EARLY DEVELOPMENT IN

BIRDS
By Kamran Saeed Paracha
Cleavage
• Fertilization of the chick egg occurs in the oviduct, before the albumen and
shell are secreted to cover it.
• Like the egg of the zebrafish, the chick egg is telolecithal, with a small disc
of cytoplasm—the blastodisc—sitting atop a large yolk.
• Like fish eggs, the yolky eggs of birds undergo discoidal meroblastic
cleavage.
• Cleavage occurs only in the blastodisc, which is about 2-3 mm in diameter
and is located at the animal pole of the egg.
• The first cleavage furrow appears centrally in the blastodisc; other
cleavages follow to create a single-layered blastoderm (Figure 8.1).
• As in the fish embryo, the cleavages do not extend into the yolky
cytoplasm, so the early-cleavage cells are continuous with one another and
with the yolk at their bases
• Thereafter, equatorial and vertical cleavages divide the blastoderm into a
tissue 5-6 cell layers thick, and the cells become linked together by tight
junctions.
• Between the blastoderm and the yolk of avian eggs is a space called the
subgerminal cavity, which is created when the blastoderm cells absorb
water from the albumen ("egg white") and secrete the fluid between
themselves and the yolk.
• At this stage, the deep cells in the center of the blastoderm appear to be
shed and die, leaving behind a 1-cell-thick area pellucida; this part of the
blastoderm forms most of the actual embryo.
• The peripheral ring of blastoderm cells that have not shed their deep cells
constitutes the area opaca. Between the area pellucida and the area opaca
is a thin layer of cells called the marginal zone (or marginal belt*).
• Some marginal zone cells become very important in determining cell fate
during early chick development.
• Discoidal mcroblastic cleavage in
a chick egg.
• Avian eggs include some of the
largest cells known to science
(inches across), but cleavage
takes place in only a small
region, the blastodisc.
• (A-D) Four stages viewed from
the animal pole (the future
dorsal side of the embryo).
• (E) An early-cleavage embryo
viewed from the side.
Gastrulation of the Avian Embryo
• The hypoblast
• By the time a hen has laid an egg, the blastoderm contains
some 20,000 cells.
• At this rime, most of the cells of the area pellucida remain at
the surface, forming an "upper layer," called the epiblast,
while other area pellucida cells have delaminated and
migrated individually into the subgerminal cavity to form
hypoblast islands (sometimes called the primary hypoblast
or polyinvagination islands), an archi-pelago of disconnected
clusters of 5-20 cells each.
• Shortly thereafter, a sheet of cells derived from deep yolky cells at the
posterior margin of the blastoderm migrates anteriorly beneath the
surface.
• This sheet of migrating cells pushes the hypoblast islands anteriorly,
thereby forming the secondary hypoblast, or endoblast (Figure 8.2B-E)
• The resulting two-layered blastoderm (epiblast and hypoblast) is joined
together at the marginal zone of the area opaca, and the space between
the layers forms a blastocoel.
• Thus, although the shape and formation of the avian blastodisc differ from
those of the amphibian, fish, or echinoderm blastula, the overall spatial
relationships are retained.
• The amniotes have evolved is a set of extra-embryonic tissues; the
hypoblast, the yolk sac, and the amnion.
• The avian embryo comes entirely from the epiblast; the hypoblast does not
contribute any cells to the developing embryo.
• The hypoblast cells form portions of the external membranes, especially
the yolk sac and the stalk linking the yolk mass to the endodermal digestive
tube.
• Hypoblast cells also provide chemical signals that specify the migration of
epiblast cells.
• However, the three germ layers of the embryo proper (plus the amnion)
are formed solely from the epiblast.
Figure
• Formation of the chick blastoderm. The left column depicts a
diagrammatic midsagittal section through part of the blastoderm. The
middle column depicts the entire embryo viewed from the ventral
side.
• This shows the migration of the primary hypoblast and the secondary
hypoblast (endoblast) cells. The right column shows the entire
embryo seen from the dorsal side.
• (A-C) Events prior lo laying of the shelled egg,
• (A) Stage X embryo, where islands of hypoblast cells can be seen, as well as a
congregation of hypoblast cells around Koller's sickle.
• (B) Bv stage XII just perior to primitive streak formation the hypoblast island
cell have coalesced to form the primary hypoblast layer, which meets
endoblast cells and primitive streak cells at Koller's sickle.
• (C) By stage XIII, the secondary hypoblast cells migrate anteriorly.
• (D) By stage 2 (6-7 hours after the egg is laid), the primitive streak cells form a third
layer that lies between the hypoblast and epiblast cells.
• (E) By stage 3 (up to 13 hours post laying), the primitive streak has become a definitive
region of the epiblast, with cells migrating through it to become the mesoderm and
endoderm.
The primitive streak
• The major structural characteristic of avian, reptilian, and mammalian
gastrulation is the primitive streak;* the primitive streak becomes the
blastopore lips of amniote embryos.
• Dye-marking experiments and time-lapse cine-micrography indicate
that the primitive streak first arises from a local thickening of the
epiblast at the posterior edge of the area pellucida, called Roller's
sickle, and the epiblast above it.
• FORMATION OF THE PRIMITIVE STREAK
• The Streak is first visible as cells accumulate in the middle layer,
followed by a thickening of the epiblast at the posterior marginal
zone, just anterior to Roller's sickle (Figure 8.3A).
• This thickening is initiated by an increase in the height (thickness) of
the cells forming the center of the primitive streak.
• The presumptive streak cells around them become globular and
motile, and they appear to digest away the extracellular matrix
underlying them.
• This process allows these cells to undergo intercalation
(mediolaterally) and convergent extension.
• Convergent extension is responsible for the progression of the
streak— a doubling in streak length is accompanied b y a concomitant
halving of its width (Figure 8.3B).
• Those cells that initiate streak formation (i.e., the cells in the midline
of the epiblast , overlying Roller's sickle) appear to migrate anteriorly
and may constitute a stable cell population that directs the
movement of epiblast cells into the streak.
• As cells converge to form the primitive streak, a depression forms
within the streak. This depression is called the primitive groove, and
it serves as an opening through which migrating cells pass into the
deep layers of the embryo.
• Thus , the primitive groove is homologous to the amphibian blastopore,
and the primitive streak is homologous to the blastopore lips.
• At the anterior end of the primitive streak is a regional thickening of cells
called Hensen's node (also known as the primitive knot; Figure 8.3C).
• The center of Hensen's node contains a funnel-shaped depression
(sometimes called the primitive pit) through which cells can enter the
embryo to form the notochord and prechordal plate.
• Hensen's node is the functional equivalent of the dorsal lip of the
amphibian blastopore (i.e., the organizer) and the fish embryonic shield
• The primitive streak defines the axes of the avian embryo. It extends from
posterior to anterior; migrating cells enter through its dorsal side and move
to its ventral side; and it separates the left portion of the embryo from the
right.
• The axis of the streak is equivalent to the dorsal-ventral axis of amphibians.
• The anterior end of the streak (Hensen's node) gives rise to the prechordal
mesoderm, notochord, and medial part of the somites.
• Cells that ingress through the middle of the streak give rise to the lateral
part of the somites and to the heart and kidneys.
• Cells in the posterior portion of the streak make the lateral plate and
extraembryonic mesoderm.
• After the ingression of the mesoderm cells, epiblast cells remaining
outside, but dose to, the streak will form medial (dorsal) structures such as
the neural plate, while those epiblast cells farther from the streak will
become epidermis.
• ELONGATION OF THE PRIMITIVE STREAK
• As cells enter the primitive streak, they undergo an epithelial-to-
mesenchymal transformation, and the basal lamina beneath them breaks
down.
• The streak elongates toward the future head region as more anterior cells
migrate toward the center of the embryo.
• Cell division adds to the length produced by convergent extension, and
some of the cells from the anterior portion of the epiblast contribute to the
formation of Hensen's node.
• At the same time, the secondary hypoblast (endoblast) cells continue to
migrate anteriorly from the posterior margin of the blastoderm (see Figure
8.2E).
• The elongation of the primitive streak appears to be coextensive with the
anterior migration of these secondary hypoblast cells, and the hypoblast
directs the movement of the primitive streak.
• The streak eventually extends to 60-75% of the length of the area
pellucida.
FORMATION OF ENDODERM AND MESODERM
• As soon as the primitive streak has formed, epiblast cells begin to
migrate through it and into the blastocoel (Figure 8.4).
• The streak thus has a continually changing cell population.
• Cells migrating through the anterior end pass down into the
blastocoel and migrate anteriorly, forming the endoderm, head
mesoderm, and notochord;
• cells passing through the more posterior portions of the primitive
streak give rise to the majority of mesodermal tissues.
• The first cells to migrate through Hensen's node are those destined to
become the pharyngeal endoderm of the foregut.
• Once deep within the embryo, these endodermal cells migrate
anteriorly and eventually displace the hypoblast cells, causing the
hypoblast cells to be confined to a region in the anterior portion of
the area pellucida.
• This anterior region, the germinal crescent, does not form any
embryonic structures, but it does contain the precursors of the germ
cells, which later migrate through the blood vessels to the gonads
• The next cells entering through Hensen's node also move anteriorly,
but they do not travel as far ventrally as the presumptive foregut
endodermal cells.
• Rather, they remain between the endoderm and the epiblast to form
the prechordal plate mesoderm.
• Thus, the head of the avian embryo forms anterior (rostral) to
Hensen's node.
• The next cells passing through Hensen's node become the
chordamesoderm.
• The chordamesoderm has two components: the head process and the
notochord.
• The most anterior part, the head process, is formed by central mesoderm
cells migrating anteriorly, behind the prechordal plate mesoderm and
toward the rostral tip of the embryo (see Figures 8.3 and 8.4).
• The head process will underlie those cells that will form the forebrain and
midbrain.
• As the primitive streak regresses (see below), the cells deposited by the
regressing Hensen's node will become the notochord.
 • Migration of endodermal and
mesodermal cells through the
primitive streak.
• (A) Stereogram of a gastrulating chick
embryo, showing the relationship of
the primitive streak, the migrating
cells, and the hypoblast and epiblast
of the blastoderm.
• The lower layer becomes a mosaic of
hypoblast and endodermal cells; the
hypoblast cells eventually sort out to
form a layer beneath the endoderm
and contribute to the yolk sac.

Cells migrating through Hensen's node travel anteriorly to form the prechordal plate and notochord; those
migrating through the next anterior region of the streak travel laterally, but converge near the midline to make
notochord and somites; those from the middle of the streak form intermediate mesoderm and lateral plate
mesoderm (see the fate map in Figure 8.3). Farther posterior, the cells migrating through the primitive streak
make the extraembryonic mesoderm (not shown).
• (B) This scanning electron micrograph shows epiblast cells passing
into the blastocoel and extending their apical ends to become bottle
cells.
Regression of the primitive streak and epiboly
of the ectoderm
• Now a new phase of gastrulation begins. While mesodermal
ingression continues, the primitive streak starts to regress, moving
Hensen's node from near the center of the area pellucida to a more
posterior position (see Figure 11.12).
• The regressing streak leaves in its wake the dorsal axis of the embryo,
including the notochord. The notochord is laid down in a head-to-tail
direction, starting at the level where the ears and hindbrain form and
extending caudally to the tail bud.
• As in the frog, the pharyngeal endoderm and head mesoendoderm
will induce the anterior parts of the brain, while the notochord will
induce the hindbrain and spinal cord.
• By this time, all the presumptive endodermal and mesodermal cells
have entered the embryo and the epiblast is composed entirely of
presumptive ectodermal cells
• Finally, Hensen's node regresses to its most posterior position,
forming the anal region. At this time, all the presumptive endodermal
and mesodermal cells have entered the embryo, and the epiblast is
composed entirely of presumptive ectodermal cells.
• Chick gastrulation 24-28 hours after fertilization.
• (A) The primitive streak at full extension (24 hours). The head process (anterior
notochord) can be seen extending from Hensen's node.
• (B) Two-somite stage (25 hours). Pharyngeal endoderm is seen anteriorly, while the
anterior notochord pushes up the head process beneath it. The primitive streak is
regressing.
• (C) Four-somite stage (27 hours).
• (D) At 28 hours, the primitive streak has regressed to the caudal portion of
the embryo.
• (E) Regression of the primitive streak, leaving the notochord in its wake.
Various points of the streak (represented by letters) were followed after it
achieved its maximum length. The x axis (time) represents hours after
achieving maximum length (the reference line is about 18 hours of
incubation).
Epiboly of the ectoderm
• While the presumptive mesodermal and endodermal cells are moving inward, the
ectodermal precursors proliferate and migrate to surround the yolk by epiboly.
• The enclosure of the yolk by the ectoderm (again, reminiscent of the epiboly of
the amphibian ectoderm) is a Herculean task that takes the greater part of 4 days
to complete.
• It involves the continuous production of new cellular material and the migration
of the presumptive ectodermal cells along the underside of the vitelline
envelope.
• Interestingly, only the cells of the outer margin of the area opaca attach firmly to
the vitelline envelope.
• These cells are inherently different from the other blastoderm cells, as they can
extend enormous (500 um) Cytoplasmic processes onto the vitelline envelope.
• These elongated filopodia are believed to be the locomotor apparatous of the
marginal cells, by which the marginal cells pull other ectodermal cells around the
yolk (Schlesinger 1958).
• The filopodia bind to fibronectin, a laminar protein that is a component of the
chick vitelline envelope.
• If the contact between the marginal cells and the fibronectin is experimentally
broken by adding a soluble polypeptide similar in fibronectin, the filopodia retract
and ectodermal migration ceases.
• Thus, as avian gastrulation draws to a close, the ectoderm has surrounded the
yolk, the endoderm has replaced the hypoblast, and the mesoderm has
positioned itself between these two regions.
• Although we have identified many of the processes involved in avian gastrulation,
we are only beginning to understand the molecular mechanism by which some of
these processes are carried out.
Axis Specification and the Avian
'Organizer"
The Role of Gravity and the PMZ
• The conversion of the radially symmetrical blastoderm into a
bilaterally symmetrical structure appears to be determined by gravity.
• As the ovum passes through the hen's reproductive tract, it is rotated
for about 20 hours in the shell gland.
• This spinning, at a rate of 10-12 revolutions per hour, shifts the yolk
such that its lighter components (probably containing stored maternal
determinants for development) lie beneath one side of the
blastoderm.
• This imbalance tips up one end of the blastoderm, and that end
becomes the posterior marginal zone (PMZ) of the embryo—the part
where primitive streak formation begins (Figure 8.8).
• Specification of the chick anterior-posterior axis by gravity.
• (A) Rotation in the shell gland results in
• (B) the lighter components of the yolk pushing up one side of the
blastoderm.
• (C) This more elevated region becomes the posterior of the embryo.
• It is not known what interactions cause this specific portion of the
blastoderm to become the PMZ.
• Early on, the ability to initiate a primitive streak is found throughout
the marginal zone; if the blastoderm is separated into parts, each
with its own marginal zone, each part will form its own primitive
streak.
• However, once the PMZ has formed, it controls the other regions of
the margin. Not only do the cells of the PMZ initiate gastrulation, they
also prevent other regions of the margin from forming their own
primitive streaks
• It now seems apparent that the posterior marginal zone contains cells
that act as the equivalent of the amphibian Nieuwkoop center.
• When placed in the anterior region of the marginal zone, a graft of
PMZ tissue (posterior to and not including Roller's sickle) is able to
induce a primitive streak and Hensen's node without contributing
cells to either structure.
• Current evidence suggests that the entire marginal zone produces
Wnt8c (capable of inducing β-catenin) and that, like the amphibian
Nieuwkoop center, the PMZ produces Vgl, a member of the TGF-B
family of secreted proteins.
• Wnt8c and Vgl act together to induce expression of Nodal (another
secreted TGF-B protein) in the future embryonic epiblast next to
Roller's sickle and the PMZ .
• Thus the pattern appears similar to that of amphibian embryos.
• Recent studies suggest that Nodal activity is needed to initiate the
primitive streak, and that it is the secretion of Cerberus—an
antagonist of Nodal—by the primary hypoblast cells that prevents
primitive streak formation.
• As the primary hypoblast cells move away from the PMZ, Cerberus
protein is no longer present, allowing Nodal activity and therefore
formation of the primitive streak in the posterior epiblast.
• Once formed, however, the streak secretes its own Nodal antagonist
(the Lefty protein), thereby preventing any further primitive streaks
from forming.
• Eventually, the Cerberus-secreting hypoblast cells are pushed to the
future anterior of the embryo, where they contribute to ensuring that
neural cells in this region become forebrain rather than more
posterior structures of the nervous system.
The chick "organizer"
• The "organizer" of the chick embryo forms from cells initially located just anterior
to the posterior marginal zone.
• The epiblast and middle layer cells in the anterior portion of Koller's sickle
become Hensen's node, as described earlier. The posterior portions of Koller's
sickle contribute to the posterior portion of the primitive streak (Figure 8.9).
• Hensen's node has long been known to be the avian equivalent of the amphibian
dorsal blastopore lip, since
• (1) it is the region whose cells become the prechordal plate and
chordamesoderm,
• (2) it is the region whose cells can both induce and pattern a second embryonic
axis when transplanted into other locations of the gastrula (Figure 8.10)
• (3) it expresses the same marker genes as Spemann's organizer in amphibians
and the embryonic shield of teleost fishes, such as the transcription factor
Goosecoid.
• Formation of Hensen's node from Koller's sickle.
• (A) Diagram of posterior end of an early (pre-streak) embryo, showing the cells labeled with
fluorescent dyes in the photographs.
• (B) Just before gastrulation, cells in the anterior end of Koller's sickle (the epiblast and middle layer)
were labeled with green dye. Cells of the posterior portion of Koller's sickle were labeled with red
dye. As the cells migrated, the anterior cells formed Hensen's node and its notochord derivatives. The
posterior cells formed the posterior region of the primitive streak.
• The time after dye injection is labeled on each photograph.
• Induction of a new embryo by transplantation of Hensen's node.
• (A) A Hensen's node from a duck embryo is transplanted into the
epiblast of a chick embryo.
• (B) A secondary embryo is induced (as is evident by the neural tube)
from host tissues at the graft site.
• Hensen's node can induce neural tissue when grafted into fish,
amphibian, or mammalian embryos.
• As is the case in all vertebrates, the dorsal mesoderm is able to
induce the formation of the central nervous system in the ectoderm
overlying it.
• The cells of Hensen's node and its derivatives act like the amphibian
organizer, and they secrete bone morphogenetic protein (BMP)
antagonist proteins such as chordin, Noggin, and Nodal.
• These proteins repress BMP signaling and dorsalize the ectoderm and
mesoderm (Figure 8.11).
• However, repression of the BMP signal by these antagonists does not
appear to be sufficient for neural induction.
• Fibroblast growth factors synthesized in the hypoblast and in Hensen's
node precursor cells just prior to gastrulation appear to be critical for
preparing the epiblast to generate neuronal phenotypes.
• Although FGFs can block BMP signaling, this does not account for the
ability of FGFs to induce a transient expression of pre-neural genes in the
epiblast. These neural genes do not stay active unless they are supported
by BMP antagonists.
• Thus, FGF signaling inhibits BMPs from inducing the genes that specify
ectoderm to become epidermis, and they activate the genes that specify to
ectoderm to become neural.
• Possible contribution to chick neural induction by the inhibition of BMP signaling.
• (A) In a neurulating embryo, Noggin protein (purple) is expressed in the notochord and
the pharyngeal endoderm.
• (B) BMP7 expression (dark purple), which had encompassed the entire epiblast,
becomes restricted to the non-neural regions of the ectoderm.
• (C) Similarly, the product of BMP s gnaling, the phosphorylated form of Smadl
(recognized by antibodies to the phosphorylated form of the protein; dark brown) is
not seen in the neural plate.
Anterior-posterior patterning
• The patterning of the definitive anterior-posterior axis occurs differently for
the mesoderm and neural ectoderm, but in both cases the process involves
timing (the sequential generation of cells from a zone of undifferentiated
proliferating cells) and the influence of caudalizing molecules.
• In the ectoderm, most of the initial neural plate corresponds to the future
head region (from forebrain to the level of the future ear vesicle, which lies
adjacent to Hensen's node at full primitive streak stage).
• A small region oi neural ectoderm just lateral and posterior to the node
(sometimes called the caudal lateral epiblast) will give rise to the rest of
the nervous system, including the posterior hindbrain and all of the spinal
cord.
• As the primitive streak regresses, this latter region regresses with the
node and adds cells to the caudal end of the elongating neural plate.
• It appears that FGF signalling in the streak and paraxial (future
somite) mesoderm keeps this region young" and undifferentiated as it
regresses, and that this is antagonized by retinoic acid (RA) activity as
cells leave this zone.
• In the mesoderm, the anterior-posterior axis is also related to time.
• The entire length of the notochord at the midline is derived from cells that
are present in Hensen's node by the full primitive streak stage.
• A population of progenitor cells remains in the node; their descendants
gradually leave as the node regresses, laying down the chordamesoderm
and the ventral midline of the neural tube.
• Therefore anterior-posterior identities along the axis from the hindbrain to
the tail are specified as a function of the time of emergence from the
primitive streak and Hensen's node.
• It has been proposed that the length of time cells are resident in this region
determines which Hox genes are expressed by the cells. This pattern of Hox
gene expression can also be under the influence of the FGF and retinoic
acid gradients
Left-right axis formation
• The vertebrate body has distinct right and left sides. The heart and
spleen, for instance, are generally on the left side of the body, while
the liver is usually on the right.
• The distinction between the sides is primarily regulated by two
proteins: the paracrine factor Nodal and the transcription factor Pitx2.
• The mechanism by which nodal gene expression is activated in the
left side of the body differs among the vertebrate classes.
• The ease with which chick embryos can be manipulated has allowed
scientists to elucidate the pathways of left-right axis determination in
birds more readily than in other vertebrates.
• As the primitive streak reaches its maximum length, transcription of
the sonic hedgehog gene (shh) becomes restricted to the left side of
the embryo, controlled by activin and its receptor (Figure 8.14A).
• Activin signaling, along with BMP4, appears to block the expression of
Sonic hedgehog and to activate expression of Fgf8 protein on the
right side of the embryo.
• Fgf8 blocks expression of the paracrine factor Cerberus on the right-
hand side; it may also activate a signalling cascade that instructs the
mesoderm to have right-sided capacities.
• Meanwhile, on the left side of the body, Shh protein ativates Cerberus
(Figure 8.14B), which in this case acts with BMP to stimulate the
synthesis of Nodal protein.
• Nodal activates the pitx2 gene while repressing snail. In addition,
Leftyl in the ventral midline prevents the Cerberus signal from passing
to the right side of the embryo (Figure 8.14C,D).
• As in Xenopus, Pitx2 is crucial in directing the asymmetry of the
embryonic structures.
• Experimentally induced expression of either Nodal or Pitx2 on the
right side of the chick reverses the asymmetry or cause randomization
of asymmetry on the right or left sides.
• Model for generating left-right asymmetry in the chick embryo.
• (A) On the left side of Hensen's node, Sonic hedgehog (Shh) activates Cerberus,
which stimulate BMPs to induce the expression of Nodal. In the presence of
Nodal, the pitx2 gene is activated. Pitx2 protein is active in the various organ
primordia and specifies which side will be the left. On the right side of the
embryo, activin is expressed, along with activin receptor Ha. This activates
Fgf8, a protein that blocks expression of the gene for Cerberus. In the absence
of Cerberus, Nodal is not activated and thus Pitx2 is not expressed.
• (B) Whole-mount in situ hybridization of cerberus mRNA. This view is from the
ventral surface ("from below", so the expression seems to be on the right.
Dorsally, the expression pattern would be on the left.
• (C) Whole-mount in situ hybridization using probes for the chick nodal message
(stained purple) shows its expression in the lateral plate mesoderm only on the
left side of the embryo. This view is from the dorsal side.
• (D) Similar in situ hybridization, using the probe for pitx2 at a later stage of
development. The embryo is seen from its ventral surface. At this stage, the
heart is forming, and pitx2 expression can be seen on the left side of the heart
tube (as well as symmetrically in more anterior tissues. (A after Raya and
Izpisua-Belmonte 2004; B from Rodriguez-Esteban et