slender tubule.
4. Fusion of acrosomal tubule with egg membrane: Acrosomal tubule enters the egg after
Lytic enzyme has made a passage through the membrane.
Sperm Contents are passed: Acrosomal tubules dissolve and the other material of the sperm gets
mixed with the egg cytoplasm.
1. The nucleus of the spermatozoon moves towards the fertilization cone.
This process remains the same for other animals. It is the number and size of acrosomal tubules
which vary from species to species.
STEP 4: Ovum activation: This process starts when the sperm touches the surface of the egg
plasma membrane facilitated by acrosome filament. The fusion of the two eggs takes place and a
single mosaic membrane is formed. Thus plasma membrane of both cell become a single cell called
zygotes. A lot of changes occur in the cytoplasm of the egg. These changes have been summed up
in the following steps:
1. Fertilization Cone Formation.
2. Formation of membrane and cortical reaction.
1. Fertilization cone formation: The acrosomal filament of spermatozoa touches the egg
membrane, and the cytoplasm of the egg moves forward at the point of contact and forms a
structure that appears like a simple conical protrusion. The cytoplasm started to engulf the
spermatozoa. Spermatozoon doesn’t enter the egg cytoplasm intact instead they are spread over.
The Sperm nucleus and other sperm structures move towards the fertilization cone. The plasma
membrane of spermatozoa mixes with the egg plasma membrane to form a single entity. The
acrosomal granule stays outside the egg membrane; only peri acrosomal material enters the egg
cytoplasm.
Some variation exists in how the spermatozoon is taken into the interior of the egg. In mammals,
the entire spermatozoon penetrates the egg cytoplasm. In Nereis only the head and proximal
centriole enter the egg cytoplasm. However, as a golden rule in the majority of animals, the sperm
nucleus and mid-piece enter the egg.

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Fig. 2.6: Fertilization cone formation

2. Formation of membrane and cortical reaction: A set of physical-chemical reactions occur

inside the egg before the spermatozoa start to penetrate them. The set of physical-chemical
reactions is known as a cortical reaction. This cortical reaction role is concerned with the formation
of membrane outside the egg plasma with the sole objective to prevent the arrival of late
spermatozoa inside the egg.
The process of membrane formation varies from animal to animal. A glimpse of cortical reactions
and membrane formation in some animals has been discussed.
Sea Urchins: Upon touching of acrosomal tubule with egg surface, color changes from yellow to
white (under dark field microscopy), which travels rapidly around egg cortex. It is followed by the
formation of a fertilization cone and membrane around the egg plasma membrane. Sea Urchin
unfertilized egg has egg cortex bound by two membranes namely vitelline and inner thick plasma
membrane. A layer is formed below the plasma membrane. The fertilization membrane is formed
in the following way:
Outer vitelline membrane is separated and undergoes an expansion to become the outer layer.
During expansion cortical granules explode and release three components namely Dark, denser,
lamellar, and folded parts, Globules, and the liquid component.
All the three component form different structures namely
1. Dark, denser, lamellar, and folded part: They fuse with the inner side of the vitelline
membrane.
2. Globules: They form the surface of the hyaline layer. This hyaline help to keep the
blastomeres together.

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3. Liquid component: They fill the perivitelline space between the new egg surfaces. It
contains mucopolysaccharides and water.
All three components together form a fertilization membrane (Slow block to polyspermy)
Vertebrates: Upon touching of acrosomal tubule with the egg surface, Lytic digestive enzymes are
released by the acrosomal tubule to penetrate the egg membrane. The cortical granules are broken
into egg cytoplasm and their content becomes liquefied and comes on the surface and fills the
perivitelline space between the chorion and plasma membrane. In fishes chorion becomes
hardened. In mammals, cortical granules fill the space between egg plasmalemma and the zona
pellucida.
An animal that lacks cortical granules such as urodele amphibians, some lower order mammals. In
those animals neither a membrane is formed nor does neither any cortical reaction take place.

Theory of Activation: Several Theories have been proposed to illustrate how sperm stimulates the
eggs. They are
1. Theory of Boveri: Boveri proposed that the mature egg has no active division center. This
division center is transferred into the egg by the sperm to initiate the process of cell
division.
2. Theory of Loeb: Loeb, 1913 proposes two principles namely the lytic principle and
cytolysis principle. The lytic principle states the factors which increase the oxidation
process and the cytolysis principle deals with controlling the increased oxidation rate.
3. Theory of Bataillon: Bataillon proposed that the secretion of a substance in the
perivitelline membrane and elevation of the fertilization membrane activate the egg to start
fertilization.
4. Viscosity Theory: Heilbrunn postulated that calcium is released during fertilization which
increases the viscosity of egg cytoplasm. This increased calcium concentration starts the
development process.
5. Deinhibition Theory: Runnstrom and Brachet observe that metabolic inhibitors
accumulate in the oocyte during the maturation process. These inhibitors are removed and
eggs are deinhibited.
Step 5: Pronuclei migration and amphimixis: The sperm nucleus remains compact with
mitochondria and centrioles behind it after penetrating the membrane of the egg. The sperm
nucleus has to perform two functions to be able to perform amphimixis.

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1: Becomes pronuclei.
2: Migrate from the site of amphimixis.
1: Becomes Pronuclei: After the penetration of the egg by spermatozoon, the nucleus starts to
move inwards from the site of cone formation. The nucleus rotates by 180o to enable mitochondria
and centrioles to assume the first position. The sperm nucleus starts to swell. The chromatin
material inside the cell starts to become granular. It starts to look more like an interphase nucleus
called a male pronucleus.
2: Migrate from the site of amphimixis: Sperm centriole is surrounded by sperm aster in the egg
cytoplasm. Sperm aster starts to lead the nucleus to the site of amphimixis (fusion of male and
female gametes).
.

Fig. 2.7: Copulation Path

The egg nucleus also undergoes some changes. The Haploids nucleus of the egg fuses into one
another and forms a female pronucleus. They also swell and become vesicular. The female
pronucleus also starts to move towards the site of amphimixis.
The site of fusion lies near the center of the microlecithal and telolecithal egg or at the center of the
active cytoplasm. The path that which male pronucleus follows depends upon the effect of the
chemical released by the female pronucleus. The normal path followed by the male pronucleus is
known as the penetration path and a new path subjected to some changes is known as the
copulation path

2.4.2 POST FERTILIZATION

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Spermatozoa penetration of the egg results in a significant change in the position of cytoplasmic
constituents. Several new areas emerge within the egg. Cortical granules extrusion results in
replacing the outer egg cell surface with the inner surface with everted on the exterior.
The most significant change in the cytoplasm has been observed in ascidian and frog. A bilateral
symmetry has been established in the cytoplasm of both animals in the fertilized egg. A Zygote is
formed after the rearrangement of the cytoplasm. Displacement has been quite remarkable in the
ascidian, frog, and Styela partita. This need arise to rearrange the nucleus and genetic material
equally.
Displacement of cytoplasmic material in ascidian Styela partita:
The mature egg of Styela partita is covered with a layer of cortical cytoplasm which contains
yellow granules. As the spermatozoon enters the egg, the cytoplasm starts a violent commotion.
The cytoplasm starts to move towards the vegetal pole and arrange itself into the shape of a cap. As
sperm move towards the egg nucleus, the cytoplasm moves upward towards the direction from
where spermatozoa entered. This start to enter four different regions of the cytoplasm. They are
1. Yellow cytoplasm on one side.
2. Light color cytoplasm on the other side.
3. Slaty grey color cytoplasm containing yolk granules and mitochondria.
4. Clear and transparent Cytoplasm
This displacement separates the cytoplasm and arranges them to start the process of cleavage.

Fig. 2.8: Displacement of cytoplasmic material in ascidian Styela partita

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Fertilization ensures that the cycle of cleavage can start, which will become the first step of embryo
development. This is followed by morulation, Blastulation, Gastrulation and finally, a new
multicellular organism is brought into the world.

2.4.3 BIOCHEMISTRY OF FERTILIZATION

Metabolic activation: A series of cytoplasmic reactions take place after the sperm penetrates the
unfertilized egg. The steps involved are:
a. Changes in plasma membrane
b. Ionic changes
c. Changes in the rate of respiration
d. Co-enzyme changes
e. Rate of protein synthesis
f. Mitosis initiation
g. Breakdown of polysaccharides
h. Increase in hexose phosphate
i. Dehydrogenase
a. Changes in the plasma membrane: The sperm penetration has increased the movement of
water and other chemicals like ethylene, glycol, phosphate, K+, etc. This led to the increase of
electrical potential in the plasma membrane. It became more positive, but slowly turns negative due
to unequal distribution of chloride ions.
Adenyl cyclase, an enzyme of plasma membrane gets activated at the time of fertilization and
initiates the formation of 3’ – 5’ cyclicAMP molecule. This molecule is responsible for activating
metabolic reactions in a fertilized egg.
b. Ionic changes: A substantial level of changes in the intracellular composition of the fertilized
egg takes place at the ionic level. This is especially in sodium, potassium, and calcium. This
change in ion concentration had a great influence on the metabolism activation of the fertilized
egg.
c. Changes in the rate of respiration: The rate of respiration varies from animal to animal. In
some animals where fertilization is completed (Sea Urchin), it increases whereas in others it either
decreases (Molluscs) or remains stable (Bufo).
The increased rate is associated with the increased oxygen demand for oxidation of glycogen and
the release of numerous ATP molecules.

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d. Co-enzyme changes: Spermatozoa contain an acrosome that has a lot of oxidative enzymes.
These enzymes come inside the egg after fertilization and ensure an increase in oxidation. This
increase in oxidation is to provide energy for the development of egg and supplement other
changes necessary for it.
The following process takes place:
NADKinase
NAD + ATP NADP + ADP
e. Rate of protein synthesis: The cytoplasm of an unfertilized egg contains a lot of protein
synthesis material like DNA molecules, tRNA, mRNA, ribosomes, and proteolytic enzymes.
During fertilization, proteolytic activity increase to remove the inhibitor protein and mRNA is
unmasked to start the process of active protein synthesis.
f. Mitosis initiation: The process of active protein synthesis increases the rate of DNA synthesis.
The sperm initiate the first mitotic division (Cleavage) by contributing centriole to the egg. Though
centriole is there in the unfertilized egg, they are incapable of performing mitotic division. This
sperm centriole becomes the basics of the new organism gender as half of DNA material is
contributed by sperm and another half by egg.
g. Breakdown of polysaccharides: An increase in lactic acid concentration takes place on account
of the rapid breakdown of polysaccharides.
h. Increase in hexose phosphate: This chemical increases considerably after fertilization.
i. Dehydrogenase: The activity of the enzyme performing dehydrogenase increase significantly
after fertilization.
SIGNIFICANCE OF FERTILIZATION:
1. Maintain diploidy in the race.
2. Bring genetic variation.
3. Egg is activated to start the process of Cleavage.

2.5 SUMMARY

Sexual reproduction has evolved to improve the chance of an individual or species. This
reproduction has evolved by the fusing of male and female gametes. These gametes have evolved
with the climatic situations, adaptation, and their habitat and habitat. The male gamete called
spermatozoa has evolved as a structure where it can carry the genetic material, move through the
liquid medium, have sufficient resources to penetrate the ovum, and the mechanism to provide
energy while traversing to fertilize the egg. To facilitate them it has a head (for genetic material),

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neck (to join the tail), Middle piece (to provide energy), and tail (traversing through liquid medium
to meet ovum).
Ovum develops as a non-motile, spherical, or oval shape and it is larger than the sperms. A large
amount of cytoplasm provides the energy required to fertilize an egg for its developmental journey
to an embryo. A mechanism of protection where only one sperm of the same species can penetrate
the egg and fertilize it. It also has villi for absorbing the food material to facilitate the growth of the
cell.
Fertilization has evolved to maintain genetic variation and diploidy in the race. There are two types
of fertilization: external fertilization and internal fertilization. A significant set of factor influence
fertilization like life span of gametes, and the production of an enormous number of sperm.
mechanical juxtaposition of gametes, their synchrony in production and release of gametes, and the
mechanism by which sperm will fertilize the egg.
The process of fertilization has two different phases namely prefertilization and post fertilization.
Prefertilization refers to the process which occurs before the fusion of gametes. This
prefertilization is further made up of 5 steps namely: Bringing sperm and ova to the same place,
Capacitation and contact, Acrosome Reaction, Ovum Activation, and Pronuclei migration and
Amphimixis. Post Fertilization refers to the process which occurs after fertilization has been done.
A lot of metabolic activities take place after the sperm penetrates the unfertilized egg. They involve
changes in the plasma membrane and ionic changes. Changes in the rate of respiration, Co-enzyme
changes, Rate of protein synthesis, and Mitosis initiation. Thus these activities lead to further
development of cleavage, morulation, and gastrulation. These steps finally form a multicellular
animal.

2.6 TERMINAL QUESTIONS AND ANSWER

2.6.1 MULTIPLE CHOICE QUESTIONS:

1. Which of the following animal has the longest duration of sperm survival in the female genital
tract?
a. Rat
b. Garter Snake
c. Guppy
d. Turtle

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2. Which of the following animal has the shortest duration of sperm survival in the female genital
tract?
a. Rat
b. Hen
c. Rabbit
d. Bat
3. In Mammals ________ fertilization occurs.
a. Monospermic
b. Polyspermic
c. Depends On Species
d. Depends on climatic situation
4. Pre-Fertilization is made up ________ steps
a. 2
b. 3
c. 5
d. 4
5. A set of chemicals which are released by an egg to attract the opposite sex gametes
a. Fertilizins
b. Anti-Fertilizins
c. Glycoprotein
d. Sperm lysins
6. A set of chemicals that are released by sperm during fertilization.
a. Fertilizins
b. Anti- Fertilizins
c. Glycoprotein
d. Sperm lysins
7. A mechanism of protection where only one sperm of the same species can penetrate the egg and
fertilize it.
a. Fertilization
b. Cortical reaction
c. Capacitation
d. Sperm lysins

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8. Ova mature in the _________ of females.

a. Fallopian Tube
b. Uterus
c. Ovary
d. None of these
2.6.2 VERY SHORT QUESTION
1. Discuss the structure of sperm.
2. What is the difference between external and internal fertilization.
3. How fertilization helps in maintaining genetic variation and diploidy in the race.
4. Explain the basic structure of the ovum.
5. Illustrate the role of acrosomes in sperm.
6. Explain the process of formation of membrane and cortical reaction in fertilization.
7. Why a change in the rate of respiration occurs during fertilization?
8. Name the steps which the sperm nucleus has to perform to be able to perform Amphimixis.
9. How prefertilization is different from postfertilization.
10. Discuss fertilizin and anti fertilizin reactions mechanisms.
2.6.3 LONG QUESTIONS:
1. Give a details description of any four metabolic changes in the ovum followed by fertilization.
2. Explain the influence of physical factors like climate, temperature, humidity, etc. on
fertilization.
3. What type of metabolic changes happens during fertilization?
4. Explain the significance of the mechanical juxtaposition of gametes during the process of
fertilization.
5. Explain the process which occurs before fertilization.
6. Discuss the various type of fertilization.
7. Discuss the process of ovum activation during fertilization.
8. Explain the significance of capacitation in fertilization.
9. Explain how sperm structure has evolved to facilitate fertilization.
10. Explain the various metabolic activities that occurred during fertilization.
11. Explain the various physical, chemical, and cytological factors involved in fertilization.
Answers: 2.6.1
1(d) 2(c) 3(a) 4(c) 5(a) 6(b) 7(c) 8(c)

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2.7 REFERENCES

1. Development Biology (1997), Sinauer Gilbert 5th Edition Ass. Publ. Massachusetts.
2. Developmental Biology (2011), Wolpert 5th Edition Oxford Press.
3. Analysis of Biological Development (2000) 2nd Edition, Klaus Kolthoff McGraw- Hill Science,
New Delhi, India.
4. Introduction to Embryology (2012) 5th Edition B.I. Balinsky. Cengage Learning.
5. Development Biology (1979) N J Berrill Tata McGraw Hill.
6. Developmental Biology (2018): Shastry and Shukla 2nd Edition Rastogi Publication Meerut
7. Chordate Embryology (2007) P.S Verma, VK Agarwal 2nd Edition: S Chand Publication New
Delhi

2.8 GLOSSARY

Acrosomes: A part of a spermatozoon that is capable of releasing enzymes to break the ovum
membrane.
Anti Fertilizin: A protein released by sperm to help them identify and attach with the same species
of an egg.
Capacitation: The mechanism by which the sperm will fertilize the egg.
Corona radiate: The area of follicular cells surrounding the ovum.
Egg: The cell released by the ovary of the female which is capable to produce new organisms after
fusing with the Male sex gametes.
Fertilization: The process in which two sex gametes namely sperms and ovum fuse together to
form a new organism.
Fertilizin: A protein released by the ovum to stimulate the sperm to move towards them.
Microlecithal Egg; Egg that has little or no yolk.
Oxidation: The process by which energy-giving molecules or matter are oxidized to produce
energy.
Proteolytic enzyme: Enzyme that is capable of producing or releasing protein.
Sperm: The Male sex gametes
Telolecithal Egg: Egg that has a significant amount of yolk which remain concentrates at one
pole.

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UNIT 3: CLEAVAGE, BLASTULATION AND GASTRULATION

CONTENTS
3.1 Objectives
3.2 Introduction
3.3 Patterns of Cleavage
3.4 Determinate and Indeterminate Cleavage
3.5 Influence of Yolk on Cleavage
3.6 Metabolic Changes during Cleavage
3.7 Morulation and Blastulation in Frog, Chick and Rabbit
3.8 Types of Blastulae
3.9 Major events of Gastrulation and Fate maps
3.10 Morphogenetic movements in Frog, Chick and Rabbit
3.11 Significance of Gastrulation and Exogastrulation
3.12 Summary
3.13 Terminal Questions and Answers

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3.1 OBJECTIVES

After studying this module, you shall be able to learn and understand:
 Patterns of cleavage
 Understand how yolk influences cleavage
 Understand the significance of blastulation
 Elucidate the morulation and blastulation in frog, chick, and rabbit
 Classification of blastula
 Major events of gastrulation
 The significance of fate maps.
 Understand the morphogenetic movements in frogs, chick, and rabbits
 Understand the significance of gastrulation and exogastrulation
 Understand the different movements occurring during gastrulation.

3.2 INTRODUCTION

All sexually multicellular organism bodies have developed from a single cell formed after the
fusion of male (spermatozoa) and female gametes (ovum). The process of further cell division
starts called Cleavage just after fertilization. A series of mitotic cell divisions divide the single cell
into 16-cell called mesomeres. A total of 4 mitotic divisions are followed which converts the single
cell into 2, 4, 8, and 16 mesomeres. When the stages of 16 cells are attained they get transformed
into a multicellular structure called morula. This division is further transformed into blastula by
successive cell division. This blastula has a single layer of the blastoderm. No growth of ovum
during blastula formation. The general shape doesn’t change for the embryo except for a formation
of a cavity called the blastocoel. The chemical change of glycogen and yolk into molecules of
nuclear material like DNA, RNA, and nucleoproteins take place. The sudden cell division has
increased the number of the nucleus which leads to the unfavorable ratio between the nucleus to the
cytoplasm. This is brought to normal by the end of blastulation. Blastulation is followed by
Gastrulation where the single layer of blastoderm is transformed into two and finally three germ
layers namely ectoderm, mesoderm, and endoderm. The cell moves from one part to another and
rearranges itself to start the process of development of several parts and organs of a multicellular

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organism. Fate Map has been designed to understand which part of the blastula transformed itself
into which part of a multicellular animal.

3.3 PATTERN OF CLEAVAGE

CLEVAGE: Cleavage is a process where a series of mitotic divisions happens after egg
fertilization. The zygote undergoes a rapid succession so that the unicellular body will be
transformed into a multicellular organism. The mitotic division led to numerous smaller nucleated
cells called blastomeres which finally results in a hollow spherical body called a blastula.
CHARACTERISTICS:
1. Thousands of cells called blastomeres are formed.
2. Cleavage produces a hollow sphere of cells called a blastula.
3. Cleavage forms the cell to create tissue and organs.
4. Embryo shape and size don’t change during cleavage.
5. Cleavage brings a proposition between nuclear and cytoplasmic material.
6. Conversion of yolk, and glycogen into the cytoplasm, then into a molecule of a nuclear
substance.
PRINCIPLE OF CLEAVAGE: All cleavages follow common basic principles or laws.
They are as follows:
A. Sach's law–Sach in 1877 proposes two rules.
Rule 1: Cleavage divisions occur uniformly subjected to the uniform yolk stored in them. It will
not be uniform if the yolk is unevenly stored in them.
Rule 2: Successive Division is at a right angle to its previous division.
B. Hertwig law -O. Hertwig in 1881 proposed 2 rules:
Rule 1: Nucleus and mitotic spindle is found at the center of blastomeres.
Rule 2: Spindle fibrosis is the longest axis of the egg.
C. Pfluger'sLaw - Eduard Friedrich Wilhelm Pflüger state that the formation of spindle fibers
takes place in the region of less yolk.
D. Balfour's Law–Balfour in 1885 stated that the rate of cleavage is inversely proportional to the
amount of yolk.

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Fig 3.1: Types of Cleavage

Cleavages are of different types due to changes in egg organization. They are of the following
types namely-

Radial Cleavage: Cleavage divides the Zygote in radial symmetry. Their cleavage divisions are at
a right angle to their previous division. The successive divisions of the cell are placed just above
the previous blastomeres. Thus, the new four blastomeres are arranged just above their four
previous blastomeres. Thus, partitioning the blastula along any plane produce two identical halve.
Example Echinodermata, Chordata, Frog.

Fig 3.2: Radial Cleavage

Biradial Cleavage: Cleavage has two different patterns in the mitotic division. The First
two mitotic divisions are meridional and the third division is vertical. Thus the 8 blastomeres
formed don’t stand at a right angle to each other. Example: Polychaeta, Ctenophora.

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Fig 3.3: Biradial Cleavage

Bilateral Cleavage: This cleavage has two identical halves when the blastula is cut vertically. It
can be right or left. This cleavage occurs due to unequal holoblastic (produce blastomere of
unequal size) cleavage. The plane of bilateral symmetry is established by the plane of the first
cleavage furrow. Example: Higher Mammals, ambiphians, tunicates.

Fig 3.4: Bilateral Cleavage

Spiral Cleavage: This cleavage has a rotational movement of cell parts around the north or south
pole axis of the egg. This led to the inclination of the mitotic spindle concerning symmetry radii.
Thus, each division produces one bigger cell (macromere) and a smaller cell (micromere). The
following cleavage produces the increase in inclination which results in an arrangement in a spiral
shape. If the rotation is in a clockwise direction then it is called a dextral or right-handedCleavage,
else if in an anti-clockwise direction then it is called a sinistral or left-handed Cleavage. Examples:
Nematoda, Rotifers, Annelids, Mollusca, annelids.

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Fig 3.5: Spiral Cleavage

3.4 DETERMINATE AND INDERTERMINATE CLEAVAGE

Cleavages are also been classified according to the potential of the blastomeres to develop for
future development. They are classified as determinate and indeterminate cleavage.

Determinate Cleavage: In this cleavage, the area has been marked for the development of a
region from different parts of the egg quite early before the onset of Cleavage. In the ascidian egg,
a region marked for the development of endoderm was removed. It resulted in the absence of
endoderm when the embryo was formed later. Examples: Nematodes, Annelids, Mollusks, and
Ascidian.

Indeterminate Cleavage: In this cleavage, there is no area marked for the development of a
region. This Cleavage is quite flexible. A region generally used for the development of endoderm
was removed from fertilized eggs of sea urchins. Yet the endoderm was developed in the final
embryo. This cleavage simply cut the eggs into segments with each segment having the potential to
develop any region. Example: All Vertebrates and some species of Echinoderms.

3.5 INFLUENCE OF YOLK ON CLEAVAGE

Cleavage has also been divided on the basis ofYolk and its distribution in an egg:
The following patterns have been observed:

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Fig 3.6: Classification of Cleavage based on Yolk

1. Total or Holoblastic Cleavage

2. Meroblastic Cleavage

1. Total or Holoblastic Cleavage: The entire cell is divided by each furrow.

Fig 3.7 Total or Holoblastic Cleavage

This has been further divided into:

Equal holoblastic: This Cleavage produces blastomeres of equal size in any symmetry. This
symmetry can be radial, biradial, spiral, and bilateral. This Cleavage is found in microlecithal and
isolecithal eggs. Examples: Amphioxus, Marsupials, and placental mammals.

Fig 3.8 Equal holoblastic

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Unequal holoblastic: This Cleavage produces blastomeres of unequal size in any symmetry. This
symmetry can be radial, biradial, spiral, and bilateral. They produce small-size blastomeres called
micromeres and large-size blastomeres called macromeres. This Cleavage is found in mesolecithal
and moderately telolecithal eggs. Example: Lower Fish and amphibians.

Fig 3.9: Unequal holoblastic

2. Meroblastic Cleavage: This cleavage divided the cell partially. This result in the creation
of unequal size micromeres. This cleavage is found in those eggs which have a patch of
yolk-free cytoplasm called blastodisc. The first two or three cleavage divides the blastodisc
vertically, but never reaches the bottom of the blastodisc. The yolk part of the ovum is
never cut by furrow.

Fig 3.10: Meroblastic cleavage

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This has been further divided into:

1. Discoidal: This type of Cleavage is found in macrolecithal and telolecithal eggs. The
cleavage is restricted to the disc-shaped active cytoplasm of the animal pole. Example:
bony fish, reptiles, birds.

Fig 3.11: Discoidal cleavage

2. Superficial: This type of Cleavage is found in a centrolecithal egg. The cleavage is

restricted to the peripheral cytoplasm of an egg. The zygote nucleus divides without
cytoplasm division. So a large number of nuclei are formed and get embedded in the
cytoplasm superficial layer. Example: Arthropoda, and Insects.

Fig 3.12: Superficial Cleavage

Influence of yolk: The yolk is always an integral part of the egg. Its quantity varies significantly
from microlecithal to telolecithal egg. Cleavage occurs in the active cytoplasm. Yolk behaves
passively during cleavage. Yolk has a significant influence on cleavage by regulating the process of
mitosis by extending beyond the dense area of the egg and moving the germ layer to its final
position.
The influence of yolk can be summed by:

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1. The yolk is responsible for moving the zygotic nucleus from the geometrical center of the
egg to the less yolky cytoplasm. This results in unequal-sized blastomeres.
2. The yolk is responsible to retard the process of mitosis. Thus, its amount and distribution
affect the process of cleavage.

3.6 METABOLIC CHANGES DURING CLEAVAGE

Chemical change during cleavage: A significant change occurs during the process of
cleavage. They are namely:
1. Increased Nuclear Material: A steady increase in the genetic material (predominantly
DNA) has been seen. The cytoplasm of the egg contains mitochondria and yolk platelets aid
in an increase in nuclear material by acting as a source. A large amount of energy is
required to facilitate the movement of genetic material towards the pole. These ATP
molecules are made in ooplasm and mitochondria through glycolysis and aerobic oxidation
of yolk, glycogen, and other energy-yielding molecules. A continuous supply of
deoxyribonucleotides, ribonucleotides, purines, pyrimidines, amino acids, and ribose is
needed for the synthesis of DNA and RNA.
2. RNA Synthesis: mRNA (messenger RNA) and tRNA (Transport RNA) are synthesized in
large numbers during cleavage.
3. Synthesis of Protein: There has been a steady increase in protein synthesis during the
entire process of cleavage.

3.7 MORULATION AND BLASTULATION IN FROG, CHICK AND

RABBIT

Cleavage splits the fertilized egg into smaller cells called blastomeres. These blastomere increases
in the typical double sequence of 2, 4, 8, 16 so on. Cleavage forms the layers where each layer is
loosely joined together by a stacking gel. This heap of cohering, sticky blastomeres is known as
Morula. This has been named as its resemblance to mulberry (Morula means mulberry in Latin).
The arrangement of blastomere varies among animals. For example: In a megalecithal egg, a
planoconvex-like mass of blastomere is formed. The morula stage is followed by the next phase of
development called a blastula. Cleavage led to an increase in the number of blastomeres. This
blastomere undergoes a rearrangement which results in arranging themselves into a single cell thick

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epithelium called blastoderm. A fluid-filled space or cavity called blastocoel appears between the
blastomeres. This hollow, spherical and nonepithelial thick embryonic stage is called a blastula.
This process of creating a blastula is known as blastulation.
Morulation in Frog: Cleavage is the early stage of embryogenesis. The cleavage leads the ovum
cell partitioning from a single cell to a 32 cell using orders of 2-4-8-16 and finally 32. Cleavage in
frogs produces unequal microsomes with a smaller one (micromere) and the larger one
(megamere). The cleavage after 32 cells becomes quite difficult to follow. Micromeres divide more
rapidly as compared with megamere. This is because micromere has less or lacks yolk. The zygote
starts to appear like a mulberry-shaped solid ball of the cell. This mulberry-shaped ball of a cell is
known as a morula.

Fig 3.13: Morulation in frog

Morulation in Chick: Cleavage starts to happen after 3 hours of fertilization. In birds due to the
availability of enormous yolk, the cleavage cannot happen in one furrow. This led to the rise of
small-sized cells called mesomeres. Cleavage in birds is initially restricted to blastodisc with yolk
remaining unaffected. The first cleavage is restricted to the area around the center of the blastodisc.
It is superficial with no blastomere formed. The second cleavage just happens at the right angle to
the first cleavage. 3rd Cleavage is formed just parallel to the first and is vertical. Thus eight
blastomeres are formed with no signs of a boundary. 4 th Cleavage results in the formation of eight

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central blastomeres and eight peripheral blastomeres. It is after the 4th Cleavage a clear
demarcation of a cell is seen. The eight central blastomeres get completely separated from the yolk.
After the 4th cleavage division becomes irregular. The central blastomeres and peripheral
blastomeres start to divide rapidly. The cell of peripheral blastomere is added with central
blastomeres resulting in increased volume. These cells start to arrange themselves resulting in the
formation of a cavity called the blastocoel. The cleavage in birds is partial, teloblastic, or
meroblastic.

Fig. 3.14: Morulation in chick

Morulation in Rabbit: Cleavage in mammals tends to be holoblastic but unequal. The first
cleavage divides the cell vertically into two slightly unequal blastomeres. The second cleavage is at
the right angle to the first, dividing vertically resulting in four blastomeres. The third cleavage is
also at the right angle to the second with cleavage occurring in four blastomeres separately. Thus, a
total of 8 blastomeres are created by the end of the third cleavage. At this rate of division, the 16-
cell stage is reached. This 16-cell stage is known as morula. Fully formed morula has an outer or
superficial layer of cells called the trophoderm or trophoblast. This morula stage passes through the
oviduct to enter the uterus. Later this morula makes an attachment with the mother uterus and
absorbs liquid fluid.

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Fig. 3.15: Morulation in rabbit

Blastulation in Frog : Blastulation stages start after morulation when one cell has grown into 32
cells (blastomere). These blastomeres start to arrange at the periphery and a small fluid cavity or
space starts to form within the embryo. This cavity is known as a blastocoel or segmentation
cavity. The whole embryo formed is called a blastula. The process of formation of blastula is
known as blastulation. After the formation of the blastula, the process of formation of body parts
starts with a specific area marked within the cell. These are:
 Presumptive ectoderm: The region of the animal pole of the blastula.
 Presumptive Notochord: An area near the vegetal pole.
 Presumptive Mesoderm: An area close to the notochord.

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Fig. 3.16: Blastulation in frog

Blastulation in Chick: The morulation stage is of short duration. The cell undergoes further
division resulting in the creation of several layers with their complete boundaries. Cell present near
the periphery is not free from yolk known as marginal cells. This region is known as the zone of
the junction.
The area in the center of the blastoderm is free from yolk with four to five layers of the cell, these
cells undergoes arrangement. Space is created between blastoderm and yolk. This created space is
known as blastocoel. This region is called area pellucida and is transparent. Area pellucida form the
core of the embryo. This region is in contact with a region known as area opaca. Area opaca is
responsible for the extra-embryonic structure.
Area opaca is opaque and white. They are differentiated into three more or less distinct zones. In
birds, embryo blastomeres grow on the surface of a large yolk sphere. This blastomere forms an
outer ring with no well-defined boundary.
An inner layer within the embryo is in close contact with the yolk. Thus two types of cells are
found one with large yolk laden and another with small or yolk-free blastomeres. Yolk-laden
blastomeres accumulate the under the surface of the blastoderm with yolk free at the surface. This
led to the formation of two layers namely the upper layer and lower layer. These layers are called
epiblast and the lower layer hypoblast respectively. A thin cleft appears between the epiblast and
hypoblast. This cleft is known as blastocoels.

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Fig. 3.17: Blastulation in chick

Blastulation in Rabbit: A fluid-filled cavity appears within the morula called the blastocoel
appears. This is then known as a blastocyst or blastula. This embryo grows in size with the liquid
food getting collected in the cavity, and separating the outer layer of the small trophoblast cell from
the inner cell mass. This embryo is now known as a blastocyst. Inner cell mass grows inside
Blastocyst and becomes a knob-like thickening at one pole. This knob is known as an embryonal
knob because all parts of the embryo will be derived from it. Later this blastocyst becomes attached
to the uterus of the mother and the villi absorb food from the villi.

Fig. 3.18: Blastulation in rabbit

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3.8 TYPES OF BLASTULAE

There are six types of blastulae found in the animal kingdom with differencing on various factors
like size of the egg, amount and distribution pattern of yolk, and rate of cleavage.
1. Coeloblastula: This blastula is hollow and the blastocoel is surrounded by a single-layer
cell. Examples: Echinoderms, Amphioxus, and frog.
2. Stereoblastula: This blastula is solid and has no blastocoel. Examples: Annelids,
Molluscs, Nemerteans, and some species of planarians.
3. Discoblastula: This blastula is a multilayered flat disc at the animal pole separated by
narrow segmentation from the yolk. They are found in eggs with a large and developed
yolk. Examples: Reptiles, Birds, Prototherians, and Fishes.
4. Blastocyst: This blastula has a regular cleavage and a small cavity inside each cell. Two
types of cells are found namely the outer layer of epithelial having nutritive cells and the
inner mass of the formative cell. Example: Mammals.
5. Superficial blastula or peri blastula: This blastula has blastocoel which is filled with yolk
and surrounded by a peripheral layer of cell Example. Insects.
6. Amphiblastula: This blastula is made up of two different types of structurally different
blastomeres. Example: Amphibian

Fig. 3.19: Types of blastulae

3.9 MAJOR EVENTS OF GASTRULATION AND FATE MAPS

Gastrulation is a process of formation of germ layers. This formation involves complex cell
movements that rearrange those cells within themselves. This is one of the most important phases
of embryonic development.
Gastrulation involves the following major events:

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1. Morphogenic Movement (Movement of blastular cells or blastomeres): Blastomeres

move in a different direction to form multiple patterns to develop germ layers and start the
process of developing multicellular organisms.
2. The rate of cell division (Cleavage) is slowed down: Before reaching the stage of
Gastrulation, Cleavage has produced a sufficient number of cells, so the emphasis lay on
how to arrange those cells so that they can be used as per the requirement.
3. Types of Metabolism changes and oxidation rate increases: Gastrulation emphasized
developing germ layers and the start of developing different parts of the animal. This
requires a different type of protein synthesis. To facilitate the different proteins that are
been secreted and oxidation rate increases.
4. Nuclei control embryonic cell activities: Gastrulation starts the process of differentiation
where different parts of the animals are formed based on the genetic material. The cell
nucleus stores the genetic code and the arrangement of the blastomeres are been done on the
basics of the genetic code. Thus, the Nuclei enforce the genetic code in managing the
process of gastrulation.
5. Chemo-Differentiation is started: Gastrulation initiates the process of chemo-
differentiation where the blastomeres are subjected to a different set of chemicals and
proteins to mold the blastomere's behavior. This molding of blastomeres results in the
development of different parts of the multicellular animals.

Major Events of Fate Map: Fate map is the process of mapping which part of the blastula will
develop into which organs in the embryo. It varies from animal to animal.
Fate map in frog: In frog the area demarcated for the different part are already been demarcated
at the end of cleavage.
1. Macromeres of vegetal pole will become endoderm.
2. Micromeres of the animal pole will become ectoderm.
3. Prechordal both sides will become mesoderm.
4. Micromeres present near notochord and mesoderm becomes neurectoderm.

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Fig. 3.20: Fate Map of frog

Fate map in chicks: In chicks after the formation of hypoblast, the area marked for which organs
can be detailed:
1. The hypoblast area forms the endoderm.
2. Epiblast forms the various organ of the chick.
3. Area opaca forms the extra-embryonic membrane and blood vessels.
4. Area pellucida forms epidermal ectoderm, neurectoderm, prechordal, notochordal and
mesoderm cells.

Fig. 3.21: Fate Map of Chicks

Fate map in mammals: Fate Map in Mammals is marked after the blastocyst stage.
1. The Innermost layer forms the endoderm.
2. The embryonic disc forms the ectoderm and mesoderm.

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3. Anterior germ disc forms the epidermal ectoderm.

4. Behind the crescent, the area forms the notochord.
5. Behind the notochordal area forms a prechordal plate.
6. The trophoblast forms the ectoderm layer of the chorion.

3.10 MORPHOGENETIC MOVEMENTS IN FROG, CHICK AND

RABBIT

Morphogenetic movements occur during gastrulation:

These movements are classified according to the number of cells migrating together.
Movement by individual cell:
i. Migration – It is a movement where an individual cell moves over to the other cells.
ii. Ingression– It is the movement of individual cells from epithelium into a cavity.
Groups of cells move by: Types of Morphogenetic movement based on the direction. They are
classified as epiboly and emboly.
Epiboly - In this movement, a group of cells arranges themselves to form an outside cell layer to
cover the yolk. They do it by thinning the layers. Their movement is over ectoderm.

Fig. 3.22: Diagram of classification

Emboly: Their movement is over Endoderm and mesoderm. They are of 4 Types
i. Invagination– It is a movement where the cell moves inward.
ii. Involution - It is an inward movement of a group of cells or epithelial sheets around a
point or an edge. This is done to form an underlying layer.
iii. Delamination - In this process cells splits themselves into two different cell layers namely
epiblast (outer layer) and hypoblast (inner layer).

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iv. Convergent Extension– In this process, two or more cell rows move together intending to
elongate the structures in one dimension while shortening them in another direction.

Fig.3.23: Morphogenetic movements

3.11 SIGNIFICANCE OF GASTRULATION AND

EXOGASTRULATION

Gastrulation: The process in which a blastula or a single layer of the cell is converted into
multiple layers of cells called gastrula is known as gastrulation.
Significance:
 Gastrulation starts the process of a multicellular organism. It sets the process of developing
different parts of the body and brings the morphology changes in the embryo.
 Morphological changes bring an increased metabolic activity of the cells.
 Gastrulation brings the three primary germ layers i.e. ectoderm, mesoderm, and endoderm.
This starts the process of developing skin.
 Gastrulation shows the influence of paternal chromosomes.
 The Blastocoel cavity is transformed into archenteron.

Exogastrulation: The abnormal gastrula in which the mesoderm, notochord, and endoderm are
made to evaginate to the outside. This is just the opposite of the normal process where inward
movement takes place. In this process, the embryo never develops. It is done experimentally to the
induced disturbance in the embryo.
Significance:
1. Exogastrulation helps to understand the factors which affect normal gastrulation.

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2. Exogastrulation helps to understand how the cells behave and divide and form structure
after being subjected to certain chemicals and proteins.
3. The gained knowledge can be used in research and development so that certain
embryonically can be modified as per the needs.

3.12 SUMMARY

All multicellular organisms have evolved from a single cell which is a result of the fertilization of
sex gametes. These gametes' fusion starts to divide themselves into several mitotic divisions. These
mitotic divisions led to an increase in the number of cells from 2 to 16. This process of division is
known as cleavage. These cleavages have been different depending on the size of the egg, amount
and distribution pattern of yolk, rate of cleavage, etc.
The cleavage brings a significant change in catabolic and anabolic activities of the newly created
cells called mesomeres. All cleavage follows four basics law namely Sach’s Law, Hertwige Law,
Plugger Law, and Balfour Law. Cleavage leads to the stage of morula where each cleavage cell
arranges itself over the cell of its previous cleavage loosely joined together by a stacking gel.
Morula is a 16-celled stage arranged in the shape of a mulberry. The morula stage is short-lived.
The cell division process continues and starts to take on the next phase of development called a
blastula. Cleavage led to an increase in the number of blastomeres. This blastomere undergoes a
rearrangement which results in arranging themselves into a single cell thick epithelium called
blastoderm. A fluid-filled space or cavity called blastocoel appears between the blastomeres. This
hollow, spherical and nonepithelial thick embryonic stage is called a blastula. This process of
creating a blastula is known as blastulation where a single layer of blastoderm is created.
Gastrulation follows blastulation where a single layer of blastoderm is changed into three germ
layers i.e. mesoderm, ectoderm, and endoderm. This transformation requires Morphogenic
movement (movement of blastulae cells or blastomeres), rate of cell division (cleavage) to be
slowed, metabolism changes and oxidation rate increases, and nuclei control the embryonic cell
activities. These morphological changes can be inwards or outwards known as epiboly and emboly
respectively. A lot of research has been done to understand the behavior of the morphological
movement of blastulae cells. It is done to induce a disturbance in the embryo. This inducement of
making an embryo work differently from the normal embryo is known as exo-gastrulation. This
exo-gastrulation has assumed a significant role in understanding and developing an embryo as per
the requirement.

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3.13 TERMINAL QUESTIOS AND ANSWERS

3.13.1 MULTIPLE CHOICE QUESTIONS:

1. Cleavage produces a hollow sphere of cell called __________.
a. Morula
b.Blastomere
c.Blastula
d. Mesomere

2. Nematodes, annelids, molluscs and ascidian follows___________ cleavage.

a. Determinate
b.Indeterminate
c. Both a and b
d. None of them

3. In ______ cleavage blastula can be divided into two identical halves.

a. Biradial cleavage
b. Radial cleavage
c. Bilateral cleavage
d. Spiral cleavage

4. Cleavage is divided into ________ types based on yolk distribution in an egg.

a. 1
b. 2
c. 3
d. 4

5. This process of creating a blastula is known as:

a. Gastrulation
b. Cleavage
c. Blastulation

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d. Morulation

6. ________ blastula is made up of two different types of structurally different blastomeres.

a. Coeloblastula
b. Stereoblastula
c. Discoblastula
d.Amphiblastula

7. In ________ step of development nuclei controls the embryonic cell activities.

a. Gastrulation
b. Morulation
c. Blastulation
d.Cleavage

8. In _________ morphological movement cells splits themselves into two different cell layers
namely epiblast (outer layer) and hypoblast (inner layer).
a. Invagination
b. Involution
c.Delamination
d. Convergent Extension

9. In this process the embryo never develops. It is done experimentally to induce a disturbance in
the embryo. Who am I?
a. Cleavage
b. Gastrulation
c. Exo-Gastrulation
d. Blastulation

10. In ________ process blastocoel cavity is transformed into archenteron.

a. Cleavage
b. Gastrulation
c. Exo-Gastrulation

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d. Blastulation
Answers: 1(c), 2(a), 3(b), 4(b), 5(c), 6(d), 7(a), 8(c), 9(c), 10(b)
3.13.2 VERY SHORT QUESTION
1. Discuss the Significance of Gastrulation and Exo-Gastrulation.
2. How Epiboly is different from Emboly.
3. How Exo-Gastrulation will help in understanding how the embryo develops.
4. Name the various Law which governs Cleavage.
4. Name the various morphogenetic techniques used in the animal kingdom.
5. Write the various characteristics of cleavage.
6. Explain the morulation in the frog.
7. Name the stage where the roles of genetic material first come during the process of development
of the multicellular animal.
8. How invagination is different from involution.
3.13.3 LONG QUESTIONS
1. Explain how determinate and indeterminate cleavages are different from each other. Write
the name of animals that exhibits deterministic and indeterminate cleavage.
2. Explain the influence of yolk on cleavage.
3. What type of metabolic changes happen during cleavage?
4. Explain the significance of morulation and blastulation.
5. Explain the various type of cleavage. How they are different from each other?
6. Discuss the various types of blastulae.
7. Illustrate the significance of gastrulation. Explain the various events that happen in
gastrulation.
8. Illustrate the significance of the fate map. How fate map of frog is different from
mammals?
9. Explain the various morphogenetic movements that happen during gastrulation.
10. Explain the process of blastulation in frogs and rabbits.
11.

3.14 REFERENCES

1. Development Biology (1997), SinauersGilbert 5th Edition Ass. Publ. Massachusetts.

2. Developmental Biology (2011), Wolpert 5th Edition Oxford Press.

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3. Analysis of Biological Development (2000) 2nd Edition, Klaus Kolthoff McGraw- Hill Science,
New Delhi, India.
4. Introduction to Embryology (2012) 5th Edition B.I. Balinsky.Cengage Learning.
5. Development Biology (1979) N J BerrillTata McGraw Hill.

3.15 GLOSSARY

Archenteron: Cavity in an embryo during the gastrulation stage which forms the digestive tract.
Blastocoel: The fluid-filled cavity of the blastula.
Blastomere: cell which is the result after the division of a fertilized egg
Blastula: a 32-celled structure of an embryo that is hollow and is surrounded by an inner-filled
fluid called the blastocoel.
Cleavage: Process of mitotic cell division of fertilized egg.
Ectoderm: The outermost layer of the germ layer
Emboly: The process of movement of gastrula during gastrulation where cells move inwards to
form archenteron.
Endoderm: The innermost layer of the germ layer
Epiboly: The process of movement of gastrula during gastrulation where cells move outwards to
cover up the yolk and the remaining cell form the ectoderm.
ExoGastrulation: The process in which an embryo is induced with some disturbance to see the
behavior of the gastrula.
Gastrulation: Stage of embryo development where three germ layers are formed and the process
of development of body parts and organs starts.
Holoblastic: Cell which undergoes a complete cleavage.
Meroblastic: Cell which undergoes partial cleavage only.
Mesoderm: The middle layer of the germ layer
Morula: A 32-celled structure resembling mulberry which further makes blastula.
Trophoblast: the membrane of cells that forms the wall of a blastocyst during early pregnancy,
providing nutrients to the embryo and later developing into part of the placenta
Yolk: The Yellow and the main food of the egg

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UNIT 4: EARLY DEVELOPMENT

CONTENTS
4.1 Objectives
4.2 Introduction
4.3 Neurulation and Ectoderm origin and fate of Neural Crest Cells
4.3.1. Neurulation
4.3.2 Primary Neurulation
4.3.3Secondary Neurulation
4.4 Development of Mesoderm
4.5. Development of Endoderm
4.6 Summary
4.7 Terminal Questions and Answers

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4.1 OBJECTIVES
After studying this module, you shall be able to learn and understand:
1. The tissue, cellular, and molecular basis for neural induction and neural tube formation.
2. Explain how neuronal precursors are generated in the CNS.
3. The early changes in neural tube shape and the formation of the primary brain vesicles.
4. How two important signaling molecules, sonic hedgehog (Shh) and bone morphogenic
protein (BMP-4), regulate the expression of regional distinctions in the nervous system.
5. Where and how the neural crest forms, the origin of the migratory pathways that lead crest-
derived cells to stop specific targets.
6. Secondary Neurulation

4.2 INTRODUCTION
Neurulation is a process in which the neural plate bends up and later fuses to form the hollow tube
that will eventually differentiate into the brain and the spinal cord of the central nervous system. In
humans, it begins in the 3rd week after fertilization and requires that the top layers of the embryonic
germ disc elevate as folds and fuse in the midline. Neurulation is the embryological process that
forms the precursors of the central nervous system and occurs after gastrulation. It has established
the three primary cell layers of the embryo: ectoderm, mesoderm, and endoderm. In humans, the
majority of this system is formed via primary neurulation, in which the central portion of the
ectoderm—originally appearing as a flat sheet of cells—folds upwards and inwards, sealing off to
form a hollow neural tube. As development proceeds, the anterior portion of the neural tube will
give rise to the brain, with the rest forming the spinal cord. The epidermis, the central and
peripheral nervous systems, and some non-neuronal cells of the head and heart are derived from
ectoderm (Fig. 4.1). During the third week of gestation, a portion of the dorsal ectoderm is
specified to become neural ectoderm. This region of the embryo is called the neural plate. The
process by which the neural plate forms a neural tube is called neurulation.

4.3 NEURULATION AND ECTODERM ORIGIN AND FATE OF

NEURAL CREST CELLS

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The original ectoderm can be divided into three sets of cells: (i) the internally positioned neural
plate, (ii) the externally positioned future epidermis of the skin, (iii) and the neural crest cells that
connect the neural plate and epidermis.
At the time the neural plate becomes specified, an interaction between the surface ectoderm (SE)
and neural plate (NP) creates an intermediate structure, known as the neural crest (Fig. 4.2).

Fig.4.1 Cross sections through the forming neural tube (Human Embryology & Developmental Biology, 2 nd
edition, Carlson, B. M.)

The neural plate folds in stages to form the neural tube and the Scanning electron micrographs of
chick embryosis are depicted in Fig. 4.2. During neural tube closure, when the dorsal tips of the

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neural folds converge, these cells delaminate from the dorsal neural tube and migrate from their
original site in the neuraxis, via specific routes, to colonize peripheral targets.
At the junction of the neural and surface ectoderm, there is another population of cells, which is
called the neural crest. As the neural folds begin to appear, neural crest cells (NCCs) can be seen in
their tips through the expression of characteristic markers, like the Pax7 transcription factor. As
development proceeds and the neural folds are fused, NCCs are seen either in the topmost portion
of the neural tube or migrating along this structure’s sides towards the lower regions of the embryo.
Crest-derived cells are capable of differentiating into an astonishing number of different and
diversified cell types and tissues including Schwann cells or glial cells of the sensory, sympathetic,
parasympathetic, and enteric nervous systems, cells of the adrenal medulla, pigment cells in the
epidermis, and connective tissue components of the head (Fig. 4.3 and 4.4) yet they express only
those phenol types that are appropriate for the organ to which they have migrated.

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Fig.4.2: The neural plate folds in stages to form the neural tube. (Scanning electron micrographs of chick
embryos are provided by G. Schoen wolf.) A. Position of the neural plate about then on neural ectoderm, the
mesoderm, and the endoderm. B. Foldingof the neural plate to form the neural groove. C. Dorsal closure of
the neural folds to form the neural tube and neural crest. D. Maturation of the neural tube and its position n
relative to the axial mesodermal structure, notochord, and somites (derived from the paraxial mesoderm).
(Adapted from Jessell & Sanes, Principles of Neuroscience 4thedition, 2002, E. Kandeleditor)

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Fig. 4.3: Some derivatives of the neural crest (After Jacobson, 1991)

4.3.1. NEURULATION
Neurulation is a process in which the neural plate bends up and later fuses to form the hollow tube
that will eventually differentiate into the brain and the spinal cord of the central nervous system. In
humans, it begins in the 3rd week after fertilization and requires that the top layers of the
embryonic germ disc elevate as folds and fuse in the midline. Neurulation is the embryological
process that forms the precursors of the central nervous system and occurs after gastrulation.
4.3.2. PRIMARY NEURULATION
This term refers to the formation of the neural tube from the neural plate, situated between the
anterior and posterior neuropores (fig.4.6).
1. Neural induction formation of the neural plate: Neural induction is the first step whereby the
uncommitted or naïve ectoderm becomes committed to the neural lineage. During gastrulation,
signals from the node or its derivative, the notochord, induce commitment. Classical studies led to
the notion that inducing substances, secreted by the underlying prechordal plate and the cranial
portion of the notochordal plate, were responsible for ectodermal commitment to a neuronal
lineage by the overlying epiblast cells. There is now good evidence that neural induction’ actually
involves suppression of duction of an epidermal fate rather than induction of a neural fate so that

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the default state of the naïve ectoderm is neural, not epidermal as suggested by older studies. In
amphibians, molecules (e. g. noggin, chordin, follistatin) that inhibit the expression of bone
morphogenetic protein 4(BMP-) appear to block epidermal expression. Although the suppression
signal is generated by Hensen’s node in birds, suppression of BMP-4maynotbe the only
requirement for neural induction in mammals. The original ectoderm can be divided into three sets
of cells: (i) the internally positioned neural plate,(ii) the externally positioned future epidermis of
the skin, (iii) and the neural crest cells that connect the neural plate and epidermis. Lateral folding
or bending of the neural plate results in elevation of two walls, the neural folds, flanking a ventral
midline floor plate (composed of non-neuronal cells) of the neural groove.

Fig. 4.4: Major derivatives of the ectodermal germ layer.The ectoderm is divided into three major domains
the surface ectoderm (primarily epidermis), the neural tube (brain and spinal cord), and the neural crest
(peripheral neurons, pigment, facial cartilage). (Gilbert, DevelopmentalBiology, 6 th edition)

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The principal early morphological response of the embryonic ectoderm to neural specification is an
increase in the height of the cells destined to become components of the nervous system. These
transformed cells, now known as neuroepithelial cells or neuroectoderm, are evident as a thickened
neural plate visible on the medial dorsal surface of the early embryo.
2. Shaping of the Neural Plate: At the time of its formation, the neural plate is shaped like a spade
being relatively wide mediolaterally and short rostro caudally (Fig. 4.5). The caudal wings of the
spade flank the primitive node. During shaping, the nascent neural plate becomes narrower and
longer. Although the processes of neurulation and gastrulation can be uncoupled experimentally,
full craniocaudal formation and extension require the normal cellular movement of gastrulation.
3. Formation of the neural tube: It occurs when the two dorsolateral apical surfaces of the neural
folds meet, fuse at the dorsal midline, and separate from the overlying ectoderm. Forces generated
by the surface epithelium as it expands towards the dorsal midline cause elevation of the neural
folds and ultimately, closure of the neural tube. The bends in the medial portion of each neural fold
maintain the structure of the tube so that the lumen remains patent as the neural folds converge.

Fig. 4-5: A schematic sequence showing how the neural plate grows and changes proportions between da18
and day 20. The primitive streak shortens only slightly, but it occupies a progressively smaller proportion of
the length of the embryonic discs the neural plate and embryo grow (Larsen, 3rd edition)

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The molecular signals for primary neurulation in human embryos (Fig.4.6) remain largely
unknown but several candidate genes that perturb neurulation when mutated have now been
identified. Sonic hedgehog (Shh) is an important signaling center. Not only does it induce elevation
of neural folds but also the formation of the neural groove and floor plate. In the dorsal portions of
the future neural tube, Wnt6, secreted by the epidermal ectoderm adjacent to the neural plate and
BMPs induce slug in the future neural crest cells. The BMPs also appear to maintain the dorsal
expression of Pax transcription factors. Shh signaling from the floor plate, suppresses the
expression of dorsal Pax genes in the ventral half of the neural tube where motor neurons devlop.
Closure of the neural tube begins almost midway along the craniocaudal extent of the nervous
system of the 21-22 day human embryo (Fig. 4.6 A, B). Over the next couple of days, closure
extends both cephalically and caudally in a manner resembling the closing of a double-headed
zipper. The unclosed cephalic and caudal parts of the neural tube are called the anterior (cranial)
and posterior (caudal) neuropores.
The neuropores will ultimately close (24 days gestation for the cranial neuropore and 26days for
the caudal) so that the future central nervous system (CNS) is organized in a way that resembles an
irregular cylinder sealed at both ends. Neural tube defects occur when various parts of the neural
tube fail to close. An open posterior neuropore result causes spina bifida (Fig. 4.6 E), the severity
of which depends on the length and position of the open segment. Anencephaly (Figure 4.7 D) is a
lethal condition in which the anterior neuropore fails to close. The forebrain remains in contact
with the amniotic fluid and subsequently degenerates.

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Fig.4.6: Dorsal and ventral signaling in the early central nervous system. A. Signals from sonic hedgehog
(Shh) (orange arrows) in the no-to chord induce the floor plate. B. In the dorsal part of the future neural
tube, Wnt from the ectoderm adjacent to the neural tube induces slug in the future neural crest and
maintains Pax-3 and Pax-7 expression dorsally. Ventrally, the sonic hedgehog, now produced by the floor
plate, induces motoneurons. C. Sonic hedgehog, produced by the floor plate, suppresses the expression of
dorsal Pax genes (Pax-3 and Pax-7) in the ventral half of the neural tube. (Carlson,
HumanEmbryology&DevelopmentalBiology, 2nd edition).

Fig.4.7: Neurulation in the human embryo: (A) Dorsal and transverse sections of a 22 day human embryo
initiating neurulation. Both anterior and posterior neuropores are open to the amniotic fluid. (B)Dorsal
view of the neurulating human embryo a day later. The anterior neuropore region is closing while the
posterior neuropore remains open. (C) Regions of neural tube closure postulated by genetic evidence
(superimposed on newborn body). D) Anencephalyis caused by the failure of neural plate fusion in region 2.
(E)Spina bifida is caused by the failure of region 5 to fuse (or of the posterior neuropore to close). (C-Eafter
VanAllen et al.1993.)(Gilbert, DevelopmentalBiology, 6th edition)

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4. The neural tube forms the primordia of the central nervous system: Even before the
neuropores have closed the future brain and spinal cord are recognizable and the brain becomes
subdivided into a forebrain (prosencephalon), midbrain (mesencephalon), and hindbrain
(rhombencephalon) (Fig. 4.8 and 4.9). The increased volume of the early brain is the result of an
increase in cavity size, not tissue growth. In the chick embryo, brain volume expands 30 fold
between days 3 and 5 of development. This rapid expansion is thought to be caused by pressure
from fluid exerted against the walls of the neural tube after the surrounding dorsal tissues push in to
temporarily constrict the neural tube in the region between the presumptive brain and spinal cord
(Fig. 4.8). The concluded region reopens after the initial rapid enlargement of the brain vesicles.
Another prominent force in shaping the early nervous system is the overall bending of the cephalic
end of the embryo into a C-shape.

Fig. 4.8: Early human brain development. The three primary brain vesicles are subdivided as development
continues. At the right is a list of the adult derivatives formed by the walls and cavities of the brain. (After
Moore and Persaud1993)(Developmental Biology, 6th edition, S.Gilbert)

Soon the brain almost doubles back on itself at the cephalic flexure. At the beginning of the fifth
week, a second cervical flexure appears at the boundary between the hindbrain and the spinal cord.
By the end of the fifth week, the prosencephalon becomes further subdivided into a telencephalon
and a more caudal diencephalon with prominent optic vesicles extending from its lateral walls. The
rhombencephalon divides into the metencephalon and more caudally, the yelencephalon. These
five primary brain vesicles, plus the spinal cord, comprise the early fundamental organization of
the CNS.

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The original neural tube is lined by a ventricular zone, composed of a single layer of rapidly
dividing neural stem cells, called the neuroepithelium (sometimes known as a germinal
epithelium). All the cells of the neuroepithelium extend to the luminal surface but their nuclei are at
different heights thereby giving the structure a pseudostratified appearance. DNA synthesis (S
phase) occurs while the nucleus is positioned at the outside edge of the zone. As the cell cycle
proceeds, the nucleus migrates within the cell cytoplasm toward the lumen. Mitosis occurs at the
luminal side of the ventricular zone and the two daughter cells then continue to cycle. A cell that
has undergone its last mitotic division and is derived from a stem cell that divides parallel to the
ventricular surface. The daughter cell adjacent to the lumen remains connected to the ventricular
surface, continuing in the cell cycle, while the post-mitotic daughter migrates out of the germinal
epithelium.

5. Neuronal survival depends upon target related trophic signals: It should be noted that not all
neuroblasts survive. Of the huge number generated, nearly half are destined to undergo apoptosis
and die (Fig. 4.8). Only those neurons that make structural and functional synaptic connections
with specific targets are not eliminated. Both in CNS and PNS, neuronal survival and neuronal cell
death are under the tight developmental control of gene products secreted by target structures.
These trophic factors are required to sustain growth and survival.
6. The Neural Crest: At the time the neural plate becomes specified, an interaction between the
surface ectoderm (SE) and neural plate (NP) creates an intermediate structure, known as the neural
crest (Fig. 4.9). During neural tube closure, when the dorsal tips of the neural folds converge, these
cells delaminate from the dorsal neural tube and migrate from their original sites in the neuraxis,
via specific routes, to colonize peripheral targets. Crest-derived cells are capable of differentiating
into an astonishing number of different and diversified cell types and tissues including Schwann
cells or glial cells of the sensory, sympathetic, parasympathetic, and enteric nervous systems, cells
of the adrenal medulla, pigment cells in the epidermis, and connective tissue components of the
head, yet they express only those phenotypes that are appropriate for the organ to which they have
migrated.
7. Origin of neural crest migration pathways that lead crest-derived cell to target organs:
Studies utilizing avian chimeric embryos (Le Douarin and colleagues) have provided a great deal
of information regarding the specificity of individual neural crest migration pathways, as well as
the development potential and restriction of crest-derived phenotypes. Neural crest migration

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routes originate from specific sites along the cranial-caudal axis (neuraxis) of the dorsal neural
tube. The migration pathways lead the dividing crest-derived cells to specific end targets where
they stop dividing and differentiate into target-related phenotypes. Thus, the site in the neuraxis
from which a crest cell originates determines the target it will reach (Fig.4.9). Heterotopic (ectopic)
transplantation of crest cells into a migration pathway that they normally do not traverse, leads
them to a new target where, depending on their developmental potential, they may express a new
phenotype that is appropriate for the target they have colonized. Research conducted on
mammalian embryos suggests that except for relatively minor structural details, information
learned from birds can be directly applied to mammalian development.
The crest-derived cells that reach a target at the end of the migration pathway are different than
those that entered it. As they migrate, they encounter extracellular signaling molecules, e.g. growth
factors and trophic factors, and components of the extracellular matrix, e. g. fibronectin, laminin,
and collagen, which are conducive to their continued migration and proliferation. As they migrate,
the crest-derived cells develop appropriate receptors that allow them to interact with these
environmental cues by the time they reach their specific target, and their number has increased
significantly.

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Fig. 4-9: The neurotrophic factor hypothesis. (Adapted from Reichardt andFarinas1997) A.
Neuronsextendaxonstothevicinityof target cells. B.The target cells secrete limited amounts of neurotrophic
factors.Theneurotrophic factors bind to specific cell surface receptors. Neurons that do not receive
adequate amounts of neurotrophic factor die by apoptosis. (Jessell& Sanes, Principles of
Neuroscience/2000, E. Kandeleditor).

Fig.4.10: Cross sections through the forming neural tube. A. Neural plate. B. Neural fold, C. Neural folds
appose and D. Neural tube complete (Human Embryology & Developmental Biology, 2nd edition, Carlson,
B.M.)

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8. The cranial neural crest: Crest-derived cells in the head region produce the craniofacial
mesenchyme that differentiates into cartilage and bone, cranial neurons and glia, and connective
tissues of the face. Other cells enter pathways traversing pharyngeal structures where they give rise
to such diversified cells as those of the thymus, odontoblasts of the tooth primordia, and the bones
of the middle ear and jaw (Fig. 4.11).

Fig.4.11: Regions of the neural crest.The cranial neural crest migrates into the branchial arches and the
face to form the bone and cartilage of the face and neck. It also produces pigment and cranial nerves. The
vagal neural crest (nearsomites1-7) and the sacral neural crest (posterior to somite28) form the intrinsic
neurons of the gut. The cardiac neural crest cells arise from the neural crest near somites 1-3; they are
critical in dividing the aorta and the pulmonary artery. Neural crest cells of the trunk (about somite 6
through the tail) make the sympathetic neurons, and a subset of these (at the level of somites18-24) form the
medullary portion of the adrenal gland. (After Le Douarin 1982.) (Developmental Biology, 6thedition, S.
Gilbert)

9. Genetic potential, developmental restriction, differentiation: Developmental regulation of

neural crest cell differentiation requires activation and expression of appropriate transcription
factors and receptors. Some populations of neural crest-derived cells are pluripotent and although

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they are capable of generating a remarkable number of differentiated cell types, their phenotypic
repertoire is limited to the expression of those gene products that are appropriate for the target to
which they have migrated. Heterotopic transplantation of these cells reveals their greater
phenotypic capacity. Other crest-derived cells may constitute a more restricted population of stem
cells. There are only a limited number of options in their genetic repertoire. Finally, some pre-
migratory crest cells appear to be programmed for a specific developmental fate or if they are not
committed before leaving the neuraxial crest, they are inhibited from further developmental
expression during their migration.
4.3.3. SECONDARY NEURULATION
Secondary neurulation is a morphological process described since the second half of the 19th
century; it accounts for the formation of the caudal spinal cord in mammals including humans. A
similar process takes place in birds. This form of neurulation is caused by the growth of the tail bud
region, the most caudal axial region of the embryo. Experimental work in different animal species
leads to questioning dogmas widely disseminated in the medical literature. Thus, it is established
that the tail bud is not a mass of undifferentiated pluripotent cells but is made up of a juxtaposition
of territories whose fate is different. The lumens of the two tubes generated by the two modes of
neurulation are continuous.

Fig. 4.12: Axial section of chick embryo from rostral (A) to caudal (E). This figure shows the first historical
illustration of secondary neurulation in chick embryos. Multiple lumens can be evidenced in secondary
neural tubes (A and B). C: The primordium of the spinal cord is formed as a solid cellular structure.D and
E: Doros-ventral gradient of epithelialization (Adopted from Catala, 2021).

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There seem to be multiple cavities in the human embryo, but discrepancies exist according to the
authors. Finally, the tissues that generate the secondary neural tube are initially located in the most
superficial layer of the embryo. These cells must undergo internalization to generate the secondary
neurectoderm. A defect in internalization could lead to an open neural tube defect that contradicts
the dogma that a secondary neurulation defect is closed by definition. Caudal to the posterior
neuropore, the neural tube is formed by the process of secondary neurulation (Fig. 4.12).A rod-like
condensation of mesenchymal cells forms beneath the dorsal ectoderm of the tailbud. Within the
mesenchymal rod, a central canal forms by cavitation.This central canal becomes continuous with
the one formed during primary neurulation and closure of the posterior neuropore. Because of the
diminished development of the tail bud in humans, secondary neurulation is not a prominent
process (Martin Catala, 2021).
PRIMARY VS SECONDARY NEURULATION
While primary neurulation forms most of the central nervous system in humans, a small area of the
posterior spinal cord results from a distinct process called secondary neurulation. In this region,
rather than having three distinct cell sheets, the embryo contains a mixture of loosely-packed cells
covered by a thin layer of ectoderm. Some of these “loose” cells condense to generate a rod-like
structure called the medullary or neural cord. This cord eventually hollows out, and merges with
the more anterior primary neural tube, forming a continuous structure. Although secondary
neurulation plays a relatively minor role in the formation of the human central nervous system,
defects in this process can still have developmental consequences, such as certain types of spina
bifida.

4.4 DEVELOPMENT OF MESODERM

The mesoderm generates all the organs between the ectodermal wall and endodermal tissues.
During the third and fourth weeks of embryonic development the mesoderm is established as the
2nd germ layer. The mesodermal cells are organized into 4 regions: the axial mesoderm of the
prechordal plate and notochord, paraxial mesoderm, intermediate mesoderm and lateral plate
mesoderm. Each of these undergoes some form of segmentation. The most evident and complete
segmentation occurs in the paraxial trunk mesoderm, where each segment becomes an entirely
separate somite. Much of the paraxial and lateral plate mesoderm develops into mesenchyme, an
embryonic connective tissue. The derivatives of mesenchyme are connective tissue proper,

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cartilage, bone and blood. The cardiovascular and lymphatic systems are derived from mesoderm
as well. Part of the paraxial mesoderm gives rise to all skeletal muscle cells. The intermediate
mesoderm gives rise to most of the urogenital system. Part of the lateral plate mesoderm develops
into the lining of the pericardial, pleural and peritoneal cavities.

Mesoderm Formation: Gastrulation is a series of cell movements that transforms the bilaminar
germ disc (epiblast and hypoblast) into a 3 layered embryo (ectoderm, mesoderm, and endoderm,
Fig. 4.13). Not only are the 3 germ layers established but cells also become committed to
endodermal or mesodermal lineages during this process. The critical factors that determine the
different fates of the mesodermal cell populations are: 1) the point of entrance of the epiblast cells
into the primitive streak and 2) the direction of their subsequent migration. Depending on these 2
events, mesodermal cells can form tissues as varied as muscle, heart, kidney, or bone. After
gastrulation the mesodermal sheet on either side of the notochord is a connected layer of
undifferentiated mesenchymal cells (Fig. 4.13). During the third week, this undifferentiated
mesoderm will begin to condense on both sides of the notochord to form the 1) paraxial, 2)
Intermediate and 3) lateral plate mesoderm (Fig. 4.14). Starting on day 20 at what will be the base
of the skull, the paraxial mesoderm (just lateral to the notochord; also called the segmental plate)
begins to condense in a cranial to caudal direction (Fig. 4.15). These condensations will become
the somites. Lateral to the paraxial mesoderm is the intermediate mesoderm. As the embryo begins
to fold the intermediate mesoderm will lose its connection with the segmental plate and condense
to form a solid mass of tissue running most of the length of the embryo. The intermediate
mesoderm will give rise to the kidneys (two embryonic forms and the final adult form) and most of
the urgenital tract, including gonadal tissue but excluding the primordial germ cells.

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Fig. 4.13: Diagrammatic view with cross sections ofembryo during gastrulation

Fig. 4.14. Sections through a 17-day embryo showing the differentiation of the mesoderm on either side of
the midline. (A) Early on day 17, the mesoderm has begun to differentiate into paraxial, intermediate, and
lateral plate mesoderm. (B) Sagittal cutaway showing the rod-like condensations ofparaxial and
intermediate mesoderm. The dotted line marks the plane of thetwo transverse sections. (C) Later on day
17, the lateral plate begins to vacuolate to form the rudiment of theintraembryonic coelom.

4.5 DEVELOPMENT OF ENDODERM

Animals have bodies of diverse shapes with internal collections of organs of unique morphology
and function. Such sophisticated body architecture is elaborated during embryonic development,

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whereby a fertilized egg undergoes a program of cell divisions, fate specification, and movements.
One key process of embryogenesis is determination of the anteroposterior (AP), dorsoventral
(DV), and left-right (LR) embryonic axes. Other aspects of embryogenesis are specification of the
germ layers, endoderm, mesoderm, and ectoderm, as well as their subsequent patterning and
diversification of cell fates along the embryonic axes. These processes occur very early during
development when most embryos consist of a relatively small number of morphologically similar
cells arranged in simple structures, such as cell balls or sheets, which can be flat or cup shaped.
Gastrulation is a fundamental phase of animal embryogenesis during which germ layers are
specified, rearranged, and shaped into a body plan with organ rudiments. The term gastrulation,
derived from the Greek word gaster, denoting stomach or gut, is a fundamental process of animal
embryogenesis that employs cellular rearrangements and movements to reposition and shape the
germ layers, thus creating the internal organization as well as the external form of developing
animals. Gastrulation is a complex series of cell movements that:
a. Rearranges cells, giving them new neighbors. These rearrangements put cells in a new
environment, with the potential to receive new signals.

b. Results in the formation of the 3 germ layers that will form most of the subsequent embryo:
Ectoderm, Endoderm and Mesoderm.
The following general types of morphogenetic movements have been recognized:
a. Individual cells move by:
i. Migration -movement of individual cells over other cells or matrix.
ii. Ingression -movement of individual cells or small groups from an epithelium into a cavity.
b. Groups of cells move by:
i. Invagination -local inward buckling of an epithelium
ii. Involution -inward movement of a cell layer around a point or edge
iii. Epiboly -spread of an outside cell layer to envelop a yolk mass or deeper layer
iv. Delamination -splitting 1 cell sheet into 2 or more parallel sheets.
v. Convergent Extension -elongation of a cell layer in one dimension with shortening in another.
Epiboly: In the late blastula, the anterior half consists of micromeres which constitute the
ectoderm while the posterior megameres constitute the endoderm. The germ ring forms the
mesoderm. During epiboly the ectoderm overgrows backwards on the endoderm; ultimately the
entire embryo (except for the small area called the yolk plug) is covered by the ectoderm. In other
words the pigmented micromeres (animal half) grow over the megameres (vegetative half). The

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reason for overgrowth is the rapid rate of division of micromeres.

Invagination: A small depression is formed in the region occupied by the grey crescent area. This
depression grows inwards and forms the archenteron or gastrocoel or secondary body cavity. The
outer opening of the gastrocoel is called the gastropole. As the gastrocoel increases in size the
blastocoel gets reduced. Ultimately only a slit like semicircular cavity indicates the remnants of the
blastocoel. The blastopore meanwhile becomes expanded and becomes ring shaped.

Involution: During this process the cells which have grown backward during epiboly now roll
inside at the margin of the blastopore. The endoderm is the first to roll inside. The cells of the
notochord and mesoderm which were formed outside now migrate over the lip of blastopore and
become internal and arrange themselves on the roof, sides and the floor of the archenteron. The
notochord cells are found on the roof along the midline. While the endoderm forms the anterior,
lateral and ventral walls, the mesoderm forms wing like extensions in the archenteron.

Convergence: Convergence means the movement of cells towards a particular point. The
presumptive cells of the notochord and mesoderm located on the surface of the blastula move
towards the blastopore or primitive streak.

Infiltration: This involves the detachment of individual cells or groups of cells from the surface
of the blastula and their falling into the blastocoels. In the blastocoels they arrange themselves as a
single layer.

Divergence: It refers to the migration of involuted cells from the blastopore or primitive streak. In
divergence the cells move in different directions from a sngle point. The involuted cells of
notochord and mesoderm migrate and diverge from the blastopore and primitive streak to their
future positions within the developing embryo.

Ingression: Ingression involves movement of individual or groups of cells from the external layer
of blastula into the blastocoels. It is categorized into two types-unipolar and multipolar.
Unipolar ingression: in which individual cells migrate inwards at one end of a blastoderm.eg.
Porifera and Coelenterata
Multipolar ingression: in which individual cells migrate inwards from all points of the
blastocoels. Eg. Echidna

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Delamination: The word delamination means mass separation of groups of cells from other cell
groups. The separation of endodermal, mesodermal and notochordal cells from each other in teleost
fishes is a good example for delamination. According to a widely accepted view the endoderm
formation in birds takesplace by delamination.

Germ Layer theory and derivatives of germ layers: In 1817, Pander described a trilaminar
condition of the chick blastoderm. Later this trilayer concept was proved true for many types of
embryos and this concept became an accepted embryological principle. Towards the end of 19th
centuary the terms ectoderm, endoderm and mesoderm were introduced to refer to the outer, inner
and middle layers of the embryo respectively. The adult organs do not arise directly from the cells
derived by the cleavages of the zygote. The embryonic cells are at first arranged into layers called

germ layers from which various organs are formed. This concept is known as germ layer theory.
Tissues and organs of animals arise from layers, or blocks, of embryonic cells called primary germ
layers. Their development from a nondescript form in the early embryo to their form in late
embryonic through adult stages is called differentiation. Ectoderm, gives rise to the outer body
wall. Endoderm forms the inner lining of the digestive cavity. Mesoderm gives rise to tissues
between ectoderm and endoderm. Undifferentiated mesoderm develops into muscles, blood and
blood vessels, skeletal elements, and other connective tissues

Table 4.1: Derivatives of three germinal layers

Ectoderm Mesoderm Endoderm

Nervous tissue Connective tissues, Dermis of the skin Gut tract lining
and Muscles
Epidermis of the skin Circulatory system, Excretory Digestive glands
structures Reproductive structures
Sensory organs Bones, tendons and ligaments Respiratory tract lining

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Fig. 4.15 a: Morphogenetic cellular movement during gatrulation

Fig. 4.15 b: Morphogenetic cellular movement during gatrulation

4.6 SUMMARY
Neurulation is a process in which the neural plate bends up and later fuses to form the hollow tube
that will eventually differentiate into the brain and the spinal cord of the central nervous system. In
humans, it begins in the 3rd week after fertilization and requires that the top layers of the embryonic
germ disc elevate as folds and fuse in the midline. Neurulation is the embryological process that
forms the precursors of the central nervous system and occurs after gastrulation. It has established
the three primary cell layers of the embryo: ectoderm, mesoderm, and endoderm. In humans, the
majority of this system is formed via primary neurulation, in which the central portion of the
ectoderm—originally appearing as a flat sheet of cells—folds upwards and inwards, sealing off to
form a hollow neural tube. As development proceeds, the anterior portion of the neural tube will

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give rise to the brain, with the rest forming the spinal cord. The epidermis, the central and
peripheral nervous systems, and some non-neuronal cells of the head and heart are derived from
ectoderm. During the third week of gestation, a portion of the dorsal ectoderm is specified to
become neural ectoderm. This region of the embryo is called the neural plate. The process by
which the neural plate forms a neural tube is called neurulation. Neurulation is the embryological
process that forms the precursors of the central nervous system and occurs after gastrulation.
Primary neurulation refers to the formation of the neural tube from the neural plate, situated
between the anterior and posterior neuropores. The primary neurulation forms most of the central
nervous system in humans, a small area of the posterior spinal cord results from a distinct process
called secondary neurulation. In this region, rather than having three distinct cell sheets, the
embryo contains a mixture of loosely-packed cells covered by a thin layer of ectoderm. Some of
these “loose” cells condense to generate a rod-like structure called the medullary or neural cord.
This cord eventually hollows out, and merges with the more anterior primary neural tube, forming
a continuous structure. Although secondary neurulation plays a relatively minor role in the
formation of the human central nervous system, defects in this process can still have developmental
consequences, such as certain types of spina bifida.
The mesoderm generates all the organs between the ectodermal wall and endodermal tissues.
During the third and fourth weeks of embryonic development the mesoderm is established as the
2nd germ layer. The mesodermal cells are organized into 4 regions: the axial mesoderm of the
prechordal plate and notochord, paraxial mesoderm, intermediate mesoderm and lateral plate
mesoderm. Each of these undergoes some form of segmentation. The most evident and complete
segmentation occurs in the paraxial trunk mesoderm, where each segment becomes an entirely
separate somite. Much of the paraxial and lateral plate mesoderm develops into mesenchyme, an
embryonic connective tissue. The derivatives of mesenchyme are connective tissue proper,
cartilage, bone and blood. The cardiovascular and lymphatic systems are derived from mesoderm
as well. Part of the paraxial mesoderm gives rise to all skeletal muscle cells. The intermediate
mesoderm gives rise to most of the urogenital system. Part of the lateral plate mesoderm develops
into the lining of the pericardial, pleural and peritoneal cavities.

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Animals have bodies of diverse shapes with internal collections of organs of unique morphology
and function. Such sophisticated body architecture is elaborated during embryonic development,
whereby a fertilized egg undergoes a program of cell divisions, fate specification, and movements.
One key process of embryogenesis is determination of the anteroposterior (AP), dorsoventral (DV),
and left-right (LR) embryonic axes. Other aspects of embryogenesis are specification of the germ
layers, endoderm, mesoderm, and ectoderm, as well as their subsequent patterning and
diversification of cell fates along the embryonic axes. These processes occur very early during
development when most embryos consist of a relatively small number of morphologically similar
cells arranged in simple structures, such as cell balls or sheets, which can be flat or cup shaped.
Gastrulation is a fundamental phase of animal embryogenesis during which germ layers are
specified, rearranged, and shaped into a body plan with organ rudiments. The term gastrulation,
derived from the Greek word gaster, denoting stomach or gut, is a fundamental process of animal
embryogenesis that employs cellular rearrangements and movements to reposition and shape the
germ layers, thus creating the internal organization as well as the external form of developing
animals.

4.7 TERMINAL QUESTIONS AND ANSWERS

Q.1 What is neurulation. Explain in detail.
Q.2 what are the steps in neurulation, describe in points and illustrate in diagrams.
Q.3 Write a short note on the importance of Neurulation.
Q.4 What is secondary neurulation?
Q.5 Explain how the neural tube formed.
Q.6 Explain the process of mesoderm formation.
Q.7 Write the short note on morphogenetic cellular movements.

4.8 REFERENCES
Martin Catla. Overview of secondary neurulation. J Korean Neurosurg Soc 64 (3): 346-358,

Carlson B.M. (1999) Human embryology and developmental biology. 2nd ed. St.Louis: Mosby.

Jacobson, M. (1991). The Neural Crest and Its Derivatives. In: Developmental Neurobiology.

Springer, Boston, MA. https://doi.org/10.1007/978-1-4757-4954-0-4.

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Kandel E. R., Schwartz J. H. and Jessell T., M., 2000. Principles of Neural Science, 4th
ed. McGraw-Hill, New York. ISBN 0-8385-7701-6

Gilbert, Scott F. (1st ed. 1985; 12th ed. 2019). Developmental biology. Sunderland, MA: Sinauer
Associates.

Larsen W. J. Human embryology. New York: Churchill Livingston; 1993.

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UNIT-5 ORGANOGENESIS AND ORGANIZER CONCEPT

CONTENTS
5.1 Objectives
5.2 Introduction
5.3 Development of organs in chick
5.3.1. Development of Brain
5.3.2. Development of Eye
5.3.3. Development of Heart
5.4. Embryonic Induction
5.4.1. Determination of the Primary (1°) Organ Rudiments
5.4.2. Mesoderm induction occurs before primary embryonic induction
5.4.3. Secondary Induction
5.5. Primary Organiser and its Morphological Differentiation
5.6. Concept of organizer
5.7. Nature of Inductive Signal (Possible mechanism of neural induction)
5.8. Competence
5.9 Summary
5.10 Terminal Questions and Answers

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5.1 OBJECTIVES
This chapter will assist in understanding:
1. The mechanism of chick brain, eye, and heart development.
2. The process of embryonic induction.
3. Primary organizers and their morphological differentiation.
4. Primary organizer's point of origin and inductive contact.
5. The inductive signal's nature (Possible mechanism of neural induction).
6. Concept of competence.

5.2 INTRODUCTION
The development of an egg into a fully grown chick appears to be a wonderful magical event.
Despite being extremely complex, the process of creating a chick from an egg cannot be
understood without a rational understanding of the embryology of the growing embryo. Before the
chick hatches from the egg, it is incubated for three weeks.
Since some embryonic development has already taken place at the moment of egg laying, it is
halted until favorable environmental circumstances are created for the incubation to resume. At
first, all cells are identical, but throughout time, they begin to differentiate into distinct types, such
as some becoming critical organs and others turning into wings or legs.
A pointed, thicker layer of cells known as a "Primitive Streak" that develops the embryo's
longitudinal axis can be observed in the caudal, or tail, region when incubation begins (by 18 hours
of incubation). The head and backbone of the chick are derived from it. Along with these
structures, the precursor to the digestive tract, blood islands that subsequently give rise to the
vascular or blood system, and the beginning of eye development all appear.
On the second day of incubation, the blood islands that originally emerged on the first day of
incubation begin to connect to each another to build the vascular system, while the creation of the
heart occurs simultaneously somewhere else. On the 44th hour of incubation, the circulatory
system and heart are linked, and the heart starts to beat. An embryonic circulatory system for the
embryo and a vitelline system supplying the egg are both created at this stage. The liver also
develops. The endoderm begins to give rise to the interior lining epithelium of the respiratory,

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immunological, and digestive systems on the same day. By the twelfth day, the digestive system is
fully developed and the organs begin to visualize.
By the conclusion of the third day of incubation, the limb buds for the wings and legs are apparent.
Additionally, the beak begins to form. Torsion and flexion continue for the entire fourth day of
incubation, twisting the chick's entire body 90 degrees, causing it to lie down with its left side on
the yolk. As the embryo's head and tail move toward one another, it assumes a "C" form. Mouth,
tongue, and nasal pits develop from the respiratory system and alimentary canal. Even though it's
outside the body, the heart is growing and can be seen beating by cracking open an egg. The tissue
that eventually becomes the respiratory organs is undifferentiated and disordered at this stage.
Other internal organs also continue to grow at the same time. As a result, by the end of the fourth
day of incubation, the chick embryo has grown all of its vital organs.
The embryo develops quickly, and by the seventh day of incubation, the heart is entirely enclosed
in the thoracic cavity. The wings and feet's digits are visible. The immune system starts taking
shape as the spleen, thymus, and cloacal bursa began to appear on the tenth day. After the tenth day
of incubation, feather and feather tracts are apparent, and the differentiation of the respiratory
system is also complete. The beak also becomes harder. Syrinx, however, is not noticed until the
19th day. On the fourteenth day, when the claws are formed, the embryo starts to migrate towards
the hatching position. On the twentieth day, when the beak pierces the air cell and pulmonary
breathing starts, the embryo is in the hatching position.
After 21 days of incubation, the chick finally starts to break out from the shell by poking its beak
through the air cell. By this point, the allantois, which had served as the chick's lungs, is beginning
to dry out. At this point, the chick is using its lungs. As the chick continues to thrust its head
outward, the egg teeth (sharp horny structures) on the upper beak and the muscles on the back of
the neck cut the shell. The process continues as the chick shifts positions and continues to cut the
shell until its head pops out of the cracked shell. Following that, it kicks itself free from the bottom
of the shell. The bird becomes fatigued after such a lengthy workout, and it sleeps while its naval
openings mend and it dries from the bottom. It gets strength once more and begins to move. Within
a few days of the chick's hatching, the horny cap falls off the beak.
Due to the economic significance and expansion of the use of this animal model in studies like
genetics, the study of chicken organogenesis based on germ layers is exceedingly complex and
understudied. As a result, it is crucial to understand chicken embryology.

5.3 DEVELOPMENT OF ORGANS IN CHICKS

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Development of brain, eye and heart: The telolecithal egg of the chick undergoes discoidal
meroblastic cleavage after fertilization in the oviduct, during which the blastoderm is separated into
a tissue that is 5–6 cell layers thick by equatorial and vertical cleavages. Tight junctions are
typically used to connect these cells. The subgerminal cavity is a compartment located between
the blastoderm and the yolk
(Fig.5.1). At this point, the deep cells in the blastoderm's center die and are shed, leaving an area
of pellucida that is one cell thick. The majority of the embryo is made up of this portion of the
blastoderm. The area opaca is made up of the outer ring of blastoderm cells that have not yet lost
their deep cells. The marginal zone is a thin layer of cells that lies between the area pellucida and
the area opaca (or marginal belt). During the early stages of chick development, certain marginal
zone cells play a crucial role in determining cell destiny.

Fig.5.1. Chick embryo in blastula stage (source: https://biologyease.com)

When a hen lays an egg, it has about 20,000 cells. The majority of the cells in the area of
pellucida form the epiblast at the surface, but some of the remaining cells have delaminated and
moved separately into the submarginal cavity to create polyinvagination islands or primary
hypoblast. After a short period, a layer of cells from the blastoderm's posterior boundary moves
anteriorly to join the poly invagination islands, forming the secondary hypoblast. Epiblast and
hypoblast, the two layers of the blastoderm, are fused at the edge of the area opaca, and the space
between the layers creates a blastocoel. Thus, the bird embryo completely comes from the epiblast.
The growing embryo receives no cells from the hypoblast. The hypoblast cells, on the other hand,
are responsible for the formation of some of the exterior membranes, particularly the yolk sac and
the stalk connecting the yolk mass to the endodermal digestive tube. The epiblastic cells develop

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into the three germ layers of the embryo proper, as well as a sizeable portion of the extraembryonic
membrane.
5.3.1. DEVELOPMENT OF BRAIN
The ectoderm is given instructions to build the nervous system and the epidermis, as is now widely
accepted. A section of the dorsal ectoderm is designated as neural ectoderm, and its cells can be
identified by their columnar shape. The neural plate is the name of this area of the
embryo. Neurulation is the process by which this tissue develops into a neural tube, the beginning
of the central nervous system. An embryo going through this process is referred to as
a neurula (Fig.5.2).
Formation of the neural tube: There are primarily two methods for creating a neural tube. The
cells that surround the neural plate instruct the neural plate cells to multiply, invaginate, and pinch
off from the surface to form a hollow tube during primary neurulation. The neural tube develops
during secondary neurulation from a solid cord of cells that enters the embryo and then cavitates
to form a hollow tube. Different vertebrate classes utilize these processes of formation to different
extents.

Fig.5.2: Neurulation in Chick. (A) Primitive streak bearing embryo, neural plate epidermis ; (B) Antero-
posterior extension of neural plate formation of the neural folds and shortening of the primitive streak

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(stage 7-8); (C)Internal anatomy of the embryo (stage 6-8); (D) Surface view of the embryo of the stage
having 3-4 pairs of somites; (E) Internal anatomy of stage 8 embryo;(F) Surface view of the embryo of 9-10
pairs of somites. (source:https://www.notesonzoology.com/vertebrates/chick/development-of-chick-with-
diagram-vertebrates-chordata-zoology/8645)

Primary neurulation: Fig. 5.3 shows the chick's major neurulation events. The original ectoderm
is split into three groups of cells during primary neurulation: the neural tube, which will eventually
give rise to the brain and spinal cord, the skin's epidermis, and the neural crest cells. The neural
crest cells develop at the area that joins the neural tube and epidermis before migrating elsewhere.
These cells produce the skin's pigment cells, peripheral neurons, and glia, as well as several other
cell types. In mammals, birds, reptiles, and amphibians, the main neurulation mechanism seems to
be comparable. A U-shaped neural groove develops in the center of the neural plate shortly after it
forms, dividing the future right and left sides of the embryo. The neural plate's margins thicken and
travel upward to produce the neural folds. The neural folds move toward the embryo's midline and
eventually join to form the neural tube, which lies beneath the ectoderm above. The neural crest is
formed from the cells at the dorsalmost end of the neural tube. Distinct parts of the body have
slightly different processes for neurulation to take place. The inductive link between the pharyngeal
endoderm, prechordal plate, and notochord and its covering ectoderm is reflected in how the head,
trunk, and tail of the neural tube each form their respective regions. The formation of the neural
plate, shaping of the neural plate, bending of the neural plate to form the neural groove, and closing
of the neural groove to form the neural tube are the four distinct but spatially and temporally
overlapping stages of primary neurulation that occur in the head and trunk regions.

Fig.5.3.Neurulation in Chick development

(Source:https://biology.kenyon.edu/courses/biol114/Chap14/Chapter_14B.html)

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Formation and shaping of the neural plate: When the pharyngeal endoderm in the head area and
the underlying dorsal mesoderm signal the ectodermal cells above them to expand into columnar
neural plate cells, neurulation occurs. The cells of the potential neural plate can be distinguished
from the flatter pre-epidermal cells around them by their elongated shape. The neural plate contains
up to 50% of the ectoderm. The inherent motions of the epidermal and neural plate areas mold the
neural plate.
The neural plate enlarges along the anterior-posterior axis and contracts to produce a tube-like
shape when bent later. The neural plate expands and contracts via convergent expansion in both
amphibians and amniotes, intercalating several layers of cells into a few layers. The neural plate
cells also divide preferentially in the rostral-caudal (beak-tail; anterior-posterior) orientation. Even
if the affected tissues are isolated, these events will still take place. In an isolated neural plate, the
cells converge and spread out to form a narrower plate, but they do not roll up into a neural tube. It
will, however, develop into tiny neural folds in culture if the "boundary region"—which contains
both presumed epidermis and neural plate tissue—is isolated (Fig.5.4).
Bending of the Neural Plate: Where the neural tube touches the surrounding tissues, hinge areas
are formed, causing the neural plate to bend. In these areas, the presumed epidermal cells stick to
the lateral neural plate margins and push them in the direction of the midline (Fig.5.4). The cells in
the neural plate's midline are known as medial hinge point (MHP) cells in both birds and
mammals. They come from Hensen's node's anterior midline and the area of the neural plate
immediately in front of it. The MHP cells form a hinge that creates a furrow at the dorsal midline
and anchors to the notochord beneath them. The MHP cells become shorter and more wedge-
shaped when attached to the notochord. The MHP's side cells do not experience this
alteration. Soon later, two more hinge regions develop furrows close to where the neural plate
connects to the remaining ectoderm. The surface ectoderm of the neural folds serves as an anchor
for these areas, which are known as the dorsolateral hinge points (DLHPs). These cells also grow
taller and develop a wedge shape.
Changes in cell shape are closely related to cell wedging. Both microtubules and microfilaments
are implicated in these modifications in the DLHPs. The neural plate is initially furrowed, and then
it bends around these hinge areas. Each hinge serves as a center, controlling how the cells rotate
around it. Extrinsic factors are also in action in the meanwhile. The surface ectoderm of the chick
embryo, which thrusts in the direction of the embryo's midline, is another force that bends the
neural plate (Figure 5.4.). It may also be essential for the neural tube to invade internally rather

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than externally for the neural plate to adhere to the underlying mesoderm and the assumed
epidermis to migrate. The neural folds are produced by the neural tube being furrowed and the
presumed epidermis being pushed toward the center.

Fig.5.4. Formation and shaping of the neural plate

(Adapted from Developmental-Biology-7thed-sf-Gilbert-pdf)

Closure of the neural tube: As the paired neural folds are brought together at the dorsal midline,
the neural tube closes. The cells from the two folds combine when the folds cling to one another.
The cells at this juncture give rise to neural crest cells in some species. The neural tube at the
dorsal part of the bird does not close before the neural crest cells move from that area. However, in
mammals, the cranial neural crest cells—which develop into the facial and neck structures—
migrate as the neural folds rise, or before the neural tube closes. In contrast, the crest cells in the
spinal cord region wait until the closure has taken place. Not all areas of the ectoderm experience
the neural tube's closure at once. This is most evident in vertebrates with elongated body axes
before neurulation, such as birds and mammals. While gastrulation is still occurring in the embryo's
caudal (tail) area, neurulation is well underway in the cephalic (head) region. Changes in the shape
of the tube leading to regionalization of the neural tube as well. The wall of the tube is broad and

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thickest towards the cephalic end, where the brain will develop. The distinct brain compartments
are defined by a succession of swells and constrictions in this area. The neural tube, however,
continues to be a straightforward tube from the caudal to the head area that tapers out toward the
tail. The anterior neuropore and the posterior neuropore are the two open ends of the neural tube.
Mammals' neural tube closure begins at many locations along the anterior-posterior axis, in
contrast to chicks, where neurulation begins at the level of the future midbrain and "zips up" in
both directions.

Fig.5.6. Stereogram of developing chick embryo (24 hr stage)

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)

The neural tube eventually separates from the surface ectoderm to form a closed cylinder. It is
believed that the expression of several cell adhesion molecules mediates this separation. Although
the neural tube-forming cells initially produce E-cadherin, they stop making this protein as the
neural tube develops in favor of N-cadherin and NCAM (Fig.5.7). The surface ectoderm and neural
tube tissues stop adhering to one another as a result.
Secondary neurulation: A medullary cord is created during secondary neurulation, and the cord is
then hollowed out to form a neural tube. Secondary neurulation typically occurs in the neural tubes
of the lumbar (abdominal) and tail vertebrae in frogs and chicks. It can be viewed as a continuation

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of gastrulation in both situations. In the frog, the cells of the dorsal blastopore lip continue to
develop ventrally rather than involuting into the embryo. Precursors for both the posteriormost
component of the neural plate and the posterior portion of the notochord can be found in the region
that is developing near the tip of the lip, known as the chordoneural hinge. The 1.2 mm diameter,
somewhat spherical gastrula grows in this area, becoming a linear, and 9 mm long tadpole. The
blastopore cells that line the neurenteric canal are directly descended from the dorsal blastopore lip,
which is located at the tip of the tail.
The distal component of the neurenteric canal becomes the ependymal canal (i.e., the neural tube
lumen), while the proximal portion unites with the anus (Fig.5.8).

Fig.5.7: Showing neural tube cells producing N-cadherin and E-cadherin

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)

Fig.5.8: Secondary neurulation

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)
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Development of the neural tube: Three distinct processes simultaneously separate the neural tube
into the different regions of the central nervous system. The neural tube and its lumen expand and
contract to form the brain and spinal cord's chambers at the gross anatomical level. The diverse
functional regions of the brain and spinal cord are formed at the tissue level by the rearrangement
of the cell populations within the neural tube wall. The neuroepithelial cells themselves finally
differentiate into the many types of nerve cells (neurons) and supporting cells (glia) present in the
body at the cellular level.
The anterior-posterior axis: The neural tube of the early mammalian has a straight structure.
The anterior part of the tube, however, is experiencing significant modifications even before the
posterior portion of the tube has developed. The neural tube expands here, forming the forebrain
(prosencephalon), midbrain (mesencephalon), and hindbrain (rhombencephalon).
The optic vesicles have spread laterally from each side of the growing forebrain by the time the
posterior end of the neural tube closes. The front telencephalon and the more
caudal diencephalon are divisions of the prosencephalon. The diencephalon eventually gives rise
to the thalamic and hypothalamus brain areas that receive neuronal input from the retina, whereas
the telencephalon eventually gives rise to the cerebral hemispheres. The cerebral aqueduct finally
develops from the lumen of the mesencephalon, which does not further split. A
posterior myelencephalon and a more anterior metencephalon separate from the
rhombencephalon. The medulla oblongata, whose neurons produce the nerves that control
respiratory, gastrointestinal, and cardiovascular motions, finally develops from the
myelencephalon. The cerebellum, the area of the brain in charge of controlling posture, balance,
and movement, develops from the metencephalon. The rhombencephalon forms a segmental
pattern that identifies the locations from where particular nerves emerge. The rhombencephalon is
divided into smaller compartments by recurring swellings known as rhombomeres. These
rhombomeres serve as distinct developmental "territories" since cells can freely mingle within each
one, but not with cells from other rhombomeres. Furthermore, the developmental fate of every
rhombomere is unique. Every rhombus will develop ganglia, which are collections of neuronal cell
bodies whose axons make a nerve. The chick, in which the first neurons occur in the even-
numbered rhombomeres, r2, r4, and r6, has been the subject of the most thorough research on the
development of the cranial nerves from the rhombomeres. The fifth (trigeminal) cranial nerve is
made up of neurons from the r2 ganglia; the seventh (facial) and eighth (vestibuloacoustic) cranial

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nerves are made up of neurons from r4, and the ninth (glossopharyngeal) cranial nerve is made up
of neurons from r6.
The early embryonic brain balloons at an astonishing rate and scale, and it does so predominantly
due to an increase in cavity size rather than tissue growth. The brain volume in the developing
chick embryo increases thirty-fold between days three and five. This quick expansion is assumed to
be brought on by the fluid inside the neural tube exerting positive fluid pressure against the walls of
the tube. With this pressure, the surrounding dorsal tissues push in to compress the neural tube at
the base of the brain as the neural folds close in the region between the presumed brain and the
presumed spinal cord. The putative brain region and the prospective spinal cord are effectively
divided by this blockage (Fig.5.10)

Fig.5.10.Early development of the brain in chick embryo (Source: https://www.notesonzoology.com/wp-

content/uploads/2016/07/clip_image008-49.jpg)

The dorsal ventral axis: The dorsal-ventral axis determines the polarity of the neural tube. For
example, in the spinal cord, the ventral region is home to the motor neurons, whereas the dorsal
region is where the spinal neurons receive information from sensory neurons. Numerous
interneurons in the middle act as communication hubs. Signals emanating from the neural tube's
immediate environment cause it to become polarised. The epidermis imposes the dorsal pattern,
while the notochord induces the ventral pattern.
Ventral patterning of the neural tube: It indicates that external tissues act as a mediator in the
specification of the ventral neural tube. The Sonic hedgehog protein, which most likely originates
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from the notochord, is one agent of ventral specification. Retinoic acid, which most likely
originates from the nearby somites, is another agent that specifies the types of ventral neural cells.
The protein sonic hedgehog creates a gradient, and differing concentrations of it lead to the
emergence of various cell types. The medial hinge cells are induced by the secreted Sonic
hedgehog to develop into the neural tube's floor plate. Sonic hedgehog, which produces a gradient
with the highest concentration at the most ventral part of the neural tube, is likewise secreted by
these floor plate cells. The cells near the floor plate that are exposed to high levels of Sonic
hedgehog develop into ventral (V3) neurons, while the cells after them that are exposed to slightly
lower levels of Sonic hedgehog develop into motor neurons. The V2 and V1 interneurons are the
next two groups of cells, which receive progressively less of this protein. The various sonic
hedgehog concentrations work by triggering the expression of various transcription factors in
various populations of neurons. By activating the genes whose protein byproducts give the cell its
identity, these transcription factors in turn activate the genes. It's also possible that sonic hedgehog
suppresses the production of genes that make transcription factors for the dorsal neural tube.
Otherwise, the neural tube would express these genes throughout.
Dorsal patterning of the neural tube: Proteins from the TGF-superfamily (Transforming Growth
Factors), in particular the bone morphogenetic proteins 4 and 7 (BMP4 AND BMP7), dorsalin, and
activin determine the dorsal destiny of the neural tube. The epidermis is where BMP4 and BMP7
were initially identified. The epidermis creates a secondary signaling center by stimulating BMP4
expression in the neural tube's roof plate cells, just as the notochord does with the floor plate cells
on the ventral side of the neural tube. In neighboring cells, the roof plate's BMP4 protein triggers a
cascade of TGF-superfamily protein. As a result, different cell types are exposed to various TGF-
superfamily protein quantities at various times (Most dorsal exposed to more factors at higher
quantities and sooner). Different types of transcription factors are induced by the TGF-superfamily
proteins at different distances from the roof plate, which confers distinct identities on the cells.
Tissue architecture of the central nervous system: The brain's neurons are arranged into layers
(called cortexes) and clusters (called nuclei), each of which has distinct connections and functions.
A germinal neuroepithelium with one cell layer of thickness makes up the original neural tube.
This is a layer of neural stem cells that divide quickly.
It is known as the neuron's birthday since this vertical division marks the last time the latter cell
will divide. Birthdays of various glial and neuronal cell types occur at various times. The cells with
the earliest birthdays travel over the shortest distances, according to labeling at various
developmental stages. The more superficial parts of the cortex are formed by the migration of cells

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with later birthdays through these layers. The places occupied by these neurons outside the
germinal neuroepithelium determine the subsequent differentiation.
Spinal cord and medulla organization: The migrating cells create a second layer surrounding the
initial neural tube as the cells close to the lumen keep dividing. As new cells from the germinal
neuroepithelium are introduced to it, this layer thickens more and more. The germinal epithelium is
now referred to as the ventricular zone (and, later, the ependyma), and this new layer is known as
the mantle (or intermediate) zone. Mantle zone cells can develop into glia and neurons. The
neurons link with one another and project axons away from the lumen, resulting in a marginal zone
devoid of cells. Many of the axons in the marginal zone eventually develop myelin sheaths made of
glial cells, which give them a whitish appearance. As a result, the axonal, peripheral layer is
frequently referred to as the white matter, whereas the mantle zone, which contains the neuronal
cell bodies, is frequently dubbed the grey matter. This fundamental three-zone arrangement of the
ependymal, mantle and marginal layers is maintained throughout development in the spinal cord
and medulla. A butterfly-shaped structure made of white matter gradually surrounds the grey
matter (mantle), and both are then covered in connective tissue. The sulcus limitans, a longitudinal
groove, separates the neural tube into dorsal and ventral halves as it develops. While the ventral
region is responsible for numerous motor actions, the dorsal portion receives input from sensory
neurons (Fig.5.11).

Fig.5.11.Showing basic five-part anatomy of Chick brain

(Source: https://www.notesonzoology.com/zoology/development-of-brain-in-chick/2697)

Cerebellar organization: The three-zone pattern changes in the brain as a result of cell migration,
altered neuronal growth, and selective cell death. Some neural progenitors enter the marginal zone

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of the cerebellum to create nuclei, which are collections of neurons. As a relay station between the
outer layers of the cerebellum and other regions of the brain, each nucleus functions as a single
functional unit. Some neural progenitors can also migrate away from the germinal epithelium in the
cerebellum. Near the outermost limit of the neural tube, these precursor cells, known as
neuroblasts, move to the cerebellum's growing outer surface and establish a new germinal zone
called the external granule layer. Neuroblasts multiply at the one to two cells thick outer edge of
the external granule layer. Postmitotic neuroblasts, which are the ancestors of the granule neurons
that make up the majority of the cerebellar cortex, are found in the inner compartment of the
external granule layer. The internal granule layer is created when these granule neurons migrate
back into the growing cerebellar white matter. The cerebellum's original ependymal layer also
produces a diverse range of neurons and glial cells, including the recognizable and large Purkinje
neurons. In addition to being essential for the cerebellum's electrical system, granule neurons are
supported by Purkinje neurons. Sonic hedgehog, which is secreted by the Purkinje cell, promotes
the division of granule neuron progenitors in the external granule. Each Purkinje neuron has a
massive dendritic arbor that extends like a tree above a cell body that resembles a bulb. More than
any other neuron investigated, a typical Purkinje neuron can create up to 100,000 connections
(synapses) with other neurons. Additionally, each Purkinje neuron releases a thin axon that joins to
neurons in the deep cerebellar nuclei. For the cerebellum to operate properly, a spatial organization
must be developed. The Purkinje cells, the sole output neurons of the cerebellar cortex, are
eventually regulated by all electrical impulses. The right cells must differentiate at the right time
and place for this to happen. The reciprocal recognition between glia and neuroblasts is at the heart
of this intricate and interesting neural-glial relationship. Several proteins, including the adhesion
protein astrotactin, help the neuron to stay attached to the glial cell.
Cerebral organization: The cerebral cortex also modifies the neural tube's three-zone layout. Two
separate organizational systems exist in the cerebrum. It is initially arranged vertically into layers
that communicate with one another, similar to the cerebellum. The second zone of neurons is
created at the brain's outer surface by certain neuroblasts from the mantle zone migrating via glial
processes through the white matter. The neocortex is the name given to this new layer of grey
matter. The six layers of neuronal cell bodies that make up the neocortex ultimately stratify; the
adult forms of these layers are not finished until middle childhood. Each layer of the neocortex is
unique from the others in terms of its functional characteristics, the kinds of neurons it contains,
and the connections that those neurons form. For instance, neurons in layer 6 give their principal
output back to the thalamus, while neurons in layer 4 receive their major input from the thalamus.

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Second, there are around 40 horizontally structured regions in the cerebral cortex that control a
variety of physically and functionally diverse activities. For example, layer 6 neurons of the "visual
cortex" project axons to the medial geniculate nucleus of the thalamus, while layer 6 neurons of the
auditory cortex, which is located more anteriorly than the visual cortex, do the opposite. The
cerebral cortex is not clonally defined in either its vertical or horizontal organization. The majority
of the neuronal precursors produced in the ventricular (ependymal) zone migrate outward along
glial processes to form the cortical plate at the outer surface of the brain just after final mitosis. The
growing cortex arises from stem cell mixtures. The layer closest to the ventricle is made up of the
neuronal precursors with the earliest "birthdays," as is the case throughout the rest of the brain. The
cortex's outermost layers are formed by neurons that move farther apart later. An "inside-out"
gradient of development is created by this procedure. Any of the cortical layers' neurons (and glial
cells) can develop from a single stem cell in the ventricular layer.
5.3.2. DEVELOPMENT OF EYE
The neural tube interacts with a series of epidermal thickenings known as the cranial ectodermal
placodes to form the principal sensory organs of the skull.
The mechanism of eye development: The nearby prospective head ectoderm interacts with the
involuting endoderm and mesoderm at gastrulation to give the head ectoderm a predisposition
toward lens formation. However, not every piece of the head ectoderm finally develops into a lens,
and the lens and retina must have a precise spatial relationship. The optic vesicle is responsible for
placing the lens in respect to the retina and triggering the head ectoderm's latent lens-forming
potential. It emerges from the diencephalon and, upon coming into contact with the head ectoderm,
causes the development of a lens placode, which later invaginates to create the lens. The two-
walled optic cup that the optic vesicle transforms into has two layers differentiating in various
directions. The cells of the outer layer eventually differentiate into the pigmented retina because
they can create melanin pigment, one of the few tissues besides neural crest cells that can do so.
Fast cell division gives rise to a variety of glia, ganglion cells, interneurons, and light-sensitive
photoreceptor neurons being produced by the inner layer's cells. These cells make up the neuronal
retina as a whole. Neurons are called retinal ganglion cells to have axons that carry electrical
signals to the brain. At the base of the eye, their axons converge before proceeding down the optic
stalk. The optic nerve is the term used for this stalk.
There is evidence that the anterior tip of the neural plate expresses a cluster of the transcription
factors Six3, Pax6, and Rx1. The bilateral regions that make up the optic vesicles will later arise
from this one domain. The Pax6 protein seems to be particularly crucial for the growth of the lens

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and retina. It seems to be a feature shared by photoreceptive cells across all phyla. The eyes appear
to be the most susceptible organs to Pax6 deficiency, even though the murine forebrain, hindbrain,
and nasal placodes are all expressed.
The secretion of sonic hedgehog determines how the single eye field is divided into two bilateral
fields. The single median eye field won't split if the sonic hedgehog gene is altered or if the
protein's processing is prevented. A single eye in the middle of the face (often below the nose) is
the consequence, known as cyclopia. The prechordal plate's sonic hedgehog protein divides the
field by suppressing Pax6 expression in the center of the embryo.
Neural differentiation of the retina: The neural retina develops into a layered array of several
neuronal types, similar to the cerebral and cerebellar cortices (Fig.5.12). The rod and cone
photoreceptor cells, the ganglion cell bodies and the bipolar inter-neurons that relay electrical
inputs from the rods and cones to the ganglion cells are all included in these layers.

Fig.5.12 Showing Rods and Cones photoreceptor

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)

Numerous horizontal neurons conduct electrical impulses in the plane of the retina, amacrine
neurons (which lack long axons), and Müller glial cells preserve the integrity of the retina. The
striated, laminar pattern of the neural retina is created during the earliest stages of retinal formation
by cell division in a germinal layer, migration, and differential cell death of the resultant cells. At
least three different types of neurons, or two different types of neurons and a glial cell, can develop
from a single neuroblast precursor cell from the retinal germinal layer.
Differentiation of lens and cornea: The lens placode folds and touches the fresh ectoderm as it
continues to develop into a lens. The transparent cornea is then formed by the lens vesicle by the
induction of the ectoderm. Here, physical characteristics are crucial to the eye's growth. To ensure
that the cornea has the proper curvature and can focus light onto the retina, intraocular fluid

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pressure is required. A ring of scleral bones, which are most likely descended from the neural crest,
serves as an inelastic constraint to maintain intraocular pressure. Changes in cell structure and
shape as well as the production of translucent, lens-specific proteins known as crystallins are
required for the differentiation of the lens tissue into a transparent membrane capable of directing
light onto the retina (Fig.5.13). Under the influence of the neural retina, the cells at the inner
section of the lens vesicle expand and eventually transform into lens fibers. These fibers during
their process of development produce crystallins, which eventually fill the cell and lead to the
nucleus extrusion. As they keep expanding, the crystallin-synthesizing fibers eventually cover the
gap between the two layers of the lens vesicle. An ongoing germinal epithelium is made up of the
anterior lens vesicle cells. These dividing cells travel toward the vesicle's equator, where they pass
through the equatorial area and start to lengthen as well (Fig.5.13).

Fig.5.13: Showing differentiation of lens

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)
As a result, there are three distinct zones in the lens: an anterior zone of proliferating epithelial
cells, an equatorial zone of cellular elongation, and a posterior and middle zone of fiber cells that

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contain crystalline. Due to the ongoing laying down of fibers, this arrangement endures throughout
the animal's lifetime. It takes the adult chicken two years to transition from an epithelial cell to a
lens fiber. The iris, a pigmented and skeletal tissue, is located directly in front of the lens. The iris
muscles regulate the pupil's size. Part of the iris is derived from the ectodermal layer, in contrast to
the other muscles of the body, which are produced from the mesoderm. Specifically, the optic cup
section that forms this area of the iris is continuous with the neural retina but does not produce
photoreceptors.
5.3.3. DEVELOPMENT OF HEART
One of the lateral plate mesoderm's greatest accomplishments is the circulatory system. The
circulatory system, which is made up of a heart, blood cells, and a complex network of blood
vessels, nourishes the growing vertebrate embryo. The heart is the first functional organ and the
circulatory system is the first functional component of the growing embryo. The splanchnic
mesoderm sections on each side of the body interact with surrounding tissue to become specialized
for heart development, giving rise to the vertebrate heart.
Specification of heart tissue and fusion of heart rudiments: The embryo in amniote vertebrates
is a flattened disc, and the lateral plate mesoderm does not completely envelop the yolk sac. The
early primitive streak, which starts slightly posterior to Hensen's node and extends approximately
halfway down its length, is where the alleged heart cells also known as the cardiogenic mesoderm,
originate. As they go through the streak, these cells divide to generate two clusters of mesodermal
cells that are lateral to and at the same level as Hensen's node (Fig.5.14). These two clusters are the
source of the cells that make up the endothelium lining of the heart, the cushion cells of the valves,
the Purkinje conducting fibers, and the atrial and ventricular muscles.

Fig.5.14: Showing heart development

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)

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The presumptive heart cells travel anteriorly between the ectoderm and endoderm toward the center
of the embryo when the chick embryo is just 18–20 hours old, maintaining close touch with the
endodermal surface. The migration of these cells stops when they get to the anterior gut tube's
lateral walls. Foregut endoderm gives the movement direction. The cardiac mesoderm moves in the
opposite direction when the heart region endoderm rotates about the rest of the embryo. An
anterior-to-posterior concentration gradient of fibronectin is the endodermal component in charge
of this migration.
Some cardiogenic cells are also designated by the endoderm and primitive streak to develop into
heart muscles. The migrating mesodermal cells that will become the heart are induced to produce
the Nkx2-5 transcription factor by Cerberus and an unidentified substance, presumably BMP2 in
the anterior endoderm. Nkx2-5 is an essential protein that directs the mesoderm to develop into
cardiac tissue and triggers the production of other transcription factors (especially members of the
GATA and MEF2 families). These transcription factors stimulate the expression of genes that code
for proteins particular to the heart muscle (such as cardiac actin, atrial natriuretic factor, and the
alpha myosin heavy chains).
The heart's cells gradually become defined, with the ventricular cells doing so before the atrial
cells. The two advancing heart-forming primordia each experience separate cell differentiation. The
cells start to display N-cadherin on their apices and unite to form an epithelium as they move. The
endocardium (lining of the heart along the blood vessels) is formed when a small population of
these cells detach from the epithelium and downregulate N-cadherin, while the myocardium is
made up of epithelial cells. The myocardium will develop into the heart's pumping muscles for the
whole life of the organism.

Fig.5.15: Shows the development of the heart

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)

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Numerous heart valves are made by endocardial cells, which also govern the positioning of neural
tissue in the heart and secrete the proteins that control myocardial growth. The splanchnic
mesoderm folds inward during neurulation to create the foregut. The myocardium eventually
becomes one tube after this movement pulls the two cardiac tubes together. A condition known as
"cardio Bifida" causes a distinct heart to develop on each side of the body. For a brief period, the
two endocardia share this same tube; however, they also fuse. The previously paired coelomic
chambers now combine to create the body cavity that houses the heart. At about 29 hours of the
development of a chick, this fusion takes place. The apertures of the vitelline veins in the heart are
formed by the unfused posterior sections of the endocardium. These veins transport the yolk sac's
nutrients into the sinus venosus. The blood then enters the atrial portion of the heart through a flap
that resembles a valve. Truncus arteriosus contractions hasten blood flow into the aorta. The heart
starts to beat while the paired primordia are still joining together. The sinus venosus acts as the
contraction's pacemaker. A wave of muscular contraction propagates up the tubular heart from
where contractions start. This allows the heart to begin pumping blood even before the intricate
valve system is fully developed. Heart muscle cells can naturally contract on their own. By the
fourth day, the ECG of a chick embryo is similar to that of an adult. In the embryo, these
contractions are controlled by electrical inputs from the medulla oblongata via the vagus nerve.

Fig.5.16. Looping and Formation of Herat Chambers in Chick Embryo

(Source: https://www.notesonzoology.com/zoology/development-of-heart-in-chick/2705)

Looping and formation of heart chambers: The heart is a two-chambered tube with an atrium
and a ventricle in a three-day-old chick embryo. The unaided eye may observe the amazing cycle

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of blood entering the lower chamber and being pumped out through the aorta in the chick embryo.
The heart's looping changes the heart tube's original anterior-posterior polarity into the right-left
polarity found in adults. As a result, the area of the heart tube that will eventually become the right
ventricle is located ahead of the area that will eventually become the left ventricle. The left-right
patterning proteins are required for this looping. Nkx2 5 controls the Hand1 and Hand2
transcription factors within the heart primordium. Although it appears that the Hand proteins are
produced throughout the early heart tube, once looping begins, Hand1 is limited to the left ventricle
and Hand2 to the right. Without these proteins, looping is disrupted, and the ventricles are unable
to develop appropriately. The Pitx-2 transcription factor is essential for correct cardiac looping and
is only activated on the left side of the lateral plate mesoderm. It may also control the expression of
proteins like the extracellular matrix protein lectin to control the physical strain of the various heart
components. The Xin gene may mediate the cytoskeletal alterations required for heart looping and
is also activated by transcription factors Nkx2-5 and MEF2C. The various transcription factors that
becomes limited to either the anterior or posterior region of the heart tube help to define the
separation of the atrium from the ventricle. When cells from the myocardium release a substance
(likely transforming growth factor-β3) that triggers cells from the neighboring endocardium to
separate and enter the hyaluronate-rich cardiac jelly between the two layers, the tube is partitioned
into a distinct atrium and ventricle (Fig.5.16 A-D)

5.4 EMBRYONIC INDUCTION

New shapes and forms are created during gastrulation, which involves irreversible movements also
known as morphogenetic movements. During gastrulation, when distinct sections of the blastoderm
are displaced and brought into new spatial connections to one another, the organization of the
embryo as a whole appears to be defined to a great extent. Components of various origins can
interact more readily because groups of cells that were far apart in the blastula come close together.
The sliding of cells (presumptive mesoderm) into the interior and their positioning on the dorsal
side of the archenteron (in the archenteric roof), in direct contact with the overlying ectoderm, are
of major importance in development and subsequent differentiation, particularly in vertebrate
development. Though ectoderm lacks the capacity for any form of progressive development during
the beginning of gastrulation, it attains that ability only after invagination, when chordamesoderm
is directly beneath it. The overlaying ectoderm divides into the neural plate by the effect of dorsal
mesoderm and dorsal mesoderm eventually differentiates into the notochord, prechordal

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mesoderm, and somites. Overlying ectoderm is developed into the skin via lateral
mesoderm. Embryonic induction refers to the effect that some embryonic organs exert over
groups of cells to direct their growth in a particular direction. These gastrulation movements are
caused by the combined actions of the embryo's components rather than by the embryo as a whole.
It must be assumed that there are inducing chemicals with specialized activity because the
consequences of induction vary depending on the organ's rudiments. For instance, the neural plate
from the same type of ectoderm is not differentiated by the lateral mesoderm, but the skin is.
However, as some differentiations may be brought about by a combination of two or more inducing
chemicals or the same inducing substance may have different effects on various tissues, the number
of inducing substances need not be the same as the number of distinct types of tissues and organs.
The gradient distribution of just two inducing substances, the neuralizing substance, and the
mesodermalizing substance, along the length of the embryo, might regulate the regional
organization of the complete vertebrate body. The mesodermalizing substance is concentrated at
the posterior end and diminishes toward the anterior end whereas the neuralizing substance is
concentrated at the anterior end and gradually declines toward the posterior end. The relative
proportions of the two inducing substances at any given time during the embryo determine how
differentiated the induced structures will be. When used alone, the neuralizing and
mesodermalizing substances only produce nervous tissue (the future forebrain) and mesodermal
structures respectively.
Although close closeness between the interacting pieces is necessary for induction, physical touch
is not required. A diffusible chemical released by the activating cells is the inciting influence (the
inductor). The mesoderm is induced by a big molecule, most likely a protein or nucleoprotein.
Transplanting mammalian tissues into frog embryos or transplanting chick embryo tissue into
rabbit embryos have both been used to induce the development of main organs in vertebrates of
many different classes.
5.4.1. DETERMINATION OF THE PRIMARY (1°) ORGAN RUDIMENTS
Induction is responsible for the formation of several early organ structures in vertebrates, in
addition to the division of the ectoderm into the neural plate and epidermis. The adjacent somites
and nephrotomes, which appear to work in concert to cause the formation of limb rudiments from
the lateral plate mesoderm, are induced by the notochord. Therefore, embryonic induction is the
consequence of contact between an inducing tissue and a responsive tissue, which causes the
responding tissue to shift the direction of its differentiation. This is most likely the only process
responsible for cell differentiation and cell organization into tissues and organs in vertebrates. The

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phenomenon is credited to Spemann in 1901 and Lewis in 1904, who proved that in some species
of Rana, the development of a lens from the ectoderm is influenced by the brain's underlying optic
lobe. A heteroplastic transplanting between two species of newts from the genus Triturus was
carried out by Spemann (1921). He transferred a portion of one early gastrula embryo's
presumptive neural ectoderm into another embryo's prospective epidermis area. The tissue that was
implanted transformed into the epidermis. On the other hand, a portion of the prospective
epidermis that was implanted into the presumptive neural ectoderm grew into a component of the
neural tube. Therefore, at the early gastrula stage, the fates of presumptive neural tissue or
prospective epidermis are not fixed. If the donor embryo used is in a late stage of gastrulation as
opposed to an early stage of gastrulation, completely different outcomes are seen. A fragment of a
late gastrula's neural plate can be transplanted into another area of the embryo, where it will grow
into nerve tissue. An epidermal patch forms inside the nervous system if a portion of the potential
epidermis is transplanted into the area of the neural plate. The range of possibilities for the tissues'
fate is gradually reduced throughout gastrulation.
In an experiment by Spemann and Mangold (1924), the dorsal lip of an unpigmented Triturus
cristatus embryo was transplanted to the ventral region of a pigmented Triturus taeniatus gastrula.
This caused the pigmented host cells to create a secondary axis (Spemann, 1938). The 1935 Nobel
Prize in Medicine or Physiology went to this T 2 experiment. Even in this unsuitable environment,
involution took place. Axis induction, when a secondary axis develops with a stomach, neural tube,
notochord, and somites, also took place. It was seen due to pigment difference that a large portion
of the secondary axis is made from host tissue. As a result, the graft had an impact on the
neighboring cells' ability to develop into particular organs. This effect was called ‘Embryonic
induction. The component that is the source of the influence is known as the inductor. The
secondary axis and the primary axis are always parallel. Therefore, secondary axis orientation is
determined by the host. A parallel secondary axis is also revealed by transplant experiments using
avian Henson's Nodes. This experiment demonstrated that the chordamesoderm, the underlying
tissue, is what induces the neural system (presumptive notochord and somite mesoderm).

Fig.5.17: Embryonic Induction (https://www.biology-pages.info/S/Spemann.html)

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Because it can start the development of a secondary embryo when transplanted, Spemann dubbed
the dorsal lip of the blastopore the Primary Organiser. It is now also known as the Spemann
organizer.

Fig.5.18: The Spemann Organizer (https://www.biology-pages.info/S/Spemann.html)

In urodele amphibians, the primary organizer was shown to exist. Since then, it has been
discovered that the chordamesoderm, or archenteron roof, can induce the nervous system and
sensory organs in the majority of vertebrate species. Due to the specificity of the inductors in each
region, the numerous portions that are induced to develop do so in a systematic way.
1. Head organs are induced to develop by the head inductor, which is an anterior chordamesoderm
segment.
2. The tail bud and trunk organs are induced to form by the chordamesoderm's posterior
component, the trunk inductor.
3. The chordamesoderm crosses the blastopore's dorsal lip first at the anterior end and the posterior
end passes over in the last. Consequently, an early gastrula stage embryo's dorsal lip functions as a
head inductor and induces head organs to form.
5.4.2. MESODERM INDUCTION OCCURS BEFORE PRIMARY EMBRYONIC
INDUCTION
It has been discovered that the mesoderm is induced first, then the neural induction. Animal,
vegetative, and equatorial or marginal cells make up the three different types of cells. Their

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locations on the blastula are indicated by these names. Nieuwkoop (1969) cultivated explants from
these 3 areas separately or jointly and observed the following outcomes:

Explant Tissue Formed

Animal cells Forms epidermis
Marginal cells Forms mesoderm
Vegetal cells Forms endoderm
Animal cells+Vegetal cells Forms mesoderm

Mesoderm appears to be induced by a variety of molecules from many types of embryonic or adult
cells like cultured mammalian cells, guinea pig bone marrow, carp swim bladder, and chick
embryos. They all share the property of containing growth factors. One cell type releases growth
factors that affect other cell types in different ways. This characteristic is in line with what is
anticipated of an inducer.
5.4.3. SECONDARY INDUCTION
As the organism develops, inductive interactions lead to the emergence of numerous more cell
types. This is known as secondary induction. The two types of inductive interactions are seen, as
permissive and instructive. The inducing tissue reportedly provides precise information to
commit cells to a new developmental route in an instructive interaction. Cairns and Saunders
(1954) demonstrated in the chick that the overlying ectoderm evolved under the origin of the
mesoderm from various locations of the leg or wing into foreign regions. The growing wing
ectoderm will create thigh feathers above the grafted mesoderm if a piece of thigh mesoderm is
transplanted under the ectoderm of the wing. The reacting tissue must be relatively unknown for
such an interaction to occur. The parts of the embryo can develop into other parts in experimental
situations in addition to having a clear typical fate, known as potential significance. Prospective
potency refers to a portion of an early embryo's capacity to develop into more than one type of
tissue. Determination is the process by which potential possibilities are reduced and the fate of the
embryonic tissues is fixed. A tissue is said to be determined once the determination has taken place
in it. Its potential potency is now more restricted.
Responding cells in permissive interactions are already prepared and ready to differentiate; all they
need is a signal from the inducing tissue to enable them to realize their full potential. The pancreas
for example grows as a gastrointestinal expansion, although the mesoderm must participate in its

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formation. Rutter and his colleagues (1964) discovered that the pancreas endoderm would
differentiate when a different form of mesenchyme, from the salivary gland, was substituted. Using
early, pureed embryos and somites, which typically develop muscle and cartilage cells, this
endoderm differentiation was also seen (embryo extract). Therefore, it appears that everything is
set up at the point where the pancreatic endoderm is visible, to the point where a relatively non-
specific cue will finish the differentiating process. The neural tube is located inside the secondary
axis and is principally induced in the ectoderm by the overlying mesoderm.

5.5 PRIMARY ORGANISER AND ITS MORPHOLOGICAL

DIFFERENTIATION

An embryonic interaction, also known as an organizer, is the process by which one biological
tissue transmits a chemical that influences another embryonic section to create a structure that
would not otherwise be feasible. This type of embryonic tissue is known as an inductor, and the
substances that an inductor secretes are known as evocators. The term "responsive tissue" refers to
tissue that reacts to an evocator operating on it. The action taken by the inductor via the evocator is
referred to as induction activity or organizer action. This induction process has a substantial impact
on the protein synthesis mechanism of sensitive tissues, which causes cells that produce certain
structures to become more active. In 1924, the organizing idea for Spemann's experiment was
initially put forth. German embryologist Hans Spemann and his student Hilde Mangold conducted
a transplanting experiment on Triturus cristatus, an Urodela belonging to the class Amphibia, in
1924.
Spemann grafted a portion from the dorsal lip region of the early gastrula of Rana sp. (donor
embryo) to the lateral lip of the early gastrula of Triturus cristatus (host embryo). The transplanted
piece's cells entered the gastrula and formed the notochord and somites. The dorsal lip of the
blastopore in this embryo creates the neural groove, notochord, mesoderm, etc. Likewise, the
transplanted tissue has an impact on the mesoderm, the neural groove, and the notochord. That is,
the second set of the notochord, nerve cord, and mesoderm are generated in the same embryo. In
this instance, chemical compounds produced by the donor tissue caused the host embryo to develop
neural grooves, notochords, and other structures. Donor tissue and the constructed neural groove
included pigments. They noticed that a larva with two heads had formed after gastrulation was
finished. One head was produced naturally throughout development, and the other was stimulated
by donor tissue. It was seen under the microscope that the host embryo's tissue served as a
secondary set to build the notochord, renal tubules, gut, and other structures. Such secondary
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structures would not have formed if the donor tissue had not been grafted. From this experiment,
the researchers deduced that the donor's dorsal lip had a significant impact on the tissue, changing
how the host tissue developed. Spemann referred to this process of influencing other tissues as
induction, and the tissue that caused the other tissues to be affected was known as
the inductor or organizer.
Primary organizer: Spemann continued his grafting studies using tissues from different zones of
the gastrula and discovered that it was only the dorsal lip of the early gastrula that could create a
complete embryo while all tissues from other regions failed to do so. He designated the dorsal lip
as the organizer since it organizes the embryo's developmental process. He asserts that the dorsal
lip stimulates the development of the neural tube, which in turn stimulates the development of the
eyes. He referred to the dorsal lip, or chordamesoderm, as the major or the primary organizers
because it is made of chordamesoderm and serves largely as an inducer.
Secondary, tertiary, and quaternary organizers: Primary organs start to form as gastrulation
progresses as a result of the primary organizer's induction, and these early stages of organ
development are referred to as organ rudiments. These organ rudiments may function as organizers
on their own, in which case they are referred to as secondary organizers. Further development
may be induced by tissues that are created as a result of secondary organizer activity. They are
therefore referred to as tertiary organizers. The primary organizer is the starting point for these
subsequent levels of organizer activity. The development of the eye in amphibians, chicks, etc.,
serves as vivid instances of how these organizers function sequentially. First, eye developing cells
are formed within the forebrain as a result of the forebrain's induction action. Outside of the
forebrain, these cells protrude as a vesicle known as optic vesicles. Through the lateral
mesenchyme, this vesicle develops till it reaches the epidermis. The outer layer of the vesicle
invaginates to form a double-layered optic cup as soon as it comes into touch with the epidermis.
Sensory cells make up the optic cup's inner layer, whereas pigmented cells make up the outer layer.
Together, they make up the retina. Between the optic cup and the epidermis, the chemical
substances that the optic cup secretes cause the formation of the lens. The optic cup, therefore,
serves as a secondary organizer. Lens and retina together induce to make the cornea, thus they
together act as the tertiary organizer.

5.6 CONCEPT OF ORGANIZER

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5.6.1. ORIGIN OF PRIMARY ORGANISER

In 1924, the organizing idea for Spemann's experiment was initially put forth. Spemann grafted a
portion from the dorsal lip region of the early gastrula of Rana sp. (donor embryo) to the lateral lip
of the early gastrula of Triturus cristatus (host embryo). The transplanted piece's cells entered the
gastrula and formed the notochord and somites. The dorsal lip of the blastopore in this embryo
creates the neural groove, notochord, mesoderm, etc. Likewise, the transplanted tissue has an
impact on the mesoderm, the neural groove, and the notochord. That is, the second set of a
notochord, nerve cord, and mesoderm are generated in the same embryo. In this instance, chemical
compounds produced by the donor tissue caused the host embryo to develop neural grooves,
notochords, and other structures. Colored pigments were present in both the donor tissue and the
artificially made neural groove. They noticed that a larva with two heads had formed after
gastrulation was finished. One head was produced naturally throughout development, and the other
was stimulated by donor tissue. It was seen under the microscope that the host embryo's tissue
served as a secondary set to build the notochord, renal tubules, gut, and other structures. Such
secondary structures would not have formed if the donor tissue had not been grafted. From this
experiment, the researchers deduced that the donor's dorsal lip had a significant impact on the
tissue, changing how the host tissue developed. Spemann referred to this process of influencing
other tissues as induction, and the tissue that caused the other tissues to be affected was known as
the inductor or organizer.
5.6.2. INDUCTIVE INTERACTIONS:
Organs are intricate structures made up of several tissue kinds. For instance, light enters the
vertebrate eye through the translucent corneal tissue and is focussed by the lens tissue (the diameter
of which is regulated by muscle tissue), and then impinges on the neuronal retinal tissue. It is
impossible to alter the exact arrangement of tissues in this organ without affecting how well it
works. One group of cells influencing the behavior of an adjacent group of cells, leading them to
modify their form, mitotic rate, or fate, is how such coordination in the development of organs is
achieved. Proximate interaction, also known as induction, is the term used to describe this type of
proximity interaction between two or more cells or tissues with diverse histories and qualities.
Every inductive contact has at least two elements. The inducer, or tissue that generates the signal
(or signals) that alter the other tissue's cellular behavior, is the first part of the system. The
responder is the second element, the tissue being stimulated.

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5.7 NATURE OF INDUCTIVE SIGNAL (POSSIBLE MECHANISM

OF NEURAL INDUCTION)

Reciprocal and sequential inductive events: The reciprocal character of many inductive
interactions is another aspect of induction. Once the lens has developed, further tissues may be
induced. The optic vesicle itself is one of these reacting tissues. The inducer now changes into the
induced. The optic vesicle transforms into the optic cup under the action of substances secreted by
the lens, and the wall of the optic cup differentiates into two layers, the pigmented retina and the
neuronal retina (Fig.5.19). Reciprocal inductions are interactions like this.
The lens is also causing the ectoderm above it to develop into the cornea at the same time. The
ectoderm that forms the cornea has developed a special capacity to react to inductive impulses, in
this case, the signals from the lens, just as the lens-forming ectoderm. The corneal ectodermal cells
are induced by the lens to become columnar and secrete many layers of collagen. This collagen
matrix allows neural crest mesenchymal cells to enter the region and secrete a collection of
proteins, including the enzyme hyaluronidase, that advance the cornea's differentiation. The
hormone thyroxine, a third signal, causes the tissue to become translucent and dry. As a result,
there are several reasons for each induction as well as successive inductive occurrences.

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5.19. Neural Inductions and their interactions

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)

Instructive and permissive interactions: There are two main categories of inductive
contact:
(a) Instructive interaction: For new gene expression to begin in the responding cell, a signal from
the inducing cell is required. The responding cell could not differentiate in that specific manner
without the inciting cell. An instructive interaction, for instance, is when the optic vesicle is
experimentally positioned below a new section of the head ectoderm and induces that region of the
ectoderm to produce a lens. Three underlying principles define the majority of instructional
interactions:
1. Responding tissue B develops in a specific way when tissue A is present.
2. Responding tissue B does not form in such a way in the absence of tissue A.
3. Tissue B does not develop in that way when tissue A is not there but tissue C is.
(b) Permissive interaction: In this kind of contact, the responding tissue already possesses all of
the features that are intended to be produced; all that is required is a setting that permits the

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production of these characteristics. For instance, the development of many tissues depends on the
presence of a fibronectin- or laminin-containing solid substrate. The expression of what has been
determined to be expressed is all that the fibronectin or laminin accomplishes; it does not change
the sort of cell that will be formed.
Epithelial-mesenchymal interactions: The interactions of sheets of epithelial cells with nearby
mesenchymal cells are some of the well-studied examples of induction. Such interactions are
known as Epithelial-mesenchymal interactions. Connected cells give rise to sheets and tubes,
which are the forms of epithelia that can be formed from any germ layer. Unconnected, loosely
packed cells are referred to as mesenchyme. The neural crest or mesoderm is the source of
mesenchymal cells. Epithelial-mesenchymal interactions are among the most significant natural
processes since epithelium and the related mesenchyme make up every organ. Table 6.1 includes a
list of some examples.
Table 5.1: Some epithelial- mesenchymal interactions

Regional specificity of induction: The spatial specificity of induction is the first characteristic of
epithelial-mesenchymal interactions. The outer epidermis, an epithelial tissue formed from
ectoderm, and the dermis, a mesenchymal tissue derived from mesoderm, are the two primary
tissues that makeup skin. The condensed dermal mesenchyme reacts by secreting substances that
cause the chick epidermis to form regionally specific cutaneous structures in response to the
underlying dermal cells receiving a signal from the chick epidermis to form condensations likely
by secreting sonic hedgehog and TGF- 2 proteins. The large wing feathers, the fine thigh feathers,
or the scales and claws of the feet are examples of these structures. The embryonic epithelium and
mesenchyme can be separated from one another and combined in various ways in laboratories.

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Genetic specificity of induction: The genetic specificity of induction is the second characteristic
of epithelial-mesenchymal interactions. While the mesenchyme may tell the epithelium which gene
sets to activate, the epithelium's ability to follow these instructions depends on its genome.
Through trials involving the transfer of tissues from one species to another, this characteristic was
revealed. Hans Spemann and Oscar Schotté (1932) in an experiment, transplanted flank
ectoderm from an early frog gastrula to the region of a newt gastrula that would later become
elements of the mouth. Similarly, they implanted presumptive mouth areas of frog embryos with
presumptive flank ectodermal tissue from a newt gastrula. The mouth region of salamander and
frog larvae has a quite different anatomy. The frog tadpole produces mucus-secreting glands and
suckers, but the salamander larva possesses club-shaped balancers beneath its mouth (Fig.5.20).

Fig.5.20.Genetic specificity of induction

(Source:https://learninglink.oup.com/access/content/barresi-12e-student-resources/barresi-12e-further-
development-4-4-from-feathers-to-claws-and-frogs-to-newts-further-your-understanding-of-induction)

The salamander has a set of calcareous teeth in its jaw, whereas the frog tadpole also has a horny
jaw without teeth. The transplants produced chimera larvae. The frog tadpoles possessed
salamander teeth and balancers, and the salamander larvae had mouths like those of frogs. To put it
another way, the ectoderm was told to create a mouth by the mesodermal cells, but instead, it
created the only mouth that it "knew" how to create, regardless of how unsuitable it was. As a
result, mesenchymal tissue signals can be transmitted across species boundaries. Frog signals are

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recognized by salamanders, and mammalian inducers are recognized by chick tissue. But the
epithelium reacts differently depending on the species. As a result, species specificity is typically
regulated by the responsive epithelium, whereas organ type specificity (such as feather or claw) is
typically regulated by the mesenchyme within a species. Changes in the way an organism responds
to a certain inducer can result in significant evolutionary changes.
Paracrine Factors: Juxtacrine interactions occur when the cell membrane interacts with receptor
proteins on a neighboring cell surface. The phenomenon is known as a paracrine interaction, and
the diffusible proteins are known as paracrine factors or growth and differentiation
factors (GDFs) when they can disperse across short distances to affect neighboring cells.Paracrine
factors are secreted into the immediate areas around the cell that produces them, in contrast to
endocrine factors (hormones), which travel through the blood to act. The embryo inherits a
relatively small "tool kit," and builds the heart, kidneys, teeth, eyes, and other organs using many
of the same proteins. Furthermore, the same proteins are used throughout the animal kingdom; the
elements involved in developing the eye or heart of the Drosophila are quite similar to those
responsible for developing mammalian organs. Based on their structural similarities, four main
families can be formed from many of these paracrine components. These families include the TGF-
superfamily, the Hedgehog family, the Wingless (Wnt) family, and the fibroblast growth factor
(FGF) family.
The fibroblast growth factors: There are now more than a dozen structurally similar members of
the fibroblast growth factor (FGF) family. FGF1 and FGF2 are also known as acidic and basic
FGF, respectively, while FGF7 is also known as the keratinocyte growth factor. Vertebrates have
more than a dozen different FGF genes that can produce hundreds of different protein isoforms by
altering their RNA splicing or initiation codons in various tissues. The fibroblast growth factor
receptors are a group of receptor tyrosine kinases that can be activated by FGFs (FGFRs). Proteins
called receptor tyrosine kinases penetrate the cell membrane (Fig.5.21). The part of the protein that
binds the paracrine factor is on the extracellular side. There is a latent tyrosine kinase on the
intracellular side (i.e., a protein that can phosphorylate another protein by splitting ATP). The
dormant kinase is activated and phosphorylates specific proteins in the receptive cell when the FGF
receptor binds an FGF. The proteins can now carry out new tasks because they have been activated.
FGFs have been linked to the development of mesoderm, axon extension, and angiogenesis (the
production of blood vessels). Even while FGFs are frequently interchangeable, their different
expression patterns offer them distinct roles. FGF8 is crucial for the development of the midbrain
and limbs, whereas FGF2 is particularly significant in angiogenesis.

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Fig.5.21.The receptor of tyrosine kinase

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)

The Hedgehog Family: The Hedgehog proteins are a class of paracrine factors that the embryo
frequently employs to stimulate specific cell types and establish tissue borders. The Drosophila
hedgehog gene has at least three homologues in vertebrates: the sonic hedgehog (shh), desert
hedgehog (dhh), and Indian hedgehog (ihh). The Sertoli cells of the testes express desert hedgehog,
and mice homozygous for a null allele of dhh have poor spermatogenesis. Indian hedgehog plays a
crucial role in postnatal bone formation and is expressed in the gut and cartilage. Of the three
vertebrate homologues, Sonic Hedgehog is the most frequently encountered. Only the amino-
terminal two-thirds of the molecule, which is produced by the notochord, is secreted. This peptide
is in charge of shaping the neural tube such that sensory neurons develop from the dorsal neurons
and motor neurons from the ventral neurons. The somites are patterned by Sonic Hedgehog so that
the region closest to the notochord develops into the cartilage of the spine. Also, Sonic Hedgehog
plays a role in the development of the left-right axis in chicks, the beginning of the anterior-
posterior axis in limbs, the induction of regionally specialized digestive tube differentiation, and
the induction of feather creation. Wnt and FGF proteins are two paracrine factors that frequently
collaborate with a sonic hedgehog. Sonic hedgehog, FGF4, and other paracrine factors are
concentrated in the area of the developing tooth where cell interactions are forming the teeth's
cusps.
The Wnt family: A class of glycoproteins high in cysteine is known as the Wnts. Vertebrates
contain at least 15 members of this family. Their name is a combination of the names of two

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vertebrate homologs of the Drosophila segment polarity gene, integrated and wingless.
While Wnt1 appears to be active in driving the dorsal cells of the somites to become muscle, Sonic
Hedgehog appears to be vital in patterning the ventral section of the somites (causing the cells to
become cartilage). Wnt proteins are also used in numerous stages of the development of the
urogenital system and are crucial in determining the polarity of insect and vertebrate limbs.
The TGF-β superfamily: There are over 30 structurally related members of the TGF-b
superfamily, and they regulate some of the most important interactions in development. The
proteins encoded by TGF-β superfamily genes are processed such that the carboxy-terminal region
contains the mature peptide. These peptides are dimerized into homodimers (with themselves) or
heterodimers (with other TGF-β peptides) and are secreted from the cell. The TGF-β superfamily
includes the TGF-β family, the activin family, the bone morphogenetic proteins (BMPs), the Vg1
family, and other proteins, including glial-derived neurotrophic factor (necessary for kidney and
enteric neuron differentiation) and Müllerian inhibitory factor (which is involved in mammalian
sex determination). TGF-β family members TGF-β1, 2, 3, and 5 are important in regulating the
formation of the extracellular matrix between cells and for regulating cell division (both positively
and negatively). TGF-β1 increases the amount of extracellular matrix epithelial cells make (both by
stimulating collagen and fibronectin synthesis and by inhibiting matrix degradation). TGF-βs may
be critical in controlling where and when epithelia can branch to form the ducts of kidneys, lungs,
and salivary glands. The effects of the individual TGF-β family members are difficult to sort out
because members of the TGF-β family appear to function similarly and can compensate for losses
of the others when expressed together. Moreover, targeted deletions of the Tgf-β1 gene in mice are
difficult to interpret, since the mother can supply this factor through the placenta and milk.
The members of the bone morphogenetic proteins (BMP) family were originally discovered by
their ability to induce bone formation; hence, they are the bone morphogenetic proteins. Bone
formation, however, is only one of their many functions, and they have been found to regulate cell
division, apoptosis (programmed cell death), cell migration, and differentiation. BMPs can be
distinguished from other members of the TGF-β superfamily by their having seven, rather than
nine, conserved cysteines in the mature polypeptide. The BMPs include proteins such as Nodal
(responsible for left-right axis formation) and BMP4 (important in neural tube polarity, eye
development, and cell death). The Drosophila decapentaplegic protein is homologous to the
vertebrate BMP4, and human BMP4 can replace the Drosophila homologue, rescuing those flies
deficient in decapentaplegic protein (Dpp).

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Other paracrine factors: The majority of the paracrine factors belong to one of the four groups
indicated above, while some have few or no close relatives. Although they do not belong to the
families indicated above, factors including epidermal growth factor, hepatocyte growth factor,
neurotrophins, and stem cell factor all play significant roles in development. Erythropoietin,
cytokines, and interleukins are a few other components that are almost entirely involved in the
development of blood cells. For instance, activin can trigger several sets of genes at various doses
and can diffuse throughout a wide range of cell diameters. However, it's likely that the Vg1, BMP4,
and Nodal proteins only affect their immediate surroundings. These factors may cause these
neighbors to express more nearby short-range factors, which could start a chain reaction of
paracrine inductions. There is autocrine regulation in addition to endocrine, paracrine, and
juxtacrine regulation. When the same cells that secrete paracrine substances also react to them,
autocrine regulation takes place. In this instance, a molecule that the cell has its receptor for is
created by the cell. Placental cytotrophoblast cells produce and secrete platelet-derived growth
factor, which has a receptor on its membrane. Although autocrine regulation is uncommon, it can
be observed in these cells. The outcome is that tissue proliferates explosively.

5.8 COMPETENCE

Not all tissues are capable of responding to the inducer's signal. For instance, the Xenopus
laevis optic vesicle, or presumed retina, can cause the formation of lens tissue when it is positioned
beneath the head ectoderm in an ectopic location, or somewhere other than where it normally
develops. This appears to be a unique ability of the optic vesicle, making it an inducer. The optic
vesicle will, however, prevent the flank or abdomen of the same organism's ectoderm from
responding if it is positioned beneath it. Only the head ectoderm is capable of creating a lens in
response to signals from the optic vesicle. Competence is the capacity to react to a particular
inductive signal. Competence is an actively gained condition rather than a passive state. For
instance, the Pax6 protein appears to be crucial in enabling the ectoderm to react to the inductive
signal from the optic vesicle in the developing chick and human eye. Pax6 expression is only
present in the head ectoderm, which can develop lenses in response to the optic vesicle, and not in
other parts of the surface ectoderm.

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Fig.5.22. Showing the role of Pax6 as a competence factor

(Adapted from developmental-biology-7thed-sf-gilbert-pdf)

It is not required by the tissue that induces it. Although it is believed that the anterior portions of
the neural plate generate Pax6 expression, it is unknown how this occurs in the embryo's anterior
ectoderm. Ectodermal tissue can be given the ability to respond to the optic vesicle inducer by
being incubated close to anterior neural plate tissue. Therefore, the lens cannot be caused by a
single factor. The pharyngeal endoderm and heart-forming mesoderm, which support the lens-
forming ectoderm throughout the early- and mid-gastrula stages, maybe the initial inducers,
according to studies on amphibians. The following signals, such as one that encourages Pax6
production in the anterior ectoderm, may be produced by the anterior neural plate. Thus, although
the anterior ectoderm has already been induced by at least two different stimuli, the optic vesicle
appears to be the inducer.

5.9 SUMMARY

The process of creating a chick from an egg cannot be understood without a rational understanding
of the embryology of the growing embryo. Before the chick hatches from the egg, it is incubated
for three weeks. At this stage, all cells are identical, but throughout the time they begin to
differentiate into distinct types. By the third day of incubation, the limb buds for the wings and legs
are apparent. Even though it is not contained within the body, the heart is still getting bigger. The
immune system starts taking shape as the spleen, thymus, and cloacal bursa began to appear. When
a hen lays an egg, it has about 20,000 cells. During chick development, certain marginal zone cells
play a crucial role in determining cell destiny. Epiblast and hypoblast, the two layers of the
blastoderm, are fused at the edge of the area opaca. Neurulation is the process by which this tissue
develops into a neural tube. An embryo going through this process is referred to as a neurula.

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Different vertebrate classes utilize these processes of formation to different extents. A U-shaped
neural groove develops in the center of the neural plate shortly after it forms, dividing the right and
left sides of the embryo. The neural plate contains up to 50% of the ectoderm. It expands and
contracts via convergent expansion in both amphibians and amniotes. In an isolated neural plate,
the cells converge and spread out to form a narrower plate, but they do not roll up into a neural
tube.
The neural folds are produced by the neural tube being furrowed and the presumed epidermis being
pushed toward the center. The cells at this juncture give rise to neural crest cells in some species.
This is most evident in vertebrates with elongated body axes, such as birds and mammals.
Neural tubes are hollowed out during secondary neurulation in frogs and chicks. In the frog, cells
of the dorsal blastopore lip continue to develop ventrally rather than involuting into the embryo.
The distal component of the neurenteric canal becomes the ependymal canal ( neural tube lumen).
The early embryonic brain balloons at an astonishing rate and scale. The brain volume in the
developing chick embryo increases thirty-fold between days three and five.
The rhombencephalon forms a segmental pattern that identifies the locations from where particular
nerves emerge. Every rhombus will develop ganglia, which are collections of neuronal cell bodies.
The dorsal-ventral axis determines the polarity of the neural tube. The Sonic hedgehog protein,
which most likely originates from the notochord, is one agent of ventral specification. Retinoic acid
and TGF-superfamily determine the dorsal destiny.

The brain's neurons are arranged into layers (called cortexes) and clusters (called nuclei), each of
which has distinct connections and functions. Different types of transcription factors are induced
by the TGF-superfamily proteins at different distances from the roof plate, which confers distinct
identities on the cells. Migrating cells create a second layer surrounding the initial neural tube as
the cells close to the lumen keep dividing. As new cells from the germinal neuroepithelium are
introduced to it, this layer thickens more and more. A butterfly-shaped structure made of white
matter gradually surrounds the grey matter (mantle). The cerebellum is a relay station between the
outer layers of the brain and other regions. Some neural progenitors migrate from the germinal
epithelium to create nuclei. The internal granule layer is created when these granule neurons
migrate back into the white matter. Neurons are initially arranged vertically into layers that
communicate with one another, similar to the cerebellum. Adult forms of these layers are not
finished until middle childhood.

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Neurons in layer 6 give their principal output back to the thalamus, while those in layer 4 do the
opposite. Sonic hedgehog determines how the single eye field is divided into two bilateral fields.
The Pax6 protein seems to be particularly crucial for the growth of the lens and retina. The eyes
appear to be the most susceptible organs to Pax6 deficiency, even though the forebrain, hindbrain,
and nasal placodes are all expressed. Changes in cell structure and shape are required for the
differentiation of the lens tissue into a transparent membrane capable of directing light onto the
retina.
It takes the adult chicken two years to transition from an epithelial cell to a lens fiber. The iris, a
pigmented and skeletal tissue, is located directly in front of the photoreceptors. The embryo in
amniote vertebrates is a flattened disc, and the lateral plate mesoderm does not completely envelop
the yolk sac. The early primitive streak, which starts slightly posterior to Hensen's node and
extends approximately halfway down its length, is where the alleged heart cells originate. The heart
is a two-chambered tube with an atrium and a ventricle in a three-day-old chick embryo. A
condition known as "cardio bifida" causes a distinct heart to develop on each side of the body. By
the fourth day, the ECG of a chick embryo is similar to that of an adult.
A pitx-2 transcription factor is essential for correct cardiac looping and is only activated on the left
side of the lateral plate mesoderm. The Xin gene may mediate the cytoskeletal alterations required
for heart looping and is also activated by transcription factors Nkx2-5 and MEF2C. Gastrulation
movements are caused by the combined actions of the embryo's components rather than by the
embryo as a whole. It must be assumed that there are inducing chemicals with specialized activity.
Transplanting mammalian tissues into frog embryos or chick embryo tissue into rabbit embryos can
induce the development of main organs in vertebrates. Induction is most likely the only process
responsible for cell differentiation and cell organization into tissues and organs in vertebrates. The
phenomena are credited to Spemann in 1901 and Lewis in 1904, who proved that in some species
of Rana, the development of a lens from the ectoderm is influenced by the brain's underlying optic
lobe. In urodele amphibians, the primary organizer, or archenteron roof, can induce the nervous
system and sensory organs in the majority of vertebrate species. Spemann dubbed the dorsal lip of
the blastopore the Primary Organiser because it can start the development of a secondary embryo
when transplanted. Mesoderm appears to be induced by a variety of molecules from many types of
embryonic or adult cells like cultured mammalian cells, guinea pig bone marrow, carp swim
bladder, and chick embryos.
Cairns and Saunders (1954) demonstrated that the overlying ectoderm evolved following the origin
of the mesoderm from various locations of the leg or wing. An embryonic interaction, also known

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as an organizer, is the process by which one biological tissue transmits a chemical that influences
another embryonic section to create a structure that would not otherwise be feasible. Responding
cells are already prepared and ready to differentiate; all they need is a signal from the inducing
tissue to enable them to realize their full potential. Donor tissue caused the host embryo to develop
neural grooves, notochords, and other structures. They noticed that a larva with two heads had
formed after gastrulation - one naturally occurring and the other stimulated by the transplanted
tissue. In 1924, Max Spemann grafted a portion of the dorsal lip region of the early gastrula
of Rana sp. (donor embryo) to create a complete embryo. He asserts that this region's development
stimulates the development of the neural tube, which leads to the formation of the eyes. They
noticed that a larva with two heads had formed after gastrulation was finished. One head was
produced naturally throughout development, and the other was stimulated by donor tissue.
The lens-forming ectoderm that forms the cornea has developed a special capacity to react to
inductive impulses. Once the lens has developed, further tissues - such as the optic vesicle - may be
induced. There are several reasons for each induction as well as successive inductive occurrences.
The interactions of sheets of cells with nearby mesenchymal cells are some of the most well-
studied examples of induction. Connected cells give rise to sheets and tubes, which are the forms of
epithelia that can be formed from any germ layer. Unconnected, loosely packed cells are referred to
as mesenchyme. The mouth region of salamander and frog larvae has quite a different anatomy.
The ectoderm was told to create a mouth by the mesodermal cells, but instead, it created the only
mouth that it "knew" how to create. As a result, mesenchymal signals can be transmitted across
species boundaries. The elements involved in developing the eye or heart of the Drosophila are
quite similar to those responsible for developing mammalian organs. There are now more than a
dozen structurally similar members of the fibroblast growth factor (FGF) family.
FGFs have been linked to the development of mesoderm, axon extension, and angiogenesis. Sonic
hedgehog is a class of glycoproteins high in cysteine. This peptide is in charge of shaping the
neural tube such that sensory neurons develop from the dorsal neurons and motor neurons from the
ventral neurons. Sonic hedgehog, FGF4, and other paracrine factors are concentrated in the area of
the developing tooth where cell interactions are forming the teeth's cusps. The members of the bone
morphogenetic proteins (BMP) family were originally discovered by their ability to induce bone
formation. Bone formation is only one of their many functions, and they have been found to
regulate cell division, apoptosis (programmed cell death), cell migration, and differentiation. Some
paracrine factors are almost entirely involved in the development of blood cells. Activin, for
instance, can trigger several sets of genes at various doses and can diffuse throughout a wide range

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of cell diameters. Autocrine regulation takes place when the same cells that secrete these
substances also react to them. Ectodermal tissue can be given the ability to respond to the optic
vesicle by being incubated close to anterior neural plate tissue. Pax6 protein appears to be crucial in
enabling the ectoderm to react to the inductive signal from the optic vesicle.

5.10 TERMINAL QUESTION AND ANSWERS

5.10.1. MULTIPLE CHOICE QUESTIONS

1. How many days does it take for a chicken egg to hatch?
a. 19 Days
b. 28 Days
c. 24 Days
d. 21 Days
2. What is the part of the beak of the chick called that it uses to break out of the shell?
a. Comb
b. Pig
c. Beak
d. Egg tooth
3. Which of the tissue develops from the ectoderm of an embryo?
a. Nervous tissue
b. Muscular tissue
c. Connective tissue
d. None of the above
4. In the chick development of wing feathers, thigh feathers and claws depends on epithelial
specificity conferred by induction from mesenchymal components from different sources of
dermins which could be attributed to?
a. Autocrine interaction
b. Regional specificity of interaction
c. Regional activation by hormones
d. Inactivation of genetic interaction
5. Bones of vertebrates are derived from embryonic:
a. Ectoderm
b. Epiderm

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c. Mesoderm
d. Endoderm
6. What would happen as a result of a transplantation experiment in a chick embryo where the leg
mesenchyme is placed directly beneath the wing apical ectodermal ridge (AER)?
a. Distal hind limb structures develop at the end of the limb.
b. A complete hind limb will form in the region where the forelimb should be
c. The forelimb would form normally
d. Neither a forelimb nor a hind limb would form since the cells are already determined
7. The ability of cells to respond to a specific inductive signal is called
a. Regional specificity of induction
b. Competence
c. Juxtracrine signaling
d. Instructive interaction
Answers: 5.10.1: 1.d, 2.d, 3.a, 4.b, 5.b, 6.a, 7.b

5.10.2. LONG ANSWER TYPE QUESTIONS

Q.1. Write in detail about the Primary organizer and its morphological differentiation.
Q.2.What is embryonic induction? What are its various types?
Q.3. Write about various inductive interactions and factors affecting them.
Q. 4. Describe primary and secondary neurulation.
Q.5. Elucidate the mechanism of neural plate formation and its shaping.
Q.6. Explain the process of the development of a neural tube.
Q.7. How the tissues are organized in Central Nervous System? Explain.
Q.8. Write in detail about the development of the heart.

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UNIT 6: REGENERATION AND METAPLASIA

CONTENTS
6.1 Objectives
6.2 Introduction
6.3 Distribution of regenerative ability
6.4 Polarity in Regeneration
6.5 Mechanism of regeneration of Amphibian limb and lens
6.6 Metaplasia
6.7 Super-regeneration and heteromorphosis
6.8 Summary
6.9 Terminal Questions and Answers

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6.1 OBJECTIVES

After studying this unit, you will be able to:

1. Understand the phenomenon of regeneration in animals
2. Types of regeneration
3. Distinguish between physiological regeneration and reparative regeneration
4. Explain the terms autotomy and metaplasia.

6.2 INTRODUCTION

Regeneration can be defined as the natural ability of the organisms to replace worn-out parts,
repair or renew damaged or lost parts of the body, or reconstitute the whole body from a small
fragment during the post-embryonic life of an organism.
Regeneration thus is also a developmental process that involves growth, morphogenesis, and
differentiation.
Many organisms show the ability or power of regeneration. The power to regenerate the lost
parts of the body is found throughout the animal kingdom, having a wide diversity in its
potency. Regeneration is a fundamental phenomenon of life. It is dispensable for the survival of
the organism. Richard L. Gross has described it aptly as “if there is no regeneration there could
be no life, if everything is regenerated there would be no death, all organisms exist between these
two extremes.” Among animals, the power of regeneration was first discovered in Hydra
by Abraham Trembley.
If the tail of a house lizard is cut, the missing part develops again from the remaining part of the
tail. In some cases, regeneration is so advanced that an entire multicellular body is reconstructed
from a small fragment of tissue.
Our body spontaneously loses cells from the surface of the skin and is replaced by newly formed
cells, this is due to regeneration. Regeneration can be defined as the natural ability of organisms
to replace worn-out parts, repair or renew damaged or lost parts of the body, or reconstitute the
whole body from a small fragment during the post-embryonic life of an organism. Regeneration
is thus also a developmental process that involves growth, morphogenesis, and differentiation.

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Types of Regeneration:
In physiological Regeneration, there is a constant loss of many types of cells due to wear and
tear caused by day-to-day activities. The replacement of these cells is known as physiological
regeneration Eg: The replacement of Red Blood cells (R.B.Cs). The worn-out R.B.Cs is
deposited in the spleen and new R.B.Cs is regularly produced from the bone marrow cells. To
note further the life span of R.B.C's is only 120days and this process of regeneration is
continuous.
Replacement of Epidermal Cells of the Skin: The cells from the outer layers of the epidermis are
regularly peeled off by wear and tear. These are constantly being replaced by new cells, added by
the malpighian layer of the skin.

2. Reparative regeneration: This kind of regeneration, as the name suggests, involves the
repair of a wound or replacement of a body part removed intentionally or due to injury.
This type of regeneration may include restoration of parts of an organ or an organ as in the
regeneration of the eye and lens in amphibians or parts of the whole organism as in limbs of
urodeles, or it may be the regeneration of an entire organism from a pan detached from the parent
body as you will see in hydra.
The power of this type of regeneration is not found uniformly in all animals. Some have great
powers of such regeneration; in others, it is limited to varying degrees and in yet others, it is not
found at all.
Example: Regeneration of limbs in Salamanders, regeneration of lost tail in lizard, healing of the
wound, replacement of damaged cells etc.

Autotomy: In some animals like starfish (Asterias), some part of the body is broken off on
being threatened by a predator. This phenomenon of self-mutilation of the body is called
autotomy
Example: Crabs break off their leg on approaching the enemy, Holothurians throw off their
internal viscera, Starfish breaks off an arm etc.

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Regenerative capacity in animal group: The capacity of regeneration varies in its extent in
various animal groups. Regenerative capacity is very high among protozoans, sponges, and
coelenterates.
Invertebrates: Regeneration was first discovered in hydra by Trembley (1740). Even 1/1000th
part of the body regenerates into new organisms. In Phylum Porifera (sponges), the whole body
can be reconstructed from isolated body cells. The cells rearrange and reorganize to form a
double-layered sponge body wall.
Phylum Coelenterates (Hydra and Planaria) are the best examples of regeneration. Small
fragments of the body can give rise to a whole animal. In Hydra polyp may be cut into two or
more parts and each part will grow into a new fully developed part. The anterior cut of the body
regenerates the part of the posterior end with an adhesive end and foot while the posterior cut
end reconstitutes the mouth and tentacles. (See fig 6.1)

Fig 6.1: Regeneration in Hydra (Online sources)

When a Planaria is cut into many pieces, each part regenerates into a whole individual. The
power of regeneration in Planaria is maximum near the anterior end while minimum near the
posterior end. It is cut across lengthwise and each part of the body can regenerate the missing

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half. If a planarian can be cut from the middle part of the body through the pharynx, it can
regenerate a new one. (See fig 6.2).

Fig 6.2: Regeneration in Planaria (Online source)

Some annelids like earthworms can regenerate some segments removed from the anterior and
posterior ends of the body. Some molluscs can regenerate only the eyes and heads while squids
can also regenerate their arms. Many arthropods (e.g., spiders, crustaceans, insect larvae, etc)
can regenerate limbs only. Regeneration is faster in the young than in the adults. A regenerated
part may not always be similar to the part lost. This type of regeneration is called
heteromorphosis. Echinoderms (like starfish, brittle star, and sea Lilly) exhibit autotomy. They
can regenerate arms and parts of the body.

Vertebrates:
Fishes: Lamprey can regenerate its lost tail. Some fishes can regenerate parts of their fins.
Amphibians: The regeneration power is well marked in urodele amphibians like salamanders,
newts, and their axolotl larvae. They can regenerate limbs, tails, external gills, jaws, and parts of
the eye like the lens and retina. Tail and limb regeneration is found in the larval stages of frogs
and toads.

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Reptiles: Lizards exhibit autotomy. When threatened, the lizard detaches its tail near the base to
confuse its predator and later regenerates a new tail. The new tail differs from the old one in its
shape, absence of vertebrae, and the kind of scales covering it.
Birds: Regeneration is restricted to parts of the beak.
Mammals: Regeneration is restricted to tissues only. External parts are not regenerated. Skin
and skeletal tissues possess great power of regeneration. The liver has the maximum capacity for
regeneration. If one kidney is damaged or removed, the other enlarges to compensate for the lost
kidney. This is called compensatory hypertrophy.
Types of Regeneration based on Cellular Mechanism:
Based on cellular mechanisms regeneration can be of two types:
1. Morphallaxis: In this type, regeneration occurs mainly by the remodeling of existing tissues
and the re-establishment of boundaries, thus involving very little new growth. As a result, the
regenerated individual is much smaller initially. It subsequently increases its size and becomes
normal after feeding. This type of regeneration is known as morphallaxis or morphallactic
regeneration. Example: Regeneration of hydra from a small fragment of its body. (See fig 6.3)

Fig 6.3: Fragmentation in Planaria (Online source)

2. Epimorphosis: In this type, regeneration involves the dedifferentiation of adult structures to
form an undifferentiated mass of cells. They are highly proliferating and accumulate under the
epidermis, which has already expanded. Within two days, the bulge transforms into a conical
hump. This lump of dedifferentiated cells along with the epidermal covering is called

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regeneration bud or regeneration blastema. The dedifferential cells continue to proliferate and
finally redifferentiate to form a rudiment of the limb. The rudiment eventually transforms into a
limb. This type of regeneration is known as epimorphosis or epimorphic regeneration.
Example: Limb regeneration in amphibians.
3. Heteromorphosis or heteromorphic regeneration: When a different organ develops from
the one that has been removed, the phenomenon is called heteromorphosis. Eg: In shrimp
Palinurus, the eye is regenerated, if it is removed from the eye stalk. But if the eye is removed
along with optic ganglion, instead of the eye an antenna-like organ is regenerated. This type of
regeneration is exhibited by lower animals.
4. Super regeneration: The development of a superfluous number of organs or parts of the
body (eg. Heads, tail limbs) as a result of regeneration is known as super regeneration. When a
deep incision is made on the head end of planaria or earthworm, additional heads will develop.
Incisions in the middle part cause the development of both heads and tails.
5. Wolffian regeneration: It is a special kind of regeneration found in urodeles and anurans. In
Newt, Triturus, if the lens of the eye is removed, a new lens is formed from the uninjured iris.
The original lens is developed from epidermal ectoderm but the regenerating lens, formed from
the iris is neuroectodermal in origin. Thus regeneration of a part of an organ from tissue other
than its original embryonic tissue is called Wolffians regeneration, named after the discoverer
Wolf (1935).

Fig 6.4: Regeneration of a Limb of a Newt (Online source)

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Mechanism of Regeneration: Regeneration is a complex process that involves histological

and physiological events.
Regeneration of a Limb of a Newt: The mechanism of regeneration in salamander involves the
following stages.
 Wound healing: The epidermal cells from the edges of the wound migrate and spread
over the exposed surface. This is known as wound healing.
 Blastema formation: A few days later, undifferentiated cells accumulate inside the
epidermis, resulting in a bulge. This is known as regeneration bud or blastema.
 Redifferentiation and morphogenesis: The blastema develops rudiments of the lost
organ, like the digits which grow into new digits.
 Growth: The regenerated limb increases and attains the size of a normal limb.
In planarians and Hydra, there are undifferentiated cells called neoblasts which multiply and then
migrate from the deeper parts of the body to the cut surface.

6.3 DISTRIBUTION OF REGENERATIVE ABILITY

Although regeneration is found throughout the animal kingdom, the ability to regenerate lost
parts differs greatly in various groups of animals.
Regeneration in micro-organisms and Protozoans: The single cell marine alga called
Mermaid’s Cap (Acetabularia), which has a 50-mm length and its body part as small as 1/100th
of the total size, is capable of regenerating new individuals.

Regeneration in invertebrates:
(1) The process of regeneration is quite pronounced in sponges. The whole organism can be
reconstituted even from a few undifferentiated archaeocytes.
(2) Regeneration ability is very high in coelenterates. Trembley discovered the regeneration in
Hydra polyp in 1740 by cutting it into two or more parts; as a result, each part reconstitutes itself
into new complete individuals of miniature size.

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(3) Except planarians, Platyhelminthes do not regenerate to any great extent. When planarians
cut across or length-wise, each part of the body will regenerate the missing half.
(4) Nemerteans have a great regenerating ability and a complete worm can be formed even
starting from a very small fragment.
(5) Regenerating ability is very low in nematodes in which only closure of the superficial wound
is possible by a high degree of cell differentiation up to a fixed limit of some cells.
(6) In Annelida, if any oligochaete is cut into two halves, the posterior one regenerates the
anterior end with the mouth and the anterior half regenerates the new posterior end.
(7) Regeneration is relatively poor in Mollusca. In gastropods, eye stalks with eyes as well as
parts of the head or the foot may be regenerated.
(8) Regeneration is limited to the renewal of the lost part in Arthropoda. In most crustaceans,
limbs may regenerate at any stage of development, while in insects limb regeneration occurs
only in larval stages and regenerated limb often does not reach normal limb size.
(9) Among echinoderms, the starfishes, brittle stars, and sea lilies can regenerate arms and parts
of the disc.

Regeneration in vertebrates: Regeneration power is restricted to fins in fishes. But in

amphibia, regenerative power is highly specialized and most spectacular in urodele amphibians
(newts, salamanders) in larval as well as adult stages. Tails, external gills, upper and lower jaws,
lens, and retina of the eye can regenerate.
In newts and salamanders, repair starts in limbs by spreading the epidermis over the wound
followed by the closing of the wound. When the tails of reptiles like lizards are broken off, a new
tail regenerates which differs from the original one because of simplified vertebral columns and
scales of a different type. In birds, only the beak can be regenerated. In mammals, except few
marsupials, the regeneration power of limbs is very poor.

6.4 POLARITY IN REGENERATION

Generally, some animals show a clear example of polarity in Regeneration. The best example
is Hydra (Coelenterate) which has a base for attachment, a long body, and a set of tentacles at the
anterior end around the mouth. If we cut a Hydra into two parts, the anterior end will form the

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tentacles and the lower end will form the base for attachment. Thus, there is a distinct polarity in
the hydroids body. The upper end of each piece forms tentacles, while the lower end forms the
base. This polarity is similar to the animal-vegetal polarity in the sea urchin eggs and the
anterior-posterior polarity in the amphibian limbs. However, the polarity of the regenerating part
is not fixed like that of the polarity in embryonic systems.
The polarity of the hydra can easily be changed by treating it into two cut ends of a polarized
segment with oxygen. If we cut pieces in a chamber, dividing it into two, having different
oxygen concentrations, the polarity is completely reversed, the posterior end which would
normally develop into a base, forms the tentacles. Thus, it is evident that the organization within
that is liable to change and be reversed by the application of different concentrations of oxygen
at two ends.
Factors affecting the regeneration: As already stated, regeneration is the ability of an organism
to regain lost parts. The regenerative ability of an organism is mainly by its developmental
stages. There are many external and internal factors controlling regeneration. Some of them are
discussed here:
Temperature: This is the main controlling factor. Temp. Directly affect the rate of regeneration.
If the temperature is too low, the regeneration process also goes very slowly. The increase of
temperature to a certain limit accelerates the regeneration process. But it should not be very
much high as it is lethal to all the regenerative processes, for example, Planaria larva: this does
not regenerate at 30C but the regenerative activities are most active at 290C. Although a
temperature of 320C appears to be lethal for these types of animals.
Oxygen (O2): This is also a very important factor for regeneration power among animals. The
amount of O2 supply affects the rate of regeneration. It increases the rate of regeneration.
Food: Food does not show any important effect on regenerative ability. Fasting animals
regenerate at the expense of their stored food. However, the maximum quantity of food
accelerates regeneration to a certain extent.
Chemicals: Chemicals, especially Beryllium showed a great reaction as a controlling agent.
Traumatization with a needle is not sufficient to boost regeneration in the adult frog. Here, the
application of the hypertonic salt solution to the wound after amputation gives encouraging
results. While Beryllum salts applied to the amputated surface suppresses regeneration. It binds
the substance released from the damaged cells at the wound that cause regeneration.

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6.5 MECHANISM OF REGENERATION OF AMPHIBIAN LIMB

AND LENS

Regeneration of amputated amphibian limbs is perhaps the most dramatic example of reparative
growth among vertebrates. In anuran amphibians (frogs and toads), regeneration is restricted to
the developing larval limb, but limb regeneration occurs throughout life in many, if not most,
urodele species (newts and salamanders). Unlike fin and tail regeneration, the process in limbs
includes reproduction, not only of a complex musculature and vasculature but also a skeleton of
articulated endochondral bones with the original anterior/posterior patterning of the autopods
(hands or feet). Regeneration occurs via several overlapping phases, including wound closure,
dedifferentiation, cell proliferation and migration, growth, patterning, and differentiation.
Understanding this process involves sorting out how the events elicited by the trauma of
amputation set the stage for and integrate with the morphogenetic events and growth that allow
the replacement structures of the limb to form.
Salamander limb regenerates in the following stages:

(1) Wound healing: After amputation of limb, the epidermal cells surrounding the wound
migrate and spread over the exposed surface. This stops the bleeding. This is known as wound
healing. This layer of epidermis proliferates to form apical ectodermal cap.

(2) Blastema formation: After some days cells below the epidermis start dedifferentiation.
These dedifferentiated cells accumulate inside the epidermis. Due to this accumulation a bulge or
outgrowth form termed as blastema.

(3) Redifferentiation and morphogenesis: The blastema cells continue to divide. The specific
pattern and axis (dorsal-ventral, anterior-posterior) will form in the growing blastema. Thus the
cells of the blastema redifferentiate develop into different structure of the limb. Edge grows out
into new digits.

(4) Growth: New blood and nerve supply develops in the growing limb. The regenerated limb
increases in size and attain the normal length.

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Fig. 6.5: Regeneration of limb of Salamender (Source: Essential Developmental Biology, Slack J. W.)

Mechanism of regeneration of amphibian lens: It is a special kind of regeneration found

in urodeles and anurans. In Newt, Triturus, if the lens of the eye is removed, a new lens is
formed from the uninjured iris. The original lens is developed from epidermal ectoderm but the
regenerating lens, formed from the iris is neuroectodermal in origin. Thus regeneration of a part
of an organ from tissue other than its original embryonic tissue is called Wolffians regeneration,
named after the discoverer Wolf (1935).
It regenerates in the following stages:
a) After the removal of injury of the lens the dorsal region of iris thickens and a cleft arises
between inner and outer lamellae of the iris.
b) Amoeboid cells moved from the stroma into the cleft followed by marked increase in the
RNA and DNA synthesis as well as of mitotic cell division.
c) The pigmented cells of the dorsal region are engulfed by amoeboid cells.
d) The formed non pigmented cubical cells from hollow epithelial vesicle and extend inner
and outer lamellae.

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e) The inner wall cells of the vesicle elongated into the lumen and form primary lens fibre.
f) Later on the lens especific crystalline proteins are formed.
g) The primary lens fibres push to the front of vesicle to form the nucleus behind the lens
epithelium which forms the secondary lens fibre.

Fig. 6.6: (a) Stages of regeneration of eye lens from the dorsal part of the iris after lens surgical
extirpation in the newt. IPECs—iris pigment epithelial cells, LR—lens regenerate, DL—differentiated
lens. (b) Histological pictures of newly forming lens (grey and black nuclei—inclusion of 3H- thymidine
labelled cells) (Source: JBD)

6.6 METAPLASIA

Metaplasia refers to the replacement of a mature, differentiated cell type by another mature,
differentiated cell type that does not typically occur in the tissue in which it is found. Metaplasia
typically occurs as a response to chronic irritation of cells, which can be environmental or
pathological. Metaplasia itself is a benign, non-cancerous condition; however, if left untreated,
the cells undergoing metaplasia can become dysplastic (i.e., atypical in shape and size), which
can eventually lead to cancer. Metaplasia can be better understood with the below example;

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Intestinal metaplasia: Intestinal metaplasia refers to a transformation in cell type in the upper
digestive tract, which includes the stomach and esophagus. The nonkeratinized squamous
epithelium that typically covers the esophagus transforms into nonciliated columnar epithelial
cells. This condition is also known as Barrett's esophagus and is a consequence
of gastroesophageal reflux disease (GERD), which occurs as a result of stomach acid flowing
backward into the esophagus. Barrett's esophagus can be reversed by treating the underlying
GERD. However, if the condition persists, esophageal cells can become dysplastic. On the other
hand, intestinal metaplasia that occurs in the stomach is typically associated with a bacterial
infection known as Helicobacter pylori (H. pylori). H. pylori can affect the protective mucus
lining of the stomach, allowing the acidic contents to irritate the underlying stomach epithelial
cells, ultimately leading to metaplasia.
Causes of metaplasia: More often, metaplasia is caused by stressors (i.e. stomach acid) that
initiate the transformation into a new type of cell that is better adapted to handle the
increased stress. More specifically, intestinal metaplasia can be caused by H. pylori infection,
alcohol consumption, and chronic acid reflux. Squamous cell metaplasia of the respiratory tract
is typically induced by smoking which turns into the accumulation of toxins. Human
papillomavirus is the leading cause of cervical metaplasia and is mainly transmitted through
sexual contact.
Metaplasia reversibility: Metaplasia is reversible. Removing the offending stimulus, such
as smoking cessation in the case of respiratory squamous metaplasia or administering antibiotics
and acid-reducing proton pump inhibitors in the case of intestinal metaplasia. While metaplasia
is a risk factor for cancer, it is not cancer. If metaplasia undergoes another stage of
transformation, the cells will become dysplastic. Dysplastic cells are considered
a precancerous cell type and, if left untreated, will typically become cancerous.

6.7 SUPER-REGENERATION AND HETEROMORPHOSIS

Super regeneration: The development of a superfluous number of organs or parts of the body
(such as Heads, tails, and limbs) as a result of regeneration are known as super regeneration.
When a deep incision is made on the head end of planaria or earthworm, additional heads will
develop. Incisions in the middle part cause the development of both heads and tails.

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Heteromorphosis: Heteromorphosis refers to situations where an organ or tissue is different

from the expected, either because of (embryonic) development anomalies, or after
reparative regeneration following a trauma. The difference includes an abnormal location or an
abnormal shape. It should not be confused with homeosis, which means a big change in the
tissue structure of an organ. Heteromorphosis is an example of the imperfection of some
manifestations of the regenerative capacity.
Many organisms from protozoans to the chordate may have heteromorphosis examples, but it is
easier to find in lower forms of animals:
 Earthworm: distortion of polarity: replacement of removed tail with the head end
 Actinia: development of a cut into a second mouth
 Decapods: the replacement of removed eyes with antennae

6.8 SUMMARY

Regeneration can be defined as the natural ability to live organisms to replace worn-out parts,
repair or renew damaged or lost parts of the body, or reconstitute the whole body from a small
fragment during the post-embryonic life of an organism. Regeneration thus is also a
developmental process that involves growth, morphogenesis, and differentiation. Many
organisms show the ability or power of regeneration. If the tail of a house lizard is cut, the
missing part develops again from the remaining part of the tail. In some cases, regeneration is so
advanced that an entire multicellular body is reconstructed from a small fragment of tissue. Our
body spontaneously loses cells from the surface of the skin and is replaced by newly formed
cells. This is due to regeneration.

6.9 TERMINAL QUESTIONS AND ANSWERS

6.9.1 VERY SHORT QUESTIONS

Q 1. What is regeneration?
Ans. The process of repair and renewal of lost parts of the body is known as regeneration.
Q. 2. What is heteromorphic regeneration?

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Ans. In heteromorphic regeneration, the regenerated organ is different from the one that has been
renewed.
Q. 3. What is Blastema?
Ans. A few days after the healing of the cut or wound, the epidermis covering the wound bulges
out forming a stumpy outgrowth. This is known as blastma or regeneration bud.
Q. 4. Name the factors which affect regeneration.
Ans. Temp., food, oxygen concentration, radiation, electric current, and seawater.
Q. 5. Which factor affects the metabolism of carbohydrates and proteins?
Ans. Injury.
Q. 6. What is responsive tissue?
Ans. This is the tissue that responds to a morphogen of the inductor.
Q. 7. What is embryonic induction?
Ans. Embryonic induction is the process in which one tissue causes the differentiation of another
tissue during the development of an animal embryo.
Q. 8. What is necessary for regeneration in oliates?
Ans. Macromolecules
6.9.2 MULTIPLE CHOICES QUESTIONS
1. Regeneration is similar to:
a. autotomy
b. differentiation
c. Cleavage
d. division
2. Morpahallaxis occurs only in:
a. higher animal groups
b. lower animals group
c. both a and b
d. none of these
3. In Salamander complete limb is regenerated within:
a. 35 days
b. 45 days
c. 50 days

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d. 75 days
4. Entire organs cannot regenerate in:
a. Fishes
b. Mammals
c. Birds
d. Reptiles
5. Autonomy is seen in the tail of:
a. Tadpole
b. Rat
c. Gecko
d. All of these
6. All the physiological changes are under:
a. Neural control
b. Hormonal control
c. Both a and b
d. None of these
7. Salamander and Axolotl larva regenerate:
a. Limbs, eye structure and intestine
b. Jaws and external gills
c. Both a and b
d. Trunk
8. Regeneration in animals was reported by:
a. T. H. Huxley
b. A. G. Trembley
c. T. H. Morgan
d. T. Muller
9. Tissue regeneration is found in:
a. Mammals b. Reptiles
c. Birds d. Amphibians
10. Regeneration in micro-organisms and protozoans cannot take place without:
a. Micronucleus

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b. Nucleolus
c. Flagella
d. Macronucleus
11. Various types of cells aggregate into masses and organize into a whole organism in:
a. Coelentrates
b. Sponges
c. Vertebrates
d. Protozoans
12. Among annelids, the following animals never regenerate:
a. Earthworm
b. Allolobophora
c. Leech
d. Polychaetes
13. Destructive metabolic phase in regeneration is:
a. Catabolic
b. Anabolic
c. Respiratory
d. Excretory
14. Metaplasia is:
a. Non-reversible change
b. Reversible change
c. Both a and b
d. None of these
15. What are the common symptoms of metaplasia:
a. Loss of appetite
b. Weight loss
c. Belching
d. All of the above
16. Which of the following are the types of epithelial metaplasia:
a. Squamous metaplasia
b. Columnar metaplasia

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c. Both a and b
d. None of the above
17. What kind of response does metaplasia give:
a. Adaptive response
b. Non-adaptive response
c. Both a and b
d. None of the above
Answers: (1)a (2)b (3)d (4)b (5)c (6)b (7)c (8)b (9)a (10)d (11)b (12)c (13)a (14)b
(15)d (16)c (17)a

6.9 REFERENCES

Carlson B.M. (1999). Human embryology and developmental biology. 2 nd ed. St.Louis: Mosby.
Arora, M.P. (1997). Embryology,1 st edition. Himalayan Publishing house.
Verma, P.S. and Agarwal, V.K. (2007). Chordate Embryology. S.Chand & Company ltd.

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UNIT 7: METAMORPHOSIS

CONTENTS
7.1 Objectives
7.2 Introduction
7.3 Kinds of metamorphosis
7.4 Metamorphosis in Amphibia
7.5 Physiological and biochemical changes during metamorphosis
7.6 Hormonal control of metamorphosis
7.7 Summary
7.8 Terminal questions &answers
7.9 Glossary
7.10 References

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7.1 OBJECTIVES

After studying this module, you shall be able to learn and understand:
 To understand metamorphosis.
 Types of metamorphosis.
 Physiological and biochemical changes during metamorphosis.
 Hormonal control of metamorphosis.


7.2 INTRODUCTION

The egg of an animal at rest if provided with an enormous amount of food is smaller than the
sexually mature adult. The egg, so, does not directly develop into an organism the size of an
adult. The environment has adopted many methods to overcome this complication. Some of
these methods can be usually classified into the following three categories:
1. Direct development: In lots of animals, the hatched organism resembles an adult, without that
it is small in size. It is usually called a juvenile. In such cases, only growth and sexual
development occur in the juvenile after its hatching, e.g., certain Turbellaria, Ascaris,
Pheretima, reptiles, and birds. In the eggs during a large amount of yolk (macrolecithal eggs of
reptiles and birds), the growth is direct through no larval stage and no metamorphosis, since the
supply of food is sufficient for the whole development.
2. Viviparity: In a few animals, the hatched organism grows and develops inside the body of the
mother. It draws its food supply and oxygen from her with the help of a placenta. The remaining
growth of a newborn takes place after birth. For example, in eggs with very little yolk
(microlecithal eggs of the placental mammals), the development is direct without a larval stage.
3. Indirect development: In many animals, the egg develops into an unusually different
organism from the adult. Such an organism leads a self-regulating free life and is known as a
larva. Therefore these are with yolk but that is not enough for the complete development,
e.g., Herdmania, Amphioxus, frog, sea urchin, and insects. The larval stage is free-swimming or
free living and feeds excitedly to store food material for additional development. Compared with
their adults, the larvae habitually have different habitats and a different way of life. Larvae can
be so different from adults that in the past some were inaccurately given a species or a generic

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status. For example, the first instar larva of Meloidae (entomophagous, oil, or blister beetle) was
originally given the generic status of Triungulinus. The process of the transformation of larvae
into an adult is termed metamorphosis. (Gr., metamorphoun= to transform).
Definition of metamorphosis: Metamorphosis is a post-embryonic extension of the
developmental potential and involves dramatic changes in habit, habitat, morphology,
physiology, and behavior of the larva so that it is transformed into an adult having an entirely
different habitat and structure.
Metamorphosis is a widespread developmental phenomenon that is usually connected with a
theatrical change in habitat and succeeding way of life, such as the change from a planktonic to a
benthic existence way of the sea-urchin, from an aquatic to a terrestrial survival in frogs and
toads, and from non-flying to a flying continued existence in insects. Such changes in
atmosphere and behavior demand the equally rapid transformation of the structure and function
of the living technology. Metamorphic change during the development cycle is an acceleration or
absorption of essentially the same basic process feature of most forms of development. Mainly, it
consists of differential destruction of definite tissues, accompanied by an increase in growth and
demarcation of other tissues.
Occurrence of metamorphosis: Metamorphosis occurs in the majority of metazoan phyla,
starting with Porifera (amphiblastula) and ending with Amphibia (tadpole). Animals with no
direct development exhibit a range of larvae that undergo metamorphosis. The procedure of
metamorphosis in different animal groups differs equally like alteration and in the mode of
causation of the entire sequence, so, it is not possible to explain their metamorphosis in general
terms.

7.3 KIND OF METAMORPHOSIS

Among the chordates take place the following two basic types of metamorphosis:
1. Retrogressive metamorphosis: it occurs in ascidians wherever only the larvae
(tadpoles) have chordate features, the adults, adapted to a sessile existence, lose all larval
locomotory organs and thus give up all traces of chordate relationships.
Metamorphosis is the shape that varies in form during postembryonic development and in
several cases, signals a theatrical change in the habitat of the animal such as a change from a

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pelagic to benthic survival. Metamorphosis of the ascidian larva is unique and begins about
explosively. It involves the transformation of an active, non-feeding, pelagic, lecithotrophic (i.e.,
that feeds on its food on its yolk reserves) and tailed larva having many advanced features such
as axil notochord, dorsal neural tube, and particular sense organs, into an insert, sedentary or
sessile, simple (primitive) and planktotrophic filter- feeding adult through only a pharynx with
stigmata and endostyle, regularly indicating the chordate features of adult ascidian. This type of
metamorphosis which shows degenerative or retrogressive changes starting from larva to adult is
called retrogressive metamorphosis. It involves the following two types of change.
1. Retrogressive metamorphic changes: They involve the destruction and disappearance of
some of the larval structures such as follows:
i) Long tail of a larva with a caudal fin shortens and finally disappears.
ii) Caudal muscles, nerve cord, and notochord disappear as they break down and are consumed
by phagocytes. Thus, with the resorption of the tail go the most obvious traces of tunicate’s
chordate affinity.
iii) Larval sense organs (the ocellus and the otolith) are lost and the sensory vesicle is
transformed into an adult cerebral ganglion.
iv) Adhesive papillae disappear completely.
The biological occurrence in which the structures which are related only to the embryo or larva
and have no consequence for the adult animal, are lost at hatching, birth, or metamorphosis, is
called caenogenesis (Cohen, 1967). It allows larvae to develop quite different environments from
the adults of some species.

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Fig.7.1: Ascidia sp. Metamorphosis- free-tailed larva into a fixed ascidian

2. Progressive growth: Cells in the larval head give rise to adulthood. Some larval features are
retained and developed into the particular adult structures:
i) Due to the loss of the tail, the trunk becomes pear-shaped and four larger ectodermal ampullae
grow out of its four corners. These ampullae firmly anchor the metamorphosing tadpole to the
substratum. Rapidly two smaller ectodermal ampullae appear dorsolaterally.
ii) Anterior region among the point of attachment (adhesive papillae) and mouth exhibits rapid
growth, whereas the original dorsal side with atriopore stops growth. This causes shifting of
mouth through 90°. The body too rotates so that the general form of the adult sessile organism is
unspecified.
iii) Adult neural glands and nerve or cerebral ganglion are produced by the neural tube and trunk
ganglioncome to lie mid-dorsally between mouth and atriopore. The trunk ganglion itself persists
as a visceral nerve.
iv) With the absorption of its test covering, the mouth becomes functional and filter-mode of
feeding by inward ciliary water currents.

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v) Pharynx greatly enlarges, develops blood vessels and stigmata multiply rapidly, forming the
branchial sac.
vi) Stomach enlarges, intestine elongates and gets curved and liver develops.
vii) Atrial cavity becomes more extensive.
viii) Circulatory system with heart and pericardium develops.
ix) Gonads and gonoducts develop from larval mesodermal cells.
x) Test or tunic spreads to cover the entire animal, becomes thick, tough, and vascular, and
attaches the animal by forming a foot if essential.
Therefore, the foregoing metamorphosis changes mark the beginning of a sedentary, actively
feeding, sexual adult form.
Conclusion of retrogressive metamorphosis: As an outcome of retrogressive
metamorphosis, the free-swimming photo-positive and geo-negative ascidian tadpole larva
change into fixed, inactive geo-positive and photo-negative adults. The chordate characteristics
of larva similar to the notochord, nerve cord, and sense organs are lost in adults.

2. Progressive metamorphosis: The frog similar to other amphibians and insects has a
progressive metamorphosis (in different contrast to the retrogressive metamorphosis of the
Ascidians).In the progressive metamorphosis of the frog, the metamorphosis is connected with a
transition from an aquatic to a terrestrial mode of life and from an herbivorous to carnivorous
manner of feeding. It has a task to change an aquatic, herbivorous, tailed tadpole larva into a
terrestrial, carnivorous, and tailless frog. This alteration or metamorphosis involves many
structural, biochemical, and physiological changes. These changes comprise the destruction of
existing structures, construction of new structures, and modification of larval structures. At the
cellular level, metamorphosis is accomplished by cell death, cell proliferation, and cell
differentiation. Many genes active in larva become switched off, while many genes immobile in
larva start their movement. While metamorphosis requires a harmonious expression of many
genes in different tissues, it is controlled by hormones such as TH or thyroid hormones (e.g.,
thyroxine) In Rana tigrina the whole process of metamorphosis is completed within a week
(Agarwal and Niazi, 1977). It involves the following two types of changes:
Anatomical Metamorphic changes: Such changes fall into the following three categories:

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A. Destructive or regressive changes: The organs or structures that are essential during
larval life but no longer desirable in the adults are altered or may disappear completely:
1. The long tail of the tadpole with its dorsal and ventral fin-folds is resorbed and disappears
without a trace.
2. The internal gills are resorbed, gill clefts are closed and the peribranchial cavities disappear.
The opercular fold too falls off. Some aortic arches are reduced.
3. The horny teeth, labial rings, and horny lining of jaws are shed with the larval skin.
4. Intestine reduced from 9 times body length to 2 times body lengths.
5. Lateral line sensory system disappears and the mauthner cells of the brain also degenerate.
6. Some blood vessels disappear.
7. Resorption of different organs takes place due to autolysis, i.e., production of the lysosomal
enzymes and hydrolases (cathepsin, collagenase, nucleases, etc.). Amoebid macrophages of local
mesodermal origin engulf (phagocytose) the debris of the decomposed cells.
B. Constructive or progressive changes: These involve the growth and morphogenesis of
the following structures:
1. The limbs increase in size and differentiate further.
2. The middle ear develops in connection with the pharyngeal pouch (the pouch situated between
the mandibular and the hyoid arch).
3. The tympanic membrane develops, supported by the circular tympanic cartilage.
4. The eyes protrude on the dorsal surface of the head and develop eyelids and nictitating
membranes.
5. The tongue develops from the floor of the mouth.
6. The quadrate cartilage is rotated backward so that there is a significantly increased gap
enabling the frog to prey upon large insects.
7. With the changes in the organs of respiration, consequent changes take place in the vascular
system. The afferent and efferent vessels develop their direct associations and more and more
blood passes into the lungs. The heart becomes three-chambered. Aortic arches take on the
pattern of the adult frog.
C. Adaptation of larval structures (Adaptive change): Following organs of the frog
function both in the larva and the adult, but change their differentiation during metamorphosis:

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1. Change in the hardening, pigmentation, and change for dehydration takes place in the skin.
The skin of the tadpole is enclosed with a double-layered epidermis. During metamorphosis, the
number of cells in the epidermis increases, and the surface layers develop into cornified.
Multicellular mucous and serous glands build up as pockets sinking from the surface into the
subcutaneous connective tissue layer, the dermis. The pigmentation of the skin to changes and
new patterns of colors appear.
2. In a tadpole the intestine is very long, as in mainly herbivorous animals, becomes significantly
foreshortened and most of the coils which it forms in the tadpole become straightened out.
3. In the head, widening of mouth, growth of the adult-type jaw, and repositioning of eyes take
place. The snout assumes the adult shape.
4. Trunk becomes narrower than the head.
5. The larval pronephros (kidneys) modify into mesonephric kidneys of an adult.
6. Erythropoietic site changes from the kidney to the spleen.
7. The brain becomes more highly differentiated.
8. At the cellular level, cell change is evident in eyelids, limbs, lungs, tongue, eardrum, skin,
liver, pancreas, and intestine. Perhaps no cell or tissue or organ remains completely unchanged.
9. There is no change in the lungs throughout metamorphosis.

7.4 METAMORPHOSIS IN AMPHIBIA

Amphibians provide the best example of metamorphosis in vertebrates. In them, metamorphosis

incorporates the following ecological, morphological, physiological, and biochemical changes.
Ecological Metamorphosis Changes: In amphibians, ecologically, metamorphosis is
associated with a transition from an aquatic to a terrestrial mode of life. Superimposed on this
change of environment, a change in feeding habit occurs in the anuran amphibians. For example,
the tadpoles of most frogs and toads feed on the vegetable matter- particles of plants, living and
decaying- which they scrap off from submerged objects with the aid of the horny teeth
surrounding their mouth. Some anurans are detritus feeders and others as the tadpoles of the
clawed toad Xenopus are plankton feeders. Adult frogs and toads are carnivorous, living on
insects, worms, and small vertebrate animals such as small frogs, birds, rodents, etc., the latter is
caught overpowered, and swallowed by them. In the case of urodele amphibians (i.e.,

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salamanders, newts, etc.), however, there is no substantial change of diet, the larvae being as
carnivorous as the adults though naturally, they feed on smaller animals (mainly crustaceans and
worms).
Morphological Metamorphic Changes: The changes in the organization or morphology
of the animal during metamorphosis are in part progressive and in part regressive and may be
grouped into three categories:
1. The organ or structures necessary during larval life but redundant in the adults are reduced and
may disappear completely.
2. Some organs develop and become functional only during and after metamorphosis.
3. A third group of structures while present and functional both before and after metamorphosis
become changed to meet the requirements of the adult mode of life.
From a morphological point of view, anuran amphibians undergo extensive metamorphic
changes because the degree of difference between their larvae and adults remains much more
profound. The metamorphosis of anuran amphibians includes the following changes in the
organization.
1. Regressive metamorphic changes of Anura: Certain adaptive structures formed during
embryonic development, namely, the ventral suckers, external gills, and long tail along with fin
folds of the tadpole larvae are resorbed during early functional life. Further, gill clefts are closed,
peribranchial cavities disappear and the horny teeth of the perioral disc are shed, as well as the
horny lining of the jaws. Further, the shape of the mouth changes, the local tube becomes
shortened and reduced, and some blood vessels of precociously formed structures, are not a part
of the true metamorphic event which occurs much later. They disappear when they have served
their purpose.
2. Progressive metamorphic changes of Anura: The progressive or constructive
metamorphic changes involve the progressive development of the limbs, which increase in size
and differentiation. The fore-limbs, which in frogs develop under the cover of the opercular
membrane, breakthrough to the exterior. The gill arches become modified into the hyoid
apparatus. The middle ear develops in connection with the first pharyngeal pouch. The tympanic
membrane develops and is supported by the circular tympanic cartilage. The eyes protrude on the
dorsal surface of the head and develop eyelids. The tongue is developed from the floor of the
mouth.

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Fig.7.2: Metamorphosis in frog

3. Organs that exist both in larva and adult of Anura: The organs which function both in
the larva and the adult, but change their differentiation during metamorphosis are primarily the
skin, the intestine, and the brain. The skin thickens and becomes more glandular by possessing
multicellular mucous and serous glands, attains an outer keratinized layer, and acquires a
characteristic pattern of pigmentation. While the intestine which is very long in tadpole as in
most herbivorous animals becomes proportionately shorter and most of the coils that it forms in
the tadpole become straightened out. The brain becomes more highly differentiated.
At the cellular level cell modifications are evident in eyelids, limbs, lungs, tongue, eardrum,
operculum, skin, liver, pancreas, and intestine. Perhaps no cell or tissue or organ of Anura
remains unaffected during metamorphosis remains very rapid and takes only a few days.
Urodele amphibians go through less unusual ecological and morphological metamorphic
changes. For instance, in them the tail is retained, only the fin folds disappear. The branchial
apparatus is compact the external gills become resorbed and the gill clefts closed. The visceral

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skeleton becomes very much reduced. The head changes its shape, becoming more oval. The
progressive metamorphic changes occur mostly in the skin and the eyes. The skin becomes
cornified and multicellular skin glands become distinguish. The pigmentation of the skin
changes. The eyes bulge more on the dorsal surface of the head and develop lids. The legs and
intestine suffer no change. In urodeles, therefore the metamorphosis is more gradual and may
take up several weeks.
It is worth mentioning that lungs do not undergo drastic changes during metamorphosis in both
anurans and urodeles, they develop very gradually and become fully functional in the larval state.
Long before metamorphosis, the larvae of frogs and salamanders start coming up to the surface
and gulping air into their lungs and thus supplementing their aquatic respiration. This may be of
considerable importance when the larvae develop in stagnant and polluted waters, as is often the
case.
Time of metamorphosis of Amphibians: Frogs metamorphose after different periods of
growth according to the species and it was early known that the achievement of significant
species-specific size was much more essential than the duration of growth. Thus in nature,
American bullfrog tadpoles metamorphose at the end of the third summer-growth season in the
north except at the end of the second summer-growth season in the south, after having attained a
certain size, the time required depending on the mean environmental conditions. Further, it has
also long been known that the addition of iodine to water or feeding with thyroid gland tissue
causes metamorphosis to occur earlier at a smaller size, while the elimination of iodine from the
diet postpones it, from the first, therefore the iodine- containing thyroxin of the thyroid gland has
been studied intensively about the timing of metamorphosis and the metabolic effect of the
hormone.

7.5 PHYSIOLOGICAL AND BIOCHEMICAL CHANGES DURING

METAMORPHOSIS

Morphological changes during metamorphosis go through the following physiological changes:

1. In the frog, the endocrine function of the pancreas starts at metamorphosis and this is
associated with the increased role of the liver in the earnings of carbohydrates (glycogen).

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2. A thoughtful change takes place in the excretory mechanism. In the tadpole larva, the end
creation of nitrogen metabolism is ammonia, which is easily disposed of by diffusion in an
aquatic medium except in a terrestrial animal it might accumulate and becomes precarious since
of its high toxicity. Metamorphosed frogs, though, excrete most of their nitrogen in the
appearance of urea and only a small amount as ammonia. The changeover occurs in the late
stages of metamorphosis and is, of course, due to the changed function of the liver which
performs the synthesis of urea (Munro, 1939). The biosynthesis of urea from bicarbonate and
ammonia involves several enzymes and their activity which is found to be related to the level of
thyroxin hormone in the blood rising rapidly during the metamorphic peak.
3. The liver of the froglet starts the secretion of a copper-binding serum protein called
ceruloplasmin (Goel, 1984).
4. In the skin begins the synthesis of melanin and serotonin.
5. Peptic activity starts in the stomach of the frog let for the digestion of animal tissue.
6. In the eyes, the modification takes place in the visual pigments- the larval porphyropsin is
replaced by adult rhodopsin.
7. In red blood cells, the respiratory pigment hemoglobin also changes. Thus, larval hemoglobin
that has a higher affinity for oxygen and sensitivity to acid (i.e., independent from the pH) is
replaced by adult hemoglobin which has a lower affinity for oxygen and shows the Bohr effect
with pH changes (i.e., highly sensitive to acid).
According to Freiden (1961), the biochemical metamorphic alterations may have direct or
indirect adaptive value relating to the transition from freshwater to land. Among the most
important adaptive changes are the shift from ammonotelism to ureotelism, the increase in serum
albumin and other serum proteins, and the alteration in the properties and biosynthesis of
hemoglobins.
The development of certain digestive enzymes and augmentation of respiration also contribute to
the achievement of the differentiation process. Throughout metamorphosis, many more
important chemical developments may be secondary to primary morphological or cytological
alteration which aid in the adjustment to the land. These comprise changes in carbohydrates,
lipid, nucleic acid, and nitrogen metabolism. Major change occurs in water balance, visual
pigments (vitamin A), pigmentation, and tail metabolism.

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7.6 HORMONAL CONTROL OF METAMORPHOSIS

The simultaneous changes in so many parts of the animal’s body through metamorphosis
propose the survival of some common reason for all the transformations. It has been found
that the common cause is a hormone released in large quantities from the thyroid gland of the
animal which incoming the stage of metamorphosis. The first identification of this was obtained
when Gundernatsch (1912) fed some frog tadpoles on the dried and powdered sheep thyroid
gland and observed that they metamorphosis precociously. The confirmation that the thyroid
hormone is the source of metamorphosis in normal development was further given by the
following two experiments: In an experiment when the rudiment of the thyroid gland was
removed in frog embryos in the tail bud stage, then, it was found that the operated tadpoles were
possible and showed normal growth but failed to metamorphosis (Allen.1918). The thyroid-less
tadpoles continued to grow and attained a much larger size than normal. It was thus proved that
metamorphosis cannot set in without a stimulus coming from the thyroid gland. In the final
experiment, thyroid-less tadpoles were provided with thyroid hormone either by feeling them on
the thyroid gland or by immersing them in water containing soluble extracts from thyroid glands.
The tadpoles treated in this way immediately proceeded to metamorphosis, thus showing that
their thyroid glands are not essential so long they are supplied with thyroid hormone (Allen,
1938). Related experiments were carried out on urodele amphibians by Marx (1935).
The process of metamorphosis is essentially under hormonal control. Hormones from the
hypothalamus (e.g., neurosecretions like TRF or thyrotropin-releasing factor and PIH or
prolactin-release-inhibiting factor or hormone), the hypophysis (e.g., TSH or thyroid-stimulating
hormone and the PH or prolactin hormone) and the thyroid (e.g., thyroid hormones such as
thyroxin) regulate the process of metamorphosis. Thyroxin hormone affects the tissues directly,
causing the degeneration and necrosis of some cells and stimulating the growth and
differentiation of others. Iodine is not only essential for the working of thyroxin hormone; it has
been found that iodine alone can cause a metamorphosis in the frog. If the tadpoles are kept in
water containing the elements iodine or if a weak solution of iodine is injected or an iodine
crystal is implanted in a tadpole, it undergoes a metamorphosis.
Certain ecological factors also affected the process of metamorphosis in the frog. These factors
include starvation, temperature, crowding, illumination, chemicals, and dietary supplements.

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Throughout metamorphosis, synchronized changes in all body parts propose the survival of
hormones released in large quantities from the thyroid gland of the animal. This indication was
predictable by Gundernatsch (1912) whereas who fed some frog tadpoles on the dried and
powdered sheep thyroid gland and observed their metamorphosis precociously. Thyroid hormone
is the cause of metamorphosis in usual development was further proved experimentally.
The amphibian metamorphosis is below neuroendocrine control, connecting neurosecretory cells
in the brain (the hypothalamus) and two endocrine glands, the pituitary (anterior pituitary) and
the thyroid. The start of metamorphosis could be an environmental signal touching the larval
brain through the nervous system, or there may be an endogenous ‘clock’ in the hypothalamus.
In a way, the hypothalamus integrates the information received from the body through
environmental information.
Neurosecretory cells in the hypothalamus are stimulated to create TRF or thyroid-releasing factor
which stimulates the anterior pituitary gland to produce a TSH or thyroid-stimulating hormone
which causes an orderly increase of thyroid secretion. An increase in thyroid hormone then trips
the arranged sequence of tissue changes that transform the tadpole larva into the frog.
Another pituitary hormone, called prolactin is also found to be involved as an inhibitor in the
overall control of metamorphosis. Developmental control is affected by a balance between
inhibition and disinhibition rather than stimulation at the level of endocrine action. Thyroid
hormones are also known to affect the process of protein synthesis at the levels of transcription
and translation and to have a role in cytodifferentiation.

7.7 SUMMARY

Metamorphosis is a post-embryonic extension of the developmental potential and involves

dramatic changes in habit, habitat, morphology, physiology, and behavior of the larva so that it is
transformed into an adult having an entirely different habitat and structure. Metamorphosis is a
widespread developmental phenomenon that is usually connected with a theatrical change in
habitat and succeeding way of life, such as the change from a planktonic to a benthic existence
way of the sea-urchin, from an aquatic to a terrestrial survival in frogs and toads, and from non-
flying to a flying continued existence in insects. Such changes in atmosphere and behavior
demand an equally rapid transformation of the structure and function of the living technology.

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Metamorphic change during the development cycle is an acceleration or absorption of essentially

the same basic process feature of most forms of development. Mainly, it consists of differential
destruction of definite tissues, accompanied by an increase in growth and demarcation of other
tissues.
Metamorphosis occurs in the majority of metazoan phyla, starting with Porifera (amphiblastula)
and ending with Amphibia (tadpole). Animals with no direct development exhibit a range of
larvae that undergo metamorphosis. The procedure of metamorphosis in different animal groups
differs equally like alteration and in the mode of causation of the entire sequence, so, it is not
possible to explain their metamorphosis in general terms.
In species with small eggs, a larval stage is interposed as feeding adaptation to support the
continued development of adult structures. As soon as adult tissues mature, the larva undergoes
dramatic metamorphic changes as larval tissues are shed and transformed into an adult.
Two basic types of metamorphosis:
1. Retrogressive metamorphosis: it occurs in ascidians wherever only the larvae (tadpoles)
have chordate features, the adults, adapted to a sessile existence, lose all larval locomotory
organs and thus give up all traces of chordate relationships.
2. Progressive metamorphosis: It occurs in amphibians and includes a change of simple larval
organization into the more complex organization of the adult.
The process of metamorphosis is essentially under hormonal control. Hormones from the
hypothalamus (e.g., neurosecretions like TRF or thyrotropin-releasing factor and PIH or
prolactin-release-inhibiting factor or hormone), the hypophysis (e.g., TSH or thyroid-stimulating
hormone and the PH or prolactin hormone) and the thyroid (e.g., thyroid hormones such as
thyroxine) regulate the process of metamorphosis. Thyroxine hormone affects the tissues
directly, causing the degeneration and necrosis of some cells and stimulating the growth and
differentiation of others. Iodine is not only essential for the working of thyroxine hormone; it has
been found that iodine alone can cause a metamorphosis in frogs. If the tadpoles are kept in
water containing the elements iodine or if a weak solution of iodine is injected or an iodine
crystal is implanted in a tadpole, it undergoes a metamorphosis.

7.8 TERMINAL QUESTIONS AND ANSWERS

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7.8.1 Multiple Choice Questions:

1. In amphibians, metamorphosis is done by:
a) Pituitary gland
b) Thyroid gland
c) Adrenal gland
d) Pineal gland
2. Another word for metamorphosis is:
a) Survival
b) Evolution
c) Modelling
d) Change
3. Retrogressive metamorphosis occurs in:
a) Urochordata
b) Cephalochordates
c) Vertebrates
d) Cyclostomes
4. Retrogressive metamorphosis is found in:
a) Amphioxus
b) Ascidia
c) Rana tigrina
d) Protochordata
5. The ascidian tadpole larva undergoes:
a) Progressive metamorphosis
b) Retrogressive metamorphosis
c) Partial metamorphosis
d) Complete metamorphosis
6. What is metamorphosis?
a) Process of transforming a larva into an adult
b) Process of transforming a pupa into an adult
c) Process of modification
d) Process of regeneration

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7. Another term for a fertilized ovum is:

a) Placenta
b) Sperm
c) Fallopian
d) Zygote
Answers: 1 b, 2 d, 3 a, 4 b, 5 b, 6 a), 7 d)

7.8.2. Short Answer Question:

1. Definition of metamorphosis.
2. What are two basic types of metamorphosis?
3. Explain retrogressive metamorphosis.
4. Write about the ecological metamorphosis changes in amphibians.

7.8.3. Long Answer Question:

1. What is metamorphosis? Describe the hormonal control of metamorphosis in amphibians.
2. Discuss the role of various hormones that control metamorphosis in a frog.
3. Explain the significance of metamorphosis.
4. Discuss the morphological, anatomical, and biochemical changes involved in the
metamorphosis of the tadpole of the frog.
5. Discuss the role of various hormones that control metamorphosis in the frog.

7.9 GLOSSARY

Amnion: (Gk. amnion=foetal membrane) An extraembryonic inner, fluid-filled sac composed of

a thin double membrane that surrounds the embryo in reptiles, birds, and mammals. It provides a
kind of private aquarium to the embryo and protects it from mechanical shocks such as pressure,
abrasion, irritation, and desiccation.
Amniote egg: an egg that is isolated from the environment by a more or less impervious shell
during the period of its development and which is completely self-sufficient, requiring only
oxygen from the outside.

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Embryo: The early developmental stage of an organism produced from a fertilized egg. In the
mammal, the later embryonic stage is called a fetus.
Fetus: An unborn or unhatched vertebrate that has passed through the earliest development
stages.
Macrolecithal: referring to a large amount of yolk stored in the egg, e.g., eggs of insects,
reptiles, birds, and monotreme mammals.
Mesolecithal refers to a moderate amount of yolk stored in the egg.
Metamorphosis: Abrupt transition from larval to adult; it includes certain morphological,
anatomical, physiological, and behavioral, hormonally regulated changes in the larval form to
transform it into the adult form.
Microlecithal: referring to a small amount of yolk stored in the egg, e.g., Amphioxus, eutherian
mammals.

7.10 REFERENCES

1. Chordate Embryology Developmental Biology by P.S. Verma & V.K. Agarwal

2. Development Biology (1997), Sinauer Gilbert 5th Edition Ass. Publ. Massachusetts.
3. Arora, M.P. (1997). Embryology,1 st edition. Himalayan Publishing house.
4. https://www.yourarticlelibrary.com/science/process-of-metamorphosis-in-amphibians-and-its-
hormonal-control/23158

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UNIT 8: TERATOGENESIS

CONTENTS
8.1 Objectives
8.2 Introduction
8.3 Types of teratogenesis
8.3.1 Mechanisms of genetic and environmental teratogenesis
8.3.1.1 Genetic teratogenesis in animals
8.3.1.2 Environmental teratogenesis
8.3.2 Phenocopies
8.3.3 Developmental mechanisms of teratogenesis
8.4 Summary
8.5 Terminal questions &answers
8.6 References

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8.1 OBJECTIVES

After going through this unit you shall be able to:

 Understand the concept of teratogenesis
 Types of teratogenesis.
 What are phenocopies?
 Developmental mechanisms of teratogenesis.

8.2 INTRODUCTION

Prenatal toxicity characterized by structural or functional defects in the developing embryo or

fetus is known as Teratogenesis. It too includes intrauterine growth retardation, death of the
embryo or fetus, and transplacental carcinogenesis (in which chemical exposure of the mother
initiates cancer development in the embryo or fetus, resulting in cancer in the offspring after
birth).
Intrauterine human development has three stages: implantation, post-implantation, and fetal
development. The first two stages of implantation and post-implantation are the embryonic
stages and last through the first eight weeks after conception. The fetal development stage begins
in the ninth week and continues to birth.
The developmental stage depending on chemical contact with the mother can result in different
degrees of toxicity in the embryo or fetus. In the preimplantation phase, a toxic chemical can kill
some of the cells in the blastocyst, resulting in the death of the embryo. Throughout the post-
implantation period, chemical-induced cell death leads to one of two outcomes. If death is
controlled by those cells undergoing active cell division at the moment, the analogous organs are
affected, resulting in malformation. If the cell death is widespread with no significant replication
by the remaining cells to sustain life, the embryo dies. During the third, fetal, period, chemical
damage can retard growth or, if severe sufficient, kill the fetus.
A congenital malformation is a gross structural present at birth. Its incidence is about 2.5% in all
infants born. But, only half of these deformities are noticeable at the time of delivery, most of the
remainder coming to light during the first postnatal year. The term congenital anomaly is

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reserved for a minor congenital disorder such as a deformed finger or ear lobe. Anomalies are
found in a further 2.5 percent of live-born infants.
Individuals of a species (including humans and other animals) exhibiting minor deviation
variation from each other are considered normal. Individuals that show gross deviation from each
other are considered normal due to congenital malformations and are known as monsters or
terata. The abnormal development or formation of terata is called teratogenesis and the science
which is concerned with the investigation of terata and teratogenesis is called teratology.
Teratogenesis is the formation of an abnormal organism. A teratogen is any manager that
physically or chemically alters developmental processes and produces inherited deformities. The
nature of the teratogen and the developmental stage during which the variation occurs is critical
to the type and severity of abnormality it will produce. Biological factors such as the organism’s
gestation process, developmental pathways, and life-cycle characteristics also control the
exposure and effect of a teratogen. Mechanical disruptors, environmental factors, and chemical
contaminants are the primary categories of teratogens disturbing wildlife species. The
introduction of an embryo, fetus, or larva to a teratogen may result in death, structural
malformation, functional disorder, or growth retardation. The most commonly described
teratogenic effects taking place in ecosystems are external malformations. Wild organisms have
always been subject to teratogenic insult; however, current anthropogenic activities have
increased the prevalence of deformities. Herein, the current state of teratogenesis knowledge
concerning amphibians, reptiles, birds, fishes, mammals, and invertebrates is discussed with
emphasis on structural malformations resulting from exposure to chemical contaminants.

8.3 TYPES OF TERATOGENESIS

Teratogenesis is Genetic teratogenesis and environmental teratogenesis. Genetic teratogenesis

has been studied both in human beings and animals.
Genetic teratogenesis in human beings: Abnormal genes may be inherited from one or both
parents and they may be dominant or recessive. In the majority of the afflicted individuals,
however, a Mendelian pattern of inheritance cannot be observed, the abnormalities merely occur
more frequently among relatives than in the general population. In those occurring among
relatives, there may be a complex interplay between several genes.

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Deformities due to abnormal dominant genes are rare. In most of these, the skeleton is affected
and the deformities include achondroplasia (insufficient growth of long bones), and
arachnodactyly (abnormally long hand and foot bones).
A teratogen is a substance that may lead to birth defects in an embryo or fetus. During
pregnancy, contact with certain chemicals, infections, and drugs may increase the risk that a
person will miscarry or that the embryo or fetus could have a developmental abnormality.
Alcohol and smoking are two common teratogens. Contact with either of them can lead to
developmental anomalies, miscarriage, stillbirth, preterm labor, and a variety of other pregnancy
complications.
The contact of teratogens with pregnancy or a fetus depends on numerous factors. The timing
and length of exposure, the stage of pregnancy when the exposure happened, whether a parent’s
genes make them more at risk, and the type of agent they were exposed to all contribute to the
risk. Teratogens commonly fall under the following categories:
1. Drugs
2. Infection
3. Physical Agents
4. Environmental Toxins
5. Maternal Health Conditions
8.3.1 Mechanisms of genetic and environmental teratogenesis
8.3.1.1 Genetic teratogenesis in animals: Genetic teratogenesis can be considered under the
following two headings:
1. Gene-phene relationship. Several different genes can cause the same terata, though not
necessarily by the same route. For example, there are more than twenty genes that affected eye
color in Drosophila melanogaster. The mutants causing the same defect may be either recessive
or dominant. For example, in fowl, the trait of rumplessness (absence of tail) is controlled by
either a recessive or dominant gene. In some cases, the same mutation may behave as recessive
or dominant depending on the genetic background. Thus, in mice, the fused gene (for fusion or
absence of ribs and or absence of tail) is dominant in Mus musculus but recessive in Mus
musculus bactrianus.
The penetrance (i.e., the proportion of affected individuals in a population) and expressivity (i.e.,
degree of effect) of mutant genes is dependent on both genetic and environmental factors. For

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example, fowl carrying rumples gene can be selectively bred to produce a ‘normal’ tail
phenotype. Similarly, in Drosophila carrying the Bar eye gene, the size of the eye and the
number of facets in the eye decreases by about 100 facets with an increase in temperature (15° to
31°C) during the development.
2. Autophene, allophone, and pleiotropy. Not all genetic terata are the result of the intrinsic
action of genes in the affected tissue. The following two examples will make the point clear:
(1) Creeper mutation (cp/cp) in fowl affects the limbs (called phocomelia or abnormally short
limbs) and the eyes (called microphthalmia or small eye), the embryo does not survive till
hatching. Transplants of the cp/cp limb rudiments in the normal hosts produce phocomelia limbs.
However, transplants of cp/cp eye rudiments in the normal hosts produce normal eyes.
Therefore, the cp gene intrinsically affects limb development (it is called autophene) but only
indirectly affects eye development (it is called allophone).
(2) The multiple effects of one gene (pleiotropy) are also now better understood. Any given gene
mutation essentially affected the production or structure of one transcribed RNA molecule. The
translation product of this RNA (i.e., mRNA), the defective protein, may ultimately result in
various defects due to the correlation of various biochemical reactions in the body (i.e., by a
pedigree of causes), e.g., death in rats due to grey lethal mutant gene and sickle cell anemia in
human beings.

8.3.1.2 Environmental teratogenesis: Almost any environmental factor can be teratogenic.

The environmental factor may be either biological (e. g., viruses) or non-biological such as
physical factors (e.g., temperature, irradiation, mechanical disturbance) and chemical factors
(e.g., drugs, environmental chemicals, and dietary imbalances or malnutrition).
1. Teratogenesis due to infection: The most dangerous known teratogenic organism is the virus
of German measles (rubella). Contraction of the disease by the mother during the first month of
pregnancy appears to carry about a 50 percent risk of producing a congenital abnormality, during
the second month the risk is about 25 percent, and during the third month about 7 percent. The
virus tends to affect the developing eyes, ears, and palate. The triads of congenital cataracts
(blindness), heart disease, and deafness have become known as the rubella syndrome. Rubella
also knows to cause terata such as mental deficiency due to a very small brain, cleft palate, and
hare-lip.

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Cytomegalovirus (CMV) infection is found all over the world. Like the rubella virus, it passes
through the placenta to infect the fetus. The CMV is show signs of disease. It may cause
deafness, mental retardation, epilepsy, liver disease or cerebral palsy (paralysis due to damage to
the brain), or a variable combination of these. Other teratogenic organisms are toxoplasmosis and
syphilis. Syphilis is known to cause stillbirth or other congenital abnormalities.
2. Teratogenesis due to Drugs: The potential danger of new drugs to the fetuses was
exemplified by two unrelated drugs, thalidomide and meclizine. Thalidomide is a mild sedative
tranquilizer that was prescribed for use in pregnancy in many European countries in the late
1950s. The German scientist Lenz (1961) reported a possible connection between this drug and
an increased frequency of a human congenital abnormality of the limbs known as Amelia (no
limbs) and the closely related phocomelia (no development of long bones of limbs and flipper-
like hands or feet attached directly to the trunk). A daily intake of thalidomide for one week
during early pregnancy was sufficient to induce limb defect. From 1959-1961 thousands of
babies in West Germany and hundreds in other countries such as Japan, were born with partial or
complete absence of limbs or limbs with defects. This has led to extreme caution in the
introduction of new drugs for commercial distribution, for pregnant mothers, since they may
cause irreparable harm to human embryos.
Antimitotic drugs used in cancer therapy would be especially harmful to the rapidly growing
embryo. However, only aminopterin (a folic acid antagonist) has proved to be teratogenic in
man. This chemical has been used to bring about abortion. When it fails to induce abortion, the
offspring is likely to show multiple malformations. Teratogenic effects of certain other drugs
such as quinine (for malaria), busulphan (for leukemia), and chlorambucil (for Hodgkin’s
disease) have also been reported.
3. Teratogenesis due to radiation: It is recognized that the use of roentgen rays or radium in the
treatment of pregnant patients having pelvic tumors, is possible to produce skeletal abnormalities
in the fetuses. The contact of pregnant women with severe atomic radiation, as in the Hiroshima
and Nagasaki explosions, led to a fetal death rate of about 40 percent. The infants who survived
tended to show confirmation of brain-cell damage.
4. Teratogenesis due to autoimmunization: Significant interest was aroused by the
examination that mothers of infants born without thyroid glands, i.e., thyroid cretins, may have
antithyroid antibodies in their blood. This observation suggests that products of embryonic organ

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primitive may cross the placenta, inducing s maternal antibody production the antibodies may
return to the embryo and interfere with organ differentiation.
5. Teratogenesis due to malnutrition: It is a well-known fact that deficiency of various
vitamins causes several diseases in man the deficiency of vitamin A causes night blindness, the
deficiency of vitamin D leads to abnormal development of bone and teeth and the deficiency of
vitamin K causes abnormalities in the clotting of blood. It has been studied that if rats, rabbits,
and other animals are given a diet deficient in vitamin A, B, and D, they may produce various
teratogenic effects such as hare- lip, cleft palate, spina bifida, skeletal defects, brain defects, etc.
Likely, such vitamin malnutrition in pregnant mothers of the human species may also cause
similar congenital malformation.
Sensitive period of the teratogen: The developmental stage at which the embryo is treated by a
teratogen has great relevance to teratogenesis. In general, the very early stages of development
are not much affected by the teratogen, possibly due to the considerable regulatory capacity of
the embryo. Similarly, the later stages of development, when maturation and growth of organs
occur, are also comparatively resistant to teratogens. The main teratogenic period starts with the
creation of germ layers and continues up to organogenesis. For example, in rats trypan blue (20
mg intravenous injection to the mother) and actinomycin D (0.3 mg/kg body weight to the
mother) are maximally teratogenic on the 8th and 9th day of gestation, respectively. Both
chemicals are ineffective after 10th day and only minimally teratogenic before the 6th day of
gestation.
Specificity of the teratogen: Almost any teratogen can produce almost any terata if applied at
the right time in the experimental animal. However, each teratogen produces its typical
syndrome. For example, the micromelia (short limb) in fowl can be caused by a deficient or
imbalanced diet, (i, e., riboflavin (or biotin deficiency), insulin, thallium, boric acid, pilocarpine,
propanediol-1, 3 sulphanilamides or serine sulfate. A similar situation occurs in the case of
tetraptera (four wings) in Drosophila melanogaster, i.e., phenocopies of tetraptera can be
obtained by treating the early embryo with either ether or 40°C heat shock.
However, the types of micromelia produced in fowl by different teratogens are only superficially
similar.
The type of malformation produced by a teratogen is also dependent on the developmental stage
of an embryo at the time of treatment. For example, insulin (2 units in yolk sac), causes

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rumplessness in the young fowl embryo (24 hours) but micromelia and abnormal beak in the
older embryos (70 to 170 hours) of fowl. Thus, each organ system appears to have its sensitive
period for a given teratogen.
Teratogenic dose: A teratogen may be quite ineffective at a very low dose, while at its high
doses it may be lethal to all embryos. A teratogenic dose range comprises doses that produce at
least some malformed and living embryos. It is also necessary to discriminate between the
teratogenic and the toxic effects of an agent. By definition, an agent is toxic, if it causes
embryonic death by direct action and not through teratogenic interaction. For example, trypan
blue (50 mg/kg) injected into 8.5-day pregnant rats affects 65 percent of the embryos. But with
advancing gestation, out of these 65 percent of the embryos the number of dead one increases at
the expense of live and malformed individuals. Moreover, when trypan blue is injected outside
the teratogenic period, it does not result in appreciable deaths. Trypan blue therefore is a
teratogenic and not toxic chemical.
Interaction of Teratogens with other Environmental Factors: The effect of any teratogen
depends on other factors present in the environment of an organism. Many types of
communication between environmental factors do occur. For example, the teratogenic effects of
insulin on a 96 120- hour-old fowl embryo are almost completely by the simultaneous injection
of nicotinamide.
The synergistic effects of agents are also well known. The incidence of both the micromelia and
abnormal beak in the insulin-injected fowl embryos is significantly enhanced by chlorpromazine,
a non- teratogenic but slightly toxic chemical. Likewise in another type of interaction the non-
teratogenic dos of two teratogens, sulphanilamide and 6-aminonicotinamide, can cause
teratogenesis when injected altogether.

8.3.2 Phenocopies
A phenocopy is a variation in phenotype (generally referring to a single trait) that is caused by
environmental conditions (often, but not essentially, during the organism’s development), such
that the organism’s phenotype matches a phenotype that is determined by genetic factors. It is
not a mutation type, while it is non-hereditary.

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The term was coined by Richard Goldschmidt in 1935. He used it to refer to forms, produced by
some experimental procedure, whose form duplicates or copies the phenotype of some mutant or
combination of mutants.
Examples:The larvae of Drosophila melanogaster have been originating to be particularly
exposed to environmental factors which generate phenocopies of known mutations; these factors
include temperature, shock, radiation, and various chemical compounds. In the fruit
fly, Drosophila melanogaster, the normal body color is brownish gray with black margins. A
hereditary mutant for this was discovered by T.H. Morgan in 1910 where the body color is
yellow. This was a genotypic character that was constant in both the flies in all environments.
However, in 1939, Rapoport discovered that if larvae of normal flies were fed with silver salts,
they develop into yellow-bodied flies irrespective of their genotype. The yellow-bodied flies
which are genetically brown are a variant of the original yellow-bodied fly.
An environmental teratogen can cause a normal genotype to produce a phenotype similar to an
already known mutant that is a phenocopy of the mutant. Landauer has proposed that a true
phenocopy should satisfy two criteria:
(1)The development stage affected by the mutant gene and the environmental teratogen must be
the same that is both should cause terata by altering the same development pathway.
(2) Modifications in the genetic background should have similar effects on the incidence and
expressivity of both the mutant gene and the environmental teratogen. On these criteria, the
insulin-induced rumplessness appeared to be a true phenocopy of the recessive rumples mutant.
In both cases, the abnormal tail structures are formed and later degenerate.
8.3.3 Developmental mechanisms of teratogenesis
Each phase of development is prone to a defect. The chronological nature of development is
based on networks of interacting systems, what happens at one stage is essential to all
consequent stages. Defects initiated at an early stage may be expressed at later stages because
intermediate steps are abnormal. The magnitude of a defect will depend on the stage of
development at which it originates and the development process that is a specific target. Lesions
arising at early stages usually have more cells are affect (Grant, 1978).
Since in terata any organ and any tissue may be affected so the mode of production of terata is
much varied. The development processes that may be affected by teratogenesis include
competence, induction (evocation), determination, histogenesis and morphogenesis, cell growth,

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cell division, cell death, and cell locomotion. The genetic and molecular studies on development
indicate that each teratogen affects the developmental processes by essentially affecting cell
metabolism and gene expression. This is brought about by several independent or inter-related
mechanisms such as:
1) Production of defective, deficient, or no proteins
2) Production of proteins at an inappropriate developmental juncture
3) Production of defective, deficient, or no rRNA or tRNA of a particular kind or
4) Alteration in membrane permeability.
For example, in the case of sickle cell anemia, the production of a defective protein (β chain of
hemoglobin) leads to sickle cell syndrome.

8.4 SUMMARY

A congenital malformation is a structural or anatomical abnormality present at birth. Congenital

malformations may be caused by genetic factors or environmental insults or a combination of the
two that arise during prenatal development. Most common congenital malformations display
multifactorial inheritance with a threshold effect and are determined by an arrangement of
genetic and environmental factors. During the two weeks of gestation, teratogenic agents
generally kill the embryo rather than cause congenital malformation. Major malformations are
more common in early embryos than in newborns; however, the most severely affected embryo
is spontaneously aborted through the first six to eight weeks of gestation. During organogenesis
between days 15 to 60, teratogenic agents are more probable to cause major congenital
malformations.
Teratogenesis is the formation of an abnormal organism. Teratogens are any manager that
physically or chemically alters developmental processes and produces inherited deformities. The
nature of the teratogen and the developmental stage during which the variation occurs is critical
to the type and severity of abnormality it will produce. Biological factors such as the organism’s
gestation process, developmental pathways, and life-cycle characteristics also control the
exposure and effect of a teratogen. The introduction of an embryo, fetus, or larva to a teratogen
may result in death, structural malformation, functional disorder, or growth retardation. The most

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commonly described teratogenic effects taking place in ecosystems are external malformations.
Wild organisms have always been subject to teratogenic insult; however, current anthropogenic
activities have increased the prevalence of deformities. Herein, the current state of teratogenesis
knowledge concerning amphibians, reptiles, birds, fishes, mammals, and invertebrates is
discussed with emphasis on structural malformations resulting from exposure to chemical
contaminants.

8.5 TERMINAL QUESTIONS AND ANSWERS

8.5.1. Multiple Choice Questions:

1. What is a teratogen?
a) A noxious substance found in soil that crosses the maternal placental barrier
b) A hormonal trigger that stimulates excessive cell growth in the embryo
c) An allergen that affects both mother and fetus
d) A substance or environmental influence that affects the development of the fetus and results in
physical abnormalities
2. A teratogenic action is:
a) Toxic action on the
b) Negative action on the fetus causing fetal malformation
c) Toxic action on the blood system
d) Toxic action on kidneys
3._______are substances that can harm the fetus.
a) Testosterone
b) Telomeres
c) Teratogens
d) Testifiers
4. Which of the following is NOT a teratogen?
a) Testosterone
b) Nicotine
c) Radiation

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d) Alcohol
5. Exposure to some maternal hormones before birth may result in the child having:
a) Borderline personality disorders in later life
b) Lower levels of emotional response
c) An insecure attachment
d) None of these
Answers: 1d, 2 b, 3 c, 4 a, 5 a
8.5.2. Short Answer Question:
1. What is environmental teratogenesis?
2. What are Phenocopies?
3. Explain genetic teratogenesis in animals.
8.5.3. Long Answer Question:
1. What is teratogenesis?
2. Describe the genetic bases of teratogenesis.
3. Describe the Developmental mechanisms of teratogenesis.

8.6 REFERENCES

1. Chordate Embryology Developmental Biology by P.S. Verma & V.K. Agarwal

2. Some text adopted by Wikipedia
3. Goldschmidt. R., 1935. Gen und Ausseneigenschaft. I. Zeitschr. ind. Abstl. 69: 38-69
4. https://www.sciencedirect.com/topics/earth-and-planetary-sciences/teratogenesis
5. https://www.verywellhealth.com/teratogens-5118058

8.7 GLOSSARY

Malformation: Abnormality occurring during the formation of the structure (during the
gestation period), resulting in a complete or partial absence or alteration of normal structure
conformation. Results as a morphological defect of an organ, part of an organ, or a larger region,
in severe situations with defects in all the body.

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Teratogen: A product that could promote a birth defect. It could be biological, environmental,
toxic, chemical, or physics.
Teratology: Study of defects that could be congenital malformations or anomalies, present at
birth and can be structural, behavioral, functional, or metabolic disorders.

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