Severe neural tube defects due to failure of closure initiation can arise without abnormality of neuroepithelial convergent extension

Planar cell polarity (PCP) signalling is vital for initiation of neural tube closure in mice, with diminished convergent extension (CE) cell movements leading to a severe form of neural tube defect (NTD), termed craniorachischisis (CRN). Some human NTDs are also associated with PCP gene mutations, but affected individuals are generally heterozygous, whereas PCP homozygosity or compound heterozygosity is needed to produce CRN in mice. This suggests human NTDs may involve other genetic or environmental factors, that interact with partial loss of PCP function. We found that reduced sulfation OF glycosaminoglycans (GAGs) interacts with heterozygosity for the Lp allele of Vangl2 (a core PCP gene), to cause CRN in mice. Here, we hypothesised that this GAG-PCP interaction may regulate convergent extension movements, and hence lead to severe NTDs in the context of only partial loss of PCP function. Both Lp and null alleles of Vangl2 gave similar findings. Culture of E8.5 embryos in the presence of chlorate (a GAG sulfation inhibitor), or enzymatic cleavage of GAG chains, led to failure of NT closure initiation in the majority of Lp/+ embryos, whereas few +/+ littermates exhibited CRN. The chlorate effect was rescued by exogenous sulphate. Surprisingly, DiO labeling of the embryonic node demonstrated no abnormality of midline axial extension in chlorate-treated Lp/+ embryos that developed CRN. In contrast, positive control Lp/Lp embryos displayed severe convergent extension defects in this assay. Morphometric analysis of the closure initiation site revealed abnormalities in the size and shape of somites that flank the closing neural tube in chlorate-treated Lp/+ embryos. We conclude that severe NTDs involving failure of closure initiation can arise by a mechanism other than faulty neuroepithelial convergent extension. Matrix-mediated expansion of somites, flanking the closing neural tube, may be required for closure initiation.