FORMATION OF THE NEURAL TUBE: PRIMARY AND SECONDARY NEURULATION

During neurulation, which is conventionally divided into primary and secondary phases, the epiblast is induced to form neural ectoderm (Fig. 8-1). The first morphological sign of primary neurulation is a dorsal thickening in the anterior ectoderm concomitant with regression of the primitive streak. This elliptical region of specialized thickened ectoderm is referred to as the neural plate. Subsequently, the neural plate undergoes a shaping which converts it into a more elongated key-hole-shaped structure with broad anterior and narrow posterior regions. The main driving force of neural plate shaping seems to come from convergent extensions that cause a net medially directed movement of cells with intercalation in the midline. This leads to lengthening and narrowing of the neural plate. Neural plate shaping is followed by the development of two lateral elevations, the neural folds, on either side of a depressed midregion referred to as the neural groove. In pigs and cattle the neural folds become clear during the third week of development. Development of the anterior neural folds seems to be highly dependent on the underlying mesenchyme which proliferates and expands markedly, with a notable increase in its extracellular spaces, as the elevation of the folds begins. In the spinal part of the neural tube, expansion of the paraxial neural fold elevation is not accompanied by elevation of mesenchyme.

Fig. 8-1: Development of the neural tube and the neural crest. The lateral edges of the neural plate are elevated and become the neural folds. The depressed midregion of the neural plate is called the neural groove. The neural folds continue to rise, appose in the midline and fuse to create the neural tube, which becomes covered by future epidermal ectoderm. As the neural folds rise and fuse, cells at the lateral border of the neuroectoderm (neural crest cells) begin to dissociate from their neighbours, undergo an epithelio-mesenchymal transition, and leave the neuroectoderm. 1: Surface ectoderm; 2: Neural plate; 3: Neural groove; 4: Neural crest; 5: Neural tube; 6: Spinal ganglion; 7: Anterior neuropore; 8: Posterior neuropore; 9: Notochord; 10: Primitive node; 11: Primitive streak; 12: Somites.

The neural folds continue to elevate, appose in the midline, and, eventually, fuse to create the neural tube which becomes covered by the surface ectoderm that will develop into the future epidermis. This process is facilitated by the cytoskeleton (microfilaments and microtubuli) of neural plate cells, and by extrinsic forces from underlying paraxial and notochordal tissues. This primary neurulation creates the brain and most of the spinal cord.

In the tail bud, the neural tube is formed by secondary neurulation. This is a different mechanism, without neural folding, in which the spinal cord initially forms as a solid mass of epithelial cells, and a central lumen develops secondarily by cavitation. The transition from primary to secondary neurulation occurs at the future upper sacral level.

Bending of the neural plate and apposition of neural folds

The bending of the neural plate, which is observed during primary neurulation, occurs at three principal sites: the median hinge point (MHP), overlying the notochord, and the paired dorsolateral hinge points (DLHP) at the points of attachment of the surface ectoderm to the outside of each neural fold. The MHP is induced by signals from the notochord and is the sole site of bending in the upper spinal neural plate.

Gradually, the neural folds approach each other in the midline, where they eventually fuse. The actin microfilaments of the neuroepithelial cells play a significant role in anterior neurulation. This has become apparent from mouse mutant data and from studies using cytochalasin (a drug that disassembles actin microfilaments) which, in several species, have shown that neural tube closure, especially in the anterior part, is highly dependent on the actin cytoskeleton. Spinal neurulation, on the other hand, is more resistant to the disruption of actin microfilaments.

Cellular protrusions extend from apical cells of the neural folds as they approach one another in the dorsal midline and interdigitate as the folds come into contact. This allows a first cell-cell recognition and provides an initial adhesion pending later establishment of permanent cell contacts.

Fusion of neural folds

Fusion begins in the cervical region and proceeds anteriorly and posteriorly from there. This fusion is mediated by cell-surface glycoconjugates. As a result of these processes, the neural tube is formed and separated from the overlying ectodermal sheet. Until fusion is complete, the anterior and posterior ends of the neural tube communicate with the amniotic cavity via two openings, the anterior and posterior neuropores. Closure of the anterior neuropore occurs in the bovine embryo at approximately Day 24 (18 to 20 somite stage), whereas the posterior neuropore closes two days later (25 somite stage). Neurulation is then complete. The central nervous system is represented at this time by a closed tubular structure with a narrow posterior portion, the anlage of the spinal cord, and a much broader cephalic portion, the primordium of the encephalon. During neurulation, the neuroepithelium is entirely proliferative; cells do not begin to exit the cell cycle and start neuronal differentiation until after the neural tube closure is complete.

Apoptosis during neurulation

During neurulation, cell proliferation is also accompanied by some degree of apoptosis in the neuroepithelium. The rate of apoptosis appears to be finely tuned and it seems to be equally detrimental if the intensity of apoptosis is increased or decreased. Excessive apoptosis disturbs anterior neurulation by leaving too few normally functioning cells for morphogenesis, but there are also data indicating that reduced cell death from too little apoptosis can lead to neural tube closure defects.

Apoptosis at the tips of the neural folds may serve a special function. After opposing neural folds have made contact and adhered to each other, midline epithelial remodelling by apoptosis breaks the continuity between the neuroepithelium and surface ectoderm. Inhibition of apoptosis produces spinal neural tube defects, probably by preventing this dorsal midline remodelling.

NEURAL CREST

Formation of neural crest

As the neural folds elevate, cells of the lateral border or crest of the neuroepithelium, the neural crest, undergo an epithelio-mesenchymal transition as they leave the neuroectoderm by active migration into the underlying mesoderm (Fig. 8-1). Induction of neural crest cells requires interactions between neural and overlying surface ectoderm. Neural crest cells are specified as the result of an inductive instruction by the surface ectoderm. The instruction is possibly mediated by a gradient of bone morphogenetic protein 4 (BMP4), BMP7, and Wnt. Bone morphogenetic proteins secreted by surface ectoderm initiate this process. Thus stimulated, neural crest cells express slug, a transcription factor of the zinc finger family that characterizes cells that break away from an embryonic cell layer to migrate as mesenchymal cells.

Neural crest cell migration and neurulation are temporally and spatially related in the anterior parts of the neural tube, but in the midbrain and hindbrain, neural crest cells begin to detach from the apices of the neural folds and start to migrate well in advance of neural tube closure. In contrast, in the spinal cord region, migration of neural crest cells does not begin until several hours after spinal neural tube closure is complete.

Migration of neural crest cells

The pathway by which the neural crest cells leave the neural tube depends on the region. Migrating crest cells give rise to a heterogeneous array of cells and tissues. Neural crest cells that leave the anterior parts of the neural folds, before closure of the neural tube in this region, contribute to the craniofacial skeleton and other mesenchymal derivatives, but can also differentiate into several other cell types including neurons of the cranial ganglia, Schwann cells and melanocytes Table 8-1.

Table 8-1: Major derivatives of the cranial and circumpharyngeal neural crest

Sensory nervous system	Ganglia of trigeminal nerve (V), facial nerve (VII), glossopharyngeal nerve (superior ganglion), vagus nerve (jugular ganglion)
Autonomic nervous system	Parasympathetic ganglia: ciliary, ethmoidal, sphenopalatine, submandibular, visceral
Autonomic nervous system	Satellite cells of sensory ganglia, Schwann cells of peripheral nerves, leptomeninges of prosencephalon and part of mesencephalon
Endocrine cells	Carotid body, parafollicular cells of thyroid
Pigment cells	Melanocytes
Mesectodermal cells	Cranial vault (squamosum and part of os frontale), nasal and orbital bones, part of the otic capsule, palate, maxilla, visceral cartilage, part of external ear cartilage
Connective tissue	Dermis and adipocytes of the skin, cornea of the eye, odontoblasts, stroma of glands (thyroid, parathyroid, thymus, salivary, lachrymal), outflow tract of heart, cardiac semilunar valves, walls of aorta and aortic-arch derived arteries
Muscle	Ciliary muscle; dermal smooth muscles, vascular smooth muscle