Embryology, Gastrulation

Jeremy Muhr; Kristin M. Ackerman.

Introduction
This article will give a brief overview of gastrulation, a critical process during week 3 of
human development. Gastrulation is defined as an early developmental process in which an
embryo transforms from a one-dimensional layer of epithelial cells (blastula) and
reorganizes into a multilayered and multidimensional structure called the gastrula. In
reptiles, avians, and mammals, which are triploblastic organisms, gastrulation derives a
three tissue-layered organism composed of endoderm, mesoderm, and ectoderm; each
germ layer corresponds to the development of specific primitive systems during
organogenesis. In addition to setting the embryo up for organ formation, gastrulation
provides a mechanism to develop a multileveled body plan that demarcates anatomical axis
formation with dorsal/ventral and cranial/caudal axis (also termed anterior or
rostral/posterior, respectively), retention of global left/right symmetry, and the loss of bilateral
symmetry in specific systems (e.g., heart).

Development
After fertilization, the single-celled zygote will undergo multiple mitotic cleavages of the
blastomeres to change from a two-celled to a 16-celled ball or morula. The morula begins
as a solid mass of totipotent blastomeres but then undergoes compaction and cavitation to
transform into the blastula (non-mammalian term) or blastocyst (human development).
Within the blastocyst, two tissue layers differentiate: an outer shell, known as the
trophoblast, and an inner collection of cells termed the inner cell mass (ICM). Cells within
the outer ring/shell bind together via gap junctions and desmosomes to undergo compaction,
which ultimately forms a water-tight ring/shell called the trophoblast.[1] The outer trophoblast
will develop into structures that provide nutrients, help the growing embryo implant in the
uterine lining, and become part of the placenta. Additionally, the trophoblast cells are
essential in the cavitation of the solid morula into a hollowed-ball of cells with an internal
cavity. Trophoblast cells utilize the active transport of sodium ions and osmosis of water to
form a fluid-filled cavity known as a blastocoel.[2]

The cells remaining after cavitation/blastocoel formation are pluripotent ICM progenitor cells,
which will give rise to the distinctive formation of the fetus. Rather than being an arrangement
of a solid sphere of cells, the inner cell mass is pushed off to one side of the sphere formed
by the trophoblast. Together the trophoblastic layer, blastocoel, and ICM define the human
blastocyst.[3] From zygote to blastocyst formation, the organism has been surrounded by
the zona pellucida, which is a layer of the extracellular matrix that plays a role in the
protection and prevention of implantation into the uterine tubes. During blastocyst formation,
the zona pellucida begins to disappear from the blastocyst, allowing the ball of cells to
proliferate, differentiate, change shape, and eventually implant into the uterine wall.
During implantation, the trophoblastic layer, which surrounds the blastocyst, further
differentiates into two functionally distinct layers. The outer trophoblast, known as the
syncytiotrophoblast, releases digestive enzymes to assist with implantation to the
endometrium. This layer also releases human chorionic gonadotropin (hCG, necessary in
regulating progesterone secretion), the protein used in many pregnancy tests.[4] The inner
trophoblast layer, known as the cytotrophoblast, is a single sheet of cells surrounding the
extraembryonic mesoderm. Within the cytotrophoblast is the ball of ICM, and during the
second week of human development, the ICM cells spread into a flattened tissue layer and
differentiate into a two-layered tissue containing epiblast (columnar epithelial cells) and the
hypoblast (cuboidal epithelial cells), which are together known as the bilaminar disc.[5] The
formation of the bilaminar disc sets the dorsal/ventral axis as the epiblast cell layer is
positioned dorsal to hypoblast. The anatomical location of the bilaminar disc is found
between the amniotic cavity and the primitive yolk sac. The cells of the epiblast stretch to
form a semi-sphere known as the amniotic cavity, while the cells of the hypoblast extend to
surround the yolk sack. On the hypoblast is a raised area of columnar cells known as the
prechordal plate; this is the earliest delineation of cranial from caudal. Development of the
bilaminar disc directly precedes gastrulation, where the end goal during week 3 of
development is to transform the human blastocyst into a multilayered gastrula with
endoderm, mesoderm, and ectoderm.

Cellular
The beginning of gastrulation is marked by the appearance of a groove in the caudal end of
the epiblast layer known as the primitive streak.[6] Thus, the formation of primitive steak
firmly establishes the cranial/caudal axis. The primitive streak initially forms via a thickening
of cells near the connecting stalk. As cells proliferate and migrate toward the midline of the
embryo, the thickening elongates to become linear in shape, thus the term primitive steak.
The cranial end of the embryo seems to play an important role in beginning the process of
gastrulation. At the cranial end of the primitive streak, epiblast cells ingress at a greater rate
forming a circular cavity known as the primitive pit. As the primitive streak and pit elongate,
migrating epiblast cells join the streak at the cranial end, forming a mass of cells called the
primitive node, which becomes the primary tissue organizer where transcription factors and
chemical signaling drive induction of tissue formation. Known factors in primitive streak
formation include TGFB, WNT, Nodal, and BMPs and are discussed in more detail in the
molecular section.

Epithelial cells in the lateral edge of the epiblast layer undergo an epithelial to mesenchymal
cellular transition to delaminate (detach) and migrate down/into the primitive streak.[7] The
movement of epiblastic mesenchymal cells down primitive streak is known as ingression.
The first set of cells to move down primitive streak integrate into the hypoblast layer and
transform into endoderm, the first of the three germ layers. The second set of cells to detach
and ingress will fill in the space between the endoderm and epiblast layer to form the second
germ layer termed mesoderm. Multiple mesodermal structures will develop: cells that move
into the body stalk will help to form extraembryonic mesoderm, and later the umbilical cord,
cells passing through primitive pit become notochord or paraxial mesoderm, and other cells
coming through streak become lateral plate or extraembryonic mesoderm. Finally, the
remaining epiblast cells will transform into the final germ layer, ectoderm. Cell proliferation
and ingression continue in all directions as the embryo grows; however, the primitive streak
will always expand directionally from the caudal to the cranial end and then regress in the
opposite fashion. Regression occurs after the formation of the intra-embryonic mesoderm,
and the primitive streak should completely disappear by the end of the fourth week — a lack
of primitive streak regression results in clinical abnormalities.

After the three germ layers have formed, the newly produced structure (trilaminar disc or
gastrula) is primed for organ system formation, which is highly reliant upon direct
interaction/communication and induction events between the endoderm, mesoderm, and
ectoderm. Cells continue to invaginate through what is now called the primitive node. The
cells begin to form a hollow tube extending from the cranial end to the prechordal plate,
known as the notochordal process. As the embryo continues to grow in each direction, the
notochordal process grows longer until it fuses with the endoderm to form the notochordal
plate. Once the fusion is complete, there is a free passageway between the amniotic cavity
and the yolk sac, known as the neurenteric canal.[8] It is theorized that the neurenteric canal
forms as a way to maintain pressure equilibria between both chambers. Later in
development, the two edges of the notochordal plate will then fuse, becoming a solid
mesoderm rod known as the notochord. The notochord is one of the most important features
in embryology. It is a mesodermal structure that not only provides structural support but
marks the midline of the embryo. It will provide chemical and physical interactions with the
dorsal lying ectoderm to specialize a portion of that ectoderm into neuroectoderm to derive
the nervous system.

Biochemical
RNA helicase A (RHA) can function as a helicase with both RNA and DNA. The sequence
and biochemical conservation of RHA and its homologs to humans suggests an
evolutionarily conserved function. Normal gastrulation depends on RNA helicase A activity,
as lack of proper RHA signaling results in ectodermal cell death with clear alterations in
differentiation.[9]

The differentiation of pluripotent stem cells to lineage-specified cells within the endoderm,
mesoderm, and ectoderm is marked by down-regulation of pluripotency markers (Oct 4,
Nanog, Sox 2, etc.) in conjunction with the activation of lineage-specific gene expression,
including microRNAs. MicroRNAs (miRNAs) have demonstrated to be enriched in germ
layers, specifically targeting TGFB to promote mesoderm and restrict or block
neuroectoderm.[10]
Molecular
Primitive Streak
The initiation of the primitive streak is based upon a system of signaling pathways working
to both positively and negatively regulate downstream expression. The combination of
TGFB, WNT, Nodal, and BMPs are all important in primitive streak
development.[11][12][13][14][15] The interplay between Wnt and TGFB signaling seems to
be the inducer of the formation of the primitive streak. Specifically, Vg1 (a member of the
TGFB family) has been shown to induce streak formation and to prevent formation with Vg1
misexpression at the posterior marginal zone.[16] Vg1 acts on Nodal to continue the
chemical cascade to streak formation. To ensure the proper location of the streak on the
epiblast, the hypoblast releases antagonists of Nodal signaling.[17] Additionally, the
induction of streak formation can be regulated by Wnt factors; not only has upregulated Wnt
induced streak formation, but the use of Wnt antagonists such as Dkk-1 and Crescent
prevents the formation of the streak.[11] Finally, BMP signaling has been shown to regulate
streak formation. Towards the streak itself, BMP concentration is low, with the surrounding
embryo exhibiting higher levels of active BMP. In addition to this, BMP inhibitors cause the
formation of a streak in chick embryos. As seen in BMP and other signaling, concentration
gradients are typical through most of the gastrulation process, where the different
concentrations of signaling factors allow for cells to differentiate into unique tissues.

Endoderm
Endoderm is the embryonic precursor to the thyroid, lungs, pancreas, liver, and intestines,
which evolve from four consecutive steps developmental steps: proliferation and induction
of pluripotent stem cells, the separation of stem cell-derived endoderm versus mesoderm
germ layers, anterior-posterior patterning, and bifurcation of liver and pancreas. Cells near
the anterior portion of the primitive streak will express Forkhead box A2 (Foxa2) to become
definitive endoderm (DE). The DE will pattern itself into the foregut, midgut, and hindgut via
mesodermal induction during embryonic folding with foregut cells expressing Hhex, Sox2,
and Foxa2 and the hindgut expressing different homeobox genes Cdx1, Cdx2, and Cdx4.
The upregulation of TGF-beta signaling promotes pancreas formation with BMP and
FGF/MAPK signaling to specify the liver.[18] The specification of the respiratory bud starts
with the expression of the Nbx1-2gene. Complex signaling between the respiratory bud
epithelium and mesoderm involves FGF and FGFR interactions to promote the growth of
the respiratory bud. [19]

Mesoderm
Epiblast cells invaginating through the primitive streak that express high levels of a fibroblast
growth factor (FGF2) are fated down a path towards becoming mesodermal cells, but more
specifically, they will end up paraxial, intermediate, or lateral plate mesoderm, which will
correlate to different tissues as the embryo continues to develop.[20]
Notochord
Progenitor cells from the node/pit migrate to initiate notochord formation along with epiblast
cells from the floor plate of the amniotic cavity filling in the notochord to form a thick rod-like
structure down the midline of the embryo. Providing support and serving as an induction
center for surrounding cells, the notochord in vertebrates extends throughout the entire
length of what will be the vertebral column and reaches as far as the midbrain. Notochord
develops first, and then mesodermal cells grow medially to surround it. The notochord is
only present in developing organisms with the primary goal of patterning the tissues
surrounding them. Notochord secretes Sonic Hedgehog, Chordin, and Noggin in a
morphogenic gradient pattern (highest concentration is near the notochord with diffusion
outward), which binds to receptors on target cells to induce specification and differentiation
events in the neural plate, somites, and ectoderm.[21]

Mesoderm divides into three main categories: (par)axial, intermediate, and lateral
mesoderm, which are the embryonic precursors to a large variety of cells and tissues,
including smooth, cardiac, and skeletal muscle, kidney, reproductive organs, the muscles of
the tongue, and the pharyngeal arches muscle, connective tissue, bone, cartilage, dermis
and subcutaneous layer of the skin, dura mater, vascular endothelium, blood cells, microglia,
and adrenal cortex.

Paraxial mesoderm-Paraxial mesoderm cells first organize to form somitomeres. As the

somitomeres develop into somites in a cranial to caudal fashion, the outer cells undergo a
mesenchymal to epithelial transition, which serves as a distinct boundary between individual
somites. Individual somites then separate into cranial and caudal portions, followed by the
cranial portion of each fusing with the caudal portion of the somite directly anterior to it.
Distinct regions of each somite (sclerotome, dermatome, myotome) become specific tissue
and cell types as the body matures. The skull, vertebral column, and brain meninges develop
from the mesoderm surrounding the neural tube and notochord.
The intermediate mesoderm connects the paraxial mesoderm with the lateral plate and
differentiates into urogenital structures.
Lateral plate mesoderm splits into parietal (somatic) to aid in lateral body fold wall formation,
and visceral (splanchnic) layers are involved in gut tube formation.

Ectoderm
The interplay between bone morphogenic proteins (BMP’s) and Hox genes is integral to the
differentiation of the remaining epiblast tissue into the ectoderm. This is especially important
in regards to what will become the neuroectoderm, setting up the brain and spinal cord, as
well as the surface ectoderm.[13]
The notochord is the main inductive tissue to delineate neuroectoderm from the remaining
ectoderm that will become skin.[22] The entire presumptive ectoderm plate expresses BMP
and TGF-beta. Noggin and Chordin secretion from the notochord diffuses into the ectoderm
directly anterior to the notochord and binds to receptors in the overlying ectoderm to block
BMP. The blockade of BMP specifies the tissue to neural ectoderm, while the remaining
ectoderm, which still expresses BMP, will become skin.

Function
Gastrulation occurs during week 3 of human development. The process of gastrulation
generates the three primary germ layers (ectoderm, endoderm, mesoderm), which primes
the system for organogenesis and is one of the most critical steps of development. The
endoderm is the innermost layer, which gives rise to the gastrointestinal tract, the lining of
the gut, liver, pancreas, and portions of the lungs and glandular tissues. The mesoderm
derives the musculoskeletal system, including connective tissue, the non-epidermal portions
of the integumentary system, the circulatory system, the kidney, and the internal sex organs.
The ectoderm is the outer layer of the embryo, which gives rise to the external ectoderm
(epidermis, hair, nails) and the neuroectoderm (neural crest and neural tube-brain and spinal
cord), along with the lens of the eyes and the inner ear. Another important function of
gastrulation is to establish directionality within the developing embryo. Cranial/caudal
directionality becomes established by the placement of the prechordal plate and the path of
the primitive groove, and the establishment of the dorsal/ventral axis is by the layering of the
epiblast and hypoblast (discussed above).

Mechanism
Gastrulation involves a complex series of cellular morphogenesis, cellular movements, and
cell signaling via transcription factors, chemical morphogenic gradients, and differential gene
expression to allow for the induction of germ cell layer formation that orchestrates the
initiation of eventual organ system development.

Conclusion
In conclusion, gastrulation is a crucial point in embryonic development: during this process
an essentially spherical blastula transforms into a cylindrical structure with a head and tail
and three distinct embryonic walls. From the ectoderm will originate the skin, the nervous
system and the sensory structures of the eyes, ears and nose. From the mesoderm will
originate the bone, muscular and circulatory systems. From the endoderm, the lining
epithelia will originate, such as the digestive and respiratory systems.
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