Gdnf and endothelin-3 regulate colorectal enteric nervous system development

Gdnf 和内皮素 3 调节结直肠肠神经系统发育

• 批准号: 8072025
• 负责人: ALLAN M GOLDSTEIN
• 金额: $ 37.73万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072025
• 关键词: Address; Affect; Animal Model; Award; Biological Assay; Biological Models; Birds; Cecum; Cells; Child; Chimera organism; Chronic; Cloaca Chamber; Colon; Colonic Aganglionosis; Colorectal; Complex; Congenital Disorders; Congenital Megacolon; Data; Defect; Development; Diagnosis; Disease; Distal; Embryo; Embryonic Development; Endothelin B Receptor; Endothelin-3; Enteral; Enteric Nervous System; Equilibrium; Etiology; Excision; Ganglia; Gastrointestinal tract structure; Gene Expression; Gene Silencing; Genes; Genetic; Goals; Health; Hindgut; Human; Immunohistochemistry; In Vitro; Intestinal Diseases; Intestinal Motility; Intestinal Obstruction; Intestines; Large Intestine; Ligands; Modeling; Molecular; Motor Activity; Mutation; Nerve; Nervous system structure; Neural Crest; Neural Crest Cell; Neural tube; Neuroglia; Neuronal Differentiation; Neurons; Neuropathy; Newborn Infant; Organ Culture Techniques; Pathway interactions; Patients; Phenotype; Population; Quail; RNA Interference; Retroviridae; Rodent; Role; Signal Pathway; Signal Transduction; Staging; Stem cells; Testing; Work; base; cost; improved; in vivo; inhibitor/antagonist; insight; loss of function; migration; mouse model; nervous system development; overexpression; prevent; progenitor; research study; vector

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.

描述（由申请人提供）：肠神经系统（ENS）是肠壁中存在的神经元和神经胶质细胞的复杂网络，对于调节肠道蠕动至关重要。 ENS 发育异常是先天性巨结肠症的根本原因，先天性肠道疾病是由于肠神经节缺失引起的，通常位于结直肠远端，导致新生儿严重肠梗阻。 Ret 和内皮素受体 B (EdnrB) 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病例的原因。然而，这些关键通路如何在体内发挥作用来调节结直肠中 ENS 的发育，以及为什么突变会导致人类结直肠神经节缺失症，人们知之甚少。该提案的主要目标是确定调节结直肠 ENS 发育的分子机制，以便确定治疗先天性巨结肠症和其他先天性肠道神经病的潜在靶点。我们假设 Ret 和 EdnrB 信号传导协同作用，影响 ENS 祖细胞穿过盲肠和泄殖腔区域时的迁移、增殖和分化，并且该功能对于 ENS 在结肠的定植至关重要。为了检验这一假设，我们将使用禽类胚胎来激活和抑制体内基因表达，以研究这些信号通路的作用。鸟类模型系统的主要优点是在整个胚胎发生过程中易于进行实验操作，并且能够比啮齿类动物更快、成本更低地进行遗传功能获得和功能丧失研究。我们建议使用具有复制能力的逆转录病毒进行基因过表达，使用基于载体的 RNAi 进行基因沉默和器官培养测定。具体目标 I 将确定 EdnrB 信号传导在远端肠 ENS 形成过程中迷走神经和骶神经 ENS 祖细胞的迁移、存活、增殖和分化中的作用。具体目标 II 将通过重点关注该通路对 ENS 祖细胞穿过盲肠和泄殖腔区域的迁移和增殖的功能，来研究 Ret 信号传导在结直肠无神经节细胞病病因学中的作用。具体目标 III 将使用结直肠无神经节细胞病的器官培养模型，该模型是通过抑制远端肠道的 EdnrB 信号传导而生成的。 Ret 活性的激活剂和抑制剂将被添加到 EdnrB 缺陷的肠道中，以便通过调节这两条途径之间的活性平衡来挽救无神经节表型。这些实验将确定 Ret 和 EdnrB 信号在远端 ENS 中的作用，为结直肠 ENS 发育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道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残疾疾病的儿童的能力。