激活T细胞核因子5在海鞘脊索管腔形成中的渗透调控机制

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.

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。

海洋动物可以利用渗透调控机制塑造自身的功能器官，然而其如何利用渗透调控机制影响渗透敏感的组织器官发育的研究却十分缺乏。海鞘在脊索管腔形成过程中，脊索细胞向内分泌高渗腔液并形成胞外管腔，为研究渗透调控影响组织发育提供了很好的切入点。本项目以海鞘脊索腔液发生与管腔膨胀过程为模式，研究渗透调控分子机制在海洋动物器官形成过程中的角色。本研究发现，渗透调控关键基因激活T细胞核因子5（NFAT5）在形成腔液的萨氏海鞘定位于尾芽幼体脊索细胞核中，而在不形成腔液的柄海鞘尾芽幼体期不表达。通过CRIPSR/Cas9技术敲除海鞘NFAT5发现，突变脊索细胞顶端膜向外隆起而无法正常分泌腔液并完成管腔膨胀过程，同时细胞无法正常变形及迁移，从而导致脊索无法连通为管腔结构，表明NFAT5参与脊索管腔形成的调控过程。细胞学实验显示，海鞘NFAT5可响应渗透压变化而入核或出核，显性负效应突变后NFAT5则无法做出响应，暗示海鞘NFAT5对脊索腔液形成的调控作用是通过渗透压响应途径实现的。比较转录组分析发现细胞外基质蛋白与溶质转运蛋白为可能的NFAT5下游基因。利用染色和显微镜观察，明晰了海鞘脊索胚胎的细胞外基质分布、结构与组成，通过抑制剂实验表明胶原蛋白参与脊索形态维持。开展了萨氏海鞘SLC6 家族基因的鉴定、表达及功能研究。发现长非编码RNA在海鞘脊索管腔腔液形成过程中可能发挥调控作用。以上研究为建立NFAT5及其下游基因调控海鞘脊索腔液发生的信号网络提供了依据，并为了解海洋动物的渗透调控机理和器官发生过程提供了借鉴。