Chapter 5

Gastrulation: Formation of Embryonic Mesoderm and Endoderm

Introduction to Gastrulation
Gastrulation is a key process in the third week of gestation that results in the formation of all
three primary germ layers: ectoderm, mesoderm, and endoderm. This process begins with the
formation of the primitive streak on the surface of the epiblast and continues with the migration
of cells, which ultimately give rise to various tissues and organs.

Formation of the Primitive Streak

 Initial Formation: The primitive streak initially forms as a faint structure. By the 15th to
16th day of embryonic development, the streak becomes more pronounced and is
characterized by a narrow groove flanked by bulging areas on either side.
 Cephalic End – The Primitive Node: At the head (cephalic) end of the primitive streak,
there is a slightly raised area known as the primitive node, which surrounds a small
depression called the primitive pit.

Cell Migration and Invagination

 Cell Movement Toward the Streak: Epiblast cells migrate toward the primitive streak.
Upon reaching the streak, the cells undergo invagination—becoming flask-shaped,
detaching from the epiblast, and moving beneath it.
 Invagination Process: This inward migration is essential for the formation of the three
germ layers. Cells that move inward displace the hypoblast to form the endoderm, while
others form the mesoderm. Cells that remain in the epiblast will form the ectoderm.

Regulation of Gastrulation

 Fibroblast Growth Factor 8 (FGF8): The migration and differentiation of cells during
gastrulation are regulated by FGF8, a growth factor secreted by the streak cells. FGF8
controls cell movement by downregulating E-cadherin (a protein that usually holds
epiblast cells together), thus facilitating invagination and mesoderm formation.
 BRACHYURY (T) Expression: FGF8 also plays a role in regulating BRACHYURY
(T) gene expression, which is essential for mesoderm formation.

Development of the Germ Layers

  Endoderm Formation: Some of the invaginated cells displace the hypoblast to form the
embryonic endoderm.
 Mesoderm Formation: Other cells lie between the epiblast and the newly formed
endoderm, forming the mesoderm.
 Ectoderm Formation: The cells remaining in the epiblast give rise to the ectoderm.

Lateral and Cranial Spread of Cells

 As more cells migrate between the epiblast and hypoblast, they begin to spread laterally
and cranially.
 Contact with Extraembryonic Mesoderm: Cells also extend beyond the edges of the
disc, making contact with the extraembryonic mesoderm that covers the yolk sac and
amnion.

Prechordal Plate and Its Significance

 The prechordal plate forms as cells migrate cranially from the primitive node and are
located between the notochord and the oropharyngeal membrane.
 Function of the Prechordal Plate: This structure plays a crucial role in the induction of
the forebrain.

Oropharyngeal Membrane

 The oropharyngeal membrane marks the future opening of the oral cavity and consists of
tightly adherent ectoderm and endoderm cells. This membrane will play a key role in the
formation of the mouth during later stages of development.

Key Points to Remember

1. Gastrulation establishes the three primary germ layers: ectoderm, mesoderm, and
endoderm.
2. The process begins with the formation of the primitive streak and the primitive node.
3. FGF8 regulates cell movement and mesoderm formation during invagination.
4. Cells of the epiblast form the ectoderm, mesoderm, and endoderm through migration.
5. The prechordal plate and oropharyngeal membrane have significant roles in the
development of the brain and oral cavity.
Review Questions

1. What are the three primary germ layers formed during gastrulation?
2. Describe the role of FGF8 in the process of gastrulation.
3. What is the significance of the prechordal plate in early embryonic development?
4. How does the oropharyngeal membrane contribute to the formation of the oral cavity?
5. Explain the process of invagination during gastrulation and its importance for germ layer
formation.

Formation of the Notochord

Introduction to the Notochord Formation

The notochord is a critical structure during early embryonic development that plays a vital role in
the formation of the axial skeleton and the development of the neural tube. It is formed by a
specific movement of cells known as invagination and intercalation. This structure serves as a
signaling center for the development of surrounding tissues, particularly for inducing the
formation of the axial skeleton.

Prenotochordal Cell Migration

 Invagination of Prenotochordal Cells: The process begins when prenotochordal cells

from the primitive node undergo invagination and migrate cranially along the midline of
the embryo.
 Contact with the Prechordal Plate: These migrating prenotochordal cells move until
they reach the prechordal plate, an important region located just caudal to the
oropharyngeal membrane.

Formation of the Notochordal Plate

 Intercalation in the Hypoblast: As the prenotochordal cells reach the midline, they
become intercalated (inserted) into the hypoblast, which briefly forms a two-layer
structure called the notochordal plate.
 Replacement by Endoderm: As the hypoblast is replaced by endoderm cells migrating
through the primitive streak, the cells of the notochordal plate proliferate and detach from
the endoderm.

Formation of the Definitive Notochord

 Proliferation and Detachment: The cells of the notochordal plate proliferate, detach
from the endoderm, and form a solid cord of cells known as the definitive notochord.
  Role of the Notochord: The notochord underlies the developing neural tube and serves
as a signaling center for the formation of the axial skeleton, influencing the development
of surrounding tissues. It plays a crucial role in the early formation of the body’s
structural framework.

Elongation of the Notochord

 Cranial Formation: The notochord elongates dynamically, with the cranial (head) end
forming first.
 Caudal Addition: The caudal (tail) regions of the notochord are added as the primitive
streak shifts caudally, progressively forming the entire length of the notochord from the
cranial end to the caudal end.

Neurenteric Canal

 Temporary Connection: The notochord and prenotochordal cells extend from the
cranial region to the prechordal plate and caudally to the primitive pit. At the site where
the primitive pit forms an indentation in the epiblast, the neurenteric canal temporarily
connects the amniotic and yolk sac cavities. This canal is only transient and plays a role
in early embryo development.

Cloacal Membrane

 Formation of the Cloacal Membrane: At the caudal end of the embryonic disc, the
cloacal membrane is formed, similar in structure to the oropharyngeal membrane. It
consists of tightly adherent ectoderm and endoderm cells without any mesoderm in
between.
 Function of the Cloacal Membrane: This membrane marks the future location of the
anus and plays a role in the development of the lower body structures.

Formation of the Allantois

 Development of the Allantois: Around the 16th day of development, a small

diverticulum called the allantoenteric diverticulum (or allantois) forms from the posterior
wall of the yolk sac.
 Function of the Allantois: In lower vertebrates, the allantois serves as a reservoir for
excretory products. In humans, it remains rudimentary and is thought to play a minor role
 in the development of the bladder and other urinary structures, though it is not fully
functional.

Key Points to Remember

1. Prenotochordal cells invaginate from the primitive node and migrate cranially to form the
notochord.
2. The notochordal plate forms temporarily before the definitive notochord is established.
3. The notochord plays a critical role in signaling the development of the axial skeleton and
neural tube.
4. The neurenteric canal temporarily connects the amniotic and yolk sac cavities during
early development.
5. The cloacal membrane forms at the caudal end and is important for the development of
the anus and lower structures.
6. The allantois, though rudimentary in humans, may play a role in bladder development.

Review Questions

1. What are the steps involved in the formation of the notochord?

2. How do prenotochordal cells contribute to the formation of the notochordal plate and
definitive notochord?
3. What is the function of the notochord during early embryonic development?
4. Describe the role of the neurenteric canal in embryogenesis.
5. What is the significance of the cloacal membrane, and how does it contribute to the
development of the body?
6. What is the allantois, and what role does it play in human development?

Establishment of the Body Axes

Introduction to the Body Axes

The establishment of the body axes is one of the earliest events in embryonic development. The
body axes, which define the structure and orientation of the organism, include:

1. Anterior-Posterior (A-P) Axis: Defines the head-tail orientation (cranial to caudal).

2. Dorso-Ventral (D-V) Axis: Defines the back (dorsal) and belly (ventral) orientation.
3. Left-Right (L-R) Axis: Defines the asymmetrical positioning of organs on the left and
right sides of the body.

These axes are established early, starting from the late morula to blastocyst stages, with the
anterior-posterior (A-P) and dorso-ventral (D-V) axes being determined first. The left-right (L-R)
axis is established later.
Anterior-Posterior (A-P) Axis Development

 Cranial-End Visceral Endoderm (AVE): During the bilaminar disc stage, the anterior
visceral endoderm (AVE) at the cranial end of the embryo migrates towards the future
head region.
 Gene Expression in the AVE: The AVE expresses specific genes critical for head
development, including:
o OTX2, LIM1, and HESX1 (transcription factors).
o Cerberus and Lefty1 (secreted proteins from the TGF-β family).

These genes inhibit Nodal expression at the cranial end, helping to establish the anterior region
of the embryo.

Nodal and Primitive Streak Formation

 Nodal Expression: At the caudal end, the absence of cerberus and lefty1 allows Nodal
expression to continue, which is essential for maintaining the primitive streak.
 Primitive Streak: The primitive streak is a key structure in gastrulation, and its
formation is driven by Nodal signaling. It helps to establish dorsal and ventral mesoderm
and organizes the head and tail regions.

Bone Morphogenetic Protein 4 (BMP4) and Mesoderm Patterning

 BMP4 Secretion: BMP4 is secreted throughout the embryonic disc. It promotes the
ventralization of mesoderm, which contributes to kidney, blood, and body wall mesoderm
(lateral plate mesoderm).
 BMP4 Antagonists: The node produces Chordin, Noggin, and Follistatin, which
antagonize BMP4 and dorsalize the mesoderm, leading to the formation of the notochord,
somites, and somitomeres (cranial mesoderm).

Role of Goosecoid and the Node in Head Development

 Goosecoid Gene: Goosecoid, activated by the transcription factor Goosecoid, is involved

in regulating head development. It plays a role in inhibiting BMP4 and maintaining the
dorsal mesoderm that forms the notochord.
 Head Malformations: Over- or under-expression of Goosecoid can lead to severe
malformations in the head region, including duplications or abnormal conjoined twins.
BRACHYURY Gene and Mesoderm Formation

 BRACHYURY Gene: The BRACHYURY (T) gene, expressed in the node and
notochord precursor cells, is essential for proper mesoderm formation, particularly in the
middle and caudal regions of the embryo.
 Function: This gene encodes a transcription factor that regulates mesodermal cell
migration through the primitive streak. A deficiency in BRACHYURY results in a
shortened embryonic axis, known as caudal dysgenesis.

Establishment of the Left-Right (L-R) Axis

 FGF8 and Nodal Expression: As the primitive streak forms, FGF8 induces Nodal
expression in the node and primitive streak. The expression of Nodal becomes restricted
to the left side of the embryo.
 Serotonin (5-HT): On the left side, serotonin accumulates, activating the transcription
factor MAD3, which restricts Nodal expression to the left side.
 Midline Genes: Genes like SONIC HEDGEHOG (SHH), LEFTY1, and ZIC3 are
involved in maintaining the midline and preventing Nodal from crossing over to the right
side of the embryo.

PITX2 and Left-Sided Organ Development

 PITX2 Gene: Nodal signaling on the left side activates the expression of PITX2, a
homeobox-containing transcription factor that is crucial for establishing left-sidedness in
organs like the heart, stomach, and gut.
 Laterality Defects: Abnormal expression of PITX2 on the right side can lead to laterality
defects such as situs inversus (reversed organ positioning) and dextrocardia (heart
positioned on the right side).

Role of 5-HT and Cilia in Left-Right Asymmetry

 Serotonin (5-HT): The neurotransmitter 5-HT is critical in establishing the left-right

asymmetry by accumulating on the left side of the embryo.
 Cilia in the Node: Cilia in the node may generate a leftward flow of fluids or signaling
gradients that contribute to establishing the L-R axis. Disruptions in this signaling
pathway can result in various laterality defects.
Key Points to Remember

1. The A-P and D-V axes are determined early, while the L-R axis is established later.
2. Nodal signaling is crucial for the formation and maintenance of the primitive streak,
which influences mesoderm formation.
3. BMP4, in conjunction with its antagonists like Chordin and Noggin, helps pattern the
mesoderm into different regions.
4. The Goosecoid gene regulates head development and works by inhibiting BMP4 activity.
5. BRACHYURY gene is essential for mesoderm migration and axis elongation.
6. PITX2 is a key factor in establishing left-sidedness in organs.
7. Disruptions in serotonin signaling or cilia function can result in laterality defects like
situs inversus.

Review Questions

1. How is the anterior-posterior axis established during early embryogenesis?

2. What role does Nodal signaling play in the formation of the primitive streak?
3. Describe the function of BMP4 and how its activity is regulated during mesoderm
formation.
4. How does the Goosecoid gene contribute to head development?
5. What is the role of the BRACHYURY gene in mesoderm formation and embryonic axis
elongation?
6. How is left-right asymmetry established in the embryo, and what genes are involved?
7. What are the consequences of abnormal PITX2 expression on the development of left-
sided organs?
8. How does serotonin contribute to establishing the left-right axis in the embryo?

Fate Map Established During Gastrulation

Introduction
During gastrulation, cells of the epiblast migrate through the primitive streak, and their ultimate
fates are determined. This process is crucial in establishing the germ layers (ectoderm,
mesoderm, and endoderm) and organizing the tissues that will form various parts of the body.

Fate of Epiblast Cells During Gastrulation

1. Cranial Region of the Node:

o Cells that ingress through the cranial region of the primitive node contribute to the
prechordal plate and notochord.
2. Lateral Edges of the Node:
 o Cells migrating at the lateral edges of the node, as well as from the cranial end of
the streak, become paraxial mesoderm (which will form somites).
3. Mid-Streak Region:
o Cells migrating through the middle of the streak become intermediate mesoderm
(which gives rise to kidneys and gonads).
4. Caudal Part of the Streak:
o Cells migrating through the caudal portion of the streak form lateral plate
mesoderm (which will contribute to the formation of limbs, heart, and body
walls).
5. Extraembryonic Mesoderm:
o Cells from the caudalmost part of the streak contribute to extraembryonic
mesoderm (which is involved in the formation of structures outside the embryo,
such as the placenta).

Oropharyngeal and Cloacal Membranes

 Oropharyngeal Membrane:
o The oropharyngeal membrane, which forms the future opening of the mouth, is
located at the cranial end of the embryo.
 Cloacal Membrane:
o At the caudal end, the cloacal membrane, which forms the future anus, consists of
tightly adherent ectoderm and endoderm cells with no intervening mesoderm.

Growth of the Embryonic Disc

Changes in the Shape of the Embryonic Disc

 The embryonic disc, initially flat and round, gradually becomes elongated with a broad
cephalic (head) region and a narrow caudal (tail) end.

Cephalic Growth

 The expansion of the disc occurs mainly in the cephalic (head) region. Growth in this
area is driven by the continuous migration of cells from the primitive streak in a cranial
direction.

Caudal End and Primitive Streak

 The region of the primitive streak remains largely the same size while the cephalic region
continues to grow. The primitive streak supplies new cells at the caudal end until the
fourth week.
Elongation and Differentiation

 The differentiation of germ layers occurs first in the cephalic part by the middle of the
third week, while the caudal part continues to undergo gastrulation and differentiation
until the end of the fourth week. This results in the cephalocaudal (head-to-tail)
development of the embryo.

Key Concepts

 Primitive Streak: Responsible for the migration of cells to form different tissues and
germ layers.
 Mesodermal Contributions: Different parts of the mesoderm give rise to specific tissues
such as the notochord, somites, kidneys, and heart.
 Cephalocaudal Development: The embryo develops from head to tail, with
differentiation occurring in the cranial regions first and the caudal regions last.

Questions

1. What are the fates of cells migrating through the cranial region of the node during
gastrulation?
2. How does the growth of the embryonic disc differ between the cephalic and caudal
regions?
3. What is the significance of the primitive streak in the development of the embryo?
4. How does the migration of cells through the primitive streak contribute to the formation
of the mesodermal layers?
5. Explain the concept of cephalocaudal development and how it affects the differentiation
of the embryo.

Teratogenesis Associated with Gastrulation

Introduction
Gastrulation, which occurs around the third week of embryonic development, is a critical period
for the proper formation of the body's axes and organ systems. This stage is highly sensitive to
teratogenic insults, which can result in various birth defects. Teratogens, such as certain drugs,
environmental factors, or genetic mutations, can disrupt the normal process of gastrulation,
leading to abnormalities in the development of organs and body structures.

Effects of Teratogenic Insults during Gastrulation

1. Holoprosencephaly (Craniofacial and Forebrain Defects):

 o Cause: High doses of alcohol during the third week of pregnancy can kill cells in
the anterior midline of the germ disc.
o Effects: Deficiency of the midline leads to malformations in craniofacial
structures, such as small forebrain, merged lateral ventricles, and close-set eyes
(hypotelorism).
2. Caudal Dysgenesis (Sirenomelia):
o Cause: Insufficient mesoderm formation in the caudal region of the embryo.
o Effects: Defects in the lower limbs, urogenital system, and vertebral column,
leading to fusion of lower limbs (mermaid syndrome), renal agenesis, and
imperforate anus.
o Associated Factors: Maternal diabetes and genetic mutations (e.g., mutations in
BRACHYURY [T], WNT, and ENGRAILED genes in animal models).

Tumors Associated with Gastrulation

1. Sacrococcygeal Teratomas:
o Cause: Remnants of the primitive streak persist in the sacrococcygeal region,
leading to the formation of tumors.
o Characteristics: These tumors commonly contain tissues derived from all three
germ layers (ectoderm, mesoderm, and endoderm).
o Prevalence: Most common tumor in newborns (1 in 37,000 live births), more
frequent in female fetuses.
o Potential for Malignancy: Some teratomas may become malignant.

Laterality-Associated Birth Defects

1. Situs Solitus:
o Normal positioning of the internal organs.
2. Situs Inversus:
o Definition: The organs are reversed in a mirror image arrangement.
o Risk: Individuals with situs inversus have a slightly increased risk of congenital
heart defects but do not typically have other major malformations.
o Kartagener Syndrome: Some individuals with situs inversus develop respiratory
issues such as bronchiectasis and chronic sinusitis due to abnormal cilia function.
3. Situs Ambiguus (Heterotaxy):
o Definition: Abnormal positioning of one or more organs, where organs may be
reversed or exhibit isomerism (e.g., both atria in the heart looking the same).
o Risk: High risk for midline malformations (neural tube defects, cleft palate) and
complex congenital heart defects.
o Causes: Disruption in L-R axis formation, often due to mutations in genes like
ZIC3 (X-linked) or disruptions in the serotonin signaling pathway.
4. Laterality and Midline Defects:
 o Interaction: Disruption in the establishment of both the A-P and L-R axes can
result in both midline defects and abnormal organ positioning.
o Examples: Neural tube defects, cleft palate, and other structural anomalies.

Role of Serotonin (5-HT) in Laterality

 Importance: Serotonin is critical for establishing the left-right (L-R) asymmetry in the
body.
 Disruption: Altering serotonin signaling can result in situs inversus, heterotaxy, and
heart malformations.
 Clinical Implications:
o Animal studies show that disruption of serotonin signaling can cause defects in
organ positioning.
o SSRIs (selective serotonin reuptake inhibitors, such as Prozac, Paxil, and Zoloft)
taken by pregnant women have been linked to an increased risk of heart
malformations and other birth defects in the offspring.

Key Concepts

 Teratogenic Effects: High sensitivity of the embryo during gastrulation to teratogens

like alcohol, which can disrupt the formation of midline structures.
 Caudal Dysgenesis (Sirenomelia): Insufficient mesoderm in the caudal region leads to
severe malformations of the lower body.
 Sacrococcygeal Teratomas: Tumors arising from remnants of the primitive streak.
 Laterality Defects: Conditions like situs inversus and heterotaxy arise from disruptions
in the L-R axis, often with severe associated birth defects, including heart malformations.

Questions

1. What are the potential effects of teratogenic insults, such as alcohol, during the third
week of development?
2. How does caudal dysgenesis (sirenomelia) affect the lower body structures, and what are
its potential causes?
3. Describe the formation of sacrococcygeal teratomas and their possible complications.
4. What is the difference between situs solitus, situs inversus, and situs ambiguus
(heterotaxy)?
5. How does serotonin (5-HT) signaling play a role in establishing laterality, and what are
the potential risks if this signaling is disrupted?
6. Explain how mutations in genes like ZIC3 and disruption in serotonin signaling lead to
laterality defects and other associated birth defects.
Further Development of the Trophoblast

Introduction
By the beginning of the third week of pregnancy, the trophoblast, the outer layer of cells that will
form part of the placenta, undergoes significant changes. These transformations include the
formation of villi, which evolve from primary villi into secondary and tertiary villi. The
trophoblast plays a crucial role in establishing the connection between the embryo and the
maternal endometrium, allowing for nutrient and oxygen exchange.

Stages of Trophoblast Development

1. Primary Villi:
o Structure: Consists of a cytotrophoblastic core covered by a syncytial layer.
o Function: Serves as the initial form of the villous structure, but does not yet
facilitate nutrient exchange.
2. Secondary Villi:
o Formation: Mesodermal cells penetrate the core of the primary villi and begin to
grow toward the decidua.
o Structure: These villi now contain mesoderm, marking them as secondary villi.
3. Tertiary (Definitive) Villi:
o Formation: By the end of the third week, mesodermal cells within the villus
differentiate into blood cells and small blood vessels.
o Function: The tertiary villus contains a developing villous capillary system, and
these capillaries make contact with the developing circulatory system in the
embryo, forming the connection between the placenta and embryo.
o Significance: When the heart begins to beat in the fourth week, the villous system
is already established to supply the embryo with essential nutrients and oxygen.

Cytotrophoblast and Syncytiotrophoblast Development

1. Cytotrophoblast Cells:
o Role: These cells penetrate the syncytium and progressively extend into the
maternal endometrium, establishing contact with similar villous extensions.
o Function: They form a thin outer cytotrophoblast shell around the trophoblast,
which helps anchor the chorionic sac to the maternal endometrial tissue.
2. Syncytiotrophoblast Layer:
o Role: The outer layer that covers the villi and is involved in the invasion of the
maternal endometrium.

Villi Types and Functions

 1. Stem (Anchoring) Villi:
o Definition: Villi that extend from the chorionic plate to the decidua basalis
(decidual plate).
o Function: Serve as the anchors that hold the placenta to the maternal tissue.
o Role in Placenta Formation: These villi play a crucial role in the attachment of
the placenta to the uterine wall.
2. Free (Terminal) Villi:
o Definition: Villi that branch from the stem villi.
o Function: These villi are responsible for nutrient exchange between the mother
and the embryo.

Chorionic Cavity and Connecting Stalk

1. Chorionic Cavity:
o Development: The chorionic cavity expands during this period.
o Role: This cavity is surrounded by the trophoblast and is part of the space
between the developing embryo and placenta.
2. Connecting Stalk:
o Formation: By days 19–20, the embryo is attached to the trophoblastic shell via a
narrow connecting stalk.
o Development into the Umbilical Cord: The connecting stalk eventually forms
the umbilical cord, which connects the placenta to the embryo, facilitating
nutrient, oxygen, and waste exchange.

Key Concepts

 The trophoblast undergoes significant changes, starting from primary villi and
progressing to secondary and tertiary villi, with mesodermal cells differentiating to form
blood vessels.
 The cytotrophoblast cells help anchor the placenta to the maternal tissue, while the
syncytiotrophoblast layer facilitates the invasion of maternal blood supply.
 The formation of stem and terminal villi is crucial for the attachment and nutrient
exchange functions of the placenta.
 The connecting stalk, which later forms the umbilical cord, is essential for linking the
embryo to the placenta.

Questions

1. What are the stages of trophoblast development, and how do primary, secondary, and
tertiary villi differ?
2. How do mesodermal cells contribute to the development of the villous capillary system?
3. What is the role of cytotrophoblast and syncytiotrophoblast cells in the trophoblast’s
interaction with the maternal endometrium?
4. How do stem (anchoring) villi and free (terminal) villi function differently in placental
development?
5. What is the significance of the connecting stalk, and how does it relate to the formation
of the umbilical cord?