Vangl-dependent planar cell polarity signalling is not required for neural crest migration in mammals.

The role of planar cell polarity (PCP) signalling in neural crest (NC) development is unclear. The PCP dependence of NC cell migration has been reported in Xenopus and zebrafish, but NC migration has not been studied in mammalian PCP mutants. Vangl2(Lp/Lp) mouse embryos lack PCP signalling and undergo almost complete failure of neural tube closure. Here we show, however, that NC specification, migration and derivative formation occur normally in Vangl2(Lp/Lp) embryos. The gene family member Vangl1 was not expressed in NC nor ectopically expressed in Vangl2(Lp/Lp) embryos, and doubly homozygous Vangl1/Vangl2 mutants exhibited normal NC migration. Acute downregulation of Vangl2 in the NC lineage did not prevent NC migration. In vitro, Vangl2(Lp/Lp) neural tube explants generated emigrating NC cells, as in wild type. Hence, PCP signalling is not essential for NC migration in mammals, in contrast to its essential role in neural tube closure. PCP mutations are thus unlikely to mediate NC-related birth defects in humans.

Syndecan 4 interacts genetically with Vangl2 to regulate neural tube closure and planar cell polarity.

Syndecan 4 (Sdc4) is a cell-surface heparan sulfate proteoglycan (HSPG) that regulates gastrulation, neural tube closure and directed neural crest migration in Xenopus development. To determine whether Sdc4 participates in Wnt/PCP signaling during mouse development, we evaluated a possible interaction between a null mutation of Sdc4 and the loop-tail allele of Vangl2. Sdc4 is expressed in multiple tissues, but particularly in the non-neural ectoderm, hindgut and otic vesicles. Sdc4;Vangl2(Lp) compound mutant mice have defective spinal neural tube closure, disrupted orientation of the stereocilia bundles in the cochlea and delayed wound healing, demonstrating a strong genetic interaction. In Xenopus, co-injection of suboptimal amounts of Sdc4 and Vangl2 morpholinos resulted in a significantly greater proportion of embryos with defective neural tube closure than each individual morpholino alone. To probe the mechanism of this interaction, we overexpressed or knocked down Vangl2 function in HEK293 cells. The Sdc4 and Vangl2 proteins colocalize, and Vangl2, particularly the Vangl2(Lp) mutant form, diminishes Sdc4 protein levels. Conversely, Vangl2 knockdown enhances Sdc4 protein levels. Overall HSPG steady-state levels were regulated by Vangl2, suggesting a molecular mechanism for the genetic interaction in which Vangl2(Lp/+) enhances the Sdc4-null phenotype. This could be mediated via heparan sulfate residues, as Vangl2(Lp/+) embryos fail to initiate neural tube closure and develop craniorachischisis (usually seen only in Vangl2(Lp/Lp)) when cultured in the presence of chlorate, a sulfation inhibitor. These results demonstrate that Sdc4 can participate in the Wnt/PCP pathway, unveiling its importance during neural tube closure in mammalian embryos.

Convergent extension analysis in mouse whole embryo culture.

Mutations have been identified in a non-canonical Wnt signalling cascade (the planar cell polarity pathway) in several mouse genetic models of severe neural tube defects. In each of these models, neurulation fails to be initiated at the 3-4 somite stage, leading to an almost entirely open neural tube (termed craniorachischisis). Studies in whole embryo culture have identified a defect in the morphogenetic process of convergent extension during gastrulation, preceding the onset of neural tube closure. The principal defect is a failure of midline extension, both in the neural plate and axial mesoderm. This leads to an abnormally wide neural plate in which the elevating neural folds are too far apart to achieve closure. In this chapter, we provide details of several experimental methods that can be used to evaluate convergent extension in cultured mouse embryos. We describe analytical methods that can reveal the abnormalities that characterise neurulation-stage embryos with defective planar cell polarity signalling, in particular the loop-tail (Lp; Vangl2) mutant.