Increased tissue tension caused by depletion of CLDN3 in the non-neural ectoderm causes neural tube defects in chick embryos.

Neural tube morphogenesis provides a dynamic setting in which to study epithelial cell behaviours. Here, we examined CLDN3-dependent biomechanical properties of the non-neural ectoderm during neural tube closure in chick embryos. Previous analyses of static images of chick embryos suggested that spinal neural tube closure occurs through multiple contact points or 'buttoning'. Using live imaging and transverse cuts through the neural folds we confirmed the multiple simultaneous 'buttoning' contacts along the anterior-posterior axis and showed that spinal neural fold fusion is not the result of progressive 'zippering' cell behaviours as is required in cranial and posterior neuropore regions. CLDN3 depletion decreased the rate of neural fold closure between points of contact and increased phosphorylated myosin light chain (pMLC) within the apical domain of non-neural ectoderm cells, suggesting increased actomyosin contraction. Laser ablation studies revealed that CLDN3-depleted embryos exhibited higher tension across the non-neural ectoderm during neural fold fusion. Decreasing tension with blebbistatin rescued the neural fold fusion defects in CLDN3-depleted embryos. Our data support the importance of CLDN3 to regulate tissue tension through effects on cytoskeletal dynamics in the non-neural ectoderm during neural fold fusion.