51
Chapter 5
52 Part 1 General Embryology
into the connecting stalk. This diverticulum, the allantoenteric diverticulum, or allantois, appears around the 16th day of development (Fig. 5.3A). Although in some lower vertebrates the allantois serves as a reservoir for excretion prod-ucts of the renal system, in humans, it remains rudimentary but may be involved in abnormalities of bladder development (see Chapter 16, p. 240).
ESTABLISHMENT OF THE BODY AXES
Establishment of the body axes, anteroposterior, dorsoventral, and left–right, takes place before and during the period of gastrulation. The anteropos-terior axis is signaled by cells at the ananteropos-terior (cra-nial) 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 factors cerberus and lefty, which inhibit nodal activity in the cranial end of the embryo. These genes establish the cra-nial end of the embryo before gastrulation. The primitive streak itself is initiated and maintained by expression of Nodal, a member of the trans-forming growth factor-b (TGF-b) family (Fig. 5.4). Once the streak is formed, Nodal upreg-ulates a number of genes responsible for formation of dorsal and ventral mesoderm and head and tail structures. Another member of the TGF-b family, bone morphogenetic protein 4 (BMP4), is secreted throughout the embryonic disc (Fig. 5.4).
In the presence of this protein and FGF, mesoderm
Amniotic cavity Epiblast Hypoblast
Wall of yolk sac Epiblast
Hypoblast Primitive
streak
Cut edge of amnion
Extraembryonic mesoderm Cytotrophoblast Syncytiotrophoblast
Definitive yolk sac
A
B
Oropharyngeal membrane
Figure 5.1 A. Implantation site at the end of the second week. B. Representative view of the germ disc at the end of the second week of development. The amniotic cavity has been opened to permit a view of the dorsal side of the epiblast. The hypoblast and epiblast are in contact with each other, and the primitive streak forms a shallow groove in the caudal region of the embryo.
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Chapter 5 Third Week of Development: Trilaminar Germ Disc 53
Oropharyngeal membrane
Cut edge of amnion Prenotochordal cells
Cloacal membrane Primitive node
(the organizer) Primitive streak A
Yolk sac Hypoblast
Amnioblasts Epiblast
Invaginating mesoderm cells Primitive node
Primitive streak
B
Primitive node Primitive node
Primitive streak Primitive streak
Epiblast Epiblast Hypoblast
Detaching cells
C
Figure 5.2 A. Dorsal side of the germ disc from a 16-day embryo indicating the movement of surface epiblast cells (solid black lines) through the primitive streak and node and the subsequent migration of cells between the hypoblast and epiblast (broken lines). B. Cross section through the cranial region of the streak at 15 days showing invagination of epiblast cells. The fi rst cells to move inward displace the hypoblast to create the defi nitive endoderm. Once defi nitive endoderm is established, inwardly moving epiblast forms mesoderm. C. Dorsal view of an embryo showing the primitive node and streak and a cross section through the streak. The view is similar to the illustration in B; arrow, detaching epiblast cells in the primitive streak.
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54 Part 1 General Embryology
Notochord
Notochordal plate
Intraembryonic
mesoderm Endoderm
Intraembryonic mesoderm
Extraembryonic mesoderm Endoderm
A
B
C
Primitive pit and neurenteric canal
Connecting stalk
Allantois Notochord
Prechordal mesoderm Wall of
yolk sac
Oropharyngeal membrane
Ectoderm B
Cut lines for C
Cloacal plate (membrane) Amnion
Figure 5.3 Schematic views illustrating formation of the notochord, whereby prenotochordal cells migrate through the primitive streak, become intercalated in the endoderm to form the notochordal plate, and fi nally detach from the endoderm to form the defi nitive notochord. Because these events occur in a cranial-to-caudal sequence, portions of the defi nitive notochord are established in the head region fi rst. A. Drawing of a sagittal section through a 17-day embryo. The most cranial portion of the defi nitive notochord has formed, while prenotochordal cells caudal to this region are intercalated into the endoderm as the notochordal plate. Note that some cells migrate ahead of the notochord. These mesoderm cells form the prechordal plate that will assist in forebrain induction. B. Schematic cross section through the region of the notochordal plate.
Soon, the notochordal plate will detach from the endoderm to form the defi nitive notochord. C. Schematic view showing the defi nitive notochord.
Goosecoid, chordin, noggin, follistatin, nodal
AVE
Figure 5.4 Sagittal section through the node and primitive streak showing the expression pattern of genes regulating the craniocaudal and dorsoventral axes. Cells at the prospective cranial end of the embryo in the AVE express the transcription factors OTX2, LIM1, and HESX1 and the secreted factor cerberus that contribute to head development and establish the cephalic region. Once the streak is formed and gastrulation is progressing, BMP4 is secreted throughout the bilaminar disc and acts with FGF to ventralize mesoderm into intermediate and lateral plate mesoderm. Goosecoid, expressed in the node, regulates chordin expression, and this gene product, together with noggin and follistatin, antagonizes the activity of BMP4, dor-salizing mesoderm into notochord and paraxial mesoderm for the head region. Later, expression of the Brachyury (T) gene antagonizes BMP4 to dorsalize mesoderm into notochord and paraxial mesoderm in caudal regions of the embryo.
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Chapter 5 Third Week of Development: Trilaminar Germ Disc 55
will be ventralized to contribute to kidneys (intermediate mesoderm), blood, and body wall mesoderm (lateral plate mesoderm). In fact, all mesoderm would be ventralized if the activity of BMP4 were not blocked by other genes expressed in the node. For this reason, the node is the orga-nizer. It was given that designation by Hans Spemann, who fi rst described this activity in the dorsal lip of the blastopore, a structure analogous to the node, in Xenopus embryos. Thus, chordin (activated by the transcription factor Goosecoid), noggin, and follistatin antagonize the activity of BMP4. As a result, cranial mesoderm is dorsalized into notochord, somites, and somitomeres (Fig. 5.4).
Later, these three genes are expressed in the noto-chord and are important in neural induction in the cranial region.
As mentioned, Nodal is involved in initiating and maintaining the primitive streak. Similarly, HNF-3b maintains the node and later induces regional specifi city in the forebrain and midbrain areas. Without HNF-3b, embryos fail to gastrulate properly and lack forebrain and midbrain struc-tures. As mentioned previously, Goosecoid activates inhibitors of BMP4 and contributes to regulation of head development. Over- or underexpression of this gene in laboratory animals results in severe malformations of the head region, including duplications, similar to some types of conjoined twins (Fig. 5.5).
Regulation of dorsal mesoderm formation in middle and caudal regions of the embryo is controlled by the Brachyury (T) gene expressed in the node, notochord precursor cells, and notochord. This gene is essential for cell migra-tion through the primitive streak. Brachyury encodes a sequence-specifi c DNA binding protein that functions as a transcription factor.
The DNA-binding domain is called the T-box, and there are more than 20 genes in the T-box family. Thus, mesoderm formation in these regions depends on this gene product, and its absence results in shortening of the embryonic axis (caudal dysgenesis). The degree of shorten-ing depends on the time at which the protein becomes defi cient.
Laterality (left–right-sidedness) also is estab-lished early in development and is orchestrated by a cascade of signal molecules and genes. When the primitive streak appears, FGF8 is secreted by cells in the node and primitive streak and induces expression of Nodal, but only on the left side of the embryo (Fig. 5.6A). Later, as the neural plate is established, FGF8 maintains Nodal expression in the lateral plate mesoderm, as well as LEFTY-2, and both of these genes upregulate PITX2.
PITX2 is a homeobox-containing transcription factor responsible for establishing left-sidedness (Fig. 5.6B) and its expression is repeated on the left side of the heart, stomach, and gut primordia as these organs are assuming their normal asym-metrical body positions. If the gene is expressed ectopically (e.g., on the right side), this abnormal expression results in laterality defects, including situs inversus and dextrocardia (placement of the heart to the right side; see p. 57). Simultaneously, LEFTY is expressed on the left side of the fl oor plate of the neural tube and may act as a bar-rier to prevent left-sided signals from crossing over. Sonic hedgehog (SHH) may also func-tion in this role as well as serving as a repres-sor for left-sided gene expression on the right.
The Brachyury (T) gene, encoding a transcription factor secreted by the notochord, is also essential for expression of Nodal, LEFTY-1, and LEFTY-2 (Fig. 5.6B). Importantly, the neurotransmitter serotonin (5HT) also plays a critical role in this signaling cascade that establishes laterality. 5HT is concentrated on the left side, probably because it is broken down by its metabolizing enzyme monoamine oxidase (MAO) on the right, and is upstream from FGF8 signaling (Fig. 5.6B).
Alterations in 5HT signaling result in situs inver-sus, dextrocardia, and a variety of heart defects (see Clinical Correlations, p. 57).
Genes regulating right-sided development are not as well defi ned, although expression of the Figure 5.5 Conjoined twins. If the gene Goosecoid is
overexpressed in frog embryos, the result is a two-headed tadpole. Perhaps overexpression of this gene explains the origin of this type of conjoined twins.
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56 Part 1 General Embryology
Figure 5.6 Dorsal views of the germ disc showing gene expression patterns responsible for establishing the left–right body axis. A. FGF8, secreted by the node and primitive streak, establishes expression of Nodal, a member of the TGF-b superfamily, and the nodal protein then accumulates on the left side near the node. B. Later, as the neural plate starts to form, FGF8 induces expression of Nodal and LEFTY-2 in the lateral plate mesoderm, whereas LEFTY-1 is expressed on the left side of the ventral aspect of the neural tube. These signals are dependent upon the neurotransmitter serotonin (5HT) that is upstream of FGF8 and that increases in concentration on the left because of its metabolism by MAO on the right.
Products from the Brachyury (T) gene, expressed in the notochord, also participate in induction of these three genes. In turn, expression of Nodal and LEFTY-2 regulates expression of the transcription factor PITX 2, which, through further downstream effectors, establishes left-sidedness. SHH, expressed in the notochord, may serve as a midline barrier and also represses expression of left-sided genes on the right. Expression of the transcription factor Snail may regulate downstream genes important for establishing right-sidedness.
Oropharyngeal membrane
Nodal
FGF8
Node
Cloacal membrane A
Nodal MAO
Lefty 1
Primitive node (FGF8) Primitive
streak
Cloacal membrane Oropharyngeal
membrane
FGF8 5HT
Nodal Lefty2 PITX2
Notochord (SHH)
B
Figure 5.7 Dorsal view of the germ disc showing the primitive streak and a fate map for epiblast cells. Specifi c regions of the epiblast migrate through different parts of the node and streak to form mesoderm. Thus, cells migrating at the crani-almost part of the node will form the notochord (n); those migrating more posteriorly through the node and cranicrani-almost 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).
n pm
Oropharyngeal membrane
Cloacal membrane imlpm
lpmim
eem eem
pm
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A B
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Maternal sinusoid Connecting
stalk Trophoblastic
lacunae
Extraembryonic cavity (chorionic cavity)
Extraembryonic somatopleuric
mesoderm (chorionic plate) Oropharyngeal
membrane Primary villi
Secondary yolk sac Amniotic
cavity
Exocoelomic cyst
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60 Part 1 General Embryology
Connecting stalk Outer
cytotrophoblast shell
Chorionic cavity Chorionic plate
Exocoelomic cyst Tertiary
stem villi Intervillous spaces Syncytium
Definitive yolk sac Amniotic cavity
Figure 5.12 Presomite embryo and the trophoblast at the end of the third week. Tertiary and secondary stem villi give the trophoblast a characteristic radial appearance. Intervillous spaces, which are found throughout the trophoblast, are lined with syncytium. Cytotrophoblastic cells surround the trophoblast entirely and are in direct contact with the endometrium.
The embryo is suspended in the chorionic cavity by means of the connecting stalk.
Syncytiotrophoblast
Cytotrophoblast
Mesoderm core Villous capillary
Primary villus
A B Secondary C
villus
Tertiary villus
Figure 5.11 Development of a villus. A. Transverse section of a primary villus showing a core of cytotrophoblastic cells covered by a layer of syncytium. B. Transverse section of a secondary villus with a core of mesoderm covered by a single layer of cytotrophoblastic cells, which in turn is covered by syncytium. C. Mesoderm of the villus showing a number of capil-laries and venules.
By the end of the third week, mesodermal cells in the core of the villus begin to differentiate into blood cells and small blood vessels, forming the villous capillary system (Fig. 5.11). The villus is now known as a tertiary villus or a defi ni-tive placental villus. Capillaries in tertiary villi make contact with capillaries developing in the mesoderm of the chorionic plate and in the con-necting stalk (Figs. 5.12 and 5.13). These vessels,
in turn, establish contact with the intraembry-onic circulatory system, connecting the placenta and the embryo. Hence, when the heart begins to beat in the fourth week of development, the vil-lous system is ready to supply the embryo proper with essential nutrients and oxygen.
Meanwhile, cytotrophoblastic cells in the villi penetrate progressively into the overlying syncy-tium until they reach the maternal endometrium.
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Chapter 5 Third Week of Development: Trilaminar Germ Disc 61
Here they establish contact with similar exten-sions of neighboring villous stems, forming a thin outer cytotrophoblast shell (Figs. 5.12 and 5.13). This shell gradually surrounds the tropho-blast entirely and attaches the chorionic sac fi rmly to the maternal endometrial tissue (Fig. 5.12).
Villi that extend from the chorionic plate to the decidua basalis (decidual plate: the part of the endometrium where the placenta will form; see Chapter 8) are called stem or anchoring villi.
Those that branch from the sides of stem villi are free (terminal) villi, through which exchange of nutrients and other factors will occur.
The chorionic cavity, meanwhile, becomes larger, and by the 19th or the 20th day, the embryo is attached to its trophoblastic shell by a narrow connecting stalk (Fig. 5.12). The con-necting stalk later develops into the umbilical cord, which forms the connection between the placenta and embryo.
Summary
The most characteristic event occurring during the third week is gastrulation, which begins with the appearance of the primitive streak, which has at its cephalic end the primitive node. In the region of the node and streak, epi-blast cells move inward (invaginate) to form new cell layers, endoderm and mesoderm.
Cells that do not migrate through the streak but
Maternal vessels Outer
cytotrophoblast shell
Syncytiotrophoblast
Cytotrophoblast Mesoderm core with capillaries
Chorionic plate
Chorionic cavity Connecting stalk
Intervillous space Endometrium
Figure 5.13 Longitudinal section through a villus at the end of the fourth week of development. Maternal vessels pen-etrate the cytotrophoblastic shell to enter intervillous spaces, which surround the villi. Capillaries in the villi are in contact with vessels in the chorionic plate and in the connecting stalk, which in turn are connected to intraembryonic vessels.
remain in the epiblast form ectoderm. Hence, epiblast gives rise to all three germ layers in the embryo, ectoderm, mesoderm, and endo-derm, and these layers form all of the tissues and organs (Figs. 5.2 and 5.3).
Prenotochordal cells invaginating in the primitive pit move forward until they reach the prechordal plate. They intercalate in the endo-derm as the notochordal plate (Fig. 5.3). With further development, the plate detaches from the endoderm, and a solid cord, the notochord, is formed. It forms a midline axis, which will serve as the basis of the axial skeleton (Fig. 5.3).
Cephalic and caudal ends of the embryo are established before the primitive streak is formed.
Thus, cells in the hypoblast (endoderm) at the cephalic margin of the disc form the AVE, which expresses head-forming genes, including OTX2, LIM1, and HESX1 and the secreted factor cer-berus. Nodal, a member of the TGF-b family of genes, is then activated and initiates and main-tains the integrity of the node and streak. In the presence of FGF, BMP4 ventralizes mesoderm during gastrulation so that it forms intermediate and lateral plate mesoderm. Chordin, noggin, and follistatin antagonize BMP4 activity and dorsal-ize mesoderm to form the notochord and somi-tomeres in the head region. Formation of these structures in more caudal regions is regulated by the Brachyury (T) gene (Fig. 5.4A). Laterality (left–right asymmetry) is regulated by a cascade of signaling molecules and genes. FGF8, secreted
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62 Part 1 General Embryology
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 deter-mined 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 posi-tioning 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?
by cells in the node and streak, induces Nodal and LEFTY-2 expression on the left side and these genes upregulate PITX2, a transcription factor and master gene for left-sidedness (Fig. 5.6). The neurotransmitter serotonin (5HT) also plays a role as a signal molecule upstream from FGF8.
Disruption of 5HT levels or misexpression of PITX2 results in laterality defects, such as dextro-cardia, situs inversus, and cardiac abnormalities.
Epiblast cells moving through the node and streak are predetermined by their position to become specifi c types of mesoderm and endo-derm. Thus, it is possible to construct a fate map of the epiblast showing this pattern (Fig. 5.7).
By the end of the third week, three basic germ layers, consisting of ectoderm, mesoderm, and endoderm, are established in the head region, and the process continues to produce these germ layers for more caudal areas of the embryo until the end of the fourth week. Tissue and organ dif-ferentiation has begun, and it occurs in a cepha-locaudal direction as gastrulation continues.
In the meantime, the trophoblast progresses rapidly. Primary villi obtain a mesenchymal core in which small capillaries arise (Fig. 5.12).
When these villous capillaries make contact with capillaries in the chorionic plate and connect-ing stalk, the villous system is ready to supply the embryo with its nutrients and oxygen (Fig. 5.13).
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63
T
he embryonic period, or period of organogenesis, occurs from the third to the eighth weeks of development and is the time when each of the three germ layers, ectoderm, mesoderm, and endo-derm, gives rise to a number of specifi c tis-sues 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 develop-ment, the ectodermal germ layer has the shape of a disc that is broader in the cephalic than in the caudal region (Fig. 6.1). Appearance of the notochord and prechordal mesoderm induces the overlying ectoderm to thicken and form the neural plate (Fig. 6.2A,B). Cells of the plate make up the neuroectoderm, and their induc-tion represents the initial event in the process of neurulation.
Molecular Regulation of Neural Induction
Upregulation of fi broblast growth factor (FGF) signaling together with inhibition of the activity of bone morphogenetic protein 4 (BMP4), a transforming growth factor-b (TGF-b) family member responsible for ventral-izing ectoderm and mesoderm, causes induc-tion of the neural plate. FGF signaling probably promotes a neural pathway by an unknown mechanism while it represses BMP transcription and upregulates expression of chordin and nog-gin, which inhibit BMP activity. In the presence of BMP4, which permeates the mesoderm and ectoderm of the gastrulating embryo, ectoderm is induced to form epidermis, and mesoderm forms intermediate and lateral plate mesoderm.
If ectoderm is protected from exposure to BMPs,
its “default state” is to become neural tissue.
Secretion of three other molecules, noggin, chordin, and follistatin, inactivates BMP. These three proteins are present in the organizer (primi-tive node), notochord, and prechordal mesoderm.
They neuralize ectoderm by inhibiting BMP and cause mesoderm to become notochord and par-axial mesoderm (dorsalizes mesoderm); however, these neural inducers induce only forebrain and midbrain types of tissues. Induction of caudal neural plate structures (hindbrain and spinal cord) depends on two secreted proteins, WNT3a and FGF. In addition, retinoic acid (RA) appears to play a role in organizing the cranial-to-caudal axis because it can cause respecifi cation of cranial segments into more caudal ones by regulating expression of homeobox genes (see p. 78).
Neurulation
Neurulation is the process whereby the neu-ral plate forms the neuneu-ral tube. By the end of the third week, the lateral edges of the neural plate become elevated to form neural folds, and the depressed midregion forms the neural groove (Fig. 6.2). Gradually, the neural folds approach each other in the midline, where they fuse (Fig. 6.3A,B). Fusion begins in the cervi-cal region (fi fth somite) and proceeds cranially and caudally (Fig. 6.3C,D). As a result, the neu-ral 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 anterior (cranial) and posterior (cau-dal) neuropores, respectively (Figs. 6.3C,D and 6.4A). Closure of the cranial neuropore occurs at approximately day 25 (18- to 20-somite stage), whereas the posterior neuropore closes at day 28 (25-somite stage) (Fig. 6.4B). 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 18).