渗透调控不仅是海洋动物适应高盐海水环境的重要生理过程，而且影响渗透敏感的组织器官发育。海洋模式动物海鞘就是通过分泌腔液营造高渗环境继而膨胀形成脊索管腔。申请人前期筛选调查发现渗透调控关键基因激活T细胞核因子5（NFAT5）特异性在海鞘脊索细胞中高表达，暗示其参与脊索管腔形成的调控过程。.本研究拟以海鞘脊索腔液发生与管腔膨胀过程为模式，通过CRISPR/Cas9敲除NFAT5建立突变体。利用组织特异性荧光标记和共聚焦显微镜，观察确定海鞘幼虫NFAT5突变体表型。对比分离的野生型和NFAT5突变体脊索细胞在不同渗透环境下的生理形态反应，确定NFAT5调控脊索细胞渗透压的角色。利用单细胞测序技术筛选NFAT5下游靶基因，重点研究与细胞外基质蛋白分泌及离子通道转运功能相关因子，建立NFAT5调控渗透的分子信号通路。预期将阐明脊索腔液发生与管腔膨胀的分子机制并为了解海洋动物的渗透调控机理提供借鉴。

For marine animals, osmoregulation is not only responsible for adaption in seawater, but also plays an important role during organ formation. Ciona, which is an important marine model animal, forms its notochord by secretion of hyperosmotic lumen during its early development. It has been proved that, the key nuclear factor for osmoregulation, NFAT5 was specifically and highly expressed in Ciona notochord cells, which implied its important function during notochord formation..In this study, we plan to use Ciona notochord as a model to establish the NFAT5 mutant by CRIPSR/Cas9 knockout. The mutant phenotype will be observed by laser confocal microscopy and tissue specific fluorescence labeling. The morphology and physiology changes of sorted notochord cells under different osmotic pressure will be compared between wild types and NFAT5 mutants to clarify the function of NFAT5 during osmoregulation. Single cell sequencing technology will also be used to screen the downstream genes of NFAT5. Among these candidate downstream genes, we intend to focus on extracellular matrix and transport related genes. The expected results would be able to illustrate the molecular mechanisms of notochord lumen formation and tube expansion and would help to further understand the osmoregulation mechanisms of marine animals.