The role of Aqp3b during neurulation in Xenopus laevis

Abstract

Convergent extension (CE) is an important morphological event during gastrulation formation of the notochord from axial mesoderm and during neurulation, when CE in the neural ectoderm helps the neural plate to fold into a neural tube, the precursor of the central nervous system. CE is regulated by the noncanonical Wnt/ PCP signaling pathway and any perturbation of the pathway can cause abnormalities in neural tube closure. Another driver of neural tube closure is apical constriction of the neural plate cells, which allows bending of the neural plate to form the neural tube. Aqp3b is an aquaglyeroporin that is expressed in the neural folds of Xenopus laevis embryos during neural tube closure. Using aqp3b morpholino oligonucleotides (MOs) to inhibit the translation of aqp3b in Xenopus embryos, our study revealed that aqp3b is required for the convergence of the neural folds during neural tube closure. aqp3b MO injections into a single dorsal blastomere in the animal or medial region showed neural tube closure defects (stage 17) with neural folds unable to fuse. Beta-catenin staining of these embryos illustrated that lack of apical constriction in neuroepithelial cells on the injected side prevented neural tube closure. Phalloidin staining also revealed a lack of apical constriction of neural plate cells on the injected side. In situ hybridization with en-2 probe was performed on aqp3b MO-injected embryos, which confirmed neural tube closure defects. In order to test whether the neural tube closure defects were the result of underlying gastrulation defects, embryos were injected with aqp3b MOs and analyzed by in situ hybridization with chordin probe (to visualize the dorsal mesoderm) during gastrulation (stage 12.5). When aqp3b MOs were injected medially (targeting the dorsal mesoderm), wider chordin expression was observed, indicating convergent extension defects of the dorsal mesoderm. However, when aqp3b MOs were injected into the animal region (targeting the neural ectoderm), chordin staining was largely normal, suggesting normal CE of the dorsal mesoderm. This indicated that the neural tube closure defects observed in embryos, where aqp3b MOs were targeted to the neural ectoderm, were not caused by gastrulation defects. Thus, aqp3b MO is required in the dorsal mesoderm for CE and in the neural plate for apical constriction during neural tube closure. Since these experiments did not assess whether Aqp3b is required for CE of the neural plate, XBF-2 was expressed in animal caps to give them neural character and elicit CE. Injecting aqp3b MO into XBF-2-expressing animal caps significantly reduced CE, suggesting a role for aqp3b during CE of the neural plate during neural tube closure.