Fig. 1.

Inhibition of RhoA signalling disrupts spinal neural tube closure. (A) Mouse embryos cultured for 18 h to the 24 somite stage. Exposure to 5 µM Y27632 yields an enlarged PNP (between arrowheads), open forebrain (asterisks) and defective heart looping (h) compared with the DMSO control. Inset, method of PNP length measurement, between the dashed lines. Scale bar: 0.5 mm. (B) Progressive closure of PNP is arrested after the 13 somite stage in embryos cultured for 5–6 h in Y27632 (**P<0.001 versus DMSO). Embryos exposed to 50 µM Blebbistatin or 0.05 µg/ml CytD close at the same rate as DMSO controls. (C) PNP closure is significantly delayed after culture in ROCK inhibitor Y27632 or HA-1100, for 18–20 h to the 15–19 or 20–24 somite stage (**P<0.001 versus DMSO). Embryos cultured in Blebbistatin, CytD or LatB resemble DMSO controls. (D) Immunohistochemistry (green, anti-pMLC; nuclei, DAPI) shows apically localized pMLC (arrowheads) in DMSO- and Blebbistatin-treated embryos with 19 somites. The signal is abolished by 5–6 h culture in Y27632. Grayscale insets, pMLC staining only. Scale bar: 30 µm. (E) Western blots for pLIMK and LIMK, p-cofilin and cofilin, and pMLC and MLC. The level of the phosphorylated forms are quantified as a proportion of the total, after normalizing to GAPDH. Culture for 5–6 h in Y27632 significantly reduces the relative amount of pMLC, pLIMK and p-cofilin compared with DMSO controls, whereas Blebbistatin has no effect (n=3, **P<0.001). (F) G-LISA assay of active GTP-bound RhoA (top) relative to total RhoA (below), normalized to DMSO (mean±s.d.). Y27632-treated embryos have significantly reduced RhoA activation whereas total RhoA does not differ between treatments (n=3, *P<0.001). Significance values were calculated with a Student's t-test, compared with DMSO control.