Chapter 5

Third Week of
Development:Trilaminar
Germ Disc
GASTRULATION: FORMATION
OF EMBRYONIC MESODERM AND
ENDODERM
The most characteristic event occurring during
the third week of gestation is gastrulation, the
process that establishes all three germ layers
(ectoderm, mesoderm, and endoderm) in the
embryo. Gastrulation begins with formation of the
primitive streak on the surface of the epiblast
(Figs. 5.1 and 5.2A). Initially, the streak is vaguely
defined (Fig. 5.1), but in a 15- to 16-day embryo,
it is clearly visible as a narrow groove with slightly
bulging regions on either side. The cephalic end
of the streak, the primitive node, consists of a
slightly elevated area surrounding the small primitive pit (Fig. 5.2). Cells of the epiblast migrate
toward the primitive streak (Fig. 5.2). Upon
arrival in the region of the streak, they become
flask-shaped, detach from the epiblast, and slip
beneath it (Fig. 5.2B,C). This inward movement
is known as invagination. Cell migration and
specification are controlled by fibroblast growth
factor 8 (FGF8), which is synthesized by streak
cells themselves. This growth factor controls cell
movement by downregulating E-cadherin, a protein that normally binds epiblast cells together.
FGF8 then controls cell specification into the
mesoderm by regulating Brachyury (T) expression.
Once the cells have invaginated, some displace the
hypoblast, creating the embryonic endoderm,
and others come to lie between the epiblast and
newly created endoderm to form mesoderm.
Cells remaining in the epiblast then form ectoderm. Thus, the epiblast, through the process of
gastrulation, is the source of all of the germ layers
(Fig. 5.2B), and cells in these layers will give rise to
all of the tissues and organs in the embryo.
As more and more cells move between the
epiblast and hypoblast layers, they begin to spread
laterally and cranially (Fig. 5.2). Gradually, they
migrate beyond the margin of the disc and establish contact with the extraembryonic mesoderm
covering the yolk sac and amnion. In the cephalic
direction, they pass on each side of the prechordal plate. The prechordal plate itself forms

between the tip of the notochord and the

oropharyngeal membrane and is derived from
some of the first cells that migrate through the
node in the midline and move in a cephalic direction. Later, the prechordal plate will be important
for induction of the forebrain (Figs. 5.2 and 5.3).
The oropharyngeal membrane at the cranial end
of the disc consists of a small region of tightly
adherent ectoderm and endoderm cells that represents the future opening of the oral cavity.

FORMATION OF THE
NOTOCHORD
Prenotochordal cells invaginating in the
primitive node move forward cranially in the
midline until they reach the prechordal plate
(Fig. 5.3). These prenotochordal cells become
intercalated in the hypoblast so that for a short
time, the midline of the embryo consists of two cell
layers that form the notochordal plate (Fig. 5.3B).
As the hypoblast is replaced by endoderm cells
moving in at the streak, cells of the notochordal
plate proliferate and detach from the endoderm.
They then form a solid cord of cells, the definitive notochord (Fig. 5.3C), which underlies the
neural tube and serves as the basis for the axial
skeleton. Because elongation of the notochord is
a dynamic process, the cranial end forms first, and
caudal regions are added as the primitive streak
assumes a more caudal position. The notochord
and prenotochordal cells extend cranially to
the prechordal plate (an area just caudal to the
oropharyngeal membrane) and caudally to the
primitive pit. At the point where the pit forms
an indentation in the epiblast, the neurenteric
canal temporarily connects the amniotic and
yolk sac cavities (Fig. 5.3A).
The cloacal membrane is formed at the
caudal end of the embryonic disc (Fig. 5.2A).
This membrane, which is similar in structure
to the oropharyngeal membrane, consists of
tightly adherent ectoderm and endoderm cells
with no intervening mesoderm. When the cloacal membrane appears, the posterior wall of the
yolk sac forms a small diverticulum that extends
51

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52 Part 1 General Embryology

Amniotic
cavity
Syncytiotrophoblast

Epiblast
Hypoblast

Cytotrophoblast

Extraembryonic
mesoderm

Definitive
yolk sac

Cut edge of
amnion

Oropharyngeal membrane

Primitive
streak

Wall of
yolk sac
Hypoblast

Epiblast

Figure 5.1 A. Implantation site at the end of the second week. B. Representative view of the germ disc at the end of the
second week of development. The amniotic cavity has been opened to permit a view of the dorsal side of the epiblast. The
hypoblast and epiblast are in contact with each other, and the primitive streak forms a shallow groove in the caudal region
of the embryo.

into the connecting stalk. This diverticulum, the

allantoenteric diverticulum, or allantois,
appears around the 16th day of development
(Fig. 5.3A). Although in some lower vertebrates
the allantois serves as a reservoir for excretion products of the renal system, in humans, it remains
rudimentary but may be involved in abnormalities
of bladder development (see Chapter 16, p. 240).

ESTABLISHMENT OF THE
BODY AXES
Establishment of the body axes, anteroposterior,
dorsoventral, and leftright, takes place before and
during the period of gastrulation. The anteroposterior axis is signaled by cells at the anterior (cranial) margin of the embryonic disc. This area, the

Sadler_Chap05.indd 52

anterior visceral endoderm (AVE), expresses

genes essential for head formation, including the
transcription factors OTX2, LIM1, and HESX1,
and the secreted factors cerberus and lefty,
which inhibit nodal activity in the cranial end
of the embryo. These genes establish the cranial end of the embryo before gastrulation. The
primitive streak itself is initiated and maintained
by expression of Nodal, a member of the transforming growth factor-b (TGF-b) family
(Fig. 5.4). Once the streak is formed, Nodal upregulates a number of genes responsible for formation
of dorsal and ventral mesoderm and head and tail
structures. Another member of the TGF-b family,
bone morphogenetic protein 4 (BMP4), is
secreted throughout the embryonic disc (Fig. 5.4).
In the presence of this protein and FGF, mesoderm

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Chapter 5

Third Week of Development: Trilaminar Germ Disc 53

Oropharyngeal
membrane
Cut edge of amnion
Prenotochordal cells
Primitive node
(the organizer)
Primitive streak
Cloacal membrane

A
Primitive node

Primitive streak
Epiblast
Amnioblasts

Yolk sac

Hypoblast
Invaginating mesoderm cells

Primitive node

Primitive streak

Epiblast

Detaching cells
Hypoblast

C
Figure 5.2 A. Dorsal side of the germ disc from a 16-day embryo indicating the movement of surface epiblast cells (solid black
lines) through the primitive streak and node and the subsequent migration of cells between the hypoblast and epiblast (broken
lines). B. Cross section through the cranial region of the streak at 15 days showing invagination of epiblast cells.The first cells to
move inward displace the hypoblast to create the definitive endoderm. Once definitive endoderm is established, inwardly moving
epiblast forms mesoderm. C. Dorsal view of an embryo showing the primitive node and streak and a cross section through the
streak.The view is similar to the illustration in B; arrow, detaching epiblast cells in the primitive streak.

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54 Part 1 General Embryology

Primitive pit and
neurenteric canal
Connecting stalk

Cut lines for

C
B
Ectoderm
Amnion
Wall of
yolk sac

Prechordal
mesoderm

Allantois
Notochord

Cloacal plate
(membrane)

Oropharyngeal
membrane

Notochordal plate

Intraembryonic
mesoderm

Endoderm

Intraembryonic mesoderm

Extraembryonic mesoderm
Endoderm
Notochord

Figure 5.3 Schematic views illustrating formation of the notochord, whereby prenotochordal cells migrate through the
primitive streak, become intercalated in the endoderm to form the notochordal plate, and finally detach from the endoderm
to form the definitive notochord. Because these events occur in a cranial-to-caudal sequence, portions of the definitive
notochord are established in the head region first. A. Drawing of a sagittal section through a 17-day embryo. The most
cranial portion of the definitive notochord has formed, while prenotochordal cells caudal to this region are intercalated into
the endoderm as the notochordal plate. Note that some cells migrate ahead of the notochord. These mesoderm cells form the
prechordal plate that will assist in forebrain induction. B. Schematic cross section through the region of the notochordal plate.
Soon, the notochordal plate will detach from the endoderm to form the definitive notochord. C. Schematic view showing
the definitive notochord.

Goosecoid, chordin,
noggin, follistatin,
nodal

AVE

Figure 5.4 Sagittal section through the node and primitive streak showing the expression pattern of genes regulating the
craniocaudal and dorsoventral axes. Cells at the prospective cranial end of the embryo in the AVE express the transcription
factors OTX2, LIM1, and HESX1 and the secreted factor cerberus that contribute to head development and establish the
cephalic region. Once the streak is formed and gastrulation is progressing, BMP4 is secreted throughout the bilaminar disc
and acts with FGF to ventralize mesoderm into intermediate and lateral plate mesoderm. Goosecoid, expressed in the node,
regulates chordin expression, and this gene product, together with noggin and follistatin, antagonizes the activity of BMP4, dorsalizing mesoderm into notochord and paraxial mesoderm for the head region. Later, expression of the Brachyury (T) gene
antagonizes BMP4 to dorsalize mesoderm into notochord and paraxial mesoderm in caudal regions of the embryo.

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Chapter 5

will be ventralized to contribute to kidneys

(intermediate mesoderm), blood, and body wall
mesoderm (lateral plate mesoderm). In fact, all
mesoderm would be ventralized if the activity of
BMP4 were not blocked by other genes expressed
in the node. For this reason, the node is the organizer. It was given that designation by Hans
Spemann, who first described this activity in the
dorsal lip of the blastopore, a structure analogous
to the node, in Xenopus embryos. Thus, chordin
(activated by the transcription factor Goosecoid),
noggin, and follistatin antagonize the activity of
BMP4. As a result, cranial mesoderm is dorsalized
into notochord, somites, and somitomeres (Fig. 5.4).
Later, these three genes are expressed in the notochord and are important in neural induction in
the cranial region.
As mentioned, Nodal is involved in initiating
and maintaining the primitive streak. Similarly,
HNF-3b maintains the node and later induces
regional specificity in the forebrain and midbrain
areas.Without HNF-3b, embryos fail to gastrulate
properly and lack forebrain and midbrain structures. As mentioned previously, Goosecoid activates
inhibitors of BMP4 and contributes to regulation
of head development. Over- or underexpression
of this gene in laboratory animals results in severe
malformations of the head region, including
duplications, similar to some types of conjoined
twins (Fig. 5.5).

Figure 5.5 Conjoined twins. If the gene Goosecoid is

overexpressed in frog embryos, the result is a two-headed
tadpole. Perhaps overexpression of this gene explains the
origin of this type of conjoined twins.

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Third Week of Development: Trilaminar Germ Disc 55

Regulation of dorsal mesoderm formation

in middle and caudal regions of the embryo is
controlled by the Brachyury (T) gene expressed
in the node, notochord precursor cells, and
notochord. This gene is essential for cell migration through the primitive streak. Brachyury
encodes a sequence-specific DNA binding
protein that functions as a transcription factor.
The DNA-binding domain is called the T-box,
and there are more than 20 genes in the T-box
family. Thus, mesoderm formation in these
regions depends on this gene product, and its
absence results in shortening of the embryonic
axis (caudal dysgenesis). The degree of shortening depends on the time at which the protein
becomes deficient.
Laterality (leftright-sidedness) also is established early in development and is orchestrated
by a cascade of signal molecules and genes.When
the primitive streak appears, FGF8 is secreted by
cells in the node and primitive streak and induces
expression of Nodal, but only on the left side of
the embryo (Fig. 5.6A). Later, as the neural plate
is established, FGF8 maintains Nodal expression
in the lateral plate mesoderm, as well as LEFTY2, and both of these genes upregulate PITX2.
PITX2 is a homeobox-containing transcription
factor responsible for establishing left-sidedness
(Fig. 5.6B) and its expression is repeated on the
left side of the heart, stomach, and gut primordia
as these organs are assuming their normal asymmetrical body positions. If the gene is expressed
ectopically (e.g., on the right side), this abnormal
expression results in laterality defects, including
situs inversus and dextrocardia (placement of the
heart to the right side; see p. 57). Simultaneously,
LEFTY is expressed on the left side of the floor
plate of the neural tube and may act as a barrier to prevent left-sided signals from crossing
over. Sonic hedgehog (SHH) may also function in this role as well as serving as a repressor for left-sided gene expression on the right.
The Brachyury (T) gene, encoding a transcription
factor secreted by the notochord, is also essential
for expression of Nodal, LEFTY-1, and LEFTY-2
(Fig. 5.6B). Importantly, the neurotransmitter
serotonin (5HT) also plays a critical role in this
signaling cascade that establishes laterality. 5HT
is concentrated on the left side, probably because
it is broken down by its metabolizing enzyme
monoamine oxidase (MAO) on the right, and
is upstream from FGF8 signaling (Fig. 5.6B).
Alterations in 5HT signaling result in situs inversus, dextrocardia, and a variety of heart defects
(see Clinical Correlations, p. 57).
Genes regulating right-sided development are
not as well defined, although expression of the

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56 Part 1 General Embryology

Oropharyngeal
membrane

Oropharyngeal
membrane
5HT
FGF8
Nodal
Lefty2
PITX2
Nodal

Notochord
(SHH)

Lefty 1
Node

Nodal

MAO

FGF8

Primitive node
(FGF8)

Primitive
streak

Cloacal
membrane

Cloacal membrane

Figure 5.6 Dorsal views of the germ disc showing gene expression patterns responsible for establishing the leftright
body axis. A. FGF8, secreted by the node and primitive streak, establishes expression of Nodal, a member of the TGF-b
superfamily, and the nodal protein then accumulates on the left side near the node. B. Later, as the neural plate starts to
form, FGF8 induces expression of Nodal and LEFTY-2 in the lateral plate mesoderm, whereas LEFTY-1 is expressed on the
left side of the ventral aspect of the neural tube. These signals are dependent upon the neurotransmitter serotonin (5HT)
that is upstream of FGF8 and that increases in concentration on the left because of its metabolism by MAO on the right.
Products from the Brachyury (T) gene, expressed in the notochord, also participate in induction of these three genes. In turn,
expression of Nodal and LEFTY-2 regulates expression of the transcription factor PITX 2, which, through further downstream
effectors, establishes left-sidedness. SHH, expressed in the notochord, may serve as a midline barrier and also represses
expression of left-sided genes on the right. Expression of the transcription factor Snail may regulate downstream genes
important for establishing right-sidedness.
Oropharyngeal membrane

n
pm
lpm im
eem

pm
im

lpm
eem

Cloacal membrane

Figure 5.7 Dorsal view of the germ disc showing the primitive streak and a fate map for epiblast cells. Specific regions
of the epiblast migrate through different parts of the node and streak to form mesoderm. Thus, cells migrating at the cranialmost part of the node will form the notochord (n); those migrating more posteriorly through the node and cranialmost
aspect of the streak will form paraxial mesoderm (pm; somitomeres and somites); those migrating through the next portion
of the streak will form intermediate mesoderm (im; urogenital system); those migrating through the more caudal part of the
streak will form lateral plate mesoderm (lpm; body wall); and those migrating through the most caudal part will contribute
to extraembryonic mesoderm (eem; chorion).

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Oropharyngeal
membrane

Primary
villi

Trophoblastic
lacunae

Maternal
sinusoid
Connecting
stalk
Amniotic
cavity

Secondary
yolk sac

Extraembryonic
somatopleuric
mesoderm
(chorionic plate)
Extraembryonic
cavity
(chorionic cavity)

Exocoelomic cyst

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60 Part 1 General Embryology

Mesoderm core

Syncytiotrophoblast

Villous capillary

Cytotrophoblast

Primary
villus

Secondary
villus

Tertiary
villus

Figure 5.11 Development of a villus. A. Transverse section of a primary villus showing a core of cytotrophoblastic cells
covered by a layer of syncytium. B. Transverse section of a secondary villus with a core of mesoderm covered by a single
layer of cytotrophoblastic cells, which in turn is covered by syncytium. C. Mesoderm of the villus showing a number of capillaries and venules.

By the end of the third week, mesodermal

cells in the core of the villus begin to differentiate
into blood cells and small blood vessels, forming
the villous capillary system (Fig. 5.11). The villus
is now known as a tertiary villus or a definitive placental villus. Capillaries in tertiary villi
make contact with capillaries developing in the
mesoderm of the chorionic plate and in the connecting stalk (Figs. 5.12 and 5.13). These vessels,
Tertiary
stem villi

in turn, establish contact with the intraembryonic circulatory system, connecting the placenta
and the embryo. Hence, when the heart begins to
beat in the fourth week of development, the villous system is ready to supply the embryo proper
with essential nutrients and oxygen.
Meanwhile, cytotrophoblastic cells in the villi
penetrate progressively into the overlying syncytium until they reach the maternal endometrium.

Intervillous spaces

Syncytium
Outer
cytotrophoblast
shell

Connecting
stalk

Amniotic
cavity

Definitive
yolk sac
Chorionic
plate
Chorionic
cavity

Exocoelomic cyst

Figure 5.12 Presomite embryo and the trophoblast at the end of the third week. Tertiary and secondary stem villi give
the trophoblast a characteristic radial appearance. Intervillous spaces, which are found throughout the trophoblast, are lined
with syncytium. Cytotrophoblastic cells surround the trophoblast entirely and are in direct contact with the endometrium.
The embryo is suspended in the chorionic cavity by means of the connecting stalk.

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Chapter 5

Third Week of Development: Trilaminar Germ Disc 61

Maternal
vessels

Endometrium

Intervillous
space

Outer
cytotrophoblast
shell
Syncytiotrophoblast

Cytotrophoblast
Mesoderm core
with capillaries

Chorionic plate

Connecting stalk

Chorionic cavity

Figure 5.13 Longitudinal section through a villus at the end of the fourth week of development. Maternal vessels penetrate the cytotrophoblastic shell to enter intervillous spaces, which surround the villi. Capillaries in the villi are in contact
with vessels in the chorionic plate and in the connecting stalk, which in turn are connected to intraembryonic vessels.

Here they establish contact with similar extensions of neighboring villous stems, forming a thin
outer cytotrophoblast shell (Figs. 5.12 and
5.13). This shell gradually surrounds the trophoblast entirely and attaches the chorionic sac firmly
to the maternal endometrial tissue (Fig. 5.12).
Villi that extend from the chorionic plate to the
decidua basalis (decidual plate: the part of the
endometrium where the placenta will form; see
Chapter 8) are called stem or anchoring villi.
Those that branch from the sides of stem villi are
free (terminal) villi, through which exchange
of nutrients and other factors will occur.
The chorionic cavity, meanwhile, becomes
larger, and by the 19th or the 20th day, the
embryo is attached to its trophoblastic shell by a
narrow connecting stalk (Fig. 5.12). The connecting stalk later develops into the umbilical
cord, which forms the connection between the
placenta and embryo.

Summary
The most characteristic event occurring during
the third week is gastrulation, which begins
with the appearance of the primitive streak,
which has at its cephalic end the primitive
node. In the region of the node and streak, epiblast cells move inward (invaginate) to form
new cell layers, endoderm and mesoderm.
Cells that do not migrate through the streak but

Sadler_Chap05.indd 61

remain in the epiblast form ectoderm. Hence,

epiblast gives rise to all three germ layers in the
embryo, ectoderm, mesoderm, and endoderm, and these layers form all of the tissues and
organs (Figs. 5.2 and 5.3).
Prenotochordal cells invaginating in the
primitive pit move forward until they reach the
prechordal plate. They intercalate in the endoderm as the notochordal plate (Fig. 5.3). With
further development, the plate detaches from
the endoderm, and a solid cord, the notochord,
is formed. It forms a midline axis, which will
serve as the basis of the axial skeleton (Fig. 5.3).
Cephalic and caudal ends of the embryo are
established before the primitive streak is formed.
Thus, cells in the hypoblast (endoderm) at the
cephalic margin of the disc form the AVE, which
expresses head-forming genes, including OTX2,
LIM1, and HESX1 and the secreted factor cerberus. Nodal, a member of the TGF-b family of
genes, is then activated and initiates and maintains the integrity of the node and streak. In the
presence of FGF, BMP4 ventralizes mesoderm
during gastrulation so that it forms intermediate
and lateral plate mesoderm. Chordin, noggin, and
follistatin antagonize BMP4 activity and dorsalize mesoderm to form the notochord and somitomeres in the head region. Formation of these
structures in more caudal regions is regulated by
the Brachyury (T) gene (Fig. 5.4A). Laterality
(leftright asymmetry) is regulated by a cascade
of signaling molecules and genes. FGF8, secreted

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62 Part 1 General Embryology

by cells in the node and streak, induces Nodal and

LEFTY-2 expression on the left side and these
genes upregulate PITX2, a transcription factor
and master gene for left-sidedness (Fig. 5.6). The
neurotransmitter serotonin (5HT) also plays
a role as a signal molecule upstream from FGF8.
Disruption of 5HT levels or misexpression of
PITX2 results in laterality defects, such as dextrocardia, situs inversus, and cardiac abnormalities.
Epiblast cells moving through the node and
streak are predetermined by their position to
become specific types of mesoderm and endoderm. Thus, it is possible to construct a fate map
of the epiblast showing this pattern (Fig. 5.7).
By the end of the third week, three basic germ
layers, consisting of ectoderm, mesoderm, and
endoderm, are established in the head region,
and the process continues to produce these germ
layers for more caudal areas of the embryo until
the end of the fourth week. Tissue and organ differentiation has begun, and it occurs in a cephalocaudal direction as gastrulation continues.
In the meantime, the trophoblast progresses
rapidly. Primary villi obtain a mesenchymal
core in which small capillaries arise (Fig. 5.12).
When these villous capillaries make contact with
capillaries in the chorionic plate and connecting stalk, the villous system is ready to supply the
embryo with its nutrients and oxygen (Fig. 5.13).

Sadler_Chap05.indd 62

Problems to Solve
1. A 22-year-old woman consumes large
quantities of alcohol at a party and loses
consciousness; 3 weeks later, she misses her
second consecutive period. A pregnancy test
is positive. Should she be concerned about
the effects of her binge-drinking episode on
her baby?
2. An ultrasound scan detects a large mass near
the sacrum of a 28-week female fetus. What
might the origin of such a mass be, and what
type of tissue might it contain?
3. On ultrasound examination, it was determined that a fetus had well-developed facial
and thoracic regions, but caudal structures
were abnormal. Kidneys were absent, lumbar
and sacral vertebrae were missing, and the
hindlimbs were fused. What process may have
been disturbed to cause such defects?
4. A child has polysplenia and abnormal positioning of the heart. How might these two
abnormalities be linked developmentally, and
when would they have originated? Should
you be concerned that other defects might
be present? What genes might have caused
this event, and when during embryogenesis
would it have been initiated?

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