Vangl2 function in mammalian convergent extension

 描述(申请人提供)：身体在头到臀轴的伸长是脊椎动物胚胎发生的一个基本方面，其失败与鱼、青蛙、小鼠和人类的神经管缺陷和胚胎死亡有关。在人类中，神经管缺陷影响每1000名新生儿中的1到2名，在最严重的情况下会导致婴儿早期死亡。 在不太严重的情况下，可获得终身医疗和残疾。伸长是指未来脊髓和相关中胚层的细胞在彼此之间主动移动(插入)以形成更窄、更长的组织(称为“汇聚和延伸”或CE)，从而延长胚胎身体的过程。在脊椎动物中，这种“内侧”细胞嵌入依赖于非规范的Wnt平面细胞极性(Wnt/PCP)途径，该途径由一组被认为在组织平面内极化细胞的核心蛋白质组成：膜蛋白Celsr、FrizzledFRZ和Van Gogh-like(Vangl)，以及细胞内蛋白Disheveled(DVL)和棘突(PK)。携带Celsr1、Frz3/6、Vangl2和Dvl1/2以及Ptk7和Scrib基因突变的小鼠，这两个基因与Wnt/PCP没有直接关联，表现为体轴短而宽，神经管开放，出生时死亡。一种细胞嵌入的遗传调控模型侧重于上皮细胞顶面连接的极化重塑，而另一种模型则侧重于非上皮细胞的极化“爬行”。使用高分辨率、多平面延时共聚焦显微镜同时成像正常小鼠胚胎神经上皮的顶端连接表面和基底外侧表面，并与Vangl2和Ptk7突变体进行比较，结果表明，顶端细胞边界重排和极化的基底外侧突起活动必须协同作用才能发生中外侧插入和CE。缺少Ptk7的神经细胞不能在平面上极化顶端和基底外侧细胞的行为，它们有力地但随机地插入。令人惊讶的是，在Vangl2突变体中，基础突起活动和心尖边界重排的极性都保持不变，相反，心尖邻居交换的频率显著降低。这些意想不到的观察，以及其他已发表的Wnt/PCP突变基因在脊索动物中的表型变异，表明Wnt/PCP的功能在物种和组织中是多样化的。拟议中的实验通过确定Vangl和相关蛋白Scrib如何调控神经细胞行为来探索这种可能性。首先，由于Vangl2的丢失不会影响细胞行为的平面极性，我们将测试Paralog Vangl1是否能够在没有Vangl2的情况下进行补偿以产生极性，或者是否相反地由Vangl2和Scrib的复合体来调节极性。其次，由于Vangl2特异性地影响顶端连接重排，我们将测试它是否影响神经细胞嵌入过程中基于钙粘附素的黏附动力学。这些结果将为Wnt/PCP通路在神经发育过程中调节细胞行为提供更广泛和更深入的见解，以及它们如何协调神经管的延长和关闭。

 DESCRIPTION (provided by applicant): Elongation of the body in the head-to-rump axis is a fundamental aspect of vertebrate embryogenesis whose failure is associated with neural tube defects and embryonic mortality in fish, frogs, mice, and humans. In humans, neural-tube defects affect 1 to 2 infants per 1000 births and lead in the worst cases to early infant mortality and in less severe cases to lifelong medical treatment and disability. Elongation occurs by a process in which cells of the future spinal cord and associated mesoderm actively move (intercalate) between one another to form narrower, longer tissues (called "convergence and extension' or CE), which elongates the embryonic body. In vertebrates, this "mediolateral" cell intercalation is dependent on the non-canonical Wnt planar cell polarity (Wnt/PCP) pathway, which consists of a core set of proteins thought to polarize cells within the plane of the tissue: the membrane proteins Celsr, Frizzled (Frz), and Van Gogh-like (Vangl), and the intracellular proteins Dishevelled (Dvl) and Prickle (Pk). Mice carrying mutations in the genes Celsr1, Frz3/6, Vangl2 and Dvl1/2 as well as Ptk7 and Scrib, two genes not directly associated with Wnt/PCP, exhibit a characteristic phenotype of a short, wide body axis, an open neural tube, and lethality at birth. One model of genetic regulation of cell intercalation focuses on polarized remodeling of junctions at the apical surfaces of epithelial cells and the other on polarized "crawling" of non-epithelial cells. Use of high resolution, multi-plane time-lapse confocal microscopy to simultaneously image both apical junctional and the basolateral surfaces of the neural epithelium of normal mouse embryos as compared to Vangl2 and Ptk7 mutants, showed that apical cell boundary rearrangement and polarized basolateral protrusive activity must act cooperatively for mediolateral intercalation and CE to occur. Neural cells lacking Ptk7 fail to planarly polarize both apical and basolateral cell behaviors and they intercalate vigorously but randomly. Surprisingly, polarity of both basal protrusive activity and apical boundary rearrangement is maintained in the Vangl2 mutant, and instead the frequency of apical neighbor exchange is significantly diminished. These unexpected observations, and other, published variations in phenotypes of Wnt/PCP mutant genes across the chordates, suggest that Wnt/PCP function has diversified across species and tissues. The proposed experiments explore this possibility by determining how Vangl and the related protein Scrib regulate neural cell behavior. First, because the loss of Vangl2 does not affect planar polarity of cell behavior, we will test whether the paralog Vangl1 can compensate to generate polarity in the absence of Vangl2, or whether polarity is regulated instead by a complex of Vangl2 and Scrib. Second, because Vangl2 specifically affects apical junctional rearrangement, we will test whether it affects the dynamics of cadherin-based adhesion in neural cell intercalation. The results will provide broader and deeper insights into the cellular behaviors regulated by the Wnt/PCP pathway during neural development, and how they coordinate elongation and closure of the neural tube.