Langman's Medical Embryology

Chapter 2: First Week of Development: Ovulation to Implantation 47

Figure 2.13 Changes in the uterine mucosa (endometrium) and corresponding changes
in the ovary during a regular menstrual cycle without fertilization.

uterus, and the basal layer is the only part of the endometrium that is retained
(Fig. 2.13). This layer, which is supplied by its own arteries, the basal arteries,
functions as the regenerative layer in the rebuilding of glands and arteries in
the proliferative phase (Fig. 2.13).

Summary

With each ovarian cycle, a number of primary follicles begin to grow, but
usually only one reaches full maturity, and only one oocyte is discharged
at ovulation. At ovulation, the oocyte is in metaphase of the second
meiotic division and is surrounded by the zona pellucida and some granulosa
cells (Fig. 2.4). Sweeping action of tubal fimbriae carries the oocyte into the
uterine tube.

48 Part One: General Embryology

Before spermatozoa can fertilize the oocyte, they must undergo (a) capac-
itation, during which time a glycoprotein coat and seminal plasma proteins
are removed from the spermatozoon head, and (b) the acrosome reaction,
during which acrosin and trypsin-like substances are released to penetrate the
zona pellucida. During fertilization, the spermatozoon must penetrate (a) the
corona radiata, (b) the zona pellucida, and (c) the oocyte cell membrane
(Fig. 2.5). As soon as the spermatocyte has entered the oocyte, (a) the oocyte
finishes its second meiotic division and forms the female pronucleus; (b) the
zona pellucida becomes impenetrable to other spermatozoa; and (c) the head
of the sperm separates from the tail, swells, and forms the male pronucleus
(Figs. 2.6 and 2.7). After both pronuclei have replicated their DNA, paternal
and maternal chromosomes intermingle, split longitudinally, and go through a
mitotic division, giving rise to the two-cell stage. The results of fertilization are
(a) restoration of the diploid number of chromosomes, (b) determination of
chromosomal sex, and (c) initiation of cleavage.

Cleavage is a series of mitotic divisions that results in an increase in cells,
blastomeres, which become smaller with each division. After three divisions,
blastomeres undergo compaction to become a tightly grouped ball of cells with
inner and outer layers. Compacted blastomeres divide to form a 16-cell morula.
As the morula enters the uterus on the third or fourth day after fertilization, a
cavity begins to appear, and the blastocyst forms. The inner cell mass, which
is formed at the time of compaction and will develop into the embryo proper,
is at one pole of the blastocyst. The outer cell mass, which surrounds the inner
cells and the blastocyst cavity, will form the trophoblast.

The uterus at the time of implantation is in the secretory phase, and the
blastocyst implants in the endometrium along the anterior or posterior wall. If
fertilization does not occur, then the menstrual phase begins and the spongy
and compact endometrial layers are shed. The basal layer remains to regenerate
the other layers during the next cycle.

Problems to Solve

1. What are the primary causes of infertility in men and women?

2. A woman has had several bouts of pelvic inflammatory disease and now wants
to have children. However, she has been having difficulty becoming pregnant.
What is likely to be the problem, and what would you suggest?

SUGGESTED READING

Allen CA, Green DPL: The mammalian acrosome reaction: gateway to sperm fusion with the oocyte?
Bioessays 19:241, 1997.

Archer DF, Zeleznik AJ, Rockette HE: Ovarian follicular maturation in women: 2. Reversal of estrogen
inhibited ovarian folliculogenesis by human gonadotropin. Fertil Steril 50:555, 1988.

Barratt CLR, Cooke ID: Sperm transport in the human female reproductive tract: a dynamic inter-
action. Int J Androl 14:394, 1991.

Chapter 2: First Week of Development: Ovulation to Implantation 49

Boldt J, et al: Carbohydrate involvement in sperm-egg fusion in mice. Biol Reprod 40:887, 1989.
Burrows TD, King A, Loke YW: Expression of integrins by human trophoblast and differential

adhesion to laminin or fibronectin. Hum Reprod 8:475, 1993.
Carr DH: Chromosome studies on selected spontaneous abortions: polyploidy in man. J Med Genet

8:164, 1971.
Chen CM, Sathananthan AH: Early penetration of human sperm through the vestments of human

egg in vitro. Arch Androl 16:183, 1986.
Cowchock S: Autoantibodies and fetal wastage. Am J Reprod Immunol 26:38, 1991.
Edwards RG: A decade of in vitro fertilization. Res Reprod 22:1, 1990.
Edwards RG, Bavister BD, Steptoe PC: Early stages of fertilization in vitro of human oocytes matured

in vitro. Nature (Lond) 221:632, 1969.
Egarter C: The complex nature of egg transport through the oviduct. Am J Obstet Gynecol 163:687,

1990.
Enders AC, Hendrickx AG, Schlake S: Implantation in the rhesus monkey: initial penetration of the

endometrium. Am J Anat 167:275, 1983.
Gilbert SF: Developmental Biology. Sunderland, MA, Sinauer, 1991.
Handyside AH, Kontogianni EH, Hardy K, Winston RML: Pregnancies from biopsied human preim-

plantation embryos sexed by Y-specific DNA amplification. Nature 344:768, 1990.
Hertig AT, Rock J, Adams EC: A description of 34 human ova within the first 17 days of development.

Am J Anat 98:435, 1956.
Johnson MH, Everitt BJ: Essential Reproduction. 5th ed. London, Blackwell Science Limited, 2000.
Liu J, et al: Analysis of 76 total fertilization failure cycles out of 2732 intracytoplasmic sperm

injection cycles. Hum Reprod 10:2630, 1995.
Oura C, Toshimori K: Ultrasound studies on the fertilization of mammalian gametes. Rev Cytol

122:105, 1990.
Pedersen RA, We K, Balakier H: Origin of the inner cell mass in mouse embryos: cell lineage

analysis by microinjection. Dev Biol 117:581, 1986.
Reproduction (entire issue). J NIH Res 9:1997.
Scott RT, Hodgen GD: The ovarian follicle: life cycle of a pelvic clock. Clin Obstet Gynecol 33:551,

1990.
Wasserman PM: Fertilization in mammals. Sci Am 259:78, 1988.
Wolf DP, Quigley MM (eds): Human in Vitro Fertilization and Transfer. New York, Plenum, 1984.
Yen SC, Jaffe RB (eds): Reproductive Endocrinology: Physiology, Pathophysiology, and Clinical Man-

agement. 2nd ed. Philadelphia, WB Saunders, 1986.



chapter 3

Second Week of Development:
Bilaminar Germ Disc

This chapter gives a day-by-day account of the
major events of the second week of development.
However, embryos of the same fertilization age do
not necessarily develop at the same rate. Indeed, con-
siderable differences in rate of growth have been found
even at these early stages of development.

Day 8

At the eighth day of development, the blastocyst is partially
embedded in the endometrial stroma. In the area over the em-
bryoblast, the trophoblast has differentiated into two layers:
(a) an inner layer of mononucleated cells, the cytotrophoblast,
and (b) an outer multinucleated zone without distinct cell bound-
aries, the syncytiotrophoblast (Figs. 3.1 and 3.2). Mitotic figures are
found in the cytotrophoblast but not in the syncytiotrophoblast. Thus,
cells in the cytotrophoblast divide and migrate into the syncytiotro-
phoblast, where they fuse and lose their individual cell membranes.
Cells of the inner cell mass or embryoblast also differentiate into two
layers: (a) a layer of small cuboidal cells adjacent to the blastocyst cavity,
known as the hypoblast layer, and (b) a layer of high columnar cells
adjacent to the amniotic cavity, the epiblast layer (Figs. 3.1 and 3.2).
Together, the layers form a flat disc. At the same time, a small cavity
appears within the epiblast. This cavity enlarges to become the

51

52 Part One: General Embryology

Figure 3.1 A 7.5-day human blastocyst, partially embedded in the endometrial stroma.
The trophoblast consists of an inner layer with mononuclear cells, the cytotrophoblast,
and an outer layer without distinct cell boundaries, the syncytiotrophoblast. The em-
bryoblast is formed by the epiblast and hypoblast layers. The amniotic cavity appears
as a small cleft.

Figure 3.2 Section of a 7.5-day human blastocyst (×100). Note the multinucleated ap-
pearance of the syncytiotrophoblast, large cells of the cytotrophoblast, and slit-like
amniotic cavity.

amniotic cavity. Epiblast cells adjacent to the cytotrophoblast are called am-
nioblasts; together with the rest of the epiblast, they line the amniotic cavity
(Figs. 3.1 and 3.3). The endometrial stroma adjacent to the implantation site
is edematous and highly vascular. The large, tortuous glands secrete abundant
glycogen and mucus.

Chapter 3: Second Week of Development: Bilaminar Germ Disc 53

Figure 3.3 A 9-day human blastocyst. The syncytiotrophoblast shows a large number
of lacunae. Flat cells form the exocoelomic membrane. The bilaminar disc consists of
a layer of columnar epiblast cells and a layer of cuboidal hypoblast cells. The original
surface defect is closed by a fibrin coagulum.

Day 9

The blastocyst is more deeply embedded in the endometrium, and the penetra-
tion defect in the surface epithelium is closed by a fibrin coagulum (Fig. 3.3).
The trophoblast shows considerable progress in development, particularly at
the embryonic pole, where vacuoles appear in the syncytium. When these vac-
uoles fuse, they form large lacunae, and this phase of trophoblast development
is thus known as the lacunar stage (Fig. 3.3).

At the abembryonic pole, meanwhile, flattened cells probably originating
from the hypoblast form a thin membrane, the exocoelomic (Heuser’s) mem-
brane, that lines the inner surface of the cytotrophoblast (Fig. 3.3). This mem-
brane, together with the hypoblast, forms the lining of the exocoelomic cavity,
or primitive yolk sac.

Days 11 and 12

By the 11th to 12th day of development, the blastocyst is completely embedded
in the endometrial stroma, and the surface epithelium almost entirely covers

54 Part One: General Embryology

Figure 3.4 Human blastocyst of approximately 12 days. The trophoblastic lacunae at
the embryonic pole are in open connection with maternal sinusoids in the endometrial
stroma. Extraembryonic mesoderm proliferates and fills the space between the exo-
coelomic membrane and the inner aspect of the trophoblast.

the original defect in the uterine wall (Figs. 3.4 and 3.5). The blastocyst now
produces a slight protrusion into the lumen of the uterus. The trophoblast is
characterized by lacunar spaces in the syncytium that form an intercommuni-
cating network. This network is particularly evident at the embryonic pole; at
the abembryonic pole, the trophoblast still consists mainly of cytotrophoblastic
cells (Figs. 3.4 and 3.5).

Concurrently, cells of the syncytiotrophoblast penetrate deeper into the
stroma and erode the endothelial lining of the maternal capillaries. These capil-
laries, which are congested and dilated, are known as sinusoids. The syncytial
lacunae become continuous with the sinusoids and maternal blood enters the
lacunar system (Fig. 3.4). As the trophoblast continues to erode more and more
sinusoids, maternal blood begins to flow through the trophoblastic system, es-
tablishing the uteroplacental circulation.

In the meantime, a new population of cells appears between the in-
ner surface of the cytotrophoblast and the outer surface of the exocoelomic

Chapter 3: Second Week of Development: Bilaminar Germ Disc 55

Figure 3.5 Fully implanted 12-day human blastocyst (×100). Note maternal blood cells
in the lacunae, the exocoelomic membrane lining the primitive yolk sac, and the hy-
poblast and epiblast.

cavity. These cells, derived from yolk sac cells, form a fine, loose connec-
tive tissue, the extraembryonic mesoderm, which eventually fills all of the
space between the trophoblast externally and the amnion and exocoelomic
membrane internally (Figs. 3.4 and 3.5). Soon, large cavities develop in the
extraembryonic mesoderm, and when these become confluent, they form
a new space known as the extraembryonic coelom, or chorionic cavity
(Fig. 3.4). This space surrounds the primitive yolk sac and amniotic cavity ex-
cept where the germ disc is connected to the trophoblast by the connecting stalk
(Fig. 3.6). The extraembryonic mesoderm lining the cytotrophoblast and am-
nion is called the extraembryonic somatopleuric mesoderm; the lining cover-
ing the yolk sac is known as the extraembryonic splanchnopleuric mesoderm
(Fig. 3.4).

Growth of the bilaminar disc is relatively slow compared with that of the tro-
phoblast; consequently, the disc remains very small (0.1–0.2 mm). Cells of the
endometrium, meanwhile, become polyhedral and loaded with glycogen and
lipids; intercellular spaces are filled with extravasate, and the tissue is edema-
tous. These changes, known as the decidua reaction, at first are confined to the

56 Part One: General Embryology

Figure 3.6 A 13-day human blastocyst. Trophoblastic lacunae are present at the embry-
onic as well as the abembryonic pole, and the uteroplacental circulation has begun. Note
the primary villi and the extraembryonic coelom or chorionic cavity. The secondary
yolk sac is entirely lined with endoderm.

area immediately surrounding the implantation site but soon occur throughout
the endometrium.
Day 13
By the 13th day of development, the surface defect in the endometrium has
usually healed. Occasionally, however, bleeding occurs at the implantation site
as a result of increased blood flow into the lacunar spaces. Because this bleeding
occurs near the 28th day of the menstrual cycle, it may be confused with

Chapter 3: Second Week of Development: Bilaminar Germ Disc 57

Figure 3.7 Section through the implantation site of a 13-day embryo. Note the amniotic
cavity, yolk sac, and exocoelomic cyst in the chorionic cavity. Most of the lacunae are
filled with blood.

normal menstrual bleeding and, therefore, cause inaccuracy in determining
the expected delivery date.

The trophoblast is characterized by villous structures. Cells of the cy-
totrophoblast proliferate locally and penetrate into the syncytiotrophoblast,
forming cellular columns surrounded by syncytium. Cellular columns with
the syncytial covering are known as primary villi (Figs. 3.6 and 3.7) (see
Chapter 4).

In the meantime, the hypoblast produces additional cells that migrate along
the inside of the exocoelomic membrane (Fig. 3.4). These cells proliferate and
gradually form a new cavity within the exocoelomic cavity. This new cavity is
known as the secondary yolk sac or definitive yolk sac (Figs. 3.6 and 3.7). This
yolk sac is much smaller than the original exocoelomic cavity, or primitive yolk
sac. During its formation, large portions of the exocoelomic cavity are pinched
off. These portions are represented by exocoelomic cysts, which are often
found in the extraembryonic coelom or chorionic cavity (Figs. 3.6 and 3.7).

Meanwhile, the extraembryonic coelom expands and forms a large cavity,
the chorionic cavity. The extraembryonic mesoderm lining the inside of the
cytotrophoblast is then known as the chorionic plate. The only place where
extraembryonic mesoderm traverses the chorionic cavity is in the connecting
stalk (Fig. 3.6). With development of blood vessels, the stalk becomes the
umbilical cord.

58 Part One: General Embryology

CLINICAL CORRELATES

Abnormal Implantation

The syncytiotrophoblast is responsible for hormone production (see Chap-
ter 6), including human chorionic gonadotropin (hCG). By the end of the
second week, quantities of this hormone are sufficient to be detected by ra-
dioimmunoassays, which serve as the basis for pregnancy testing.

Because 50% of the implanting embryo’s genome is derived from the
father, it is a foreign body that potentially should be rejected by the mater-
nal system. Recent evidence suggests that a combination of factors protects
the conceptus, including production of immunosuppressive cytokines and pro-
teins and the expression of an unusual major histocompatibility complex class
IB molecule (HLA-G) that blocks recognition of the conceptus as foreign tissue.
If the mother has autoimmune disease, for example systemic lupus erythe-
matosus, antibodies generated by the disease may attack the conceptus and
reject it.

Abnormal implantation sites sometimes occur even within the uterus.
Normally the human blastocyst implants along the anterior or posterior wall of
the body of the uterus. Occasionally the blastocyst implants close to the inter-
nal opening os (opening) (Fig. 3.8) of the cervix, so that later in development,

Body of Intestinal loop Mesentery
uterus
3 1
4 Uterine tube

5 2 Ampulla
6
Internal os
of uterus Fimbriae

Figure 3.8 Abnormal implantation sites of the blastocyst. 1, implantation in the ab-
dominal cavity. The ovum most frequently implants in the rectouterine cavity (Douglas’
pouch) but may implant at any place covered by peritoneum. 2, implantation in the am-
pullary region of the tube. 3, tubal implantation. 4, interstitial implantation, that is, in
the narrow portion of the uterine tube. 5, implantation in the region of the internal os,
frequently resulting in placenta previa. 6, ovarian implantation.

Chapter 3: Second Week of Development: Bilaminar Germ Disc 59

Figure 3.9 Tubal pregnancy. Embryo is approximately 2 months old and is about to
escape through a rupture in the tubal wall.

the placenta bridges the opening (placenta previa) and causes severe, even
life-threatening bleeding in the second part of pregnancy and during delivery.

Occasionally, implantation takes place outside the uterus, resulting in ex-
trauterine pregnancy, or ectopic pregnancy. Ectopic pregnancies may occur
at any place in the abdominal cavity, ovary, or uterine tube (Fig. 3.8). However,
95% of ectopic pregnancies occur in the uterine tube, and most of these are in
the ampulla (Fig. 3.9). In the abdominal cavity, the blastocyst most frequently
attaches itself to the peritoneal lining of the rectouterine cavity, or Douglas’
pouch (Fig. 3.10). The blastocyst may also attach itself to the peritoneal cov-
ering of the intestinal tract or to the omentum. Sometimes the blastocyst
develops in the ovary proper, causing a primary ovarian pregnancy. In most
ectopic pregnancies, the embryo dies about the second month of gestation,
causing severe hemorrhaging and abdominal pain in the mother.

Abnormal blastocysts are common. For example, in a series of 26 im-
planted blastocysts varying in age from 7.5 to 17 days recovered from patients
of normal fertility, nine (34.6%) were abnormal. Some consisted of syncytium
only; others showed varying degrees of trophoblastic hypoplasia. In two, the
embryoblast was absent, and in some, the germ disc showed an abnormal
orientation.

It is likely that most abnormal blastocysts would not have produced any
sign of pregnancy because their trophoblast was so inferior that the corpus
luteum could not have persisted. These embryos probably would have been

60 Part One: General Embryology

Figure 3.10 Midline section of bladder, uterus, and rectum to show an abdominal preg-
nancy in the rectouterine (Douglas’) pouch.

aborted with the next menstrual flow, and therefore, pregnancy would not
have been detected. In some cases, however, the trophoblast develops and
forms placental membranes, although little or no embryonic tissue is present.
Such a condition is known as a hydatidiform mole. Moles secrete high levels of
hCG and may produce benign or malignant (invasive mole, choriocarcinoma)
tumors.

Genetic analysis of hydatidiform moles indicates that although male and
female pronuclei may be genetically equivalent, they may be different func-
tionally. This evidence is derived from the fact that while cells of moles are
diploid, their entire genome is paternal. Thus, most moles arise from fertil-
ization of an oocyte lacking a nucleus followed by duplication of the male
chromosomes to restore the diploid number. These results also suggest that
paternal genes regulate most of the development of the trophoblast, since in
moles, this tissue differentiates even in the absence of a female pronucleus.

Other examples of functional differences in maternal and paternal genes
are provided by the observation that certain genetic diseases depend on
whether the defective or missing gene is inherited from the father or the
mother. For example, inheritance of a deletion on chromosome 15 from a fa-
ther produces Prader-Willi syndrome, whereas inheritance of the same defect
from the mother results in Angelman syndrome. This phenomenon, in which

Chapter 3: Second Week of Development: Bilaminar Germ Disc 61

there is differential modification and/or expression of homologous alleles or
chromosome regions, depending on the parent from whom the genetic ma-
terial is derived, is known as genomic imprinting. Imprinting involves auto-
somes and sex chromosomes (in all female mammals, one X chromosome
is inactivated in somatic cells and forms a chromatin-positive body [Barr
body]) and is modulated by deoxyribonucleic acid (DNA) methylation. Cer-
tain diseases, such as Huntington’s chorea, neurofibromatosis, familial cancer
disorders (Wilms’ tumors, familial retinoblastoma), and myotonic dystrophy,
also involve imprinting. Fragile X syndrome, the leading cause of inherited
mental retardation, may be another example of a condition based on imprint-
ing (see Chapter 1).

Preimplantation and postimplantation reproductive failure occurs often.
Even in some fertile women under optimal conditions for pregnancy, 15% of
oocytes are not fertilized, and 10% to 15% start cleavage but fail to implant. Of
the 70% to 75% that implant, only 58% survive until the second week, and
16% of those are abnormal. Hence, when the first expected menstruation
is missed, only 42% of the eggs exposed to sperm are surviving. Of this
percentage, a number will be aborted during subsequent weeks and a number
will be abnormal at the time of birth.

Summary

At the beginning of the second week, the blastocyst is partially embed-
ded in the endometrial stroma. The trophoblast differentiates into (a)
an inner, actively proliferating layer, the cytotrophoblast, and (b) an
outer layer, the syncytiotrophoblast, which erodes maternal tissues (Fig. 3.1).
By day 9, lacunae develop in the syncytiotrophoblast. Subsequently, maternal
sinusoids are eroded by the syncytiotrophoblast, maternal blood enters the
lacunar network, and by the end of the second week, a primitive uteropla-
cental circulation begins (Fig. 3.6). The cytotrophoblast, meanwhile, forms
cellular columns penetrating into and surrounded by the syncytium. These
columns are primary villi. By the end of the second week, the blastocyst
is completely embedded, and the surface defect in the mucosa has healed
(Fig. 3.6).
The inner cell mass or embryoblast, meanwhile, differentiates into (a) the
epiblast and (b) the hypoblast, together forming a bilaminar disc (Fig. 3.1).
Epiblast cells give rise to amnioblasts that line the amniotic cavity superior
to the epiblast layer. Endoderm cells are continuous with the exocoelomic
membrane, and together they surround the primitive yolk sac (Fig. 3.4). By
the end of the second week, extraembryonic mesoderm fills the space between
the trophoblast and the amnion and exocoelomic membrane internally. When
vacuoles develop in this tissue, the extraembryonic coelom or chorionic cavity
forms (Fig. 3.6). Extraembryonic mesoderm lining the cytotrophoblast and

62 Part One: General Embryology

amnion is extraembryonic somatopleuric mesoderm; the lining surrounding
the yolk sac is extraembryonic splanchnopleuric mesoderm (Fig. 3.6).

The second week of development is known as the week of twos: The
trophoblast differentiates into two layers, the cytotrophoblast and syncytiotro-
phoblast. The embryoblast forms two layers, the epiblast and hypoblast.
The extraembryonic mesoderm splits into two layers, the somatopleure and
splanchnopleure. And two cavities, the amniotic and yolk sac cavities, form.
Implantation occurs at the end of the first week. Trophoblast cells invade the
epithelium and underlying endometrial stroma with the help of proteolytic en-
zymes. Implantation may also occur outside the uterus, such as in the rectouter-
ine pouch, on the mesentery, in the uterine tube, or in the ovary (ectopic preg-
nancies).

Problems to Solve

1. The second week of development is known as the week of twos. Formation of
what structures support this statement?

2. During implantation, the trophoblast is invading maternal tissues, and because
it contains approximately 50% paternal genes, it is a foreign body. Why is the
conceptus not rejected by an immunologic response from the mother’s system?

3. A woman who believes she is pregnant complains of edema and vaginal
bleeding. Examination reveals high plasma hCG concentrations and placental
tissue, but no evidence of an embryo. How would you explain this condition?

4. A young woman who has missed two menstrual periods complains of intense
abdominal pain. What might an initial diagnosis be, and how would you
confirm it?

SUGGESTED READING

Aplin JD: Implantation, trophoblast differentiation and hemochorial placentation: mechanistic ev-
idence in vivo and in vitro. J Cell Sci 99:681, 1991.

Bianchi DW, Wilkins-Haug LE, Enders AC, Hay ED: Origin of extraembryonic mesoderm in exper-
imental animals: relevance to chorionic mosaicism in humans. Am J Med Genet 46:542, 1993.

Cattanack BM, Beechey CV: Autosomal and X-chromosome imprinting. Dev Suppl 63, 1990.
Enders AC, King BF: Formation and differentiation of extraembryonic mesoderm in the Rhesus

monkey. Am J Anat 181:327, 1988.
Enders AC, King BF: Development of the human yolk sac. In Nogales FF (ed): The Human Yolk Sac

and Yolk Sac Tumors. Berlin, Springer Verlag, 1993.
Enders AC, Schlafke S, Hendrickx A: Differentiation of the embryonic disc, amnion, and yolk sac

in the rhesus monkey. Am J Anat 177:161, 1986.
Hertig AT, Rock J, Adams EC: A description of 34 human ova within the first 17 days of development.

Am J Anat 98:435, 1956.
Holliday R: Genomic imprinting and allelic exclusion. Dev Suppl 125, 1990.
McMaster MT, et al: Human placental HLA-G expression is restricted to differentiated cytotro-

phoblasts. J Immunol 154:3771, 1995.

Chapter 3: Second Week of Development: Bilaminar Germ Disc 63

Monk M, Grant M: Preferential X-chromosome inactivation, DNA methylation and imprinting. Dev
Suppl 55, 1990.

Roth I, et al: Human placental cytotrophoblasts produce the immunosuppressive cytokine inter-
leukin 10. J Exp Med 184:539, 1996.

Rubin GL: Ectopic pregnancy in the United States: 1970 through 1978. JAMA 249:1725, 1983.



chapter 4

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. Gastrula-
tion begins with formation of the primitive streak on the
surface of the epiblast (Figs. 4.1–4.3A). Initially, the streak
is vaguely defined (Fig. 4.1), but in a 15- to 16-day embryo,
it is clearly visible as a narrow groove with slightly bulging
regions on either side (Fig. 4.2). The cephalic end of the streak,
the primitive node, consists of a slightly elevated area surround-
ing the small primitive pit (Fig. 4.3). Cells of the epiblast migrate
toward the primitive streak (Fig. 4.3). Upon arrival in the region
of the streak, they become flask-shaped, detach from the epiblast,
and slip beneath it (Fig. 4.3, B–D). This inward movement is known
as invagination. 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. 4.3B ), 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 cephalad (Fig. 4.3). Gradually,

65

66 Part One: General Embryology

Amniotic Syncytiotrophoblast
cavity Cytotrophoblast
Extraembryonic
Epiblast mesoderm
Hypoblast
Buccopharyngeal membrane
Definitive
yolk sac

A

Cut edge of
amnion

Primitive Wall of
streak yolk sac

B Hypoblast Epiblast

Figure 4.1 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 on 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.

they migrate beyond the margin of the disc and establish contact with the ex-
traembryonic 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 buccopharyngeal mem-
brane and is derived from some of the first cells that migrate through the
node in a cephalic direction. Later, the prechordal plate will be important for

Chapter 4: Third Week of Development: Trilaminar Germ Disc 67

Figure 4.2 A. Dorsal aspect of an 18-day embryo. The embryo has a pear-shaped ap-
pearance and shows the primitive streak and node at its caudal end. B. Photograph of
an 18-day human embryo, dorsal view. Note the primitive node and the notochord ex-
tending cranially. The yolk sac has a somewhat mottled appearance. The length of the
embryo is 1.25 mm and the greatest width is 0.68 mm.

induction of the forebrain (Figs. 4.3A and 4.4A). The buccopharyngeal mem-
brane 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 pit move forward cephalad
until they reach the prechordal plate (Fig. 4.4). 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. 4.4, B
and C ). 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. 4.4, D and E ),
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 buccopharyngeal membrane)
and caudally to the primitive pit. At the point where the pit forms an indentation

68 Part One: General Embryology

Buccopharyngeal
membrane

Cut edge of amnion

Prenotochordal cells

Primitive node

Primitive streak Cloacal membrane

A

Primitive Primitive
node streak

Epiblast Amnioblasts

Yolk
sac

Invaginating Hypoblast

B mesoderm cells

Figure 4.3 A. Dorsal side of the germ disc from a 16-day embryo indicating the move-
ment 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 invagina-
tion of epiblast cells. The first cells to move inward displace the hypoblast to create
the definitive endoderm. Once definitive endoderm is established, inwardly moving epi-
blast forms mesoderm. C. Scanning electron micrograph through the primitive streak
of a mouse embryo showing migration of epiblast (eb) cells. The node region appears
as a shallow pit (arrow). D. Higher magnification of the section in C.

in the epiblast, the neurenteric canal temporarily connects the amniotic and
yolk sac cavities (Fig. 4.4A).

The cloacal membrane is formed at the caudal end of the embryonic disc
(Fig. 4.3A). This membrane, which is similar in structure to the buccopharyn-
geal membrane, consists of tightly adherent ectoderm and endoderm cells with
no intervening mesoderm. When the cloacal membrane appears, the posterior

Chapter 4: Third Week of Development: Trilaminar Germ Disc 69

C

DD

Figure 4.3—Continued.

70 Part One: General Embryology

B C Primitive pit and
Ectoderm neurenteric canal
Amnion
Connecting stalk
Wall of Notochord
yolk sac Prechordal plate Allantois
Cloacal plate
A (membrane)

Notochordal plate

C Intraembryonic Endoderm
mesoderm

Intraembryonic mesoderm

Extraembryonic

Endoderm mesoderm

E Notochord

Figure 4.4 Schematic views and scanning electron micrographs 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. B. Scanning
electron micrograph of a mouse embryo showing the region of the buccopharyngeal

Chapter 4: Third Week of Development: Trilaminar Germ Disc 71

B

D

F

membrane (arrows). Extending posteriorly is the prenotochordal plate (arrowheads).
C. Schematic cross section through the region of the notochordal plate. Soon the
notochordal plate will detach from the endoderm to form the definitive notochord.
D. Scanning electron micrograph of a mouse embryo showing detachment of the noto-
chordal plate from the endoderm. E. Schematic view showing the definitive notochord.
F. Scanning electron micrograph of a mouse embryo showing the definitive notochord
(arrows) in close approximation to the neural tube (NT).

72 Part One: General Embryology

wall of the yolk sac forms a small diverticulum that extends into the connecting
stalk. This diverticulum, the allantoenteric diverticulum, or allantois, appears
around the 16th day of development (Fig. 4.4A). Although in some lower ver-
tebrates the allantois serves as a reservoir for excretion products of the renal
system, in humans it remains rudimentary but may be involved in abnormali-
ties of bladder development (see Chapter 14).

Establishment of the Body Axes

Establishment of the body axes, anteroposterior, dorsoventral, and left-right,
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 anterior visceral endoderm (AVE), expresses genes essential for
head formation, including the transcription factors OTX2, LIM1, and HESX1
and the secreted factor cerberus. These genes establish the cranial end of the
embryo before gastrulation. The primitive streak itself is initiated and main-
tained by expression of Nodal, a member of the transforming growth factor β
(TGF-β) family (Fig. 4.5). Once the streak is formed, a number of genes reg-
ulate formation of dorsal and ventral mesoderm and head and tail structures.
Another member of the TGF-β family, bone morphogenetic protein-4 (BMP-
4) is secreted throughout the embryonic disc (Fig. 4.5). In the presence of this
protein and fibroblast growth factor (FGF), mesoderm will be ventralized to
contribute to kidneys (intermediate mesoderm), blood, and body wall meso-
derm (lateral plate mesoderm). In fact, all mesoderm would be ventralized if
the activity of BMP-4 were not blocked by other genes expressed in the node.
For this reason, the node is the organizer. It was given that designation by

Goosecoid, chordin,
noggin, follistatin,
nodal

AVE

Figure 4.5 Sagittal section through the node and primitive streak showing the expres-
sion pattern of genes regulating the craniocaudal and dorsoventral axes. Cells at the
prospective cranial end of the embryo in the anterior visceral endoderm (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, bone morphogenetic protein (BMP-4; hatched
areas), secreted throughout the bilaminar disc, acts with FGF to ventralize mesoderm
into intermediate and lateral plate structures. Goosecoid regulates chordin expression,
and this gene product, together with noggin and follistatin, antagonizes the activity
of BMP-4, dorsalizing mesoderm into notochord and paraxial mesoderm for the head
region. Later, expression of the Brachyury (T) gene antagonizes BMP-4 to dorsalize
mesoderm in caudal regions of the embryo.

Chapter 4: Third Week of Development: Trilaminar Germ Disc 73

Figure 4.6 Node and primitive streak region removed from a mouse embryo showing
expression of nodal using in situ hybridization. Nodal is expressed in the node and
initiates and maintains the primitive streak.

Hans Spemann, who first described this activity in the dorsal lip of the blasto-
pore, a structure analogous to the node, in Xenopus embryos. Thus, chordin
(activated by the transcription factor Goosecoid ), noggin, and follistatin an-
tagonize the activity of BMP-4. As a result, cranial mesoderm is dorsalized into
notochord, somites, and somitomeres (Fig. 4.5). 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 primi-
tive streak (Fig. 4.6). Similarly, HNF-3β maintains the node and later induces
regional specificity in the forebrain and midbrain areas. Without HNF-3β, em-
bryos fail to gastrulate properly and lack forebrain and midbrain structures. As
mentioned previously, Goosecoid activates inhibitors of BMP-4 and contributes
to regulation of head development. Overexpression or underexpression of this
gene results in severe malformations of the head region, including duplications
(Fig. 4.7).

Regulation of dorsal mesoderm formation in mid and caudal regions of the
embryo is controlled by the Brachyury (T) gene (Fig. 4.8). Thus, mesoderm
formation in these regions depends on this gene product, and its absence
results in shortening of the embryonic axis (caudal dysgenesis; see p. 80).
The degree of shortening depends upon the time at which the protein becomes
deficient.

Left-right sidedness, also established early in development, is orchestrated
by a cascade of genes. When the primitive streak appears, fibroblast growth
factor 8 (FGF-8) is secreted by cells in the node and primitive streak and

74 Part One: General Embryology

Figure 4.7 Two-headed tadpole produced by injecting additional Goosecoid mRNA into
frog eggs. Similar results can be obtained by transplanting an additional node region
to eggs. Goosecoid is normally expressed in the node and is a major regulator of head
development.

Figure 4.8 Expression pattern of the Brachyury (T) gene in the notochord and primitive
streak of a mouse embryo. This gene antagonizes the activity of bone morphogenetic
protein (BMP-4) in the hindbrain and spinal cord regions and dorsalizes mesoderm to
form notochord, somitomeres, and somites (paraxial mesoderm). (Mouse embryos are
dorsiflexed into a cup shape during the period of gastrulation and neurulation.) nf,
neural fold; hp, head process; ps, primitive streak; al, allantois.

Chapter 4: Third Week of Development: Trilaminar Germ Disc 75

Buccopharyngeal
membrane

FGF8 Neural
tube
Nodal
Nodal Node Lefty2 Notochord
FGF8 (SHH, T)
PITX2
Lefty 1 NKX3.2

Nodal

Node
(FGF8)

Cloacal
membrane

AB

Figure 4.9 Dorsal views of the germ disc showing gene expression patterns respon-
sible for establishing the left-right body axis. A. Fibroblast growth factor 8 (FGF-8),
secreted by the node and primitive streak, establishes expression of Nodal, a mem-
ber of the transforming growth factor β (TGF-β) superfamily, on the left side near
the node. B. Later, as the neural plate is induced, FGF-8 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. 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 fac-
tor PITX 2, which, through further downstream effectors, establishes left sidedness.
Sonic hedgehog (SHH), expressed in the notochord, may serve as a midline barrier and
also repress expression of left-sided genes on the right. NKX 3.2 may regulate down-
stream genes important for establishing right sidedness.

induces expression of Nodal but only on the left side of the embryo (Fig. 4.9A).
Later, as the neural plate is induced, FGF-8 maintains Nodal expression in the
lateral plate mesoderm (Fig. 4.10), as well as Lefty-2, and both of these genes
upregulate PITX2, a transcription factor responsible for establishing left sided-
ness (Fig. 4.9B). Simultaneously, Lefty-1 is expressed on the left side of the
floor plate of the neural tube and may act as a barrier to prevent left-sided sig-
nals 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, another growth factor secreted by the notochord, is also
essential for expression of Nodal, Lefty-1, and Lefty-2 (Fig. 4.9B ). Genes regulat-
ing right-sided development are not as well defined, although expression of the

76 Part One: General Embryology

AB

Figure 4.10 Expression pattern of the gene nodal in a mouse showing that it is re-
stricted to the left side of the body (A) in the lateral plate mesoderm (B). Nodal, together
with Lefty, regulates downstream genes to determine left-right asymmetry.

transcription factor NKX 3.2 is restricted to the right lateral plate mesoderm
and probably regulates effector genes responsible for establishing the right side.
Why the cascade is initiated on the left remains a mystery, but the reason may
involve cilia on cells in the node that beat to create a gradient of FGF-8 toward
the left. Indeed, abnormalities in cilia-related proteins result in laterality defects
in mice and some humans with these defects have abnormal ciliary function
(see p. 79).

Fate Map Established During Gastrulation
Regions of the epiblast that migrate and ingress through the primitive streak
have been mapped and their ultimate fates determined (Fig. 4.11). For exam-
ple, cells that ingress through the cranial region of the node become notochord;
those migrating at the lateral edges of the node and from the cranial end of
the streak become paraxial mesoderm; cells migrating through the midstreak
region become intermediate mesoderm; those migrating through the more
caudal part of the streak form lateral plate mesoderm; and cells migrating
through the caudal-most part of the streak contribute to extraembryonic meso-
derm (the other source of this tissue is the primitive yolk sac [hypoblast]; see
p. 55).

Chapter 4: Third Week of Development: Trilaminar Germ Disc 77

Figure 4.11 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 cranial-most part of the
node will form the notochord (n); those migrating more posteriorly through the node and
cranial-most 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).

Growth of the Embryonic Disc

The embryonic disc, initially flat and almost round, gradually becomes elon-
gated, with a broad cephalic and a narrow caudal end (Fig. 4.2). Expansion
of the embryonic disc occurs mainly in the cephalic region; the region of the
primitive streak remains more or less the same size. Growth and elongation of
the cephalic part of the disc are caused by a continuous migration of cells from
the primitive streak region in a cephalic direction. Invagination of surface cells
in the primitive streak and their subsequent migration forward and laterally
continues until the end of the fourth week. At that stage, the primitive streak
shows regressive changes, rapidly shrinks, and soon disappears.

That the primitive streak at the caudal end of the disc continues to supply
new cells until the end of the fourth week has an important bearing on devel-
opment of the embryo. In the cephalic part, germ layers begin their specific
differentiation by the middle of the third week (Fig. 4.12), whereas in the caudal

78 Part One: General Embryology

Figure 4.12 A. Scanning electron micrograph (dorsal view) of a mouse embryo (equiv-
alent to human at approximately 18 days), showing initial elevation of the cranial neu-
ral folds (CF ). The primitive streak lies farther caudally and is obscured from view. B.
Transverse section through the embryo shown in A (see line of section). Note three germ
layers; pseudostratified columnar cells of the neuroectoderm (Ec), flattened endoderm
(En), and mesoderm (Me) sandwiched between these two layers. Asterisks, mitotic cells.

Chapter 4: Third Week of Development: Trilaminar Germ Disc 79

part, differentiation begins by the end of the fourth week. Thus gastrulation,
or formation of the germ layers, continues in caudal segments while cranial
structures are differentiating, causing the embryo to develop cephalocaudally
(Fig. 4.12).

CLINICAL CORRELATES

Teratogenesis Associated With Gastrulation

The beginning of the third week of development, when gastrulation is initiated,
is a highly sensitive stage for teratogenic insult. At this time, fate maps can
be made for various organ systems, such as the eyes and brain anlage, and
these cell populations may be damaged by teratogens. For example, high
doses of alcohol at this stage kill cells in the anterior midline of the germ disc,
producing a deficiency of the midline in craniofacial structures and resulting
in holoprosencephaly. In such a child, the forebrain is small, the two lateral
ventricles often merge into a single ventricle, and the eyes are close together
(hypotelorism). Because this stage is reached 2 weeks after fertilization, it is
approximately 4 weeks from the last menses. Therefore, the woman may not
recognize she is pregnant, having assumed that menstruation is late and will
begin shortly. Consequently, she may not take precautions she would normally
consider if she knew she was pregnant.

Gastrulation itself may be disrupted by genetic abnormalities and toxic
insults. In caudal dysgenesis (sirenomelia), insufficient mesoderm is formed
in the caudal-most region of the embryo. Because this mesoderm contributes
to formation of the lower limbs, urogenital system (intermediate mesoderm),
and lumbosacral vertebrae, abnormalities in these structures ensue. Affected
individuals exhibit a variable range of defects, including hypoplasia and fusion
of the lower limbs, vertebral abnormalities, renal agenesis, imperforate anus,
and anomalies of the genital organs (Fig. 4.13). In humans, the condition is
associated with maternal diabetes and other causes. In mice, abnormalities
of Brachyury (T), Wnt, and engrailed genes produce a similar phenotype.

Situs inversus is a condition in which transposition of the viscera in the
thorax and abdomen occurs. Despite this organ reversal, other structural ab-
normalities occur only slightly more frequently in these individuals. Approxi-
mately 20% of patients with complete situs inversus also have bronchiectasis
and chronic sinusitis because of abnormal cilia (Kartagener syndrome). Inter-
estingly, cilia are normally present on the ventral surface of the primitive node
and may be involved in left-right patterning during gastrulation. Other condi-
tions of abnormal sidedness are known as laterality sequences. Patients with
these conditions do not have complete situs inversus but appear to be predom-
inantly bilaterally left sided or right sided. The spleen reflects the differences;
those with left-sided bilaterality have polysplenia, and those with right-sided
bilaterality have asplenia or hypoplastic spleen. Patients with laterality se-
quences also are likely to have other malformations, especially heart defects.

80 Part One: General Embryology

Figure 4.13 Sirenomelia (caudal dysgenesis). Loss of mesoderm in the lumbosacral
region has resulted in fusion of the limb buds and other defects.

Tumors Associated With Gastrulation
Sometimes, remnants of the primitive streak persist in the sacrococcygeal
region. These clusters of pluripotent cells proliferate and form tumors, known
as sacrococcygeal teratomas, that commonly contain tissues derived from all
three germ layers (Fig. 4.14). This is the most common tumor in newborns,
occurring with a frequency of one in 37,000. These tumors may also arise
from primordial germ cells (PGCs) that fail to migrate to the gonadal ridge
(see p. 4).

Further Development of the Trophoblast
By the beginning of the third week, the trophoblast is characterized by pri-
mary villi that consist of a cytotrophoblastic core covered by a syncytial layer
(Figs. 3.6 and 4.15). During further development, mesodermal cells penetrate
the core of primary villi and grow toward the decidua. The newly formed struc-
ture is known as a secondary villus (Fig. 4.15).

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. 4.15). The villus is now known as a tertiary villus

Chapter 4: Third Week of Development: Trilaminar Germ Disc 81

Figure 4.14 Sacrococcygeal teratoma resulting from remnants of the primitive streak.
These tumors may become malignant and are most common in females.

Figure 4.15 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.

82 Part One: General Embryology

Figure 4.16 Longitudinal section through a villus at the end of the third week of de-
velopment. 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 intraembry-
onic vessels.

or definitive placental villus. Capillaries in tertiary villi make contact with
capillaries developing in mesoderm of the chorionic plate and in the connecting
stalk (Figs. 4.16 and 4.17). These vessels, 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. Here they
establish contact with similar extensions of neighboring villous stems, forming
a thin outer cytotrophoblast shell (Figs. 4.16 and 4.17). This shell gradually
surrounds the trophoblast entirely and attaches the chorionic sac firmly to the
maternal endometrial tissue (Fig. 4.17). 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 6) 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 (Fig. 4.18).

The chorionic cavity, meanwhile, becomes larger, and by the 19th or 20th
day, the embryo is attached to its trophoblastic shell by a narrow connecting
stalk (Fig. 4.17). The connecting stalk later develops into the umbilical cord,
which forms the connection between placenta and embryo.

Chapter 4: Third Week of Development: Trilaminar Germ Disc 83

Figure 4.17 Presomite embryo and the trophoblast at the end of the third week. Tertiary
and secondary stem villi give the trophoblast a characteristic radial appearance. Inter-
villous 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.

Summary

The most characteristic event occurring during the third week is gas-
trulation, 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 lay-
ers, endoderm and mesoderm. Hence, epiblast gives rise to all three germ
layers in the embryo. Cells of the intraembryonic mesodermal germ layer
migrate between the two other germ layers until they establish contact with
the extraembryonic mesoderm covering the yolk sac and amnion (Figs. 4.3
and 4.4).
Prenotochordal cells invaginating in the primitive pit move forward until
they reach the prechordal plate. They intercalate in the endoderm as the noto-
chordal plate (Fig. 4.4). With further development, the plate detaches from the
endoderm, and a solid cord, the notochord, is formed. It forms a midline axis,

84 Part One: General Embryology

Figure 4.18 Stem villi (SV) extend from the chorionic plate (CP) to the basal plate (BP).
Terminal villi (arrows) are represented by fine branches from stem villi.

which will serve as the basis of the axial skeleton (Fig. 4.4). Cephalic and cau-
dal 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 anterior visceral endoderm that expresses head-forming genes, including
OTX2, LIM1, and HESX1 and the secreted factor cerberus. Nodal, a member
of the TGF-β family of genes, is then activated and initiates and maintains the
integrity of the node and streak. BMP-4, in the presence of FGF, ventralizes
mesoderm during gastrulation so that it forms intermediate and lateral plate
mesoderm. Chordin, noggin, and follistatin antagonize BMP-4 activity and
dorsalize mesoderm to form the notochord and somitomeres in the head re-
gion. Formation of these structures in more caudal regions is regulated by the
Brachyury (T) gene. Left-right asymmetry is regulated by a cascade of genes;
first, FGF-8, secreted by cells in the node and streak, induces Nodal and Lefty-2
expression on the left side. These genes upregulate PITX2, a transcription factor
responsible for left sidedness.

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. 4.11).

Chapter 4: Third Week of Development: Trilaminar Germ Disc 85

By the end of the third week, three basic germ layers, consisting of ec-
toderm, 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 4th 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. 4.17). When these
villous capillaries make contact with capillaries in the chorionic plate and con-
necting stalk, the villous system is ready to supply the embryo with its nutrients
and oxygen (Fig. 4.17).

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?

SUGGESTED READING

Augustine K, Liu ET, Sadler TW: Antisense attenuation of Wnt-1 and Wnt-3a expression in whole
embryo culture reveals roles for these genes in craniofacial, spinal cord, and cardiac morpho-
genesis. Dev Genet 14:500, 1993.

Beddington RSP: The origin of the foetal tissues during gastrulation in the rodent. In Johnson MH
(ed): Development in Mammals. New York, Elsevier, 99:1, 1983.

Beddington RSP: Induction of a second neural axis by the mouse node. Development 120:613,
1994.

Beddington RSP, Robertson RJ: Anterior patterning in the mouse. Trends Genet 14:277, 1998.
Bellairs R: The primitive streak. Anat Embryol 174:1, 1986.
De Robertis EM, Sasai Y: A common plan for dorsoventral patterning in Bilateria. Nature 380:37,

1996.
Herrmann BG: Expression pattern of the brachyuria gene in whole mount TWis/TWis mutant

embryos. Development 13:913, 1991.

86 Part One: General Embryology

Holzgreve W, Flake AW, Langer JC: The fetus with sacrococcygeal teratoma. In Harrison
MR, Gollus MS, Filly RA (eds): The Unborn Patient. Prenatal Diagnosis and Treatment. Philadelphia,
WB Saunders, 1991.

King BF, Mais JJ: Developmental changes in rhesus monkey placental villi and cell columns.
Anat Embryol 165:361, 1982.

Meyers EN, Martin GR: Differences in left-right axis pathways in mouse and chick: Functions of
FGF-8 and SHH. Science 285:403, 1999.

O’Rahilly R: Developmental Stages in Human Embryos. Part A. Embryos of the First Three Weeks
(Stages One to Nine). Washington, DC, Carnegie Institution of Washington, 1973.

Stott D, Kisbert A, Herrmann BG: Rescue of the tail defect of Brachyury mice. Genes Dev
7:197, 1993.

Sulik KK, Lauder JM, Dehart DB: Brain malformations in prenatal mice following acute maternal
ethanol administration. Int J Dev Neurosci 2:203, 1984.

Supp DM, Potter SS, Brueckner M: Molecular motors: The driving force behind mammalian left-
right development. Trends Cell Biol 10:41, 2000.

Tam PPL, Bedding RSP: The formation of mesodermal tissues in the mouse embryo during gastru-
lation and early organogenesis. Development 99:109, 1987.

chapter 5

Third to Eighth Week:
The Embryonic Period

The embryonic period or period of organogenesis,
occurs from the third to the eighth weeks of devel-
opment and is the time when each of the three germ
layers, ectoderm, mesoderm, and endoderm, gives
rise to a number of specific tissues and organs. By the
end of the embryonic period, the main organ systems
have been established, rendering the major features of
the external body form recognizable by the end of the
second month.

Derivatives of the Ectodermal
Germ Layer

At the beginning of the third week of development, the ectodermal
germ layer has the shape of a disc that is broader in the cephalic
than the caudal region (Fig. 5.1). Appearance of the notochord and
prechordal mesoderm induces the overlying ectoderm to thicken and
form the neural plate (Fig. 5.2). Cells of the plate make up the neu-
roectoderm and their induction represents the initial event in the
process of neurulation.

MOLECULAR REGULATION OF NEURAL INDUCTION
Blocking the activity of BMP-4, a TGF-β family member responsible

for ventralizing ectoderm and mesoderm, causes induction of the

87

88 Part One: General Embryology

Figure 5.1 A. Dorsal view of a 16-day presomite embryo. The primitive streak and
primitive node are visible. B. Dorsal view of an 18-day presomite embryo. The embryo
is pear-shaped, with its cephalic region somewhat broader than its caudal end. C. Dorsal
view of an 18-day human embryo. Note the primitive node and, extending forward from
it, the notochord. The yolk sac has a somewhat mottled appearance. The length of the
embryo is 1.25 mm, and the greatest width is 0.68 mm.

Chapter 5: Third to Eighth Week: The Embryonic Period 89

Figure 5.2 A. Dorsal view of a late presomite embryo (approximately 19 days). The am-
nion has been removed and the neural plate is clearly visible. B. Dorsal view of a human
embryo at approximately 20 days showing somites and formation of the neural groove
and neural folds. C. Scanning electron micrograph of a mouse embryo (approximately
20-day human) showing the typical appearance of the neural groove stage. Cranial neu-
ral folds have segregated themselves into forebrain (F, prosencephalon), midbrain (M,
mesencephalon), and hindbrain (H, rhombencephalon) regions.

90 Part One: General Embryology

neural plate. Thus, in the presence of BMP-4, which permeates the mesoderm
and ectoderm of the gastrulating embryo, ectoderm becomes epidermis, and
mesoderm forms intermediate and lateral plate mesoderm. If BMP-4 is ab-
sent or inactivated, ectoderm becomes neuralized. Secretion of three other
molecules, noggin, chordin, and follistatin, inactivates this protein. These
three proteins are present in the organizer (primitive node), notochord, and pre-
chordal mesoderm. They neuralize ectoderm and cause mesoderm to become
notochord and paraxial mesoderm (dorsalizes mesoderm). However, these neu-
ral inducers induce only forebrain and midbrain types of tissues. Induction of
caudal neural plate structures (hindbrain and spinal cord) depends upon two
secreted proteins, WNT-3a and FGF (fibroblast growth factor). In addition,
retinoic acid appears to play a role in organizing the cranial-to-caudal axis be-
cause it can cause respecification of cranial segments into more caudal ones
by regulating expression of homeobox genes (see p. 105).

NEURULATION

Once induction has occurred, the elongated, slipper-shaped neural plate grad-
ually expands toward the primitive streak (Fig. 5.2, B and C ). By the end of the
third week, the lateral edges of the neural plate become more elevated to form
neural folds, and the depressed midregion forms the neural groove (Figs. 5.2,
5.3, A and B, and 5.4). Gradually, the neural folds approach each other in the
midline, where they fuse (Fig. 5.3C ). Fusion begins in the cervical region (fifth
somite) and proceeds cranially and caudally (Figs. 5.5 and 5.6). As a result,
the neural tube is formed. Until fusion is complete, the cephalic and caudal
ends of the neural tube communicate with the amniotic cavity by way of the
cranial and caudal neuropores, respectively (Figs. 5.5, 5.6A, and 5.7). Clo-
sure of the cranial neuropore occurs at approximately day 25 (18- to 20-somite
stage), whereas the posterior neuropore closes at day 27 (25-somite stage).
Neurulation is then complete, and the central nervous system is represented
by a closed tubular structure with a narrow caudal portion, the spinal cord,
and a much broader cephalic portion characterized by a number of dilations,
the brain vesicles (see Chapter 19).

As the neural folds elevate and fuse, cells at the lateral border or crest of the
neuroectoderm begin to dissociate from their neighbors. This cell population,
the neural crest (Figs. 5.3 and 5.4), will undergo an epithelial-to-mesenchymal
transition as it leaves the neuroectoderm by active migration and displacement
to enter the underlying mesoderm. (Mesoderm refers to cells derived from the
epiblast and extraembryonic tissues. Mesenchyme refers to loosely organized
embryonic connective tissue regardless of origin.) Crest cells from the trunk
region leave the neural folds after closure of the neural tube and migrate along
one of two pathways: 1) a dorsal pathway through the dermis, where they will
enter the ectoderm through holes in the basal lamina to form melanocytes in
the skin and hair follicles; and 2) a ventral pathway through the anterior half of
each somite to become sensory ganglia, sympathetic and enteric neurons,

Chapter 5: Third to Eighth Week: The Embryonic Period 91

Neural crest Dorsal
root ganglion

Sympathetic
ganglion

A

Developing Preaortic
suprarenal ganglion

gland

Enteric
ganglia

B C Urogenital

ridge

Figure 5.3 Formation and migration of neural crest cells in the spinal cord. A and B.
Crest cells form at the tips of neural folds and do not migrate away from this region until
neural tube closure is complete. C. After migration, crest cells contribute to a hetero-
geneous array of structures, including dorsal root ganglia, sympathetic chain ganglia,
adrenal medulla, and other tissues (Table 5.1). D. In a scanning electron micrograph
of a mouse embryo, crest cells at the top of the closed neural tube can be seen mi-
grating away from this area (arrow). E. In a lateral view with the overlying ectoderm
removed, crest cells appear fibroblastic as they move down the sides of the neural tube.
(S, somites).

Schwann cells, and cells of the adrenal medulla (Fig. 5.3). Neural crest cells
also form and migrate from cranial neural folds, leaving the neural tube be-
fore closure in this region (Fig. 5.4). These cells contribute to the craniofacial
skeleton as well as neurons for cranial ganglia, glial cells, melanocytes, and
other cell types (Table 5.1). Induction of neural crest cells requires an interaction
between adjacent neural and overlying ectoderm. Bone morphogenetic pro-
teins (BMPs), secreted by non-neural ectoderm, appear to initiate the induction

92 Part One: General Embryology

A

NF

B

Figure 5.4 A. Cross section through the cranial neural folds of a mouse embryo. Neu-
ral crest cells at the tip of the folds (arrow) migrate and contribute to craniofacial mes-
enchyme. B. Lateral view of the cranial neural folds of a mouse embryo with the surface
ectoderm removed. Numerous neural crest cells can be observed leaving the neural
folds (NF ) and migrating beneath the ectoderm that has been removed. Unlike crest
cells of the spinal cord, cranial crest exits the neural folds before they fuse.

process. Crest cells give rise to a heterogeneous array of tissues, as indicated
in Table 5.1 (see p. 95).

By the time the neural tube is closed, two bilateral ectodermal thickenings,
the otic placodes and the lens placodes, become visible in the cephalic region
of the embryo (Fig. 5.8B). During further development, the otic placodes in-
vaginate and form the otic vesicles, which will develop into structures needed
for hearing and maintenance of equilibrium (see Chapter 16). At approximately
the same time, the lens placodes appear. These placodes also invaginate and,
during the fifth week, form the lenses of the eyes (see Chapter 17).

In general terms, the ectodermal germ layer gives rise to organs and struc-
tures that maintain contact with the outside world: (a) the central nervous

Chapter 5: Third to Eighth Week: The Embryonic Period 93

Figure 5.5 A. Dorsal view of a human embryo at approximately day 22. Seven distinct
somites are visible on each side of the neural tube. B. Dorsal view of a human embryo
at approximately day 23. Note the pericardial bulge on each side of the midline in the
cephalic part of the embryo.

system; (b) the peripheral nervous system; (c) the sensory epithelium of the
ear, nose, and eye; and (d) the epidermis, including the hair and nails. In addi-
tion, it gives rise to subcutaneous glands, the mammary glands, the pituitary
gland, and enamel of the teeth.

Derivatives of the Mesodermal Germ Layer

Initially, cells of the mesodermal germ layer form a thin sheet of loosely
woven tissue on each side of the midline (Fig. 5.9A). By approximately the
17th day, however, cells close to the midline proliferate and form a thick-
ened plate of tissue known as paraxial mesoderm (Fig. 5.9B). More later-
ally, the mesoderm layer remains thin and is known as the lateral plate.
With the appearance and coalescence of intercellular cavities in the lateral
plate, this tissue is divided into two layers (Fig. 5.9, B and C ): (a) a layer
continuous with mesoderm covering the amnion, known as the somatic or
parietal mesoderm layer; and (b) a layer continuous with mesoderm cov-
ering the yolk sac, known as the splanchnic or visceral mesoderm layer
(Figs. 5.9, C and D, and 5.10). Together, these layers line a newly formed
cavity, the intraembryonic cavity, which is continuous with the extraem-
bryonic cavity on each side of the embryo. Intermediate mesoderm con-
nects paraxial and lateral plate mesoderm (Figs. 5.9, B and D, and 5.10).

94 Part One: General Embryology

Figure 5.6 Dorsal (A) and ventral (B) views of a mouse embryo (approximately 22-day
human). A. The neural groove is closing in cranial and caudal directions and is flanked
by pairs of somites (S ). B. The same embryo showing formation of the gut tube with
anterior and posterior intestinal portals (arrowheads), heart (H ) in the pericardial cavity
(asterisks), and the septum transversum (arrow) representing the primordium of the
diaphragm (see Chapter 11). The neural folds remain open, exposing forebrain and
midbrain regions.

Figure 5.7 A 12- to 13-somite embryo (approximately 23 days). The embryo within its
amniotic sac is attached to the chorion by the connecting stalk. Note the well-developed
chorionic villi.

Chapter 5: Third to Eighth Week: The Embryonic Period 95

TABLE 5.1 Neural Crest Derivatives

Connective tissue and bones of the face and skull
Cranial nerve ganglia (see Table 19.2)
C cells of the thyroid gland
Conotruncal septum in the heart
Odontoblasts
Dermis in face and neck
Spinal (dorsal root) ganglia
Sympathetic chain and preaortic ganglia
Parasympathetic ganglia of the gastrointestinal tract
Adrenal medulla
Schwann cells
Glial cells
Arachnoid and pia mater (leptomeninges)
Melanocytes

Figure 5.8 A. Lateral view of a 14-somite embryo (approximately 25 days). Note the
bulging pericardial area and the first and second pharyngeal arches. B. The left side of
a 25-somite embryo approximately 28 days old. The first three pharyngeal arches and
lens and otic placodes are visible.

PARAXIAL MESODERM

By the beginning of the third week, paraxial mesoderm is organized into seg-
ments. These segments, known as somitomeres, first appear in the cephalic re-
gion of the embryo, and their formation proceeds cephalocaudally. Each somit-
omere consists of mesodermal cells arranged in concentric whorls around the
center of the unit. In the head region, somitomeres form in association with

96 Part One: General Embryology

Notochord Amniotic cavity Paraxial Intermediate
Ectoderm mesoderm mesoderm

Mesoderm Intercellular
cavities in
Dorsal
aorta lateral
plate

A B

Amnion Neural groove Intermediate Somite
mesoderm
Parietal
mesoderm

layer

Visceral
mesoderm

layer

Intra-
embryonic
body cavity

Endoderm

CD

Figure 5.9 Transverse sections showing development of the mesodermal germ layer.
A. Day 17. B. Day 19. C. Day 20. D. Day 21. The thin mesodermal sheet gives rise to
paraxial mesoderm (future somites), intermediate mesoderm (future excretory units),
and lateral plate, which is split into parietal and visceral mesoderm layers lining the
intraembryonic cavity.

So S Ne

*

lc

Sp En

Figure 5.10 Transverse sections through cervical somites of mouse embryos (approx-
imately 21-day human) as visualized by scanning electron microscopy. Arrow, noto-
chord; arrowhead, neural canal; En, endoderm; Ic, intraembryonic cavity; Ne, neuroec-
toderm; S, Somite; So, somatic mesoderm; and Sp, splanchnic mesoderm.