Gastrulation: Formation of Embryonic Mesoderm and Endoderm
The most characteristic event occurring during the third week of gestation is gastrulation, the process that establishes all three germ layers (ectoderm, mesoderm, and endoderm) in the embryo. Gastrulation begins with formation of the primitive streak on the surface of the epiblast (Figs. 5.1 and 5.2A). Initially, the streak is vaguely defined (Fig. 5.1), but in a 15-to 16-day embryo, it is clearly visible as a narrow groove with slightly bulging regions on either side. The cephalic end of the streak, the primitive node, consists of a slightly elevated area surrounding the small primitive pit (Fig. 5.2). Cells of the epiblast migrate toward the primitive streak (Fig. 5.2). Upon arrival in the region of the streak, they become flask-shaped, detach from the epiblast, and slip beneath it (Fig. 5.2B,C). This inward movement is known as invagination. Cell migration and specification are controlled by fibroblast growth factor 8 (FGF8), which is synthesized by streak cells themselves. This growth factor controls cell movement by downregulating E-cadherin, a protein that normally binds epiblast cells together. FGF8 then controls cell specification into the mesoderm by regulating Brachyury (T) expression. Once the cells have invaginated, some displace the hypoblast, creating the embryonic endoderm, and others come to lie between the epiblast and newly created endoderm to form mesoderm. Cells remaining in the epiblast then form ectoderm. Thus, the epiblast, through the process of gastrulation, is the source of all of the germ layers (Fig. 5.2B), and cells in these layers will give rise to all of the tissues and organs in the embryo.
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.
A. Dorsal side of the germ disc from a 16-day embryo indicating the movement of surface epiblast cells (solid black lines) through the primitive streak and node and the subsequent migration of cells between the hypoblast and epiblast (broken lines). B. Cross section through the cranial region of the streak at 15 days showing invagination of epiblast cells. The first cells to move inward displace the hypoblast to create the definitive endoderm. Once definitive endoderm is established, inwardly moving epiblast forms mesoderm. C. Dorsal view of an embryo showing the primitive node and streak and a cross section through the streak. The view is similar to the illustration in B; arrow, detaching epiblast cells in the primitive streak.
As more and more cells move between the epiblast and hypoblast layers, they begin to spread laterally and cranially (Fig. 5.2). Gradually, they migrate beyond the margin of the disc and establish contact with the extraembryonic mesoderm covering the yolk sac and amnion. In the cephalic direction, they pass on each side of the prechordal plate. The prechordal plate itself forms between the tip of the notochord and the oropharyngeal membrane and is derived from some of the first cells that migrate through the node in the midline and move in a cephalic direction. Later, the prechordal plate will be important for induction of the forebrain (Figs. 5.2 and 5.3). The oropharyngeal membrane at the cranial end of the disc consists of a small region of tightly adherent ectoderm and endoderm cells that represents the future opening of the oral cavity.
Schematic views illustrating formation of the notochord, whereby prenotochordal cells migrate through the primitive streak, become intercalated in the endoderm to form the notochordal plate, and finally detach from the endoderm to form the definitive notochord. Because these events occur in a cranial-to-caudal sequence, portions of the definitive notochord are established in the head region first. A. Drawing of a sagittal section through a 17-day embryo. The most cranial portion of the definitive notochord has formed, while prenotochordal cells caudal to this region are intercalated into the endoderm as the notochordal plate. Note that some cells migrate ahead of the notochord. These mesoderm cells form the prechordal plate that will assist in forebrain induction. B. Schematic cross section through the region of the notochordal plate. Soon, the notochordal plate will detach from the endoderm to form the definitive notochord. C. Schematic view showing the definitive notochord.
Formation of the Notochord
Prenotochordal cells invaginating in the primitive node move forward cranially in the midline until they reach the prechordal plate (Fig. 5.3). These prenotochordal cells become intercalated in the hypoblast so that for a short time, the midline of the embryo consists of two cell layers that form the notochordal plate (Fig. 5.3B). As the hypoblast is replaced by endoderm cells moving in at the streak, cells of the notochordal plate proliferate and detach from the endoderm. They then form a solid cord of cells, the definitive notochord (Fig. 5.3C), which underlies the neural tube and serves as the basis for the axial skeleton. Because elongation of the notochord is a dynamic process, the cranial end forms first, and caudal regions are added as the primitive streak assumes a more caudal position. The notochord and prenotochordal cells extend cranially to the prechordal plate (an area just caudal to the oropharyngeal membrane) and caudally to the primitive pit. At the point where the pit forms an indentation in the epiblast, the neurenteric canal temporarily connects the amniotic and yolk sac cavities (Fig. 5.3A).
The cloacal membrane is formed at the caudal end of the embryonic disc (Fig. 5.2A). This membrane, which is similar in structure to the oropharyngeal membrane, consists of tightly adherent ectoderm and endoderm cells with no intervening mesoderm. When the cloacal membrane appears, the posterior wall of the yolk sac forms a small diverticulum that extends into the connecting stalk. This diverticulum, the allantoenteric diverticulum, or allantois, appears around the 16th day of development (Fig. 5.3A). Although in some lower vertebrates the allantois serves as a reservoir for excretion products of the renal system, in humans, it remains rudimentary but may be involved in abnormalities of bladder development (see Chapter 16).
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 factors cerberus and lefty, which inhibit nodal activity in the cranial end of the embryo. These genes establish the cranial end of the embryo before gastrulation. The primitive streak itself is initiated and maintained by expression of Nodal, a member of the transforming growth factor-? (TGF-?) family (Fig. 5.4). Once the streak is formed, Nodal upregulates a number of genes responsible for formation of dorsal and ventral mesoderm and head and tail structures. Another member of the TGF-? family, bone morphogenetic protein 4 (BMP4), is secreted throughout the embryonic disc (Fig. 5.4). In the presence of this protein and FGF, mesoderm will be ventralized to contribute to kidneys (intermediate mesoderm), blood, and body wall mesoderm (lateral plate mesoderm). In fact, all mesoderm would be ventralized if the activity of BMP4 were not blocked by other genes expressed in the node. For this reason, the node is the organizer. It was given that designation by Hans Spemann, who first described this activity in the dorsal lip of the blastopore, a structure analogous to the node, in Xenopus embryos. Thus, chordin (activated by the transcription factor Goosecoid), noggin, and follistatin antagonize the activity of BMP4. As a result, cranial mesoderm is dorsalized into notochord, somites, and somitomeres (Fig. 5.4). Later, these three genes are expressed in the notochord and are important in neural induction in the cranial region.
Figure 5.4.Sagittal section through the node and primitive streak showing the expression pattern of genes regulating the craniocaudal and dorsoventral axes.
Cells at the prospective cranial end of the embryo in the AVE express the transcription factors OTX2, LIM1, and HESX1 and the secreted factor cerberus that contribute to head development and establish the cephalic region. Once the streak is formed and gastrulation is progressing, BMP4 is secreted throughout the bilaminar disc and acts with FGF to ventralize mesoderm into intermediate and lateral plate mesoderm. Goosecoid, expressed in the node, regulates chordin expression, and this gene product, together with noggin and follistatin, antagonizes the activity of BMP4, dorsalizing mesoderm into notochord and paraxial mesoderm for the head region. Later, expression of the Brachyury (T) gene antagonizes BMP4 to dorsalize mesoderm into notochord and paraxial mesoderm in caudal regions of the embryo.
As mentioned, Nodal is involved in initiating and maintaining the primitive streak. Similarly, HNF-3? maintains the node and later induces regional specificity in the forebrain and midbrain areas. Without HNF-3?, embryos fail to gastrulate properly and lack forebrain and midbrain structures. As mentioned previously, Goosecoid activates inhibitors of BMP4 and contributes to regulation of head development. Over-or underexpression of this gene in laboratory animals results in severe malformations of the head region, including duplications, similar to some types of conjoined twins (Fig. 5.5).
Figure 5.5.Conjoined twins.
If the gene Goosecoid is overexpressed in frog embryos, the result is a two-headed tadpole. Perhaps overexpression of this gene explains the origin of this type of conjoined twins.
Regulation of dorsal mesoderm formation in middle and caudal regions of the embryo is controlled by the Brachyury (T) gene expressed in the node, notochord precursor cells, and notochord. This gene is essential for cell migration through the primitive streak. Brachyury encodes a sequence-specific DNA binding protein that functions as a transcription factor. The DNA-binding domain is called the T-box, and there are more than 20 genes in the T-box family. Thus, mesoderm formation in these regions depends on this gene product, and its absence results in shortening of the embryonic axis (caudal dysgenesis). The degree of shortening depends on the time at which the protein becomes deficient.
Laterality (left–right-sidedness) also is established early in development and is orchestrated by a cascade of signal molecules and genes. When the primitive streak appears, FGF8 is secreted by cells in the node and primitive streak and induces expression of Nodal, but only on the left side of the embryo (Fig. 5.6A). Later, as the neural plate is established, FGF8 maintains Nodal expression in the lateral plate mesoderm, as well as 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 asymmetrical body positions. If the gene is expressed ectopically (e.g., on the right side), this abnormal expression results in laterality defects, including situs inversus and dextrocardia (placement of the heart to the right side; see p. 57). Simultaneously, LEFTY is expressed on the left side of the floor plate of the neural tube and may act as a barrier to prevent left-sided signals from crossing over. Sonic hedgehog(SHH) may also function in this role as well as serving as a repressor for left-sided gene expression on the right. The Brachyury (T) gene, encoding a transcription factor secreted by the notochord, is also essential for expression of Nodal, LEFTY-1, and LEFTY-2 (Fig. 5.6B). Importantly, the neurotransmitter serotonin (5HT) also plays a critical role in this signaling cascade that establishes laterality. 5HT is concentrated on the left side, probably because it is broken down by its metabolizing enzyme monoamine oxidase (MAO) on the right, and is upstream from FGF8 signaling (Fig. 5.6B). Alterations in 5HT signaling result in situs inversus, dextrocardia, and a variety of heart defects (see Clinical Correlations).
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-? 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.
Figure 5.7.Dorsal view of the germ disc showing the primitive streak and a fate map for epiblast cells.
Specific regions of the epiblast migrate through different parts of the node and streak to form mesoderm. Thus, cells migrating at the cranialmost part of the node will form the notochord (n); those migrating more posteriorly through the node and cranialmost aspect of the streak will form paraxial mesoderm (pm; somitomeres and somites); those migrating through the next portion of the streak will form intermediate mesoderm (im; urogenital system); those migrating through the more caudal part of the streak will form lateral plate mesoderm (lpm; body wall); and those migrating through the most caudal part will contribute to extraembryonic mesoderm (eem; chorion).