The Dact/Sestd1 pathway in embryonic malformations

描述（由申请人提供）：人类出生缺陷可能是由调节细胞形状和运动的分子通路中断引起的。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初级胚层，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。多种信号传导途径有助于细胞的形状和运动，包括脊椎动物中会聚延伸（CE）运动上游的Wnt平面细胞极性（PCP）途径。在之前的资助期间，我们在小鼠Dapper/Frodo（Dact 1）基因座上使用了工程敲除，以探索其损失如何导致小鼠的一系列发育缺陷，类似于一系列主要的出生缺陷。通过这个项目，我们发现Dact 1调节货车样（Vangl）跨膜蛋白，其在原肠胚形成期间原始条纹（PS）处的上皮-间充质转化（EMT）期间对PCP起中心作用。我们现在已经为哺乳动物中剩下的两个Dact家族成员（Dact 2和Dact 3）以及Sestd 1设计了新的敲除小鼠品系，Sestd 1编码我实验室鉴定的Dact相互作用蛋白。这些动物非常接近表型Dact 1突变体，强烈支持的假设，Sestd 1的主要发育功能与Dact 1密切相关。有趣的是，Sestd 1先前已被证明有助于调节通过TRPC通道蛋白的钙通量;此外，其最接近的分子亲戚已被牵连在跨膜蛋白运输中。这与证据表明，从我的实验室工作中出现的分子途径通过定位和/或跨膜蛋白的一个子集在几个重要的信号级联的交叉点的水平的行为。在新的资助期内，我建议解决这些具体目标：（1）Sestd 1和Dact 1是否在PS的EMT期间与Scribble，Celsr 1和Vangl 2形成遗传通路;在CE期间与Dvl 2形成遗传通路;在钙信号传导期间与TRPC 4/5形成遗传通路？(2)在这些突变体的后胚胎组织中，PCP和钙通道是否都被生物化学破坏？(3)在受影响的胚胎组织中，细胞膜定位和通路成分的水平与PCP和钙信号有关吗？与之前的资助期一样，研究策略借鉴了在我的实验室中定制和组装的深度小鼠遗传、胚胎、细胞生物学和生化工具，以探索这一途径。

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.