12

Birds and Mammals

This final chapter on the processes of early development extends our

survey of vertebrate development to include the amniotes —those vertebrates whose embry-
os form an amnion, or water sac (i.e., the reptiles, birds, and mammals). Birds and reptiles
follow a very similar pattern of development (Gilland and Burke 2004; Coolen et al. 2008),
and birds are considered by modern taxonomists to be a reptilian clade (Figure 12.1A).
The amniote egg is characterized by a set of membranes that together enable the
embryo to survive on land (Figure 12.1B). First, the amnion, for which the amniote egg
is named, is formed early in embryonic development and enables the embryo to float in
a fluid environment that protects it from desiccation. Another cell layer derived from the
embryo, the yolk sac, enables nutrient uptake and the development of the circulatory sys-
tem. The allantois, developing at the posterior end of the embryo, stores waste products.
The chorion contains blood vessels that exchange gases with the outside environment. In
birds and most reptiles, the embryo and its membranes are enclosed in a hard or leathery
shell within which the embryo develops outside the mother’s body. Cleavage in bird and
How did this reptile eggs, like that of the bony fishes described in the last chapter, is meroblastic, with
only a small portion of the egg cytoplasm being used to make the cells of the embryo. The
mammalian embryo
vast majority of the large egg is composed of yolk that will nourish the growing embryo.
determine which In most mammals, holoblastic cleavage is modified to accommodate the formation of
end is its head and a placenta, an organ containing tissues and blood vessels from both the embryo and the
which its tail?

The Punchline
Birds and mammals begin development differently. Birds have a meroblastic cleavage,
where mammalian cleavage is holoblastic. The chick forms a layer of cells over a large
body of yolk, whereas the relatively yolk-free eggs of mammals form a blastocyst
containing an outer layer (that becomes part of the placenta) and an inner cell
mass composed of embryonic stem cells (that will form all the cells of the embryo).
Gastrulation is initiated at the node, a site that is most likely determined by physical as
well as chemical cues. Nodal and Wnt proteins are especially important in determining
where this takes place as well as specifying the anterior-posterior polarity of the
embryo. The node extends into the primitive streak, and the cells of the upper layer
travel to and through this structure. The cells migrating into and through the streak
become the mesoderm and the endoderm. Those remaining on the surface become
the ectoderm. The node is very similar to the dorsal blastopore lip of amphibians, and
similar molecules are involved in its formation.
380   Chapter 12 

(A) Gallus gallus Figure 12.1  The membranes of the amniote egg characterize
reptiles, birds, and mammals. (A) Phylogenetic relationships of the
AMNIOTE amniotes. Note that birds are considered reptiles by most modern
VERTEBRATES Reptiles taxonomists, but for physiological studies they are often treated
as separate taxa. (Other flying and feathered reptile groups have
not survived to the present day.) The domestic chicken (Gallus gal-
lus) is the most widely studied bird species. Among mammals, the
Birds development of the laboratory mouse Mus musculus is the most
Feathers, flight
widely studied. Both avian and mouse studies contribute to our
Amniote understanding of human development. (B) The shelled amniote egg
egg (as exemplified by the chicken egg on the left) permitted animals to
Monotremes
develop away from bodies of water. The amnion provides a “water
(egg-laying mammals:
platypus, echidna) sac” in which the embryo develops; the allantois stores wastes; and
Mammary
the blood vessels of the chorion exchange gases and nutrients from
glands
Placental mammals the yolk sac. In mammals (right), this arrangement is modified such
Placenta that the blood vessels acquire nutrients and exchange gases via a
placenta joined to the mother’s uterus rather than from the yolk sac.
Mus musculus (Chick photograph courtesy of D. McIntyre; mouse photograph ©
(B) Antagain/iStock.)

Extraembryonic
Maternal
membranes:
portion
of placenta
Yolk sac

Chorion Fetal portion

Allantois of placenta
Umbilical
Amnion cord
Shell
Embryo
Amniotic cavity Embryo
Amniotic cavity

mother. Gas exchange, nutrient uptake, and waste elimination take place through the
placenta, enabling the embryo to develop inside another organism.
WEb Topic 12.1 The extraembryonic membranes  The amniote embryo is sup-
ported by a variety of membranes that provide it with nourishment, protection, and waste
disposal services.

Ever since Aristotle first observed and recorded the details of its 3-week-long devel-
opment, the domestic chicken (Gallus gallus) has been a favorite organism for embryo-
logical studies. It is accessible year-round and is easily maintained. Moreover, at any
particular temperature, its developmental stage can be accurately predicted, so large
numbers of same-stage embryos can be obtained and manipulated. Chick organ forma-
tion is accomplished by genes and cell movements similar to those of mammalian organ
formation, and the chick is one of the few organisms whose embryos are amenable to
both surgical and genetic manipulations (Stern 2005a). Thus, the chick embryo has
often served as a model for human embryos, as has the ubiquitous laboratory mouse.
Gilbert The mouse is the mammalian model organism of choice and is the subject of many
Developmental Biology 11e, Sinauer Associates studies involving genetic and surgical manipulation. In addition, the mouse was the first
DevBio11e_12.01 Date 04-08-16 mammalian genome to be sequenced, and when it was first published many scientists
felt it was more valuable than knowing the human genome sequence. Their reasoning
was that “working on mouse models allows the manipulation of each and every gene to
determine their functions” (Gunter and Dhand 2002). We cannot do that with humans.
Human development is a subject of medical as well as general scientific interest, how-
ever, and the latter sections of this chapter will cover early human development, illus-
trating the application of many of the principles we have described in model organisms.
 Birds and Mammals   381

Early Development in Birds Figure 12.2  Discoidal meroblastic cleavage

in a chick egg. (A) Avian eggs include some
Avian Cleavage of the largest cells known (inches across), but
cleavage takes place in only a small region. The
Fertilization of the chick egg occurs in the hen’s oviduct, before the albumin yolk fills up the entire cytoplasm of the egg cell,
(“egg white”) and shell are secreted to cover it. Cleavage occurs during the first with the exception of a small blastodisc in which
day of development, while the egg is still inside the hen, during which time the cleavage and development will take place. The
embryo progresses from a zygote through late blastula stages (Sheng 2014). Like chalaza are protein strings that keep the yolky
the egg of the zebrafish, the chick egg is telolecithal, with a small disc of cyto- egg cell centered in the shell. The albumin (egg
plasm—the blastodisc —sitting on top of a large yolk (Figure 12.2A). Like fish white) is secreted onto the egg in its passage out
of the oviduct. (B) Early cleavage stages viewed
eggs, the yolky eggs of birds undergo discoidal meroblastic cleavage. Cleavage
from the animal pole (the future dorsal side of the
occurs only in the blastodisc, which is about 2–3 mm in diameter and is located
embryo). In the micrographs, the tightly apposed
at the animal pole of the egg. The first cleavage furrow appears centrally in the cell membranes have been stained with phal-
blastodisc; other cleavages follow to create a blastoderm (Figure 12.2B,C). As loidin (green). (C) Schematic view of cellulariza-
in the fish embryo, the cleavages do not extend into the yolky cytoplasm, so the tion in the chick egg during the day it is fertilized
early-cleavage cells are continuous with one another and with the yolk at their and still inside the hen. The numbers refer to the
layers of cells. (A,B after Bellairs et al. 1978, pho-
tographs from Lee et al. 2013, courtesy of J. Y.
Han; C after Nagai et al. 2015.)
(A) (C)
Shell
Chalaza
Albumin

Blastodisc 1
0 0
Vitelline
membrane

Yolk
1 2 1
0 0
Inner shell membrane

Outer shell membrane

2 3 2
1 1
0 0
Air space

Subgerminal
(B) 4 cavity
2 3 3 2
1 1

Area Area Marginal

pellucida opaca zone

Hypoblast Epiblast

Yolk
382   Chapter 12 

bases. Thereafter, equatorial and vertical cleavages divide the blastoderm into a tissue
about 4 cell layers thick, with the cells linked together by tight junctions (see Figure
12.2C; Bellairs et al. 1978; Eyal-Giladi 1991; Nagai et al. 2015). The switch from mater-
nal to zygotic gene expression occurs at about the seventh or eighth division, when
there are around 128 cells (Nagai et al. 2015).
Between the blastoderm and the yolk of avian eggs is a space called the subgermi-
nal cavity, which is created when the blastoderm cells absorb water from the albumin
(“egg white”) and secrete the fluid between themselves and the yolk (New 1956). At
VADE MECUM 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
Viewing the movies of 3-D mod-
of the actual embryo. The peripheral ring of blastoderm cells that have not shed their
els of chick cleavage and gas-
trulation in the segment on chick
deep cells constitutes the area opaca. Between the area pellucida and the area opaca
development will help you under- is a thin layer of cells called the marginal zone (Eyal-Giladi 1997; Arendt and Nübler-
stand these phenomena. Jung 1999). Some marginal zone cells become very important in determining cell fate
during early chick development.

Gastrulation of the Avian Embryo

The hypoblast
By the time a hen has laid an egg, its blastoderm contains some 50,000 cells. At this
time, most of the cells of the area pellucida remain at the surface, forming an “upper
layer” called the epiblast. Shortly after the egg is laid, a local thickening of the epiblast,
called Koller’s sickle, is formed at the posterior edge of the area pellucida. In between
the area opaca and Koller’s sickle is a beltlike region called the posterior marginal
zone (PMZ). A sheet of cells at the posterior boundary between the area pellucida and
marginal zone migrates anteriorly beneath the surface. Meanwhile, cells in more ante-
rior regions of the epiblast have delaminated and stay attached to the epiblast, to form
hypoblast “islands,” an archipelago of disconnected clusters of 5–20 cells each that
migrate and become the primary hypoblast (Figure 12.3A,B). The sheet of cells that
grows anteriorly from Koller’s sickle combines with the primary hypoblast to form the
complete hypoblast layer, also called the secondary hypoblast or endoblast ( Fig-
ure 12.3C–E ; Eyal-Giladi et al. 1992; Bertocchini and Stern 2002; Khaner 2007a,b).
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-
like cavity Thus, although the shape and formation of the avian blastodisc differs from
those of the amphibian, fish, or echinoderm blastula, the overall spatial relationships
are retained.
The avian embryo comes entirely from the epiblast; the hypoblast does not contrib-
ute any cells to the developing embryo (Rosenquist 1966, 1972). Rather, the hypoblast
cells form portions of the extraembryonic membranes (see Figure 12.1B), especially the
yolk sac and the stalk linking the yolk mass to the endodermal digestive tube. Hypo-
blast cells also provide chemical signals that specify the migration of epiblast cells.
However, the three germ layers of the embryo proper (plus the amnion, chorion, and
allantois extraembryonic membranes) are formed solely from the epiblast (Schoenwolf
1991).

The primitive streak

Although many reptile groups initiate gastrulation by migration through an amphibi-
an-like blastopore, avian and mammalian gastrulation takes place through the primi-
tive streak . This can be considered the equivalent of an elongated blastopore lip of
amphibian embryos (Alev et al. 2013; Bertocchini et al. 2013; Stower et al. 2015). Dye-
marking experiments and time-lapse cinemicrography indicate that the primitive streak
first arises from Koller’s sickle and the epiblast above it (Bachvarova et al. 1998; Lawson
and Schoenwolf 2001a,b; Voiculescu et al. 2007). As cells converge to form the primitive
 Birds and Mammals   383

MIDSAGITTAL VENTRAL DORSAL

Anterior Posterior Hypoblast islands
(A) Stage X Anterior
Area pellucida epiblast Koller’s sickle Area opaca epiblast

Dorsal

Ventral
Area opaca Posterior
Hypoblast Precursors of endoderm
islands secondary hypoblast Koller’s sickle

(B) Stage XII

Primary
hypoblast cells

(C) Stage XIII

Secondary
hypoblast cells

(D) Stage 2

Primitive streak
(middle layer)

(E) Stage 3
Primitive streak

Epiblast
Hypoblast

Blastocoel Definitive endoderm

Primitive streak

Figure 12.3  Formation of the chick blastoderm. The left column is a diagrammatic midsagit-
tal section through part of the blastoderm. The middle column depicts the entire embryo viewed
from the ventral side, showing 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 to 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) By stage XII, a
sheet of cells that grows anteriorly from Koller’s sickle combines with the hypoblast islands to form
the complete hypoblast layer. (C) By stage XIII, just prior to primitive streak formation, the formation
of the hypoblast just been completed. (D) By stage 2 (12–14 hours after the egg is laid), the primi-
tive streak cells form a third layer that lies between the hypoblast and epiblast cells. (E) By stage 3
(15–17 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. (After Stern 2004.)
Gilbert
Developmental Biology 11e, Sinauer Associates
DevBio11e_12.03 Date 02-11-16
384   Chapter 12 

(A) Anterior Figure 12.4  Cell movements of the primitive streak and fate map of the chick embryo.
Area opaca (A–C) Dorsal view of the formation and elongation of the primitive streak. The blastoderm is
seen at (A) 12–14 hours, (B) 15–17 hours, and (C) 18–20 hours after the egg is laid. (D–F) For-
Area Margin mation of notochord and mesodermal somites as the primitive streak regresses, shown at
pellucida (D) 20–22 hours, (E) 23–25 hours, and (F) the four-somite stage. Fate maps of the chick epi-
Koller’s blast are shown for two stages, the definitive primitive streak stage (C) and neurulation (F).
sickle In (F), the endoderm has ingressed beneath the epiblast, and convergent extension is seen
Posterior in the midline. The movements of the mesodermal precursors through the primitive streak at
(B) (C) are shown. (Adapted from several sources, especially Spratt 1946; Smith and Schoenwolf
1998; Stern 2005a,b.)
Area opaca
Area streak, a depression called the primitive groove forms within
pellucida
the streak. Most migrating cells pass through the primitive
Primitive groove, which serves as a gateway into the deep layers of
streak the embryo (Figure 12.4; Voiculescu et al. 2014). Thus, the
taking shape primitive groove is homologous to the amphibian blastopore,
Fate map Anterior and the primitive streak is homologous to the blastopore lip.
(C) Notochord At the anterior end of the primitive streak is a regional
Epidermal
Hensen’s ectoderm thickening of cells called Hensen’s node (also known as the
node primitive knot; see Figure 12.4C). The center of Hensen’s
Neural
ectoderm
node contains a funnel-shaped depression (sometimes called
Area
pellucida the primitive pit) through which cells can enter the embryo
Paraxial to form the notochord and prechordal plate. Hensen’s node
Area mesoderm
(somites) is the functional equivalent of the dorsal lip of the amphib-
opaca ian blastopore (i.e., the organizer)1 and the fish embryonic
Lateral plate
Primitive shield (Boettger et al. 2001).
mesoderm
groove The primitive streak defines the major body axes of the
Extraembryonic avian embryo. It extends from posterior to anterior; migrat-
mesoderm Primitive
(D) ing cells enter through its dorsal side and move to its ventral
streak side; and it separates the left portion of the embryo from
Head Prechordal
the right. The axis of the streak is equivalent to the dorsal-
process mesoderm,
notochord
ventral axis of amphibians. The anterior end of the streak—
Hensen’s Hensen’s node—gives rise to the prechordal mesoderm,
Medial somite notochord, and medial part of the somites. Cells that ingress
node
Lateral somite through the middle of the streak give rise to the lateral part
Kidney, etc. of the somites and to the heart and kidneys. Cells in the pos-
(E) terior portion of the streak make the lateral plate and extra-
Lateral plate
embryonic mesoderm (Psychoyos and Stern 1996). After the
Ectoderm of mesoderm
head fold
ingression of the mesoderm cells, epiblast cells remaining
Extraembryonic outside of but close to the streak will form medial (dorsal)
Anterior mesoderm
structures such as the neural plate, while those epiblast cells
border of
farther from the streak will become epidermis (see Figure
mesoderm Neural fold
12.4, right-hand panels).
Somite Notochord
WEb Topic 12.2 Organizing the chick node  FGFs
Hensen’s Segmental plate
and BMPs have major roles in determining the place where
node Primitive streak gastrulation is initiated.

(F) elongation of the primitive streak  As cells enter

Head fold
the primitive streak, they undergo an epithelial-to-mesen-
Neural fold
Foregut chymal transformation and the basal lamina beneath them
Somites breaks down. The streak elongates toward the future head
Notochord region as more anterior cells migrate toward the center of
Segmental 1
Frank M. Balfour proposed the homology of the amphibian blas-
plate topore and the chick primitive streak in 1873, while he was still
an undergraduate (Hall 2003). August Rauber (1876) provided
Primitive streak further evidence for their homology.
 Birds and Mammals   385

the embryo. Convergent extension is responsible for the progression of the streak—a
doubling in streak length is accompanied by a concomitant halving of its width (see
Figure 12.4B). 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 (Streit et al. 2000; Lawson and Schoenwolf 2001b).
At the same time, the secondary hypoblast (endoblast) cells continue to migrate
anteriorly from the posterior marginal zone of the blastoderm (see Figure 12.3E). The
elongation of the primitive streak appears to be coextensive with the anterior migra-
tion of these secondary hypoblast cells, and the hypoblast directs the movement of the
primitive streak (Waddington 1933; Foley et al. 2000). The streak eventually extends to
60–75% of the length of the area pellucida.

formation of endoderm and mesoderm 

The basic rule of amniote cell specification is
that germ layer identity (ectoderm, mesoderm,
or endoderm) is established before gastrulation
starts (see Chapman et al. 2007), but the speci-
fication of cell type is controlled by inductive
influences during and after migration through
the primitive streak. As soon as the primitive
streak has formed, epiblast cells begin to migrate
through it and into the blastocoel. The streak thus
has a continually changing cell population. Cells
migrating through the anterior end pass down
into the blastocoel and migrate anteriorly, form-
(A) Hensen’s node Primitive streak Epiblast Blastocoel
ing the endoderm, head mesoderm, and noto-
chord; cells passing through the more posterior
portions of the primitive streak give rise to the
majority of mesodermal tissues (Figure 12.5;
Rosenquist et al. 1966; Schoenwolf et al. 1992).

Hypoblast

Figure 12.5  Migration of endodermal and mesoder-

Endoderm
mal 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 Migrating cells
mosaic of hypoblast and endodermal cells; the hypoblast (mesoderm)
cells eventually sort out to form a layer beneath the endo-
derm and contribute to the yolk sac. Above each region
of the stereogram are micrographs showing the tracks of (B)
GFP-labeled cells at that position in the primitive streak. 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 maps in Figure 12.4).
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. (A after Balinsky 1975, photographs
from Yang et al. 2002; B from Solursh and Revel 1978, cour-
tesy of M. Solursh and C. J. Weijer.)
386   Chapter 12 

Rostral The first cells to migrate through Hensen’s node are those destined
Neural tube to become the pharyngeal endoderm of the foregut. Once deep within
Neurons
Somites 1,5 the embryo, these endodermal cells migrate anteriorly and eventually
displace the hypoblast cells, causing the hypoblast cells to be confined
Presomitic RA to a region in the anterior portion of the area pellucida. This anterior
mesoderm region, the germinal crescent, does not form any embryonic structures,
3
Floor plate Raldh2 but it does contain the precursors of the germ cells, which later migrate
1 Shh through the blood vessels to the gonads.
Paraxial RARβ 3,4 The next cells entering through Hensen’s node also move anteri-
mesoderm 1,4 Wnt8c orly, but they do not travel as far ventrally as the presumptive foregut
Notochord 4 endodermal cells. Rather, they remain between the endoderm and the
Delta1 epiblast to form the prechordal plate mesoderm (Psychoyos and Stern
Caudal lateral
2 Fgf8 1996). Thus, the head of the avian embryo forms anterior (rostral) to
epiblast 1 Hensen’s node.
Primitive The next cells passing through Hensen’s node become the chor-
streak damesoderm . The chordamesoderm has two components: the head
Caudal
process and the notochord. The most anterior part, the head process,
Raldh2/RARβ
is formed by central mesoderm cells migrating anteriorly, behind the
Fgf8 Shh prechordal plate mesoderm and toward the rostral tip of the embryo
(see Figures 12.4 and 12.5). The head process underlies those cells that
Figure 12.6  Signals that regulate axis extension in will form the forebrain and midbrain. As the primitive streak regresses,
chick embryos. In the stage 10 chick embryo, Fgf8 inhibits the cells deposited by the regressing Hensen’s node will become the
expression of the retinoic acid (RA) synthesizing enzyme notochord. In the ectoderm, most of the initial neural plate corresponds
Raldh2 in the presomitic mesoderm (1) and the expression to the future head region (from forebrain to the level of the future ear
of the RA receptor RARβ in the neural ectoderm (4), thus vesicle, which lies adjacent to Hensen’s node at full primitive streak
preventing RA from triggering differentiation in the caudal- stage). A small region of neural ectoderm just lateral and posterior to
lateral epiblast cells (those cells adjacent to the node/
the node (sometimes called the caudal lateral epiblast) will give rise
streak border and which give rise to lateral and dorsal
neural tube) and the caudalmost paraxial mesoderm (1,5). to the rest of the nervous system, including the posterior hindbrain
In addition, Fgf8 inhibits Sonic hedgehog (Shh) expression and all of the spinal cord. As the primitive streak regresses, this latter
in the neural tube floorplate, controlling the onset of ven- region regresses with Hensen’s node and adds cells to the caudal end of
tral patterning genes (1). FGF signaling is also required for the elongating neural plate. It appears that FGF signaling in the streak
expression of Delta1 in the medial portion of the caudal- and paraxial (future somite) mesoderm keeps this region “young” and
lateral epiblast cells (2) and promotes expression of Wnt8c undifferentiated as it regresses, and that this is antagonized by retinoic
(4). As Fgf8 decays in the caudal paraxial mesoderm, Wnt
acid (RA) activity as cells leave this zone (Figure 12.6 ; Diez del Corral
signaling, most likely provided by Wnt8c, now acts to pro-
mote Raldh2 in the adjacent paraxial mesoderm (4). RA et al. 2003).
produced by Raldh2 activity represses Fgf8 (1) and Wnt8c
(3,4). (After Wilson et al. 2009.) Scientists speak 12.1  Dr. Steven Oppenheimer lectures on the
development of the chick embryo.

Molecular mechanisms of migration through the primitive streak

formation of the primitive streak  The migration of chick epiblast cells to form
the primitive streak was first analyzed by Ludwig Gräper, who in 1926 made time-lapse
movies of labeled cells under the microscope. He wrote that these movements reminded
him of the Polonaise, a courtly dance in which men and women move in parallel rows
along the sides of the room, and the man and woman at the “posterior end” leave their
respective lines to dance forward through the center. The mechanism for the cellular
“dance” was revealed by Voiculescu and colleagues (2007), who used a modern version
of cinemicrography (specifically, multiphoton time-lapse microscopy) that identified
individual moving cells. They found that cells came down the sides of the epiblast to
undergo a medially directed intercalation of cells in the posterior margin where the
primitive streak was forming (Figure 12.7). And although the movement may look like
a dance from far away, “at high power, it looks like a rush hour” (Stern 2007).
This rush to the center is mediated by the activation of the Wnt planar cell polar-
ity pathway (see Chapter 4) in the epiblast next to Koller’s sickle, at the posterior edge

Gilbert
Developmental Biology 11e, Sinauer Associates
DevBio11e_12.06 Date 02-12-16
 Birds and Mammals   387

of the embryo. If this pathway is blocked, the mesoderm and (A) (B)
endoderm form peripherally instead of centrally. The Wnt path-
way in turn appears to be activated by fibroblast growth factors
(FGFs) produced by the hypoblast. If the hypoblast is rotated,
the orientation of the primitive streak follows it. Moreover, if
FGF signaling is activated in the margin of the epiblast, Wnt
signaling will occur there and the orientation of the primitive
streak will change, as if the hypoblast had been placed there.
The cell migrations that form the primitive streak thus appear
to be regulated by FGFs coming from the hypoblast, which acti-
vates the Wnt planar cell polarity pathway in the epiblast.
5 5
16 10 4 5 11 17 10 4 17 4
migration through the primitive streak  Cells migrate 16 3 11 12 10 11
15 9 3 1 6 12 18
to the primitive streak, and as they enter the embryo, the cells 14 8 2 7 13 19 15 9 1 6 18 3 17
separate into two layers. The deep layer joins the hypoblast 14 7 13 19 16 12
8 2 15 1 18
along its midline, displacing the hypoblast cells to the sides. 9 6
7 19
These deep-moving cells give rise to the endodermal organs of 14 13
Time 8
the embryo, as well as to most of the extraembryonic membranes 2
(the hypoblast and peripheral cells of the area opaca form the
rest). The second migrating layer spreads to form a loose layer of Figure 12.7  Mediolateral intercalation in the formation of the
primitive streak. Chick embryos at (A) stage 13 (immediately prior
cells between the endoderm and the epiblast. This middle layer
to primitive streak formation) and (B) stage 2 (shortly after primi-
of cells generates the mesodermal portions of the embryo and
tive streak formation). Arrows show cell displacement toward the
the mesoderm lining the extraembryonic membranes. streak and in front of it. The red area represents the streak-forming
The migration of mesodermal cells through the anterior region; in (A), the original location of this region is shown in green.
primitive streak and their condensation to form the chordame- The circled areas are represented in the lower row. Each colored
soderm also appear to be controlled by FGF and Wnt signaling. disc represents an individual cell, and the cells become mediolat-
Fgf8 is expressed in the primitive streak and repels migrating erally intercalated as the primitive streak forms. (After Voiculescu
et al. 2007.)
cells away from the streak. Yang and colleagues (2002) were able
to follow the trajectories of cells as they migrated through the
primitive streak (see Figure 12.5) and were able to deflect these
normal trajectories by using beads that released Fgf8.
Once cells migrate away from the streak, further movement of the mesodermal pre-
cursors appears to be regulated by Wnt proteins. In the more posterior regions, Wnt5a is
unopposed and directs the cells to migrate broadly and become lateral plate mesoderm
(see Chapter 18). In the more anterior regions of the streak, however, Wnt5a is opposed
by Wnt3a, which inhibits migration and causes the cells to form paraxial mesoderm (see
Chapter 17). Indeed, the addition of Wnt3a-secreting pellets to the posterior primitive
streak suppresses lateral migration and prevents the formation of lateral plate meso-
derm (Sweetman et al. 2008). By 22 hours of incubation, most of the presumptive endo-
dermal cells are in the interior of the embryo, although presumptive mesodermal cells
continue to migrate inward for a longer time.

Regression of the primitive streak and epiboly of the ectoderm

Now a new phase of development begins. As 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 (Figure 12.8). 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 tailbud. 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
Gilbert
of presumptive ectodermal cells. Developmental Biology 11e, Sinauer Associates
While the presumptive mesodermal and endodermal cells are moving inward, the Date 02-03-16
DevBio11e_12.07
ectodermal precursors proliferate and migrate to surround the yolk by epiboly. The
388   Chapter 12 

(A) (B) (C)

Pharyngeal
endoderm Head
fold
Foregut
Head process
(anterior Neural
notochord) groove
Somite
Hensen’s node

Primitive streak

Area pellucida

Primitive
Area opaca streak

(D) (E)

Lengthening of notochord
d

d'

b' Reg
res
of p sion
c rimi
c' strea tive
k
Posterior border of pellucida area
0.0 10.5 20.5
Hours

Figure 12.8  Chick gastrulation 24–28

hours after fertilization. (A) The primitive enclosure of the yolk by the ectoderm (again, reminiscent of the epiboly of the amphib-
streak at full extension (24 hours). The ian ectoderm) is a Herculean task that takes the greater part of 4 days to complete. It
head process (anterior notochord) can be
involves the continuous production of new cellular material and the migration of the
seen extending from Hensen’s node. (B)
presumptive ectodermal cells along the underside of the vitelline envelope (New 1959;
Two-somite stage (25 hours). Pharyngeal
endoderm is seen anteriorly, while the Spratt 1963). Interestingly, only the cells of the outer margin of the area opaca attach
anterior notochord pushes up the head firmly to the vitelline envelope. These cells are inherently different from the other blas-
process beneath it. The primitive streak toderm cells, as they can extend enormous (500 μm) cytoplasmic processes onto the
is regressing. (C) Four-somite stage (27 vitelline envelope. These elongated filopodia are believed to be the locomotor apparatus
hours). (D) At 28 hours, the primitive of the marginal cells, by which the marginal cells pull other ectodermal cells around
streak has regressed to the caudal por-
the yolk (Schlesinger 1958). The filopodia bind to fibronectin, a laminar protein that is
tion of the embryo. (E) Regression of the
a component of the chick vitelline envelope. If the contact between the marginal cells
primitive streak, leaving the notochord in
its wake. Various points of the streak (rep- and the fibronectin is experimentally broken by adding a soluble polypeptide similar
resented by letters) were followed after it to fibronectin, the filopodia retract and ectodermal migration ceases (Lash et al. 1990).
achieved its maximum length. The x axis Thus, as avian gastrulation draws to a close, the ectoderm has surrounded the
(time) represents hours after achieving embryo, the endoderm has replaced the hypoblast, and the mesoderm has positioned
maximum length (the reference line is itself between these two regions. Although we have identified many of the processes
about 18 hours of incubation). (A–D cour-
involved in avian gastrulation, we are only beginning to understand the molecular
tesy of K. Linask; E after Spratt 1947.)
mechanisms by which some of these processes are carried out.
Gilbert
Developmental Biology 11e, Sinauer Associates
DevBio11e_12.08 Date 02-12-16
 Birds and Mammals   389

WEb Topic 12.3 Epiblast cell heterogeneity  Although the early epiblast
appears uniform, different cells have different molecules on their cell surfaces. This vari-
ability allows some of them to remain in the epiblast while others migrate into the embryo.

Axis Specification and the Avian “Organizer”

As a consequence of the sequence in which the head endomesoderm and notochord are
established, gastrulating avian (and mammalian) embryos exhibit a distinct anterior-
to-posterior gradient. While cells of the posterior portions of the embryo are still part of
a primitive streak and entering inside the embryo, cells at the anterior end are already
starting to form organs (see Darnell et al. 1999). For the next several days, the anterior
end of the embryo is more advanced in its development (having had a “head start,” if you
will) than the posterior end. Although the formation of the chick body axes is accom-
plished during gastrulation, axis specification begins earlier, during the cleavage stage.

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 15 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, where primitive streak formation begins (Figure 12.9 ;
Kochav and Eyal-Giladi 1971; Callebaut et al. 2004).
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 (Spratt and Haas 1960; Bertocchini
and Stern 2012). 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 (Khaner and
Eyal-Giladi 1989; Eyal-Giladi et al. 1992; Bertocchini et al. 2004).
It now seems apparent that the PMZ 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 including Koller’s sickle) is able to induce a
primitive streak and Hensen’s node without contributing cells to either structure (Bach-
varova et al. 1998; Khaner 1998). Current evidence suggests that the entire marginal
Figure 12.9  Specification of the
zone produces Wnt8c (capable of inducing the accumulation of β-catenin) and that, like
chick anterior-posterior axis by grav-
the amphibian Nieuwkoop center, the PMZ cells secrete Vg1, a member of the TGF-β ity. (A) Rotation in the shell gland
family (Mitrani et al. 1990; Hume and Dodd 1993; Seleiro et al. 1996). results in (B) the lighter components
Wnt8c and Vg1 act together to induce expression of Nodal (another secreted TGF-β of the yolk pushing up one side of the
protein) in the future embryonic epiblast next to Koller’s sickle and the PMZ (Skromne blastoderm. (C) This more elevated
region becomes the posterior of the
embryo. (After Wolpert et al. 1998.)

(A) Surface view of egg (B) Cross section through egg (C) Surface view of yolk
Blastoderm Primitive
streak
P
A
X Y X Yolk Y X P A Y

Yolk rotates Albumen Blastodisc

in shell
390   Chapter 12 

and Stern 2002). 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 (Bertocchini
et al. 2004; Voiculescu et al. 2014). 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
Figure 12.10  Model for generating left-right
formed, however, the streak secretes its own Nodal antagonist—the Lefty pro-
asymmetry in the chick embryo. (A) On the left
side of Hensen’s node, Sonic hedgehog (Shh) tein—thereby preventing any further primitive streaks from forming. Eventu-
activates Cerberus, which stimulates BMPs to ally, the Cerberus-secreting hypoblast cells are pushed to the future anterior of
induce the expression of Nodal. In the presence the embryo, where they contribute to ensuring that neural cells in this region
of Nodal, the Pitx2 gene is activated. Pitx2 pro- become forebrain rather than more posterior structures of the nervous system.2
tein is active in the various organ primordia and
specifies which side will be the left. On the right Left-right axis formation
side of the embryo, activin is expressed, along
The vertebrate body has distinct right and left sides. The heart and spleen, for
with activin receptor IIa. This activates Fgf8, a
protein that blocks expression of the gene for instance, are generally on the left side of the body, whereas the liver is usually
Cerberus. In the absence of Cerberus, Nodal is on the right. The distinction between the sides is primarily regulated by the left-
not activated and thus Pitx2 is not expressed. sided expression of two proteins: the paracrine factor Nodal and the transcrip-
(B) Whole-mount in situ hybridization of Cer- tion factor Pitx2. However, the mechanism by which Nodal gene expression is
berus mRNA. This view is from the ventral sur- activated in the left side of the body differs among the vertebrate classes. The
face (“from below,” so the expression seems to ease with which chick embryos can be manipulated has allowed scientists to
be on the right). Dorsally, the expression pattern
elucidate the pathways of left-right axis determination in birds more readily than
would be on the left. (C) Whole-mount in situ
hybridization using probes for the chick Nodal in other vertebrates.
message (stained purple) shows its expression in As the primitive streak reaches its maximum length, transcription of the
the lateral plate mesoderm only on the left side Sonic hedgehog gene (Shh) becomes restricted to the left side of the embryo, con-
of the embryo. This view is from the dorsal side. trolled by activin and its receptor (Figure 12.10A). Activin signaling, along with
(D) Similar in situ hybridization, using the probe BMP4, appears to block the expression of Sonic hedgehog protein and to activate
for Pitx2 at a later stage of development. The expression of Fgf8 protein on the right side of the embryo. Fgf8 blocks expres-
embryo is seen from its ventral surface. At this
sion of the paracrine factor Cerberus on the right-hand side; it may also activate
stage, the heart is forming, and Pitx2 expression
can be seen on the left side of the heart tube a signaling cascade that instructs the mesoderm to have right-sided capacities
(as well as symmetrically in more anterior tis- (Schlueter and Brand 2009).
sues). (A after Raya and Izpisua-Belmonte 2004;
2
B from Rodriguez-Esteban et al. 1999, courtesy Conjoined twins may be formed by having by having two sources of Nodal expression
of J. Izpisúa-Belmonte; C courtesy of C. Stern; within the same blastodisc. Experimentation with chick embryos can produce two axes
D from Logan et al. 1998, courtesy of C. Tabin.) in the same blastodisc by circumventing the usual inhibition of Nodal by the Vg1-secret-
ing posterior cells (Bertocchini et al. 2004). In mammals, multiple axes can also form if
Nodal antagonists are blocked (Perea Gomez et al. 2002).

(A) (B) (C) (D)

LEFT RIGHT

Hensen’s
node Shh Activin
Shh Fgf8 BMP4

Cerberus Cerberus
Lefty

BMPs

Nodal
Snail
Pitx2
Midline
 Birds and Mammals   391

Meanwhile, on the left side of the body, Shh protein activates Cerberus (Figure
12.10B ), which in this case acts with BMP to stimulate the synthesis of Nodal pro-
tein (Yu et al. 2008). Nodal activates the Pitx2 gene while repressing Snail. In addition,
Lefty1 in the ventral midline prevents the Cerberus signal from passing to the right
side of the embryo (Figure 12.10C,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 embryo reverses the asymmetry or causes
randomization of asymmetry on the right or left sides 3 (Levin et al. 1995; Logan et al.
1998; Ryan et al. 1998).
The real mystery is, What processes create the original asymmetry of Shh and Fgf8?
One important observation is that the first asymmetry seen during the formation of
Hensen’s node in chicks involves Fgf8- and Shh-expressing cells rearranging them-
selves to converge on the right-hand side of the node (Cui et al. 2009; Gros et al. 2009).
Therefore, the differences in gene expression can be traced back to differences in cell
migration to the right and left sides of the embryo. What establishes this initial asym-
metry is still unknown, but it may be a physical displacement of cells around the node
(Tsikolia et al. 2012; Otto et al. 2014).

Early Development in Mammals

Cleavage
Mammalian eggs are among the smallest in the animal kingdom, making them dif-
ficult to manipulate experimentally. The human zygote, for instance, is only 100 μm
in diameter—barely visible to the eye and less than one-thousandth the volume of a
Xenopus laevis egg. Also, mammalian zygotes are not produced in numbers comparable
to sea urchin or frog zygotes; a female mammal usually ovulates fewer than 10 eggs at
a given time, so it is difficult to obtain enough material for biochemical studies. As a
final hurdle, the development of mammalian embryos is accomplished inside another
organism rather than in the external environment (although early embryos prior to
implantation can be cultured and observed in vitro). Most research on mammalian
development has focused on the mouse, since mice are relatively easy to breed, have
large litters, and are easily housed in laboratories.

The unique nature of mammalian cleavage

Prior to fertilization, the mammalian oocyte, wrapped in cumulus cells, is released
from the ovary and swept by the fimbriae into the oviduct (Figure 12.11). Fertiliza-
tion occurs in the ampulla of the oviduct, a region close to the ovary. Meiosis is com-
pleted after sperm entry, and the first cleavage begins about a day later (see Figure
7.32). The positioning of the first cleavage plane may depend on the point of sperm
entry (Piotrowska and Zernicka-Goetz 2001), and in mice, a sperm-borne microRNA
(miRNA-34c) is required to initiate this first cell division. This miRNA appears to bind
and inhibit Bcl-2, a protein that prevents the cell from entering the S phase of the cell
cycle (Liu et al. 2012). The two nuclei produced by this cleavage are the first nuclei to
contain the entire genome, since the haploid pronuclei enter cell division upon meeting
(see Chapter 7).
Cleavages in mammalian eggs are among the slowest in the animal kingdom, tak-
ing place some 12–24 hours apart. The cilia in the oviduct push the embryo toward the
uterus, and the first cleavages occur along this journey. In addition to the slowness of
cell division, several other features distinguish mammalian cleavage, including the
unique orientation of mammalian blastomeres relative to one another. In many but not
all mammalian embryos, the first cleavage is a normal meridional division; however,
3
In humans, homozygous loss of PITX2 causes Rieger’s syndrome, a condition characterized by
asymmetry anomalies. A similar condition is caused by knocking out the Pitx2 gene in mice (Fu
et al. 1998; Lin et al. 1999).
392   Chapter 12 

Figure 12.11  Development of a 2-Cell stage

human embryo from fertilization to
implantation. Compaction of the human Ampulla
embryo occurs on day 4, at the 10-cell Isthmus
stage. The embryo “hatches” from the Uterus Zona
Morula
zona pellucida upon reaching the uterus. pellucida
During its migration to the uterus, the First cleavage
zona prevents the embryo from prema-
turely adhering to the oviduct rather than Oviduct
traveling to the uterus.

Fertilization

Blastocyst Infundibulum

Ovary
Early stage of
Fimbriae
implantation Ovulation

in the second cleavage, one of the two blastomeres divides meridionally and the other
divides equatorially (Figure 12.12). This is called rotational cleavage (Gulyas 1975).
Another major difference between mammalian cleavage and that of most other
embryos is the marked asynchrony of early cell division. Mammalian blastomeres do
not all divide at the same time. Thus, mammalian embryos do not increase exponen-
tially from 2 to 4 to 8 cells, but frequently contain odd numbers of cells. Furthermore,
the mammalian genome, unlike the genomes of rapidly developing animals, is activated
during early cleavage and zygotically transcribed proteins are necessary for cleavage
and development. Maternally encoded proteins can persist through most of the cleavage
stages and play important roles in early development. In the mouse and goat, the activa-
tion of zygotic (i.e., nuclear) genes begins in the late zygote and continues through the
2-cell stage (Zeng and Schultz 2005; Rother et al. 2011). In humans, the zygotic genes
are activated slightly later, around the 8-cell stage (Piko and Clegg 1982; Braude et al.
1988; Dobson et al. 2004).
In order for the zygotic genes to be activated, the parental chromatin undergoes
many changes. New histones are placed on the DNA during the early cell divisions, and
the gamete-specific DNA methyl groups are removed (except for those on imprinted
genes; see Chapter 3). In both mice and human embryos, the DNA methylation of
sperm and egg chromatin is almost entirely removed. While some “imprinted
gene” methylation remains, that concerned with cell differentiation appears
(A) Echinoderm and (B) Mammal to be removed. This allows an almost “clean slate” for the newly forming
(A)–amphibian blastocyst cells. New DNA methylation patterns characteristic of totipotent
Cleavage Cleavage Cleavage Cleavage and pluripotent cells are established (Abdalla et al. 2009; Guo et al. 2014;
Gilbert
plane II plane I plane IIA plane I Smith et al. 2014). Thus, by the 16-cell stage, the genome of each cell is hypo-
Developmental Biology 11e, Sinauer Associates
methylated Date
DevBio11e_12.11 and each of these 16 cells appears to be pluripotent (Tarkowski et
02-03-16
al. 2010). The stage is now set for cell differentiation to take place.

Figure 12.12  Comparison of early cleavage in (A) echinoderms

and amphibians (radial cleavage) and (B) mammals (rotational cleav-
age). Nematodes also have a rotational form of cleavage, but they
Cleavage
do not form the blastocyst structure characteristic of mammals.
plane IIB
(After Gulyas 1975.)
 Birds and Mammals   393

(A) (B) (C) (G)

Inner cell mass
(embryonic stem cells)

(D) (E) (F)

Blastocoel Trophoblast

Figure 12.13  Cleavage of a single

mouse embryo in vitro. (A) 2-Cell stage.
Compaction
(B) 4-Cell stage. (C) Early 8-cell stage.
One of the most crucial events of mammalian cleavage is compaction. Mouse blasto- (D) Compacted 8-cell stage. (E) Morula.
meres through the 8-cell stage form a loose arrangement (Figure 12.13A–C). Follow- (F) Blastocyst. (G) Electron micrograph
ing the third cleavage, however, the blastomeres undergo a spectacular change in their through the center of a mouse blastocyst.
behavior. Cell adhesion proteins such as E-cadherin become expressed, and the blas- (A–F from Mulnard 1967, courtesy of J.
G. Mulnard; G from Ducibella et al. 1975,
tomeres gradually huddle together and form a compact ball of cells (Figure 12.13D ;
courtesy of T. Ducibella.)
Peyrieras et al. 1983; Fleming et al. 2001). This tightly packed arrangement is stabilized
by tight junctions that form between the outside cells of the ball, sealing off the inside
of the sphere. The cells within the sphere form gap junctions, thereby enabling small
molecules and ions to pass between them.
The cells of the compacted 8-cell embryo divide to produce a 16-cell morula (Fig-
ure 12.13E ). The morula consists of a small group of internal cells surrounded by a
larger group of external cells (Barlow et al. 1972). Most of the descendants of the exter-
nal cells become trophoblast (trophectoderm) cells, whereas the internal cells give rise
to the inner cell mass (ICM). The inner cell mass, which will give rise to the embryo,
becomes positioned on one side of the ring of trophoblast cells; the resulting blastocyst
is another hallmark of mammalian cleavage (Figure 12.13F,G ; see also Figure 5.5).
The trophoblast cells produce no embryonic structures, but rather form the tissues
of the chorion, the extraembryonic membrane and portion of the placenta that enables
the fetus to get oxygen and nourishment from the mother. The chorion also secretes
hormones that cause the mother’s uterus to retain the fetus, and it produces regulators
of the immune response so that the mother will not reject the embryo.
It is important to remember that a crucial outcome of these first divisions is the
generation of cells that attach the embryo to the uterus. Thus, formation of the troph-
ectoderm is the first differentiation event in mammalian development. The earliest
blastomeres (such as each blastomere of a 2-cell embryo) can form both trophoblast
cells and the embryo precursor cells of the ICM. These very early cells are said to be
totipotent (Latin, “capable of everything”). The inner cell mass is said to be pluripotent
(Latin, “capable of many things”). That is, each cell of the ICM can generate any cell
Gilbert
type in the body
Developmental but11eis, Sinauer
Biology no longer able to form the trophoblast. These pluripotent cells
Associates
of the inner cell massDate
DevBio11e_12.13 are 03-28-16
the embryonic stem cells (see Chapter 5).

WATCH DEVELOPMENT 12.1  Two videos of mammalian development, from in vitro

fertilization clinics, show the dynamics of early development.
394   Chapter 12 

Figure 12.14  Core transcriptional circuitry for the pluripotency of (A) (B)
ES cells (A) Feedforward circuit in which Oct4/Sox2 dimers activate
Nanog genes. Nanog protein then activates its own gene as well as Oct4
Nanog
genes promoting pluripotency. (B) The interconnected regulatory cir- Sox2 Oct4 Oct4
cuit whereby Oct4, Sox2, and Nanog each activate themselves and Sox2 Sox2
each other’s synthesis. (After Boyer et al. 2005.)
Nanog Nanog Nanog

ES cell
genes

Developing Questions Trophoblast or ICM? The first decision of the rest of your life
We have been discussing The philosopher and theologian Søren Kierkegaard wrote that we define ourselves
eutherian mammals, by the choices we make. It seems that the embryo already knows this. The decision
those organisms such as to become either trophoblast or inner cell mass is the first binary decision in mam-
mice and humans that malian life. Later in development, embryonic cells must lose their pluripotency and
retain the fetus during decide on what they are going to grow up to be. In the first decision, Oct4 mutually
its development. But represses Cdx2 expression, enabling some cells to be trophoblast and other cells to
what about monotreme
become the pluripotent cells of the ICM. In the second decision, each of the cells of
mammals (such as the
platypus) that lay eggs, the ICM expresses either Nanog or Gata6, thereby retaining its pluripotency (Nanog)
or marsupial mammals or becoming primitive endoderm (Gata6) (Ralston and Rossant 2005; Rossant 2016).
(such as kangaroos) that Prior to blastocyst formation, each embryonic blastomere expresses both the Cdx2
have extremely short and Oct4 transcription factors (Niwa et al. 2005; Dietrch and Hiiragi 2007; Ralston and
pregnancies? Do their Rossant 2008) and appears to be capable of becoming either ICM or trophoblast (Hiiragi
embryos have blastocysts? and Solter 2004; Motosugi et al. 2005; Kurotaki et al. 2007). However, once the decision
to become either trophoblast or ICM is made, the cell expresses a set of genes specific to
each region. The pluripotency of the ICM is maintained by a core of three transcription
factors, Oct4, Sox2, and Nanog. These proteins bind to the enhancers of their own genes
to maintain their expression while at the same time activating one another’s enhancers
(Figure 12.14). Thus, when one of these genes is activated, the other ones are too. Act-
ing in concert, Sox2 and Oct4 form a dimer and often reside on enhancers adjacent to
Nanog, activating those genes required to maintain pluripotency in embryonic stem (ES)
cells and repressing those genes whose products would lead to differentiation (Marson
et al. 2008; Young 2011). These transcription factors appear to work by recruiting RNA
polymerase II to the promoters of those genes being activated while recruiting histone
methyltransferases to those genes being repressed (Kagey et al. 2010; Adamo et al. 2011).
Only trophoblast cells synthesize the transcription factor Cdx2, which downregu-
lates Oct4 and Nanog (Strumpf et al. 2005). The activation of the Cdx2 gene in the
trophoblast cells appears Gilbert
to be regulated by the Yap protein, which in turn is a co-
factor for the transcriptionDevelopmental (Figure
factor Tead4Biology 11e,12.15A
Sinauer).Associates
Tead4 is found in the nuclei
of both the inner and outerDevBio11e_12.14 Datebut
cells of the blastocyst, 02-17-16
it is activated by Yap only in the
outer compartment. That is because Yap can enter the nucleus in the outer cells and
thereby allow Tead4 to transcribe trophoblast-specifying genes such as Cdx2 and eome-
sodermin (Eomes). In contrast, the inner cells, with each of their surfaces surrounded by
other cells, activate the gene for Lats, a protein kinase that phosphorylates Yap (Figure
12.15B). Phosphorylated Yap cannot enter the nucleus and is degraded (Nishioka et al.
2009). Therefore, in the inner cells, Tead4 cannot function and Cdx2 remains untran-
scribed (see Wu and Scholer 2016). Cdx2 blocks the expression of Oct4, and Oct4 blocks
the expression of Cdx2. In this way, the two lineages become separated.

WEb Topic 12.4 Mechanisms of compaction and formation of the

inner cell mass  What determines whether a cell is to become a trophoblast cell or
a member of the inner cell mass? It may just be a matter of chance. However, once the
decision is made, different genes are switched on.
 Birds and Mammals   395

(A) Eomes (C) Inner cell mass Hypoblast cells

Trophoblast
Tead4 Cdx2 Psx1
cells
Hand1
Trophoblast-
specifying genes

(B)

Hippo
Hippo
P
Hippo Lats
Yap Lats P
Yap

Tead4 Tead4 Trophoblast cells Integrin proteins

Cdx2
Figure 12.15  Possible pathway ini-
Outer cell Inner cell tiating the distinction between inner cell
mass and trophoblast. (A) The Tead4
transcription factor, when active, pro-
motes transcription of the Cdx2 gene.
Together, the Tead4 and Cdx2 tran-
scription factors activate the genes that
specify the outer cells to become the
trophoblast. (B) Model for Tead4 activa-
In mice, the embryo proper is derived from the inner cell mass of the 16-cell stage,
tion. In the outer cells, the lack of cells
supplemented by cells dividing from the outer cells of the morula during the transition
surrounding the embryo sends a signal
to the 32-cell stage (Pedersen et al. 1986; Fleming 1987; McDole et al. 2011). The cells of (as yet unknown) that blocks the Hippo
the ICM give rise to the embryo and its associated yolk sac, allantois, and amnion. By pathway from activating the Lats protein.
the 64-cell stage, the ICM (comprising approximately 13 cells at that stage) and the tro- In the absence of functional Lats, the
phoblast cells have become separate cell layers, neither of which contributes cells to the Yap transcriptional co-factor can bind
other group (Dyce et al. 1987; Fleming 1987). The ICM actively supports the trophoblast, with Tead4 to activate the Cdx2 gene.
In the inner cells, the Hippo pathway is
secreting proteins that stimulate the trophoblast cells to divide (Tanaka et al. 1998).
active and the Lats kinase phosphory-
Initially, the morula does not have an internal cavity. However, during a process called
lates the Yap transcriptional co-activator.
cavitation, the trophoblast cells secrete fluid into the morula to create a blastocoel. The The phosphorylated form of Yap does
membranes of trophoblast cells contain sodium pumps (an Na+-K+ ATPase and an Na+-H+ not enter the nucleus and is targeted
exchanger) that pump Na+ into the central cavity. The subsequent accumulation of Na+ for degradation. (C) Mouse blastocyst
draws in water osmotically, creating and enlarging the blastocoel (Borland 1977; Ekkert in which the Oct4 protein in the ICM is
et al. 2004; Kawagishi et al. 2004). Interestingly, this sodium pumping activity appears stained orange. The extracellular lineages
(trophoblast and hypoblast) are stained
to be stimulated by the oviduct cells on which the embryo is traveling toward the uterus
green. (A,B after Nishioka et al. 2009; C
(Xu et al. 2004). As the blastocoel expands, the inner cell mass becomes positioned on one
courtesy of J. Rossant.)
side of the ring of trophoblast cells, resulting in the distinctive mammalian blastocyst.4

Escape from the zona pellucida and implantation

While the embryo is moving through the oviduct en route to the uterus, the blastocyst
expands within the zona pellucida (the extracellular matrix of the egg that was essen-
tial for sperm binding during fertilization; see Chapter 7). During this time, the zona
pellucida prevents the blastocyst from adhering to the oviduct walls. (If such adhering
occurs—as it sometimes does in humans—it forms an ectopic, or “tubal,” pregnancy,
Gilbert
a dangerous Biology
Developmental condition becauseAssociates
11e, Sinauer an embryo implanted in the oviduct can cause a life-
DevBio11e_12.15 Date when
threatening hemorrhage 02-12-16
it begins to grow.) When the embryo reaches the uterus,
it must “hatch” from the zona so that it can adhere to the uterine wall.
4
Although the mammalian blastocyst was discovered by Rauber in 1881, its first public display
was probably in Gustav Klimt’s 1907 painting Danae, in which blastocyst-like patterns are
featured on the heroine’s robe as she becomes impregnated by Zeus (Gilbert and Braukmann
2011).
396   Chapter 12 

(A) (B) (C) Trophoblast

ICM

Endometrium
of uterus

Figure 12.16  Hatching from the zona

and implantation of the mammalian blas- The mouse blastocyst hatches from the zona pellucida by digesting a small hole in it
tocyst in the uterus. (A) Mouse blastocyst and squeezing through the hole as the blastocyst expands (Figure 12.16A). A trypsin-
hatching from the zona pellucida. (B)
like protease secreted by the trophoblast seems responsible for hatching the blastocyst
Mouse blastocysts entering the uterus.
from the zona (Perona and Wassarman 1986; O’Sullivan et al. 2001). Once outside the
(C) Initial implantation of a rhesus mon-
key blastocyst. (A from Mark et al. 1985, zona, the blastocyst can make direct contact with the uterus (Figure 12.16B,C ). The
courtesy of E. Lacy; B from Rugh 1967; endometrium —the epithelial lining of the uterus—has been altered by estrogen and
C, Carnegie Institution of Washington, progesterone hormones and has made an extensive extracellular matrix that “catch-
Chester Reather, photographer.) es” the blastocyst. This extracellular matrix is composed of complex sugars, collagen,
laminin, fibronectin, cadherins, hyaluronic acid, and heparan sulfate receptors (see
Ramathal et al. 2011; Tu et al. 2014).
After the initial binding, several other adhesion systems appear to coordinate their
efforts to keep the blastocyst tightly bound to the uterine lining. The trophoblast cells
synthesize integrins that bind to the uterine collagen, fibronectin, and laminin, and
they synthesize heparan sulfate proteoglycan precisely prior to implantation (see Car-
son et al. 1993). P-cadherins (see Chapter 4) on the trophoblast and uterine endome-
trium also help dock the embryo to the uterus. Once in contact with the endometrium,
Wnt proteins (from the trophoblast, the endometrium, or from both) instruct the tro-
phoblast to secrete a set of proteases, including collagenase, stromelysin, and plasmino-
gen activator. These protein-digesting enzymes digest the extracellular matrix of the
uterine tissue, enabling the blastocyst to bury itself within the uterine wall (Strickland
et al. 1976; Brenner et al. 1989; Pollheimer et al. 2006).

Mammalian Gastrulation
Birds and mammals are both descendants of reptilian species (albeit different reptilian
species). It is not surprising, therefore, that mammalian development parallels that of
Gilbert reptiles and birds. What is surprising is that the gastrulation movements of reptilian
Developmental Biology 11e, Sinauer Associates and avian embryos, which evolved as an adaptation to yolky eggs, are retained in the
DevBio11e_12.16 Date 02-03-16 mammalian embryo even in the absence of large amounts of yolk. The mammalian
inner cell mass can be envisioned as sitting atop an imaginary ball of yolk, following
instructions that seem more appropriate to its reptilian ancestors.

Modifications for development inside another organism

The mammalian embryo obtains nutrients directly from its mother and does not rely on
stored yolk. This adaptation has entailed a dramatic restructuring of the maternal anat-
omy (such as expansion of the oviduct to form the uterus) as well as the development of
a fetal organ capable of absorbing maternal nutrients. The origins of early mammalian
tissues are summarized in Figure 12.17. As we saw above, the first distinction is that
between inner cell mass and trophoblast. The trophoblast develops through several
 Birds and Mammals   397

Figure 12.17  Tissue and germ layer formation in the early human embryo. Days 5–9:
Implantation of the blastocyst. The inner cell mass delaminates hypoblast cells that line the
blastocoel, forming the extraembryonic endoderm of the primitive yolk sac and a bilayered
(epiblast and hypoblast) blastodisc. Days 10–12: The trophoblast divides into the cytotropho-
blast, which will form the villi, and the syncytiotrophoblast, which will ingress into the uterine
tissue to form the chorion. Days 12–15: Gastrulation and formation of primitive streak. Mean-
while, the epiblast splits into the amniotic ectoderm (which encircles the amniotic cavity) and
the embryonic epiblast. The adult mammal (ectoderm, endoderm, mesoderm, and germ cells)
forms from the cells of the embryonic epiblast. The extraembryonic endoderm forms the yolk
sac. The actual size of the embryo at this stage is about that of the period at the end of this
sentence.

Day 5 Blastocyst
Blastocyst

Day 6–7 Blastocoel

Hypoblast
Trophoblast Inner cell mass

Epiblast

Trophoblast
Day 8–9

Cytotrophoblast Primitive Epiblast

endoderm
Amniotic
(hypoblast)
cavity
Syncytio-
trophoblast
Cyto-
Amniotic trophoblast
cavity Syncytiotro- Extraembryonic Amniotic Embryonic
phoblast endoderm ectoderm epiblast

Embyronic
epiblast Syncytio-
trophoblast

Yolk sac Primitive streak

Day 10–12
Extraembryonic Extraembryonic
mesoderm endoderm Extraembryonic Embryonic Embryonic Embryonic
mesoderm mesoderm endoderm ectoderm
Day 15
Primitive
streak

Embryonic
ectoderm
Embryonic
Embryonic endoderm Extraembryonic tissues Embryonic tissues
mesoderm
398   Chapter 12 

stages, eventually becoming the chorion, the embryonically derived portion of the
placenta. Trophoblast cells also induce the mother’s uterine cells to form the mater-
nal portion of the placenta, the decidua . The decidua becomes rich in the blood
vessels that will provide oxygen and nutrients to the embryo. The inner cell mass
gives rise to the epiblast and the hypoblast (primitive endoderm). The hypoblast will
generate yolk sac cells, while the epiblast will generate the embryo, the amnion, and
the allantois.

the primitive endoderm: the mammalian hypoblast  The first segregation of

cells within the inner cell mass forms two layers. The lower layer, in contact with the
blastocoel, is called the primitive endoderm, and it is homologous to the hypoblast
of the chick embryo. The remaining inner cell mass tissue above it is the epiblast.
The primitive endoderm will form the yolk sac of the embryo, and like the chick
Figure 12.18  Mouse embryo at day hypoblast, will be used for positioning the site of gastrulation, regulating cell move-
3.5 (early blastocyst), showing the random ments in the epiblast, and promoting the maturation of blood cells. Moreover, the
expression of Nanog (blue, for the epiblast) primitive endoderm, like the chick hypoblast, is an extraembryonic layer and does
and Gata6 (red, for the visceral endoderm) not provide many (if any) cells to the actual embryo (see Stern and Downs 2012).
in the inner cell mass. In another 24 hours,
Whether a mouse ICM cell becomes epiblast or primitive endoderm may depend
the cells will sort out: the hypoblast cells will
on when the cell became part of the ICM (Bruce and Zernicka-Goetz 2010; Morris
abut the blastocoel, and the epiblast cells
will be between the hypoblast cells and the et al. 2010). Cells that become internalized in the division from 8 to 16 cells appear
trophoblast (as in Figure 12.15C). (Courtesy biased to become pluripotent epiblast cells, while the future primitive endoderm may
of J. Rossant.) be generated by cells entering the ICM during the division from 16 to 32 cells (Fig-
ure 12.18). At that stage, the blastomeres of the ICM are a mosaic of future epiblast
cells (expressing the Nanog transcription factor, which promotes pluripotency) and
primitive endoderm cells (expressing Gata6 transcription factor) a full day before
the layers segregate at day 4.5 (Chazaud et al. 2006). Levels of FGF signaling within
Figure 12.19  Amnion structure and cell the ICM determine the final identity of epiblast or primitive endoderm, with cells
movements during human gastrulation. receiving higher levels of FGF becoming primitive endoderm (Yamanaka et al. 2010).
(A,B) Human embryo and uterine connec-
The epiblast and primitive endoderm form a structure called the bilaminar germ
tions at day 15 of gestation. (A) Sagittal
disc (Figure 12.19A). The primitive endoderm cells expand to line the blastocoel
section through the midline. (B) View looking
down on the dorsal surface of the embryo. cavity, where they give rise to the yolk sac. The primitive endoderm cells contacting
Movements of the epiblast cells through the the epiblast are the visceral endoderm , while those yolk sac cells contacting the
primitive streak and the node and under- trophoblast are the parietal endoderm. The epiblast cell layer is split by small clefts
neath the epiblast are superimposed on the that eventually coalesce to separate the embryonic epiblast from the other epiblast
dorsal surface view. (C) At days 14 and 15 cells that form the amnion. Once the amnion is completed, the amniotic cavity fills
the ingressing epiblast cells are thought to
with amniotic fluid, a secretion that serves as a shock absorber as well as prevent-
replace the hypoblast cells (which contrib-
ing the developing embryo from drying out. The embryonic epiblast is thought to
ute to the yolk sac lining), and at day 16 the
ingressing cells fan out to form the meso-
dermal layer. (After Larsen 1993.)
Gilbert
Developmental Biology 11e, Sinauer Associates
(A) (B) (C) Day 14–15 Primitive
Extraembryonic
DevBio11e_12.18 Syncytiotrophoblast
Date 02-03-16 Amnionic
mesoderm cavity Epiblast groove

Primitive Node
groove

Hypoblast Endoderm

Bilaminar Day 16
Amnionic cavity germ disc Node
Yolk sac
Primitive groove

Yolk sac Hypoblast

Extraembryonic
Epiblast mesoderm Mesoderm Endoderm
 Birds and Mammals   399

contain all the cells that will generate the actual embryo and is similar in many ways
to the avian epiblast.
By labeling individual cells of the epiblast with horseradish peroxidase, Kirstie Law-
son and her colleagues (1991) were able to construct a detailed fate map of the mouse
epiblast (see Figure 1.11). Gastrulation begins at the posterior end of the embryo, and
this is where the cells of the node5 arise (Figure 12,19B,C). Like the chick epiblast cells,
mammalian mesoderm and endoderm cells originate in the epiblast, undergo epithelial-
mesenchymal transition, lose E-cadherin, and migrate through a primitive streak as
individual mesenchymal cells (Burdsal et al. 1993). Those cells arising from the node
give rise to the notochord. However, in contrast to notochord formation in the chick, the
cells that form the mouse notochord are thought to become integrated into the endo-
derm of the primitive gut (Jurand 1974; Sulik et al. 1994). These cells can be seen as a
band of small, ciliated cells extending rostrally from the node. They form the notochord
by converging medially and “budding” off in a dorsal direction from the roof of the gut.
The timing of these developmental events varies enormously in mammals. In humans,
the migration of cells forming the mesoderm doesn’t start until day 16—around the
time that a mouse embryo is almost ready to be born (see Figure 12.19C; Larsen 1993).
Cell migration and specification are coordinated by fibroblast growth factors. The
cells of the primitive streak appear to be capable of both synthesizing and responding
to FGFs (Sun et al. 1999; Ciruna and Rossant 2001). In embryos that are homozygous for
the loss of the Fgf8 gene or its receptor, cells fail to emigrate from the primitive streak,
and neither mesoderm nor endoderm are formed. Fgf8 (and perhaps other FGFs) prob-
ably control cell movement into the primitive streak by downregulating the E-cadherin
that holds the epiblast cells together. Fgf8 may also control cell specification by regulat-
ing snail, Brachyury, and Tbx6, three genes that are essential (as they are in the chick
embryo) for mesodermal migration, specification, and patterning.
The ectodermal precursors are located anterior and lateral to the fully extended
primitive streak, as in the chick epiblast and (as in the chick embryo), a single cell can
give rise to descendants in more than one germ layer. Thus, at the epiblast stage these
lineages have not yet become fully separate from one another. Indeed, in mice, some of
the visceral endoderm, which had been extraembryonic, is able to intercalate with the
definitive endoderm and become part of the gut (Kwon et al. 2008).

Scientists speak 12.2  In two videos, Dr. Janet Rossant discusses her research
on embryonic cell lineages in the mouse embryo.

WEb Topic 12.5 Placental formation and functions  In addition to providing

nutrition, the placenta is an endocrine and immunological organ, producing hormones
that enable the uterus to maintain the pregnancy and promote the development of the
mother’s mammary glands. Recent studies suggest that the placenta uses several mech-
anisms to block the mother’s immune response against the developing fetus.

Mammalian Axis Formation

Biologist and poet Miroslav Holub (1990) remarked:
Between the fifth and tenth days the lump of stem cells differentiates into the overall
building plan of the [mouse] embryo and its organs. It is a bit like a lump of iron
turning into the space shuttle. In fact it is the profoundest wonder we can still imagine
and accept, and at the same time so usual that we have to force ourselves to wonder
about the wondrousness of this wonder.

It is, indeed, wonderful, and we are just beginning to find out how really amazing it is.

5
In mouse development, Hensen’s node is usually just called “the node,” despite the fact that
Hensen discovered this structure in rabbit and guinea pig embryos.