The Dact/Sestd1 pathway in embryonic malformations

描述(申请人提供)：人类先天缺陷可能是由于调节细胞形状和运动的分子通路中断所致。细胞形状的变化和运动有助于在原肠胚形成时建立和分离三个主要胚层，将这些胚层具体化为不同的组织类型，并将组织组合成具有独特内部结构和解剖关系的器官。多种信号通路影响细胞的形状和运动，包括脊椎动物汇聚-延伸(CE)运动上游的Wnt平面细胞极性(PCP)通路。在之前的资助期间，我们使用了小鼠Dapper/Frodo(Dact1)基因座的工程敲除来探索它的丢失如何导致小鼠的一系列发育缺陷，类似于主要的出生缺陷。通过这个项目，我们发现Dact1在原肠胚形成的原始条纹(PS)的上皮-间充质转变(EMT)过程中，调节PCP中心的梵高样蛋白(Vangl)。我们现在已经为哺乳动物中剩下的两个Dact家族成员(Dact2和Dact3)以及Sestd1设计了新的敲除小鼠系，Sestd1编码了我实验室中发现的一种Dact相互作用蛋白。这些动物非常接近表型Dact1突变体，有力地支持了Sestd1的主要发育功能与Dact1密切相关的假说。有趣的是，Sestd1之前已经被证明通过TRPC通道蛋白帮助调节钙离子的流动；此外，它最接近的分子与跨膜蛋白运输有关。这与我的实验室工作中出现的分子途径通过几个重要信号级联的交叉处的跨膜蛋白子集的定位和/或水平起作用的证据是一致的。在新的资助期，我建议解决这些特定的目标：(1)在PS的EMT期间，Sestd1和Dact1是否与Scribble、Celsr1和Vangl2形成遗传途径；在CE期间与Dvl2形成遗传途径；在钙信号传递期间与TRPC4/5形成遗传途径？(2)在这些突变体的后部胚胎组织中，PCP和钙途径是否都受到生化破坏？(3)在受影响的胚胎组织中，膜的定位和途径成分的水平是否与PCP和钙信号有关？与之前的资助时期一样，该研究战略利用在我的实验室专门设计和组装的深度小鼠遗传、胚胎、细胞生物和生化工具来探索这一途径。

DESCRIPTION (provided by applicant): Human birth defects can be caused by disruption of molecular pathways that regulate cell shape and movements. Cell shape changes and movements are responsible for helping to establish and separate the three primary germ layers at gastrulation, for reification of these germ layers into different tissue types, and for combinin tissues into organs with unique internal architecture and anatomical relationships. Multiple signaling pathways contribute to cell shape and movement, including the Wnt Planar Cell Polarity (PCP) pathway upstream of convergent-extension (CE) movements in vertebrates. In the prior funded period we used an engineered knock-out at the mouse Dapper/Frodo (Dact1) locus to explore how its loss causes a spectrum of developmental defects in mice resembling a major spectrum of birth defects. Through this project we discovered that Dact1 regulates Van Gogh-like (Vangl) transmembrane proteins central to PCP during Epithelial-Mesenchymal Transition (EMT) at the Primitive Streak (PS) during gastrulation. We have now engineered novel knock-out mouse lines for the two remaining Dact family members in mammals (Dact2 and Dact3) as well as for Sestd1, which encodes a Dact-interacting protein identified in my lab. These animals very closely phenocopy Dact1 mutants, strongly supporting the hypothesis that major developmental functions of Sestd1 are allied closely with Dact1. Intriguingly, Sestd1 has previously been shown to help regulate calcium flux through TRPC channel proteins; moreover its closest molecular relative has been implicated in transmembrane protein trafficking. This agrees with evidence suggesting that the molecular pathway emerging from work in my lab acts via localization and/or levels of a subset of transmembrane proteins at the intersection of several important signaling cascades. In the renewed funding period I propose to address these Specific Aims: (1) Do Sestd1 & Dact1 form a genetic pathway with Scribble, Celsr1 & Vangl2 during EMT at the PS; with Dvl2 during CE; and with TRPC4/5 during calcium signaling? (2) Are both the PCP & Calcium pathways biochemically disrupted in posterior embryonic tissues of these mutants? (3) Are membrane localization & levels of pathway components linked to PCP & Calcium signaling in affected embryonic tissues? As in the prior funded period, the research strategy draws on deep mouse genetic, embryonic, cell biologic and biochemical tools custom-designed and assembled in my laboratory expressly to explore this pathway.