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. 2015 Jul 15;128(14):2468–2481. doi: 10.1242/jcs.164574

Fig. 7.

Fig. 7.

Contribution of adherens junction disruption and summary of cytoskeletal signalling requirements for spinal neural tube closure. (A) Immunohistochemistry (phalloidin, red; anti-β-catenin, green) reveals precise colocalization of β-catenin and phalloidin at neuroepithelial adherens junctions of E9.5 WT embryos, which is severely disrupted in Cfl1−/− littermates. Culture in Y27632 or Jasp disrupts colocalization of β-catenin and phalloidin at adherens junctions of the 15–19 somite embryos compared with DMSO controls, whereas ML-7 or Blebbistatin have no adverse effects. Culture in Y27632+Blebbistatin restores a normal staining pattern. Arrowheads, normal staining; asterisks, disrupted staining pattern. Enlargements of the boxed regions are shown for β-catenin alone and the β-catenin and phalloidin merged views. Embryos have 18–21 somites. Scale bars: 30 µm. (B) Summary of cytoskeletal signalling requirements for spinal neural tube closure, based on findings in the study. Normal closure (B1) requires signalling through RhoA, ROCK, LIMK and cofilin to regulate actin turnover and actomyosin disassembly. We suggest this is a prerequisite for both actomyosin-based neuroepithelial tension and ATPase-independent myosin contraction, the latter regulating remodelling of adherens junctions (AJs) without affecting tight junctions (TJs). Myosin ATPase-dependent contractile activity regulated by MLC phosphorylation is not limiting for spinal NT closure, as closure is not inhibited by ML-7. Failure of closure (B2) results when RhoA and ROCK signalling is inhibited by Y27632 (left arrows in B2), with apical actomyosin accumulation, probably because of enhanced cofilin activity, leading to F-actin-assembly through increased actin nucleation. Closure also fails when F-actin is stabilized (right arrows in B2), in Jasp-treated embryos or following the decrease of actin-severing activity in Cfl1 mutants, similarly leading to actomyosin accumulation. Abnormal adherens junctions and disruption of neuroepithelial tension are suggested to result from actomyosin accumulation, and prevent spinal closure.