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

Neural tube morphogenesis provides a dynamic setting in which to study epithelial cell behaviours. Members of the claudin family of tight junction proteins regulate apical epithelial cell behaviors at all steps of neural tube development. We discovered that CLDN3, expressed in the non-neural ectoderm but not the neural ectoderm, is required to mediate neural fold fusion in chick embryos, particularly in the spinal region of the embryo. Depleting CLDN3 affects apical protein localization and apical domain morphology. Here, we used live imaging to re-examine the process of neural fold fusion in the cranial and spinal regions of the embryo and assessed biomechanical parameters of the non-neural ectoderm that are dependent on CLDN3. Our live imaging confirmed previous reports that unlike neural fold fusion in the cranial region and posterior neuropore, the spinal region does not depend on progressive fusion driven by “zippering” cell behaviors but instead fuses in a multi-step process where contact occurs simultaneously at multiple points along the anterior-posterior axis. We and others refer to the process of spinal neural fold fusion as “buttoning” to highlight the differences in cell behaviors from those observed during zippering (van Straaten et al., 1993). CLDN3-depletion decreased the rate of progression of neural fold buttoning within the spinal region. Using cell segmentation analyses we confirmed that CLDN3 depletion decreased the apical cell area of cells at the edges of the neural folds but not of lateral cells in the non-neural ectoderm. CLDN3 depletion increased pMLC staining within the apical domain of the cell, coinciding with a decrease in cell area, suggesting increased epithelial tension. Laser ablation studies revealed that the non-neural ectoderm of CLDN3-depleted embryos exhibits higher tension during neural fold fusion. We showed that treatment with the myosin II inhibitor blebbistatin is sufficient to partially rescue the neural fold fusion defects in CLDN3-depleted embryos. This work provides further evidence for the importance of non-neural ectodermal tissue tension in neural fold fusion and suggests that loss of CLDN3 may alter tissue tension through cytoskeletal regulation pathways within the apical domain. This work supports that CLDN3 contributes to neural fold fusion and epithelial tissue tension during neural fold fusion via modifications to the apical cytoskeleton.