The Dact/Sestd1 pathway in embryonic malformations

描述（由申请人提供）：人类出生缺陷可由调节细胞形状和运动的分子途径的破坏引起。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，使这些胚层具体化为不同的组织类型，并将组织结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Wnt平面细胞极性（PCP）通路在脊椎动物的收敛延伸（CE）运动的上游。在之前的资助期内，我们对小鼠Dapper/Frodo （Dact1）基因座进行了工程敲除，以探索其缺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在原肠胚形成过程中原始条纹（PS）上皮-间充质转化（EMT）过程中对PCP至关重要的梵高样（Van Gogh-like, Vangl）跨膜蛋白进行调控。我们现在已经为哺乳动物中的两个Dact家族成员（Dact2和Dact3）以及Sestd1设计了新的敲除小鼠系，Sestd1编码了我实验室发现的一种Dact相互作用蛋白。这些动物与Dact1突变体的表型非常接近，有力地支持了Sestd1的主要发育功能与Dact1密切相关的假设。有趣的是，Sestd1先前已被证明有助于通过TRPC通道蛋白调节钙通量；此外，它最接近的分子亲戚涉及跨膜蛋白质运输。这与有证据表明，在我的实验室工作中出现的分子途径是通过在几个重要信号级联的交叉点定位和/或跨膜蛋白亚群的水平起作用的。在新的资助期内，我建议解决这些具体目标：(1)在PS的EMT期间，Sestd1和Dact1是否与Scribble， Celsr1和Vangl2形成遗传通路；CE时为Dvl2；和TRPC4/5在钙信号传导中的作用？(2)这些突变体胚胎后部组织的PCP和钙通路是否都被生化破坏？(3)受影响胚胎组织中膜定位和通路成分水平是否与PCP和钙信号有关？与之前的资助期一样，研究策略利用了我的实验室专门设计和组装的小鼠遗传、胚胎、细胞生物学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.