Fourth Week: Forming the Embryo

Summary

During the fourth week, the tissue layers laid down in the third week differentiate to form the primordia of most of the major organ systems of the body. Simultaneously, the embryonic disc undergoes a process of folding that creates the basic vertebrate body form, called the tube-within-a-tube body plan . A major force responsible for embryonic folding is the differential growth of different portions of the embryo. The embryonic disc grows vigorously during the fourth week, particularly in length, whereas the growth of the yolk sac stagnates and this difference is postulated to help drive body folding. Folding commences in the cranial and lateral regions of the embryo on day 22, and in the caudal region on day 23. As a result of folding, the cranial, lateral, and caudal edges of the embryonic disc are brought together along the ventral midline. The endodermal, mesodermal, and ectodermal layers of the embryonic disc fuse to the corresponding layer on the opposite side, thus creating a tubular three-dimensional body form . The process of midline fusion transforms the flat embryonic endoderm into a gut tube . Initially, the gut consists of cranial and caudal blind-ending tubes— foregut and hindgut , respectively—separated by the future midgut , which remains open to the yolk sac. As the lateral edges of the various embryonic disc layers continue to join together along the ventral midline, the midgut is progressively converted into a tube, and, correspondingly, the yolk sac neck is reduced to a slender vitelline duct . When the edges of the ectoderm fuse along the ventral midline, the space formed within the lateral plate mesoderm is enclosed in the embryo and becomes the intraembryonic coelom . The lateral plate mesoderm gives rise to the serous membranes that line the coelom—the somatic mesoderm coating the inner surface of the body wall and the splanchnic mesoderm ensheathing the gut tube.

On the dorsal surface, neurulation converts the neural plate into a hollow neural tube covered by surface ectoderm . The neural tube then begins to differentiate into brain and spinal cord . Even before the end of the fourth week, the major regions of the brain— forebrain, midbrain , and hindbrain —become apparent, and neurons and glia begin to differentiate from the neuroepithelium of the neural tube. As neurulation occurs, neural crest cells detach from the dorsal neuroepithelium and migrate to numerous locations in the body, where they differentiate to form a wide range of structures and cell types.

Somites continue to segregate from the paraxial mesoderm in cranial-caudal progression until day 30. Meanwhile, beginning in the cervical region, the somites subdivide into two kinds of mesodermal primordia: dermamyotomes and sclerotomes . Dermamyotomes contribute to the dermis of the neck and trunk, as well as to the myotomes , which form the segmental musculature of the back and the ventrolateral body wall; additionally, myotomes give rise to cells that migrate into the limb buds to form the limb musculature . Sclerotomes give rise to vertebral bodies and vertebral arches and contribute to the base of the skull.

Clinical Taster

A 22-year-old university student is surprised to learn that his 19-year-old girlfriend is pregnant. They have been having sex for only 3 months and have timed intercourse using the rhythm method of birth control , at least most of the time. On their first visit to student health services, they are told that the pregnancy is now in the eighth week and all seems normal. They decide to wait 2 months until spring break, when they will visit with their families, who live in neighboring towns, to inform them about the pregnancy.

Although both sets of parents are shocked by the news, they are supportive and arrange an immediate appointment with an obstetrician. Ultrasound examination reveals that the fetus is growing normally. However, a mass of bowel is detected protruding from the ventral (anterior) body wall into the amniotic cavity. The diagnosis of gastroschisis is made ( Fig. 4.1A ). On a follow-up visit, the young mother-to-be is very anxious. She’s concerned that perhaps she did something to cause her baby to have gastroschisis. The doctor assures her that this is not the case and that sometimes developmental events just go awry, resulting in birth defects.

The couple decides to return to school to complete the semester and then to move back home, where they can receive more intensive prenatal care. Beginning at 30 weeks of gestation, weekly ultrasounds are scheduled to examine the thickness of the bowel wall. Based on evidence that the wall is beginning to thicken and thus is becoming damaged by exposure to the amniotic fluid, labor is induced at 35 weeks. At delivery, a 3-cm opening in the abdominal wall is noted to the right of the baby’s umbilicus, along with multiple loops of protruding bowel (see Fig. 4.1B ). The newborn baby is taken immediately to surgery to return the bowel to the abdominal cavity and to repair the body wall defect. Although it is a relatively common birth defect, the cause of gastroschisis remains unknown.

Tube-Within-A-Tube Body Plan Arises Through Body Folding

Animation 4.1: Body Folding .

Animations are available online at StudentConsult .

At the end of the third week, the embryo is a flat, ovoid, trilaminar disc. During the fourth week, it grows rapidly, particularly in length, and undergoes a process of folding that generates the recognizable vertebrate body form ( Figs. 4.2 , 4.3 ). Although some active remodeling of tissue layers takes place, including localized changes in cell shape within the body folds , the main force responsible for embryonic folding is the differential growth of various tissues. During the fourth week, the embryonic disc and the amnion grow vigorously, but the yolk sac hardly grows at all. Because the yolk sac is attached to the ventral rim of the embryonic disc, the differential expansion is thought to drive the ballooning of the embryonic disc into a three-dimensional, somewhat cylindrical shape. The developing notochord, neural tube, and somites stiffen the dorsal axis of the embryo; therefore most of the folding is concentrated in the thin, flexible outer rim of the disc. The cranial, caudal, and lateral margins of the disc fold completely under the dorsal axial structures and give rise to the ventral surface of the body. The areas of folding are referred to as cranial (head) , caudal (tail) , and lateral body folds , respectively. The cranial and caudal folds are best viewed in midsagittal sections ( Fig. 4.2A–C ; arrows in B), and the paired lateral body folds are best viewed in cross sections ( Fig. 4.2D , E ; arrows in D). Although in sections these folds have different names, it is important to realize that these folds become continuous with one another as a ring of tissue at the position of the future umbilicus ( Fig. 4.2F ).

As described in Chapter 3 , the cranial rim of the embryonic disc—the thin area located cranial to the neural plate—contains the oropharyngeal membrane, which represents the future mouth of the embryo. Cranial to the oropharyngeal membrane, a second important structure has begun to appear: the horseshoe-shaped cardiogenic area , which will give rise to the heart (covered in Chapter 12 ). Cranial to the cardiogenic area, a third important structure forms: the septum transversum . This structure appears on day 22 as a thickened bar of mesoderm; it lies just caudal to the cranial margin of the embryonic disc (see Fig. 4.2A–C ). The septum transversum forms the initial partition separating the coelom into thoracic and abdominal cavities and gives rise to part of the ventral mesentery of the stomach and duodenum (covered in Chapters 11 and 14 ).

Forward growth of the neural plate causes the thin cranial rim of the disc to fold under, forming the ventral surface of the future face, neck, and chest. This process translocates the oropharyngeal membrane to the region of the future mouth and carries the cardiogenic area and septum transversum toward the future chest (see Fig. 4.2A–C ).

Starting on about day 23, a similar process of folding commences in the caudal region of the embryo as the rapidly lengthening neural tube and somites overgrow the caudal rim of the yolk sac. Because of the relative stiffness of these dorsal axial structures, the thin caudal rim of the embryonic disc, which contains the cloacal membrane, folds under and becomes part of the ventral surface of the embryo (see Fig. 4.2A–C ). When the caudal rim of the disc folds under the body, the connecting stalk (which connects the caudal end of the embryonic disc to the developing placenta) is carried cranially until it merges with the neck of the yolk sac, which has begun to lengthen and constrict (see Figs. 4.2 , 4.3 ). The root of the connecting stalk contains a slender endodermal hindgut diverticulum called the allantois (see Fig. 4.2A–C ). The fate of the allantois is covered in Chapter 15 .

Simultaneously with cranial-caudal body folding, the right and left sides of the embryonic disc flex sharply ventrally, constricting and narrowing the neck of the yolk sac (see Fig. 4.2D ). At the head and tail ends of the embryo, these lateral edges of the embryonic disc make contact with each other and then zip up toward the site of the future umbilicus . When the edges meet, the ectodermal, mesodermal, and endodermal layers on each side fuse with the corresponding layers on the other side (see Fig. 4.2D,E ). As a result, the ectoderm of the original embryonic disc covers the entire surface of the three-dimensional embryo except for the future umbilical region , where the yolk sac and the connecting stalk emerge. The ectoderm, along with contributions from the dermamyotomes, lateral plate mesoderm, and neural crest cells, will eventually form the skin (covered in Chapter 7 ).

The endoderm of the trilaminar embryonic disc is destined to give rise to the lining of the gastrointestinal tract. When the cranial, caudal, and lateral edges of the embryo meet and fuse, the cranial and caudal portions of the endoderm are converted into blind-ending tubes—the future foregut and hindgut . At first, the central midgut region remains broadly open to the yolk sac (see Fig. 4.2A–D ). However, as the gut tube forms, the neck of the yolk sac is gradually constricted, reducing its communication with the midgut. By the end of the sixth week, the gut tube is fully formed, and the neck of the yolk sac has been reduced to a slim stalk called the vitelline duct (see Fig. 4.2C , F ). The cranial end of the foregut is capped by the oropharyngeal membrane, which ruptures at the end of the fourth week to form the mouth. The caudal end of the hindgut is capped by the cloacal membrane, which ruptures during the seventh week to form the orifices of the anus and the urogenital system (covered in Chapter 14, Chapter 15, Chapter 16 through 16 ).

As described in Chapter 3 , the lateral plate mesoderm splits into two layers: the somatic mesoderm , which associates with the ectoderm, and the splanchnic mesoderm , which associates with the endoderm. The space between these layers is originally open to the chorionic cavity. However, when the folds of the embryo fuse along the ventral midline, this space is enclosed within the embryo and becomes the intraembryonic coelom (see Fig. 4.2E , F ). The serous membranes lining this cavity form from the two layers of the lateral plate mesoderm: the inside of the body wall is lined with the somatic mesoderm, and the visceral organs derived from the gut tube are invested by splanchnic mesoderm.

As a result of body folding, the tube-within-a-tube body plan is established (see Fig. 4.2E , F ). This plan consists of an embryo body design composed of two main tubes: an outer ectodermal tube forming the external layer of the skin (epidermis) and an inner endodermal tube forming the inner layer of the gut. The space between the two tubes is filled mainly with mesoderm, the lateral plate mesodermal part of which splits to form the body cavity, or coelom . The neural tube, derived from the outer ectodermal tube, becomes internalized during the process of neurulation (covered later).

In the Clinic

Anterior Body Wall Defects

Failure of the anterior (ventral) body wall to form properly during body folding or subsequent development results in anterior body wall defects. Anterior body wall defects can occur in the abdominal (common) or thoracic (rare) region.

The most common anterior body wall defects include omphalocele and gastroschisis , which when grouped together occur in 1 in 2500 live births. In both of these defects, a portion of the gastrointestinal system herniates beyond the anterior body wall in the abdominal region. However, in omphalocele ( Fig. 4.4B–D ), the bowel is membrane covered, in contrast to gastroschisis, in which the bowel protrudes through the body wall (see Fig. 4.1B ).

Two other anterior body wall defects occur in the abdominal region: prune belly syndrome (Eagle-Barrett syndrome) and exstrophy of the bladder (also known as cloacal exstrophy ). Although the anterior body wall closes in individuals with prune belly syndrome, the abdomen becomes distended by bladder outlet obstruction, and the abdominal muscles fail to develop. Consequently, there is marked wrinkling of the anterior abdominal wall. This syndrome occurs almost exclusively in males and is associated with undescended testicles, suggesting a complex etiology. In exstrophy of the bladder, the bladder epithelium is exposed on the surface of the lower abdomen, and the bladder consists of an open vesicle (reminiscent of an open neural tube defect). Exstrophy of the bladder is covered further in Chapter 15 .

Anterior body wall defects can also occur in the thoracic wall. For example, the heart can be exposed on the surface, resulting in ectopia cordis (about 5:1,000,000 live births). In the extremely rare pentalogy of Cantrell , which is considered an anterior body wall defect, five major anomalies occur together: (1) midline abdominal wall defect, (2) anterior diaphragmatic hernia, (3) cleft sternum, (4) pericardial defect, and (5) intracardiac defects such as ventricular septal defect. Thus, this defect, which occurs mainly in the thoracic region, also involves the abdominal region.

A final anterior body wall defect with a complex etiology involving multiple structures is called limb-body wall complex (LBWC; amniotic band syndrome) . At least in some cases, LBWC results from rupture of the amnion and constriction of the limbs by fibrous amniotic bands (hence its alternative name, although not all cases of LBWC exhibit amniotic bands). In addition to limb defects (covered in Chapter 20 ) and sometimes craniofacial defects (covered in Chapter 17 ) and exencephaly or encephalocele (covered later in this chapter), anterior body wall defects such as omphalocele or gastroschisis are present in LBWC.

Neurulation: Establishing Neural Tube, Rudiment of Central Nervous System

Animation 4.2: Neurulation .

Animations are available online at StudentConsult .

As covered in Chapter 3 , by the end of the third week, the neural plate consists of a broad cranial portion that will give rise to the brain and a narrower caudal portion that will give rise to the spinal cord (see Chapter 3 , Figs. 3.19 , 3.20 ). On day 22 (eight pairs of somites), the narrower caudal portion of the neural plate—the future spinal cord—represents only about 25% of the length of the neural plate. However, as somites continue to be added, the spinal cord region lengthens faster than the more cranial neural plate. By day 23 or 24 (12 and 20 pairs of somites, respectively), the future spinal cord occupies about 50% of the length of the neural plate, and by day 26 (25 pairs of somites), it occupies about 60%. Rapid lengthening and narrowing of the neural plate during this period is driven by convergent extension (covered in Chapter 3 ) of the neuroepithelium and underlying tissues; at later stages, formation and lengthening of the most caudal neuroepithelium is largely driven by the addition of new neuroepithelial cells derived from bipotent neuromesodermal stem cells (see Chapter 3 , Fig. 3.23 ).

Formation of the neural tube occurs during the process of neurulation ( Fig. 4.5 ). Neurulation involves four main events: formation of the neural plate, shaping of the neural plate, bending of the neural plate, and apposition, adhesion, and fusion of the tips of the neural folds ( Fig. 4.6 ). Formation of the neural plate was covered in Chapter 3 under the topic of neural induction. The main morphogenetic change that occurs during formation of the neural plate is apicobasal elongation of ectodermal cells to form the thickened, single-layered neural plate (see Fig. 4.6A , B ).

Shaping of the neural plate is driven mainly by convergent extension. During shaping, the neural plate narrows in its transverse plane and simultaneously lengthens in its longitudinal plane (see Fig. 4.6A , B ).

Bending of the neural plate involves formation of neural folds at the lateral edges of the neural plate, consisting of both neuroepithelium and adjacent surface ectoderm (see Fig. 4.6C ). During bending, the neural folds elevate dorsally by rotating around a central pivot point overlying the notochord called the median hinge point . The groove delimited by the bending neural plate is called the neural groove . Bending around the median hinge point resembles closing of the leaves of a book. Additional hinge points form in the cranial neural plate which bring the tips of the neural folds together at the dorsal midline (see Fig. 4.6D , E ). The presence of these dorsolateral hinge points is thought to be important owing to the larger size of the neural folds in the future brain than spinal cord. Although dorsolateral hinge points may not be required in the upper spinal cord, they are present in the lower spine. As a result of the bending, the paired neural folds are brought into apposition at the dorsal midline.

Closure of the neural tube involves the adhesion of neural folds to one another and the subsequent rearrangement of cells within the folds to form two separate epithelial layers: the roof plate of the neural tube and the overlying surface ectoderm . Forming at the dorsal neuroepithelium are neural crest cells (see Fig. 4.6F ). These arise from the neural folds by undergoing an epithelial-to-mesenchymal transition ( EMT ; discussed later in the chapter); neural crest cells are also covered later in the chapter. In humans, closure of the neural groove begins on day 22 at the future occipital and cervical region (i.e., adjacent to the four occipital somites and first cervical somite) of the neural tube (see Fig. 4.5 ). From this level, closure progresses both cranially and caudally; eventually the cranial and caudal neuropores close on day 24 and day 26, respectively.

In the Research Lab

Mechanisms of Neurulation

Tissue and Cellular Events

Neurulation, in particular shaping and bending of the neural plate, involves a number of different forces that act in concert. These forces are generated by changes in cell behavior, particularly changes in cell shape, position, and number. Some of these forces are generated within the neural plate itself, whereas other forces are generated in surrounding tissues. Forces arising within the neural plate are called intrinsic neurulation forces , as opposed to those arising outside the neural plate, which are called extrinsic neurulation forces .

The cellular basis of neurulation has been mechanistically examined most thoroughly in chick embryos (see Fig. 4.6 ) and mouse embryos. Although shaping and bending of the neural plate occur simultaneously, to understand their mechanisms it is best to consider them separately. As covered earlier in this chapter, shaping involves convergent extension ; that is, transverse narrowing and longitudinal lengthening. In addition, the neural plate thickens apicobasally during shaping as its cells get taller (i.e., change shape to high columnar), continuing the process of cell elongation initiated during neural plate formation. Apicobasal elongation requires the presence of paraxial microtubules; that is, microtubules oriented along (parallel to) the apicobasal axis of the cell. Cell elongation contributes not only to neural plate thickening but also to its narrowing, because as cells get taller, they reduce their diameters to maintain their size (this would also reduce the length of the neural plate but is compensated for by cell rearrangement and oriented cell division; covered later). However, the major factor that narrows the neural plate is not cell elongation. Rather it is cell rearrangement (also called cell intercalation ). During cell rearrangement, cells move from lateral to medial within the neural plate, thereby narrowing the neural plate and stacking up in the cranial-caudal plane, increasing the length of the neural plate. Moreover, cell division occurs rapidly during neurulation such that the neural plate continues to grow during shaping and bending. Many of these cell divisions are oriented to place daughter cells into the length of the neural plate rather than into its width, resulting in cranial-caudal extension of the neural plate. Thus, shaping of the neural plate involves changes in cell shape, position, and number within the neural plate. Experiments have shown that shaping is largely autonomous to the neural plate; that is, intrinsic forces drive neural plate shaping.

As covered earlier in this chapter, bending of the neural plate involves the formation of hinge points. The single median hinge point forms at all craniocaudal levels of the bending neural plate and paired dorsolateral hinge points form at future brain levels and in lower spinal levels. Hinge points involve localized regions where neuroepithelial cells change their shape from column-like to wedge-like and where wedge-shaped cells become firmly attached to an adjacent structure through the deposition of extracellular matrix. Thus the median hinge point cells of the neural plate are firmly attached to the underlying notochord , and the dorsolateral hinge point cells of the neural plate on each side are firmly attached to the adjacent surface ectoderm of the neural folds . Cell wedging within the hinge points is generated by both apical constriction and basal expansion. The apices of neuroepithelial cells contain a circumferential ring of microfilaments whose contraction leads to apical narrowing. In addition, bases of neuroepithelial cells simultaneously expand as the nucleus moves basally. Recall that neuroepithelial cells are dividing throughout neurulation. As these elongated cells divide, their nuclei undergo a to-and-fro movement called interkinetic nuclear migration . During the G1/S phase of the cell cycle, nuclei move basally. After DNA synthesis is completed during the S phase, cells round up at the apex of the neuroepithelium, where mitosis ( cytokinesis ) occurs. After division, cells elongate once again, and their nuclei move basally. The cell cycle of neuroepithelial cells is prolonged so that cells spend more time in G and S phases and, consequently, more time with their bases expanded. This is because each neuroepithelial cell is very narrow, except at the level where the nuclei reside. Thus basally expanded neuroepithelial cells are wedge shaped.

Historically, most studies on neurulation have focused on changes in neuroepithelial cell shapes (i.e., wedging), which generate intrinsic forces for neurulation. However, actomyosin “cables” extend from the closure point along the dorsal tips of the open neural folds. These cables join to one another and come to fully encircle the closing posterior neuropore. Evidence suggests that these actomyosin cables contribute to the mechanical force required for neural tube closure. Additionally, studies in the chick have shown that tissues lateral to the neural plate (surface ectoderm and mesoderm) generate extrinsic forces for bending of the neural plate. Like intrinsic forces that act during shaping, these extrinsic forces are generated by changes in cell behavior and involve changes in cell shape, position, and number. Lateral tissues also undergo convergent extension driven by both oriented cell division and cell rearrangement. This results in their medial expansion, which has been suggested to push the neural folds, resulting in their elevation and convergence toward the dorsal midline. For example, surface ectodermal cells transform from cuboidal to squamous (i.e., they flatten), increasing their surface area. In the mouse, physical association of the neuroepithelium and surface ectoderm also enhances neuroepithelial cell proliferation required for dorsolateral hinge point formation, and if the ectoderm is removed, neuroepithelial bending fails. However, if only a few ectodermal cells remain attached to the neuroepithelium, the dorsolateral hinge point still forms, suggesting that the surface ectoderm provides a signaling effect on lateral neuroepithelium rather than providing mechanical pushing forces on the neuroepithelium.

The cellular basis of neural tube closure, specifically fusion of the neural folds, is poorly understood. Some studies suggest that apical extracellular adhesive coats are involved, but their molecular nature remains uncharacterized. In addition, cell rearrangements occur as the neuroepithelial and surface ectoderm components of the neural folds fuse and then reorganize into new epithelial (i.e., roof of neural tube and overlying surface ectoderm). However, precisely how cells accomplish these feats remains largely unstudied.

Molecular Mechanisms

The molecular basis of neurulation is being studied increasingly. More than 200 mutations in mouse have been shown to result in defective neurulation and, consequently, to result in neural tube defects (NTDs) ; thus these mutations provide insight into which genes are involved in both normal and abnormal neurulation. Because neurulation is driven by changes in cell behavior, it is not surprising that mutation of cytoskeletal, extracellular matrix/cell adhesion, cell cycle, and cell death genes results in NTDs. Neurulation is a highly choreographed morphogenetic event that must be precisely timed and coordinated across multiple tissues. This presumably involves signaling among tissues. It is the hope of studies using mouse mutations that such signaling pathways will be identified, ultimately leading to an understanding of the molecular basis of neurulation and the formation of NTDs both in animal models and ultimately in humans.

Planar Cell Polarity Pathway and Convergent Extension

As covered early in this chapter and in Chapter 3 , convergent extension plays a major role in vertebrate gastrulation and neurulation. Recent studies have revealed that convergent extension is regulated by a non-canonical Wnt signaling pathway. During development, epithelial sheets become polarized not only apicobasally but also within the plane of the epithelium itself. In Drosophila , the planar cell polarity (PCP) pathway functions in this latter polarization of the epithelium. Thus, for example, the orientation of wing hairs is established by the PCP pathway. In vertebrates, the PCP pathway is required for proper orientation of stereociliary bundles in the outer hair cells of the mouse inner ear (covered in Chapter 18 ) and for convergent extension during gastrulation and neurulation. How are the PCP and Wnt signaling pathways related?

The Drosophila PCP pathway consists of several core proteins that collectively act to convert an extracellular polarity cue to specific changes in the cytoskeleton. These core proteins are now known to be components of the Wnt signaling pathway, and orthologs of several of the Drosophila components are conserved in vertebrates. Thus convergent extension during gastrulation and neurulation is blocked in loss-of-function mutations of the cytoplasmic protein dishevelled in Xenopus and its two orthologs in mouse (dishevelled 1 and 2). As covered in Chapter 5 , Wnt signaling involves both a so-called canonical Wnt and non-canonical Wnt pathways. The PCP pathway utilizes the non-canonical pathway in which certain Wnts, such as Wnt11, bind to their receptors (known as frizzleds). Several other proteins in mice, homologous to proteins encoded by Drosophila PCP genes, must interact in this pathway for proper signaling and, consequently, for proper convergent extension to occur. Thus developing an understanding of the PCP pathway in Drosophila has had a surprising result—a better understanding of vertebrate gastrulation and neurulation and, potentially, a better understanding of how NTDs form in humans.

Actin-Binding Proteins and Apical Constriction

Several actin-associated proteins when genetically ablated in mice result in NTDs. One of these, the actin-binding protein shroom, has received considerable study. Overexpression of shroom in cultured epithelial cells is sufficient to cause apical constriction. Shroom causes apical constriction by altering the distribution of F-actin to the apical side of epithelial cells and by regulating the formation of a contractile actomyosin network associated with apical intercellular junctions. When shroom is inactivated in Xenopus embryos, hinge point formation is drastically altered and neural tube closure fails to occur, providing further evidence for a role of cell shape changes in generating intrinsic forces important for neurulation. Mutations in members of the shroom family have been linked to human NTDs as well.

Dorsal-Ventral Patterning of Neural Tube

As the neural tube is forming, it receives signals from adjacent tissues that result in its patterning in the dorsal-ventral axis. Three tissues provide patterning signals: surface ectoderm, paraxial mesoderm, and notochord. Thus these signals originate dorsally, laterally, and ventrally, respectively ( Fig. 4.7 ).

Ventral signals are the best understood. Several microsurgical experiments in which notochords were removed (extirpated) from the ventral midline or were transplanted adjacent to the lateral wall of the neural tube revealed that the notochord was both sufficient and necessary for formation of the median hinge point and, subsequently, for formation of the floor plate of the neural tube (the floor plate derives from the median hinge point during subsequent development). Using loss-of-function and gain-of-function experiments mainly in chick and mouse, it was shown that sonic hedgehog (Shh), secreted initially by the notochord, was the signal that induced the median hinge point and floor plate. As the floor plate is induced, it also secretes Shh ( Fig. 4.8 ), which in turn induces neurons in the ventral neural tube (e.g., motoneurons in the ventral spinal cord; covered in Chapter 9 ). Shh acts as a morphogen , such that high concentrations induce ventral neurons, lower concentrations induce more intermediate neurons, and the lowest concentrations induce more dorsal neurons. Interestingly in mice, mutations that result in gain-of-function of Shh signaling in the cranial region can cause failed neural tube closure.

In addition to producing a ventral-to-dorsal concentration gradient of Shh within the neural tube, the notochord produces a ventral-to-dorsal concentration gradient of chordin, a Bmp antagonist. The chordin gradient interacts with a dorsal-to-ventral concentration gradient of Bmp produced by the surface ectoderm. Because chordin blocks Bmp signaling, Bmp signaling is robust dorsally (where chordin concentration is weak or absent and Bmp concentration is high) and is weak or absent ventrally (where chordin concentration is high and Bmp concentration is weak or absent). A high level of Bmp signaling dorsally, along with Wnt signaling by the surface ectoderm, results in the induction of neural crest cells and the roof plate of the neural tube .

The paraxial mesoderm lying adjacent to the lateral walls of the neural tube also provides patterning signals, but these are the least understood. A rostrocaudal gradient of retinoic acid, secreted by the segmenting paraxial mesoderm, specifies the axial level of the neural tube and promotes neuronal differentiation (see Fig. 4.7 ). Both gain-of-function and loss-of-function experiments in Xenopus provide support for a role for paraxial mesoderm and Fgfs in neural crest cell induction.