On about the 15th day of embryonic life, ectodermal cells proliferate to form a plate, the
primitive streak, along the surface of the embryo. A rapidly proliferating group of cells forms at one end of the primitive streak; this nodular proliferation, surrounding a small primitive pit (known as the
Hensen node), defines the cephalic end of the primitive streak. At days 15 and 16, cells enter the primitive pit and migrate inward between the ectoderm and endoderm and then course laterally and undergo epithelial to mesenchymal transition, to form the interposed mesoderm. Initially, no cells migrate in the midline; later, these mesodermal cells will join in the midline to form the notochordal process, which will eventually roll into a tube, separate from the endoderm, and become the notochord. During a process known as
intercalation, the notochordal process fuses with the endoderm, creating a communication of the central canal of the notochordal process with the yolk sac. As the central canal is already in communication with the amniotic cavity through the primitive pit, a transient communication is present all the way from the yolk sac to the amniotic sac; this communication is called the
primitive neurenteric canal. Once formed, the notochord induces the formation of a plate of ectodermal cells in the dorsal midline, beginning immediately cephalad to the Hensen node (
Fig. 9-1). Under the influence of bone morphogenetic proteins (BMPs), most of the ectoderm is prevented from differentiating into neuroectoderm (
1). However, suppression of the BMPs by antagonists such as chordin, noggin, and follistatin, emanating from the primitive node, allows neuroectoderm to form in the midline (
1,
2). The lateral edges of this midline structure, now known as the
neural plate, are contiguous with the ectoderm from which the plate has differentiated, now known as superficial or cutaneous ectoderm.
After the neural plate is formed, it is shaped into an elongated structure that is broad at the anterior (cranial) end and narrow at the posterior (caudal) end. The major driving force of the shaping is a mediolateral elongation of a group of cells, along with development of
polarized cellular protrusions that enable the cells to migrate medially and intercalate with neighboring cells close to the midline (
3). This midline convergence of cells causes an anteroposterior elongation and narrowing of the neural plate (
4,
5). This process is strongly related to the development of cell polarity (
4,
5). Along with the process of shaping, the neural plate also bends. At approximately 17 days of gestation, the lateral portions of the neural plate begin to thicken bilaterally, forming the neural folds; the process of bending elevates these folds and brings them to the dorsal midline (
6). The process involves the formation of “hinge points” at two sites: the median hinge point (MHP) in the ventral midline and extending over the rostrocaudal extent of the neural plate and the paired dorsolateral hinge points (DLHPs), which form mainly at the levels of the developing brain (
2) and lower spine. The formation of these hinge points is controlled by secretion of the signal transduction protein sonic hedgehog by the notochord as well as an inhibitory interaction between BMP2 and Noggin, particularly in the lower spine (
7). After formation of the hinge points, the more lateral aspects of the neural plate are elevated around the MHP, bringing the DLHPs upward and toward the midline (
Fig. 9-2). This elevation is accomplished by a poorly understood process called apical constriction, in which columnar cells of the neural tube are converted into wedge-shaped cells (
8). Eventually, the lateral folds contact one another in the dorsal midline and adhere to one another, with their fusion forming the neural tube (neurulation). This midline contact (also called neural fold apposition) results from constriction of the open posterior neural tube, which is biomechanically coupled to the zippering point by an F-actin network (
9). Neurulation seems to begin separately at least two different levels in humans, when cellular protrusions (possibly cilia) project medially from the most dorsal cells of the neural folds on either side. A third site of closure at the caudal end of the embryo has recently been identified in mouse embryos; if present in humans, this may account for the high incidence of spina bifida at this level (
9). Cell recognition and adhesion occur under the influence of many molecules (Ephrin-A5, EphA7, neural cell adhesion molecule, and neural cadherin among them (
3)), closing the tube at each point. Immediately following closure, the overlying ectoderm separates from the neural tissue and the edges of the ectoderm meet in the midline and fuse, forming a continuous ectodermal covering of the neural structures, with the mesenchymal cells of the neural crest migrating between the cutaneous and neural ectoderm layers (
2). Progressive folding and closure of the neural structures and separation from ectoderm then proceed both cranially and caudally from each point of initial closure, ultimately resulting in complete closure (
10,
11). The initial closure of the neural tube in humans, the
posterior neuropore, is believed to be at the hindbrain-cervical junction, from which closure extends in both directions. A second site of closure in mice is at the forebrain-midbrain boundary; this site has not been confirmed in humans. The necessity of this site even in mice is questionable, as about 80% of mice that lack the second closure point still achieve complete cranial closure (
12). The “third” site of closure occurs at the most rostral extremity of the forebrain, the lamina terminalis (
2). The exact site of the most caudal end of the neurulation-formed neural tube has been debated, but most experts believe that it is at the S2 level (
10,
11). Others point out, however, that neural tube defects are not restricted to any specific location(s) and propose that the human neural tube initially closes at a single site with closures extending from that location (
13).
Recent work has shown marked complexity of the process of neural tube closure, involving cellular events such as convergent extension (a fundamental and conserved collective cell movement that forms elongated tissues during embryonic development (
14)), apical constriction (constriction of the apical surfaces of the cells in prospective anterior portion of the dorsal visceral ectoderm (
8)), and interkinetic nuclear migration (
15), as well as precise molecular control via the noncanonical Wnt/planar cell polarity pathway, Shh/BMP signaling, and the transcription factors Grhl2/3, Pax3, Cdx2, and Zic2 (
15,
16,
17). In mammals, this process is regulated by more than 300 genes (
18); biomechanical inputs into neural tube morphogenesis have also been identified. Nutritional factors are also important; several rodent studies show that folic acid reduces neural tube closure defects (NTDs) and others have shown that inositol reduces NTDs in some strains of mice in which folate does not work (
19). No definitive results have been published at the time this chapter was being written (
19). Here,
we review these cellular, molecular, and biomechanical mechanisms involved in neural tube closure, based on studies of various vertebrate species, focusing on the most recent advances in the field.