Gdnf and endothelin-3 regulate colorectal enteric nervous system development

描述（由申请人提供）：肠神经系统（ENS）是存在于肠壁中的神经元和神经胶质的复杂网络，对于调节肠运动至关重要。ENS的异常发育是先天性巨结肠病的根本原因，先天性巨结肠病是由于肠神经节缺失引起的先天性肠道疾病，通常发生在远端结直肠，并导致新生儿严重肠梗阻。Ret和内皮素受体B（Ednr B）信号通路的缺陷是ENS发育所必需的，并且是许多先天性巨结肠症病例的原因。然而，这些关键途径如何在体内发挥作用以调节结肠直肠中ENS的发育，以及为什么突变会导致人类结肠直肠无神经节细胞症，目前还知之甚少。这项建议的主要目的是确定调节结肠直肠ENS发育的分子机制，以确定治疗先天性巨结肠和其他先天性肠神经病变的潜在靶点。我们假设，Ret和EdnrB信号协调作用，影响ENS祖细胞的迁移，增殖和分化，因为它们穿过盲肠和泄殖腔区域，并且这种功能对于ENS在结肠的定植至关重要。为了验证这一假设，我们将使用禽类胚胎在体内激活和抑制基因表达，以研究这些信号通路的作用。禽类模型系统的主要优点是易于在整个胚胎发生过程中进行实验操作，并且能够比啮齿动物更快地以更低的成本进行遗传获得和功能丧失研究。我们建议使用复制能力的逆转录病毒基因过表达，基于载体的RNAi基因沉默，和器官培养试验。具体目标我将建立的作用EdnrB信号的迷走神经和骶骨ENS祖细胞的迁移，生存，增殖和分化的远端肠ENS的形成过程中。具体目标II将审查的作用，通过集中在这个途径的功能，ENS祖细胞的迁移和增殖，因为它们穿过盲肠和泄殖腔区的结肠直肠无神经节细胞症的病因Ret信号。Specific Aim III将使用我们通过抑制远端肠中EdnrB信号传导产生的结直肠无神经节细胞症的器官培养模型。将Ret活性的激活剂和抑制剂添加到EdnrB缺陷的肠中，以通过调节这两种途径之间的活性平衡来挽救无神经节细胞表型。这些实验将确立Ret和EdnrB信号在远端ENS中的作用，为结直肠ENS发展的机制提供新的见解，并确定治疗神经肠道疾病的潜在靶点。公共卫生相关性：控制肠道功能的神经发育异常是儿童肠道疾病的主要原因。例如，先天性巨结肠症是由大肠中先天性缺乏神经细胞引起的，导致新生儿严重肠梗阻，需要手术切除受影响的肠道。该提案的目标是了解控制肠神经系统发育的机制，以提高我们诊断和治疗患有这些慢性和致残性疾病的儿童的能力。

DESCRIPTION (provided by applicant): The enteric nervous system (ENS) is a complex network of neurons and glia present in the bowel wall and critical for regulating intestinal motility. Abnormal development of the ENS is the underlying cause of Hirschsprung's disease, a congenital intestinal disorder caused by the absence of enteric ganglia, usually in the distal colorectum, and leading to severe intestinal obstruction in newborns. Defects in the Ret and endothelin receptor B (EdnrB) signaling pathways are required for ENS development and are responsible for many cases of Hirschsprung's disease. However, how these key pathways function in vivo to regulate ENS development in the colorectum, and why mutations cause colorectal aganglionosis in humans, is poorly understood. The broad objective of this proposal is to determine the molecular mechanisms that regulate colorectal ENS development in order to identify potential targets for the treatment of Hirschsprung's disease and other congenital intestinal neuropathies. We hypothesize that Ret and EdnrB signaling act coordinately to influence the migration, proliferation, and differentiation of ENS progenitor cells as they cross the cecal and cloacal regions and that this function is critically important for ENS colonization of the colon. To test this hypothesis, we will use the avian embryo to activate and inhibit gene expression in vivo in order to study the role of these signaling pathways. The major advantages of the avian model system are the ease of performing experimental manipulations throughout embryogenesis and the ability to carry out genetic gain- and loss-of-function studies more quickly and at lower cost than in rodents. We propose to use replication-competent retrovirus for gene overexpression, vector-based RNAi for gene silencing, and organ culture assays. Specific Aim I will establish the role of EdnrB signaling on the migration, survival, proliferation, and differentiation of vagal and sacral ENS progenitor cells during formation of the distal intestinal ENS. Specific Aim II will examine the role of Ret signaling in the etiology of colorectal aganglionosis by focusing on the function of this pathway on migration and proliferation of ENS progenitors as they cross the cecal and cloacal regions. Specific Aim III will use an organ culture model of colorectal aganglionosis that we generate by inhibiting EdnrB signaling in the distal intestine. Activators and inhibitors of Ret activity will be added to EdnrB-deficient intestine in order to rescue the aganglionic phenotype by modulating the balance of activity between these two pathways. These experiments will establish the role of Ret and EdnrB signaling in the distal ENS, provide new insights into mechanisms underlying colorectal ENS development, and identify potential targets for the treatment of neurointestinal disorders. PUBLIC HEALTH RELEVANCE: Abnormalities in the development of the nerves that control intestinal function are a major cause of intestinal disease in children. Hirschsprung's disease, for example, is caused by the congenital absence of nerve cells in the large intestine, leading to severe bowel obstruction in newborns and requiring surgical removal of the affected intestine. The goal of this proposal is to understand the mechanisms that control development of the intestinal nervous system in order to improve our ability to diagnose and treat children born with these chronic and disabling conditions.