Wdr18 Is Required for Kupffer's Vesicle Formation and Regulation of Body Asymmetry in Zebrafish

Correct specification of the left-right (L-R) axis is important for organ morphogenesis. Conserved mechanisms involving cilia rotation inside node-like structures and asymmetric Nodal signaling in the lateral plate mesoderm (LPM), which are important symmetry-breaking events, have been intensively studied. In zebrafish, the clustering and migration of dorsal forerunner cells (DFCs) is critical for the formation of the Kuppfer's vesicle (KV). However, molecular events underlying DFC clustering and migration are less understood. The WD-repeat proteins function in a variety of biological processes, including cytoskeleton assembly, intracellular trafficking, mRNA splicing, transcriptional regulation and cell migration. However, little is known about the function of WD-repeat proteins in L-R asymmetry determination. Here, we report the identification and functional analyses of zebrafish wdr18, a novel gene that encodes a WD-repeat protein that is highly conserved among vertebrate species. wdr18 was identified from a Tol2 transposon-mediated enhancer trap screen. Follow-up analysis of wdr18 mRNA expression showed that it was detected in DFCs or the KV progenitor cells and later in the KV at early somitogenesis stages. Morpholino knockdown of wdr18 resulted in laterality defects in the visceral organs, which were preceded by the mis-expression of Nodal-related genes, including spaw and pitx2. Examination of morphants at earlier stages revealed that the KV had fewer and shorter cilia which are immotile and a smaller cavity. We further investigated the organization of DFCs in wdr18 morphant embryos using ntl and sox17 as specific markers and found that the clustering and migration of DFC was altered, leading to a disorganized KV. Finally, through a combination of wdr18 and itgb1b morpholino injections, we provided evidence that wdr18 and itgb1b genetically interact in the laterality determination process. Thus, we reveal a new and essential role for WD-repeat proteins in the determination and regulation of L-R asymmetry and propose a potential mechanism for wdr18 in the regulation of DFC clustering and migration and KV formation.

左右（L-R）轴的正确特化对于器官形态发生至关重要。涉及节样结构内部纤毛旋转以及侧板中胚层（lateral plate mesoderm, LPM）中不对称Nodal信号的保守机制均为重要的对称性破缺事件，已得到广泛深入的研究。在斑马鱼中，背前体细胞（dorsal forerunner cells, DFCs）的聚集与迁移对于库珀囊（Kuppfer's vesicle, KV）的形成至关重要，但目前对于DFC聚集与迁移背后的分子事件仍知之甚少。WD重复蛋白（WD-repeat protein）参与多种生物学过程，包括细胞骨架组装、细胞内运输、mRNA剪接、转录调控以及细胞迁移，但目前关于WD重复蛋白在左右轴不对称决定中的功能却鲜有报道。本研究鉴定了斑马鱼中的wdr18基因并对其功能进行分析，该基因编码一种在脊椎动物中高度保守的WD重复蛋白。wdr18是通过Tol2转座子介导的增强子陷阱筛选得以鉴定的。对wdr18 mRNA表达的后续分析显示，在早期体节发生阶段，其表达首先出现在DFCs或KV前体细胞中，随后在KV中可被检测到。对wdr18进行吗啉代寡核苷酸敲低会引发斑马鱼内脏器官的左右不对称缺陷，而这一缺陷的出现早于spaw、pitx2等Nodal相关基因的表达紊乱。对早期发育阶段的wdr18敲低胚胎进行观察发现，KV中的纤毛数量更少、长度更短且无法运动，同时囊腔体积更小。本研究进一步以ntl和sox17作为特异性标记物，对wdr18敲低胚胎中DFCs的组织结构进行了分析，结果发现DFCs的聚集与迁移过程发生异常，进而导致KV结构紊乱。最后，通过联合注射wdr18与itgb1b的吗啉代寡核苷酸，本研究证实wdr18与itgb1b在左右不对称决定过程中存在遗传互作。综上，本研究揭示了WD重复蛋白在左右不对称决定与调控过程中全新且不可或缺的重要作用，并提出了wdr18调控DFC聚集与迁移以及KV形成的潜在分子机制。