Neurulation is a fundamental embryonic process that leads to the development of the neural tube, which is the precursor of the brain and spinal cord.
Building a neural tube is an extremely complex phenomenon where cells need to change in shape, migrate and differentiate to form a hollow tube from a flat sheet of thickened epithelial cells (the neural plate).
Neurulation occurs through two distinct phases: primary neurulation (weeks 3–4) that leads to the formation of the brain and most of the spinal cord till the upper sacral level, followed by secondary neurulation (weeks 5–6) that creates the lowest portion of the spinal cord including most of the sacral and all the coccygeal regions.
Primary neurulation
This process involves formation of the neural plate, shaping of the neural plate, bending and fusion of the neural plate at the midline. Each of these events is autonomous and controlled by distinct molecular pathways.
Formation of the neural plate is described as neural induction whereby the dorsal midline ectoderm is differentiated into the neuroepithelium.
Normally, bone morphogenetic proteins (BMPs) prevent the ectoderm to follow its default pathway to form neuroectoderm and instead instruct it to form epidermis.
Neural induction occurs by suppression of this epidermal fate where BMPs antagonists, including chordin, noggin and follistatin, emanating from the primitive node, allow the ectoderm to form neuroectoderm.
Other signaling pathways implicated in neural induction involve fibroblast growth factors (FGFs), canonical Wnt signaling pathway and insulin-like growth factors (IGFs).
Shaping of the neural plate involves conversion of the neural plate into an elongated structure that is broad at the cranial and narrow at the spinal regions. During shaping, the major driving force is a morphogenetic event called CE.
CE describes the narrowing and lengthening of a group of cells that could be the embryonic axis during gastrulation or the neural plate during neural tube closure. In this complex process, cells elongate mediolaterally and produce polarized cellular protrusions that enable them to move directionally and to intercalate with other neighboring cells. This change in shape and movement results in convergence toward the midline and extension of the tissue along the anteroposterior axis.
The end result is a longer body axis with more narrow mediolateral dimension leading to the conversion of an initially wide and short neural plate into a narrow and elongated one.
CE is controlled by the noncanonical Wnt/frizzled pathway, in contrast to the canonical Wnt pathway that acts through β-catenin stabilization controlling cell fate specification, and is equivalent to the so-called planar cell polarity pathway (PCP) in the fly.
Bending of the neural plate implicates formation of the neural folds at the lateral extremes of the neural plate and the subsequent elevation and convergence of these folds toward the dorsal midline.
It involves the formation of hinge points at two sites: the median hinge point (MHP) overlying the notochord and extending along the rostrocaudal axis and the paired dorsolateral hinge points (DLHPs) at the lateral sides of the folds and predominantly at the future brain levels.
The MHP is the only bending point at the upper spinal level while the DLHP form in the lower spine and cranial region (1). Studies with mouse mutants have shown that the formation of these bending points is controlled by the signal transduction protein sonic hedgehog originating from the notochord.
Following formation of the hinge points, the neural plate rotates by elevation around the MHP and convergence around the DHLP. The major morphogenetic event that takes place in bending of the neural plate is a process termed apical constriction where columnar cells in the neural tube are converted into wedge-shaped cells.
The cellular and molecular mechanisms underlying such process are poorly understood. Only two genes have been implicated in mediating this process and both are actin related: p190RhoGap, a negative regulator of Rho GTPase involved in regulating actin dynamics, and Shroom that is an actin-binding protein.
Eventually, the lateral folds come into contact at the dorsal midline and adhere to each other leading to fusion of the neural plate. This process forms the roof of the neural tube and results in the formation of two separate layers, epidermal ectoderm (that contributes to the skin of the back of the embryo) and neuroepithelium, with intervening mesenchymal cells of the neural crest.
The cellular and molecular mechanisms underlying fusion of the folds remain poorly understood. As the neural folds approach, cellular protrusions expand from apical cells and interdigitate, followed by formation of permanent cell contacts.
Mouse studies have implicated Ephrin-A5 and EphA7 receptors in this fusion process in the cranial epithelium. A recent study in Xenopus has shown that Ephrin-B1 mediates retinal progenitor movements through interaction with Dishevelled and other members of the noncanonical Wnt/PCP pathway.
Other genes implicated in fusion of the neural fold include the neural cell adhesion molecule (N-CAM) and the neural (N)-cadherin, both produced by the neural tube, and the epithelial (E)-cadherin produced by the overlying surface ectoderm. However, null mutations in N-CAM and N-cadherin in mice do not affect neural tube closure, questioning their essential role in the fusion process.
Neural tube closure is believed to proceed bidirectionally in a zipper-like fashion. Studies from mouse models have demonstrated multiple initiation sites of neural tube closure in the developing embryo.
Closure 1 is initiated at the hindbrain/cervical boundary and then proceeds rostrally into the future brain and caudally into the spinal region. Closure 2 is initiated at the forebrain/midbrain boundary and closure 3 occurs at the rostral extremity of the forebrain.
The positions of closures 1 and 3 are invariant among mouse strains while position of closure 2 is polymorphic where it could be relatively caudal or rostral in the midbrain .
Interestingly, mouse strains with a rostral position of closure 2 are highly pre-disposed to cranial NTDs (exencephaly) . In humans, few studies have demonstrated a multisite neural tube closure but the exact number of closure sites remains controversial. Closures 1 and 3 definitely occur but the occurrence of a distinct closure 2 site remains controversial. It has been suggested that this variable site for closure 2 could represent a genetic risk factor for NTDs in humans explaining the variability in NTDs frequency among different ethnic groups.
Failure of neural tube closure during primary neurulation at any level of the body axis from the brain up to the sacral spine leads to ‘open’ NTDs. Failure of closure 1 leads to craniorachischisis where the neural tube remains open throughout the brain and spinal cord. Failure of the caudal spread of fusion from closure 1 results in open spina bifida or myelomeningocele. Failure of closure 2 leads to exencephaly that by late gestation converts into anencephaly, where the skull vault is missing and the brain tissue is degenerated. Failure of closure 3 leads to anencephaly confined to the forebrain region and that is often associated with split-face malformation.
Secondary neurulation
Secondary neurulation begins after completion of primary neurulation. During this process, the neural tube is produced by the tail bud, a mass of stem cells representing the remnant of the primitive streak that are located at the caudal end of the embryo.
These stem cells undergo proliferation and condensation followed by cavitation and fusion with the central canal of the neural tube formed by primary neurulation.
Recent studies suggest that both modes of neurulation represent a continuous program with similar molecular and cellular mechanisms.
Failure of secondary neurulation leads to the less common ‘closed’ forms of NTDs where the developing neural tube fails to separate from other tissues of the tail bud.
Videos
neurulation in embryonic chick
neurulation in Xenopus sp.
References
Kibar Z, Capra V, Gros P.Toward understanding the genetic basis of neural tube defects.Clin Genet. 2007 Apr;71(4):295-310. PMID: 17470131
Copp AJ, Greene ND, Murdoch JN. The genetic basis of mammalian neurulation. Nat Rev Genet. 2003 Oct;4(10):784-93. PMID: 13679871
