Neural tube closure: cellular, molecular and biomechanical mechanisms

Abstract

Neural tube closure has been studied for many decades, across a range of vertebrates, as a paradigm of embryonic morphogenesis. Neurulation is of particular interest in view of the severe congenital malformations - 'neural tube defects' - that result when closure fails. The process of neural tube closure is complex and involves cellular events such as convergent extension, apical constriction and interkinetic nuclear migration, as well as precise molecular control via the non-canonical Wnt/planar cell polarity pathway, Shh/BMP signalling, and the transcription factors Grhl2/3, Pax3, Cdx2 and Zic2. More recently, biomechanical inputs into neural tube morphogenesis have also been identified. 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.

Keywords: Cell protrusions; Convergent extension; Cytoskeleton; Extracellular matrix; Morphogenesis; Neural tube; Neural tube defects; Neurulation; Planar cell polarity; Proteases; Spina bifida.

Fig. 1. An overview of primary and secondary neurulation.

(A) Schematic representation of primary neurulation involving elevation of the neural folds (left panel), followed by their bending (middle panel) and fusion (right panel). DLHP, dorsolateral hinge point; MHP, median hinge point; NC, notochord; NE, neuroepithelium; NNE, non-neural ectoderm; PM, paraxial mesoderm. (B) Secondary neurulation. Tail-bud cells condense (left panel) in the midline to form the medullary cord. The medullary cord undergoes epithelialization (middle panel) around a lumen (red) while the notochordal precursor remains solid, generating the secondary neural tube and notochord (right panel).

Fig. 2. Comparative schematic summary of neurulation in different vertebrates.

Key features of neurulation are shown for (A) mouse, (B) chick, Xenopus and (D) zebrafish. Cross-sections of the mouse, chick and Xenopus neural tube are shown with typical appearance in anterior and posterior embryonic regions; for zebrafish, cross-sections of the midbrain after closure and during neural keel formation are shown. The arrows in A and B indicate directions of closure. Arrowheads indicate hinge points. EVL, envelope layer; NE, neuroepithelium; NNE, non-neural ectoderm.

Fig. 3. Schematic representation of key neural tube closure regulatory mechanisms.

A number of mechanisms involved in neural tube closure (NTC) are depicted. (1) Transcriptional regulation: Grhl2 (grainyhead-like 2) regulates the expression of E-cadherin and Cldn4 (claudin 4) in non-neural ectoderm (NNE) cells during mouse cranial neurulation. (2) Protrusions: NNE cells display Rac1-dependent protrusions that make the first contact during neural fold (NF) fusion in the mouse spinal region. (3) Proteases: a pathway involving membrane-bound serine proteases (e.g. protease-activated receptor 2, Par2) is active in NNE cells. (4) Interkinetic nuclear migration (IKNM): nuclei migrate apically to divide, with daughter nuclei returning to a basal position for S phase. As neuroepithelial cell cycles are not synchronized, the neural plate (NP) is a pseudostratified epithelium. (5) Dorsolateral hinge point (DLHP) regulation: the formation of DLHPs is regulated by antagonistic interactions between bone morphogenetic protein 2 (BMP2), sonic hedgehog (Shh) and Noggin. (6) BMP and transforming growth factor (TGF) signalling: active BMP (detected by pSMAD1/5/8) and TGFβ (detected by pSMAD2/3) signalling are found along the neural ectoderm (NE) in a cell-cycle dependent manner. Antagonism between the pathways is important for the formation of the median hinge point (MHP) in chick midbrain, by affecting the localization of apical (e.g. PAR3) or basolateral (e.g. lethal giant larva; LGL) junctional proteins. (7) Planar polarized actomyosin contraction: planar cell polarity (PCP)-controlled apical constriction (actin fibres in red) causes bending along the mediolateral axis in the cranial neural tube of the chick. Basal nuclear localization causes wedge-shaped cells in the midline NP of both chick and mouse embryos. (8) Actomyosin turnover and extracellular matrix (ECM): the assembly and disassembly of apical actin filaments is under ROCK/RhoA regulation. ECM proteins (e.g. fibronectin, perlecan, glypican 4) and their receptors (e.g. integrins) affect NTC.

Fig. 4. Morphology of the mouse spinal neural tube during closure.

(A) Whole-mount staining of the closing mouse neural tube (NT) using CellMask (green) to label cell membranes, phalloidin (blue) to label F-actin and DAPI (red) to label nuclei. Optical sectioning across different levels (B-E, as indicated by dotted lines) highlights: (B) open neural folds; (C) neural folds bending at dorsolateral hinge points; (D) NT closure point; and (E) the closed NT. Scale bars: 100 μm.

Fig. 5. Cellular protrusions in the closing neural tube.

Scanning electron micrographs of mouse embryos at early (A, 10 somites) and late (B, 25 somites) stages of spinal neurulation. High magnification of the neural tube (NT) closure points (shown in the insets) reveal the varying morphology of cellular protrusions emanating from the non-neural ectoderm (NNE): filopodia characterize early stage NT fusion (A, inset), while ruffles typify the late stages of spinal closure (B, inset). Scale bars: 100 μm in A and B; 10 μm in the insets.