Roles of a Wnt activator, Ccd1, in neural development

Wnt信号在轴突形成、分化、生长、轴突导向和突触发生中起重要作用。Wnt信号通过β-连环蛋白通路、PCP通路和钙通路三条通路进行转导。虽然已经鉴定了许多参与Wnt通路的分子，但关于Wnt信号转导的调节的许多问题仍然没有解决。最近，我们发现了一个新的蛋白质Ccd 1（卷曲螺旋-DIX 1）作为第三个DIX结构域包含蛋白质从斑马鱼。我们发现，Ccd 1激活Wnt/β-连环蛋白途径在体外和体内，它调节斑马鱼胚胎的神经模式。我们还发现，许多Ccd 1异构体产生通过差异启动子使用和选择性剪接在小鼠中。其中，一个新的异构体，命名为Ccd 1A，含有一个钙调蛋白同源（CH）结构域的N-末端除了卷曲螺旋和DIX结构域中发现的原始异构体（命名为Ccd 1B）。从斑马鱼中，我们分离了第二个基因，Ccd 2，它编码A和B亚型。当在培养的细胞系中异源表达时，Ccd 1B蛋白以小泡状存在于整个细胞中，Ccd 2A蛋白与肌动蛋白纤维共定位。此外，Dishevelled（Dvl）蛋白质的细胞质定位被Ccd 1A和Ccd 1B的强制表达所改变，表明Ccd 1蛋白在细胞中与Dvl相互作用。虽然我们产生了基因敲除小鼠，但它们没有表现出任何明显的表型。当我们在斑马鱼胚胎中过表达Ccd 2时，它们表现出卷曲的尾巴向下表型，表明Ccd 1在中线信号传导中起作用。

Wnt signaling plays important roles in axis formation, differentiation, growth, axon guidance, and synaptogenesis. Wnt signal is transduced via three pathways, beta-catenin pathway, PCP pathway, and Calcium pathway. Although many molecules involved in the Wnt pathways have been identified, many questions regarding the regulation of Wnt signaling remain unresolved. Recently we identified a novel protein Ccd1 (Coiled-coil-DIX1) as third DIX-domain-containing protein from zebrafish. We showed that Ccd1 activates Wnt/beta-catenin pathway both in vitro and in vivo, and that it regulates neural patterning in zebrafish embryos. We also found that many Ccd1 isoforms are generated via differential promoter usage and alternative splicing in mouse. Among them, a novel isoform, named Ccd1A, contains one calponin homology (CH) domain in the N-terminus in addition to the coiled-coil and DIX domains found in the original isoform (named Ccd1B). From zebrafish, we isolated second gene, Ccd2, which encode both A and B subtypes. When heterogouly expressed in cultured cell lines, Ccd1B protein is present as small vesicles throughout cells, Ccd2A protein is co-localized with actin fibers. In addition, the cytoplasmic localization of Dishevelled (Dvl) protein is altered by forced expression of Ccd1A and Ccd1B, suggesting that Ccd1 protein interacts with Dvl in the cell. Although we generated knockout mice, they did not show any obvious phenotypes. When we overexpressed Ccd2 in zebrafish embryos, they showed curly tail-down phenotype, suggesting that Ccd1 plays a role in midline signaling.