Role of AKAP12 and Claudin5 in Kupffers vesicle development

Abstract

Left-right asymmetric organ development is important to establish a proper body plan of vertebrates. In zebrafish, the Kupffers vesicle (KV) is a fluid-filled sac which controls asymmetric organ development, and a properly inflated KV lumen by means of fluid influx is a prerequisite for the asymmetric signal transmission. KV is derived from the dorsal forerunner cells (DFCs), which collectively migrate from the margin of the embryonic shield to the tail bud region, and become polarized to form a rosette structure. Then, the lumen is formed at the apical point of the rosette structure and expanded by fluid influx through ion channels. Finally, nodal signal is exclusively restricted in left-lateral plate mesoderm and organ shows its laterality. However, the underlying mechanisms of the cell-cell interaction during KV formation are poorly studied. Here, I identified that akap12 was expressed in DFCs and downregulation of akap12β in zebrafish showed a failure in organ laterality that resulted from malformed KV. In addition, KV lineage cell-specific knockdown of akap12β also exhibited the organ laterality defects. In akap12β morphants, cluster of DFC was fragmented which represents a failure of cell collectivity within DFCs, and Cdh1 expression was decreased. Thus, these findings suggest that akap12β regulates KV development by maintaining cell collectivity within DFCs via Cdh1-mediated adherens junction. Furthermore, I identified that the cldn5a is highly expressed at the apical surface of KV epithelial cells and tightly seals the KV lumen. Downregulation of cldn5a in zebrafish showed a failure in organ laterality that resulted from malformed KV. In addition, accelerated fluid influx into KV by combined treatment of forskolin and 3-isobutyl-1-methylxanthine failed to expand the partially-formed KV lumen in cldn5a morphants. However, malformed KV lumen and defective heart laterality in cldn5a morphants were significantly rescued by exogenous cldn5a mRNA, suggesting that the tightness between the luminal epithelial cells is important for KV lumen formation. Thus, these findings suggest that cldn5a is required for KV lumen inflation and left-right asymmetric organ development. Taken together, these findings suggest that akap12β regulates KV development by maintaining cell collectivity within DFCs via Cdh1-mediated adherens junction and cldn5a regulates KV development by tight sealing the paracellular space between the KV epithelial cells.