Identifying long-range polarity cues using spontaneous mutations in mammals

项目摘要/摘要 神经管闭合缺陷和先天性心脏缺陷是常见的发育障碍，影响1/4。 每100名活产儿中分别有1000名和1名。在胚胎发育过程中，细胞必须能够感知 指导和协调集体行为，以避免这些严重畸形。尽管这些信号 在相邻细胞之间传递方向性的途径是已知的，揭示了 这一信息的传输在数千个单元的数量级上已经停滞不前。幸运的是，人类体内的自发突变 小型哺乳动物产生了因毛皮取向特定地区的干扰而备受珍视的动物。我 假设这些突变扰乱了长程定向线索。这项提议的目的是利用 哺乳动物表皮中识别长程定向线索的自发突变。这项工作将脱颖而出 阐明复杂先天性疾病的病因，这将有助于宫内治疗的发展 以及人造器官的产生。 让细胞传递方向信息的一个关键分子途径是平面细胞 极性(PCP)途径，如果没有该途径，就会出现严重的发育缺陷。核心路径由三个部分组成 形成不对称复合体的跨膜蛋白，这种复合体定位于细胞的两边。 通过细胞外的正反馈和细胞内的负反馈，这种不对称性得以传播 但在整个组织中导致不对称性的机制却知之甚少。虽然核心 果蝇的途径成分是通过正向遗传筛选来鉴定的，这种方法一直很少 成功地发现了全球的极性线索。老鼠的皮肤提供了一个理想的系统来识别远程 极性暗示。扩张的组织上装饰着数以千计的毛囊，这些毛囊都定向在 通过五氯苯酚驱动的过程也是如此。值得注意的是，我们发现背部皮肤被隔开了 进入影响PCP信号方向的区域域。这一数据表明，特定地区的 远程提示协同协调PCP和HF在整个组织中的定向。为了支持这一点 认为，小型哺乳动物的自发突变产生的动物具有明显的区域特异性干扰 毛囊方向。为了研究这些突变是否会影响长程极性基因，我将确定 通过基因组测序获得致病突变，并表征突变对组织的影响 使用遗传学和图像分析的极性。然后我将询问识别出的蛋白质作为 在活体和器官皮肤培养中的长程极性提示。在这项研究完成后，我们会确定 并了解在组织形成过程中协调远距离集体行为的新机制 形态发生。

Project Summary/ Abstract Neural tube closure defects and congenital heart defects are common developmental disorders, affecting 1 in 1000 and 1 in 100 live births, respectively. During embryonic development, cells must be able to sense direction and coordinate collective behaviors to avoid these severe malformations. Although the signaling pathway that imparts directionality between neighboring cells is known, uncovering the mechanisms that transmit this information on the order of thousands of cells has stalled. Fortuitously, spontaneous mutations in small mammals have given rise to animals that are prized for region specific disruptions in fur orientation. I hypothesize that the mutations disrupt long-range directional cues. The objective of this proposal is to utilize spontaneous mutations to identify long-range directional cues in the mammalian epidermis. This work will shed light on the etiology of complex congenital diseases, which will assist in the development of in utero therapies and the generation of artificial organs. A key molecular pathway that allows cells to communicate directional information is the Planar Cell Polarity (PCP) pathway, without which severe developmental defects arise. The core pathway consists of three transmembrane proteins that form asymmetric complexes that are localized to opposite sides of a cell. Through both positive extracellular feedback and negative intracellular feedback, this asymmetry is propagated locally but the mechanism that orients asymmetry across entire tissues is poorly understood. Although the core pathway components were identified through forward genetic screens in Drosophila, this method has been less successful in uncovering global polarity cues. The mouse skin provides an ideal system to identify long-range polarity cues. The expansive tissue is decorated with thousands of hair follicles (HFs) that are all oriented in the same direction through a PCP driven process. Remarkably, we showed the back skin is compartmentalized into regional domains that influence the direction of PCP signaling. This data suggests that region specific long-range cues cooperate to coordinate PCP and HF orientation across the entire tissue. In support of this idea, spontaneous mutations in small mammals produce animals with distinct, region-specific disruptions in hair follicle orientation. To investigate whether these mutations affect long-range polarity genes, I will identify the causative mutations through genomic sequencing and characterize the effect the mutations have on tissue polarity using genetics and image analysis. I will then interrogate the ability of the identified proteins to act as long-range polarity cues in vivo and in organotypic skin culture. Upon completion of this study, we will identify and understand new mechanisms that coordinate collective behaviors across great distances during tissue morphogenesis.