Fig. 7.

Partial rescue of neural tube closure in Zic2^Ku/Ku embryos by inhibitor combinations: a dual mechanism of spinal NTDs. (A) Culture from E8.5 for 18 h in the presence of the Rho kinase inhibitor Y27632 (Y27) leads to significantly reduced PNP length in Zic2^Ku/Ku embryos compared with DMSO-treated mutants (**P<0.003). However, rescue is incomplete and PNP length remains significantly greater in Y27-treated mutants than in DMSO-treated wild-type embryos (*P<0.001). Y27-treated wild-type and mutant embryos do not differ in PNP length (P=0.076). (B) DM in combination with Bleb or Y27 similarly reduces PNP length in cultured E8.5 Zic2^Ku/Ku embryos (**P=0.026 for DM+Bleb; **P=0.002 for DM+Y27) but rescue is incomplete in both cases, with treated mutant embryos having significantly greater PNP length than DMSO-treated wild-type embryos (*P<0.001). Two-way ANOVA with Holm-Sidak post-hoc tests. (C) Schematic of the proposed dual mechanism of spinal NTDs in Zic2^Ku/Ku embryos. Loss of Zic2 function releases BMP signalling from its normal regulation by noggin and other endogenous antagonists (left side of diagram). Enhanced BMP signalling suppresses DLHP formation, contributing to failure of PNP closure. This can be ameliorated by DM, an exogenous BMP inhibitor, that reinstates DLHP formation. A second mechanism also operates (right side of diagram), via dysregulated (enhanced) RhoA activation that results in excessive accumulation of actomyosin in the mutant neural plate, leading to stiffening and lack of deformability. Bleb ameliorates the actomyosin accumulation, enhancing closure, although the neural plate shows atypical dorsal bending, not normal DLHPs. A similar effect is seen with Y27632. The mutant neural plate shows dorsoventral thickening, and this appears to be largely rescued by both types of inhibition, although the underlying mechanism is unclear.