Implantation, Gastrulation &

organogenesis in mammalian
 Schematic diagram showing the derivation of
tissues in human and rhesus monkey embryos

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Stat3

Oct4

Nanog
Blood
vessels

Cdx2
 Normal implantation zone
In order that implantation can take its normal course, the blastocysts
and the uterine mucosa must be able to interact. These two,
independent structures must, therefore, undergo synchronous
changes. The implantation normally takes place in the superior and
posterior walls of the uterine body (corpus uteri) in the functional
layer of the endometrium during the secretory phase of the cycle.

1. Uterine cavity 2. Isthmus of the tube

3. Uterine tube (tuba uterina) 4. Uterine cervix (cervix uteri)
 Implantation stages
Implantation is an event that occurs early in mammalian
pregnancy in which the embryo adheres to the wall of uterus.
The blastocyst hatches out of the
Schematically, three implantation partially dissolved zona pellucida
stages can be distinguished:

Adplantation of the blastocyst on the

endometrium
Adhesion of the blastocyst to the
endometrium
Invasion of the trophoblast and
embedding

When the blastocyst emerges from the

pellucid zone on the 5th day , it comes into
contact with the maternal uterine mucosa
in that it embeds itself in the endometrium
with its embryonic pole.
1. Pellucid zone
2. Trophoblast (outer cell mass)
3. Hypoblast (part of the inner cell mass)
4. Blastocyst cavity
5. Epiblast (part of the inner cell mass)
 Preparation of uterus for implantation
Implantation window

A. Menstruation B. Proliferation
C. Secretion D. Implantation window
The adhesion can occur when beforehand the uterus has entered its secretory phase (luteinizing phase). This reception-
ready phase of the endometrium lasts 4 days (20th -23rd day) and is usually termed the "implantation window". It
follows around 6 days after the LH peak and is characterized by the appearance of small elevations at the apical pole of
the epithelial endometrium cells. One of the tasks of these elevations consists in the absorption of the uterine fluid,
which brings the blastocyst nearer to the endometrium and immobilizes it at the same time. In this stage the blastocyst
can still be eliminated by being flushed out. There is also a hypothesis that the progesterone and the oestrogen are
responsible for an oedema that already fills the flattened out uterine cavity. This is also supposed to contribute to the
blastocyst being pressed against the uterine epithelium
 Adhesion of the blastocyst to the endometrium
Hatching of the blastocyst and
adhesion on the endometrium
After the apposition of the free
blastocyst at the uterine epithelium the
microvilli on the surface of the
outermost trophoblast cells interact with
the epithelial cells of the uterus. In this
stage the blastocyst can no longer be
eliminated by a simple flushing out.
The adhesion of the blastocyst on the
endometrium arises through cell surface
glycoproteins, the specific mechanisms
of which, though, are not yet well
understood.
 Implantation stages

Implantation: 6th 7th day Implantation: 7th 8th day

1. Epithelium of the uterine mucosa 5. Epiblast

2. Hypoblast 6. Blastocyst cavity
3. Syncytiotrophoblast
4. Cytotrophoblast
 Implantation stages
Implantation: 8th day Implantation: 9th day

1. Syncytiotrophoblast (ST)
8. Amnioblasts
2. Cytotrophoblast (CT)
9. Fibrin plug
3. Epiblast
10. Trophoblast lacunae
4. Hypoblast
11. Multiplying hypoblast
5. Blastocyst cavity
6. Maternal blood capillary
7. Amniotic cavity
 Implantation stages
Implantation: 9th 10th day Implantation: 10th 11th day

1. Hypoblast growing ventrally 1. Extraembryonic reticulum

2. Eroded maternal capillaries 2. Heuser´s membrane
3. Amniotic cavity
4. Cytotrophoblast
5. Syncytiotrophoblast
6. Lacunae, filled with blood
 Molecular aspects of implantation

At present the molecular mechanisms of implantation are only

partially understood. They evoke complex cascade-like
interactions between the embryonic trophoblast cells, epithelial
cell, decidual cell, cells responsible for immune reactions and the
extra-cellular matrix (ECM) of the maternal endometrium.
These cellular interactions require various mediators such as
growth factors, proteinases and their inhibitors, the components of
the ECM, adhesion molecules (integrin, cadherin) and hormones.
Decidual cells and the endometrial glands secrete growth factors
and various other factors necessary for the implantation of the
embryo. For its part the blastocyst secretes its own growth factors
and expresses numerous receptors that enable the tissue
interactions with the uterine epithelium.
 Signal exchange during preimplantation

The embryo and the endometrium communicate with each other already before
its apposition at the uterine epithelium. After hatching, the blastocyst secretes
molecules that affect ovarian activity, mobility of the fallopian tube and the
endometrium (EPF, HCG). With the compaction of the morula, receptors appear
for the colony-stimulating factor (CSF), the epidermal growth factor (EGF), the
leukocyte inhibitory factor (LIF) and for E-cadherin.
Cadherins are calcium-dependent cell adhesion molecules that play a role in
anchoring of the blastocyst in the endometrium. At this stage the embryo also
produces interleukin 1 (L-1a und b), which will take on a key role in orienting
the embryo toward the endometrium.
The platelet-activating factor (PAF) is also produced by the embryo. Interleukin
(IL-1), the receptors for which are localized on the epithelial cells of the
endometrium during the secretory phase, is necessary for producing LIF in the
uterus.
During the preimplantation phase the density of the surface proteins (glycocalyx)
as well as the electrostatic repulsion between the blastocyst and the
endometrium decreases, facilitating implantation.
 The blastocyst and the uterine epithelium

The apposition and the adhesion of the blastocyst onto the uterine epithelium require the
secretion of factors that bind at specific receptors (one of the tissues must secrete ligands, the
others should express the receptors).
Numerous "implantation factors" are known: Interleukin 1 (IL-1), the inhibition factor for
leukocytes (LIF), the colony-stimulating factor (CSF), as well as the epithelial growth factor
(EGF) and its receptors (EGF-R).
During implantation, embryonic IL-1 binds at its receptors on the surface of the endometrium
epithelial cells.
LIF (a glycoprotein that belongs to the cytokine group) is synthesized by the uterine epithelial
cells from the 18th day of the menstruation cycle; its receptors are expressed by the blastocyst.
It appears to play a role in the differentiation of the CT to ST and assists HCG (human
chorionic gonadotropin) secretion.
EGF receptors can bind themselves to numerous ligands. In humans EGF-R are expressed
from the 4th day by the cells of the inner cell mass and by the trophoblast. Between the 4th and
the 7th day their expression is limited to the inner cell mass and the embryonic pole of the
trophoblast. This could be an explanation for the orienting of the blastocyst, which embeds
itself in the endometrium with the embryonic pole.
 Interactions between the blastocyst and
endometrium (invasion of the trophoblast)

The trophoblast behaves like a "pseudo-tumoral tissue" that infiltrates the

endometrium. After the basal membrane is destroyed, the trophoblast grows into
the decidua of the uterine tissue. Trophoblast cells secrete proteolytically active
enzymes that affect the ECM in such a way that it becomes more porous for the
invasion of the embryo. These enzymes are mainly matrix metalloproteinase
(MMP) and plasminogen-activators.
Trophoblast cells express certain integrins (cell adhesion molecules) on their
cell membranes: surface-lying cells express integrins a5b1 and a1b1, which
interact with the uterine mucosa, whereas deeper-lying cells display integrin a6
chains.
The growth of the trophoblast into the endometrium and the decomposition of
the ECM are controlled by endometrial factors (secreted by epithelial cells,
fibroblasts, macrophages and leukocytes). These factors cause autocrine and
paracrine effects in order to ease the invasion of the trophoblast.
 Gastrulation
Gastrulation is a phase after implantation, during which the
single-layered Ebiplas is reorganized into three-layered
structure. hese three germ layers are known as the ectoderm,
mesoderm, and endoderm.
Day 16
Day 10
Amniotic Ectoderm
cavity
Mesoderm
Endoderm

Embryonic
disk

Later blastocyst stage Gastrulation

Gastrulation (day 16)
 Ectoderm germ layer:
Central Nervous system and Epidermis

Day 17
1

1. Neural plate
2. Notochord
 Neurulation: The process of neural tube formation
Day 17 Day 18
Neural groove
Neural
plate

Notochord

Notochord
Neural tube
Day 21 Future Day 23 (Central
brain Nervous
Neural system)
fold

Notochord
Notochord

Nervous system formed from ectoderm

 Ectoderm germ layer: Neurulation

Embryonic disk
Ectoderm germ layer: Neurulation
 Ectoderm germ layer: Neurulation

(A) Normal development. In the neural plate stage, N- (B) No separation of the
cadherin is seen in the neural plate, while E-cadherin is neural tube occurs when
seen on the presumptive epidermis. Eventually, the N- one side of embryo is
cadherin-bearing neural cells separate from the E- injected with N-cadherin
cadherin-containing epidermal cells. mRNA, so that N-
cadherin is expressed in
the epidermal cells as
well as in the
presumptive neural tube.
 Neurulation: The process of neural tube formation

Videos of Fertilization, implantation, Gastrulation and Neurulation

 The process of Primordial germ (PG) cells formation
(In the mouse)
Fertilization
Blastocyst
Sperm

Maturity (8 weeks) Oocyte

Primordial
germ cell
precursors E 5.5 day

E 7.5 day
Genital ridge
Primordial
germ cell
E 10.5 day

Primordial
E 8.5 germ cell

Reproductive system formed from mesoderm

 Differentiation of three germ layer into organs during organogenesis

Trophoblast

ICM

Nervous
Lines of
system,
digestive Muscle,
skin, lens
(Liver Bone, Blood,
of eye
Pancreas, Connective
stomach) & tissues
Respiratory
tract (lung)

Ectoderm Endoderm Mesoderm

Many different structures are derived from the three
embryonic germ layers during organogenesis
 Summary different structures are derived from
the three embryonic germ layers during organogenesis

ECTODERM MESODERM ENDODERM

Epidermis of skin and its
derivatives (including sweat
glands, hair follicles)
and rectum
epidermis
Nervous system
digestive tract
Muscular layer of respiratory system
stomach, intestine, etc.
Excretory system bladder, and reproductive
system
systems Liver
Reproductive system Pancreas
(except germ cells)

glands
pituitary glands
 Formation of the primitive streak
Primitive streak 1 2 3 4 5 6 7

Embryonic
disk

9 8
1. Primitive groove 4. Oropharyngeal membrane 8. Endoderm
5. Cardial plate 9. Future cloacal
2. Primitive pit
membrane
6. Sectional edge of amniotic membrane
3. Primitive node
7. Mesoderm
 Explain process of gastrulation
From the 17th day a thickening of the embryonic disk begins
around the median line along the rostro-caudal axis. This median
structure (primitive streak) lengthens until it occupies roughly
half the embryo . The primitive streak arises to the proliferation
and migration of epiblast cells in the direction of the embryonic
disk's median line.

After the 19th day, the primitive streak grows through the addition
of cells at its caudal end. At the anterior end, a groove forms in the
ectoblast (primitive groove). The cranial region is strengthened by
the epiblast cells and so forms the primitive pit with the
primitive node . The head of the embryo will form at the extremity
of the embryonic disk near the primitive pit.
 Genesis of the germ layers
Depending on their origin and the Primitive
groove Epiblast
moment of their flowing in, the epiblast
cells migrate away from the primitive
streak in various directions.
The first cells that enter through the
node and the primitive groove replace
the hypoblast layer and form the
definitive endoblast.
The largest proportion of these
immigrated cells form a third germinal
layer, the intraembryonic mesoblast. Extra- Definitive Hypoblast
embryonic endoblast
mesoblast
Genesis of the germ layers

Video:
Genesis of the germ layers
 Summary of cell fates during gastrulation

The epiblast cells form the ectoblast, the

mesoblast (or chorda-mesoblast) and the
intraembryonic endoblast.

The hypoblast cells give rise to the

extraembryonic endoblast (the umbilical
vesicle and the allantois).
 The cellular adhesion molecules (CAM)
during process of gastrulation
Starting with gastrulation the epiblast cells secrete hyaluronic acid, which gets deposited in
the intercellular spaces between the epiblast and the hypoblast. This molecule is able to bind a
large amount of water to itself (up to 1000 times its own weight), is often associated with
cell migration, and plays an anti-aggregational role for the mesoblast cells. The presence of
hyaluronic acid, however, does not suffice to explain the migration of the epiblast cells away
from the primitive streak.
In vertebrate embryos it is assumed that the migration also depends on the presence of
fibronectins that are found on the basal lamina under the epiblast. Fibronectin is an
extracellular glycoprotein
The integrins:
The integrins belong to the 4 families of the Cell Adhesion Molecules (CAM): integrin,
cadherin, selectin, and the large family of the immunoglobulins. They are responsible for the
recognition and binding of two cells with each other or a cell and components of the
extracellular matrix (ECM). The kind of the connection can be homophil (two identical
molecules) or heterophil (two differing molecules bound to each other). Being transmembranal
glycoproteins, integrins insure the cohesion (aggregation) and influence the migration of cells.
They are also responsible for the organization of the cells in the tissue and the tissue in the
organs. The integrins arrange contacts with the collagen filaments in the basal membrane and
the extracellular matrix. Laminin is a ligand of the basal membrane. Fibronectin, on the other
hand, is a ligand of the intercellular substance of e.g. the mesoblast and plays a role in cell
migration.
 Development of human embryos during pre-
and post-implantation

Age in 1 2 3 3.5 4.5

days

Size in 0.1 0.15 0.1 0.15 0.1 0.15 0.1 0.15 0.15 0.2
mm
Fertilized oocyte Zygotic gene Division of Morula stage The blastocyst stage
Polar bodies activation blastomeric Campaction Inner and out cell
Description At oviduct
(tot potent) mass (ICM, TE)
Male and female Stage of polarization CDX2 expressed
pronucleus CDX2 expressed
E-cadherin At oviduct
At oviduct expression At uterus
At oviduct
 Development of human embryos during pre-
and post-implantation
Age in 4.5 6.5 7.5 8.5-9.5 10.5-12.5
days

Size in 0.15 0.2 0.15 0.2 0.15 0.2 0.15 0.2 0.15 0.2
mm
Hatching blastocyst Starting implantation Genesis of the Lacuna trophoblast Extra-embryonic
At uterus Implanting of amniotic cavity and mesoblast
Description the primary
Definitive amniotic
blastocyst on the
cavity Anlage of the
endometrium umbilical vesicle
Primary umbilical secondary umbilical
Solid trophoblast vesicle vesicle

1. Epithelium of uterus 5. Epiblast 6. Material blood capillary

2. Hypoblast 6. Blastocyst cavity 7. Amniotic cavity
3. Syncytiotrophoblast
4. cytotrophoblast
Gastrulation (day 16)
 Development of human embryos during pre-
and post-implantation
Age in 14.5 17.5 18.5 20.5 22-23
days

Size in 0.2 0.3 0.4 0.45 0.4 0.45

mm
Primitive streak, Primitive
node, Primitive groove
Description
Secondary umbilical vesicle
Allantoic diverticulum
Chordal process Pear shaped embryo
Neural plate Neurenteric canal
Genesis of the blood Neural folds form
vessel system and neural groove
formation of blood
 Development of human embryos during
post-implantation

Age in 24-25 26-28 29

days

Size in 1.5 2.5 2.0-3.5 3.0 4.5

mm
Formation of somites The 2 first pharyngeal Closure of the rostral
arches appear neuropore
Description Oropharyngeal membrane
Rupture of the pharyngeal
Allantoic diverticulum membrane Nasal placode

1-3 somites 4-12 somites 13-20 somites

 Development of human embryos during
post-implantation

Age in 30 32 33
days

Size in 3.0 5.0 4.0 6.0 5.0 7.0

mm
Closure of the caudal Buds of the lower extremities Augmented cephalo-
neuropore cervical flexure
Description Lens placode
Buds of the upper Cerebellar primordium
extremities Fourth pharyngeal arch
More than 30 somites Visible ocular primordium
21-29 somites
 Development of human embryos during
post-implantation

Age in 38 41 44
days

Size in 8.0 11 11 - 14 14-17

mm
Pigmentation of the eye External acoustic meatus The eyelids arise
Description Genesis of the foot plate Formation of the interdigital Toe primordium
Physiologic umbilical zones Gonadal gender can be
hernia Atrophy of the embryonic determined
tail in male embryos
 Development of human embryos during
post-implantation

Age in 46 49 51
days

Size in 16-18 18-22 22 - 24

mm
Lengthening and Separated fingers Subcutaneous vessel network
straightening of the of the head spreads out
Description trunk
Gonadal gender determinable
in female embryos Hands and feet come
External acoustic closer and touch each
meatus other
 Development of human embryos during
post-implantation

Age in 53 56
days

Size in 23 - 28 28-31
mm
Development of the The head makes 50%
eyelids and the external of the embryo's size
Description ear canal
Development of the external
Tragus and antitragus genital primordium
Secondary palate
Development of human embryos during
post-implantation
 Development of human embryos during
post-implantation

Video at
Implantation, gastrulation and organogenesis in human
(http://www.youtube.com/watch?v=vTvqCAbng90)
 Implantation anomalies

1. Extra-uterine gravidity (EUG) Hemorrhages (2%)

1. In the ovary
2. In the infundibulum
3. In the fallopian tube
4. In the isthmus
5. Deep in the uterus
6. Abdominal
7. In the pelvis
 Implantation anomalies
1. Extra-uterine gravidity (EUG) Hemorrhages (2%)

There are a total of 6 potential risk factors which can

lead to EUGs:

1. Infections (fallopian tube infection): 90%

2. Surgical interventions in the pelvis
3. Tobacco misuse
4. In vitro fertilization (IVF)
5. Congenital anomalies (tube malformations)
6. Endometriosis (ectopic fragments of the uterine
mucosa)
 Implantation anomalies
Placenta previa
When the implantation takes place in the lower part (5) of the
uterus, the placenta will later develop in the cervix uteri. This type
of implantation is called placenta previa.

A birth through the birth canal

would detach the placenta before
the fetus is born. This can lead to
serious hemorrhages. For this
lower part
of uterus reason, a caesarian section is
always used for enabling births in
such situations. Placenta previas
occur in 1% of all pregnancies.
 Diagram showing the timing of human monozygotic
twinning with relation to extraembryonic membranes

TE cells Embryo Yolk sac

Amnion

ICM cells Chorion

2 Chorion
Splitting occurs before the
formation of the trophoblast,
so each twin has its own
chorion and amnion.

2 Amnion
 Diagram showing the timing of human monozygotic
twinning with relation to extraembryonic membranes

1 Chorion

2 Amnion

Splitting occurs after trophoblast formation but

before amnion formation, resulting in twins having
individual amnionic sacs but sharing one chorion.
 Diagram showing the timing of human monozygotic
twinning with relation to extraembryonic membranes
1 Chorion

1 Amnion

Siamese
Splitting after amnion Twins
formation leads to twins
in one amnionic sac and
a single chorion.
 Summary diagram showing the timing of human monozygotic
twinning with relation to extraembryonic membranes

(A) Splitting occurs before the formation of the trophoblast, so each twin has its own chorion and
amnion. (B) Splitting occurs after trophoblast formation but before amnion formation, resulting in
twins having individual amnionic sacs but sharing one chorion. (C) Splitting after amnion formation
leads to twins in one amnionic sac and a single chorion
 Monozygotic twinning: Conjoined Twins

Two-Headed Boy
northern Italy on October 4, 1877.

Types of conjoined twins. University of Chicago Press, Chicago, 1982.

Production of chimeric mice
 Aggregation of Embryos

2-embryos 3-embryos