Exploring neural crest stem cell-derived enteric neurogenesis in post-embryonic development and regeneration

项目摘要/摘要 我是加州大学洛杉矶分校的胃肠病专家，我致力于研究肠道神经发生，以更好地 介绍肠神经疾病的治疗进展，如先天性巨结肠、食道 失弛缓症和胃轻瘫。在历史上，肠神经系统(Ens)被认为是由一个 终末分化神经元的有限池。ENS的这一概念支持了治疗方法 对于主要寻求改善症状但不能纠正潜在疾病的肠道神经病 病理生理学。最近，一种新的肠道来源的鉴定对这一概念提出了挑战。 位于肠道外的神经前体细胞，称为神经脊干细胞(NCSCs)。 这份提案概述了一项为期5年的研究和职业发展计划，这将使我做好准备，成为 从事高水平科学研究的独立内科医生兼科学家。我的目的是阐明 神经干细胞介导的肠神经发生在发育中的起源、迁移动力学和分子信号 和再生。我将用斑马鱼作为我的模型生物，因为它克服了面临的几个限制 通过其他模式，并服从创新的技术。目标1将对胚胎后肠道进行调查 应用原位杂交和单细胞RNA测序获得常驻ENS祖细胞。我的前期工作 支持肠道中不存在ENS前体细胞。Aim 2将使用可诱导的Cre转基因株系 和延时共聚焦显微镜来确定神经干细胞的起源和迁移动力学 肠道神经元。这一目标将评估非公务员制度委员会通过发展和发展对环境和社会服务的持续贡献 成人期。我的初步数据提供了NCSC介导的肠神经发生的证据，并支持 这是这个目标的基础。最后，目标3将探讨5HT4受体激动剂在促进NCSC- 在发育和再生过程中介导的肠神经发生。斑马鱼对双光子激光是敏感的 细胞消融，通过消融单个肠道神经元来实现特定于ENS的损伤。我的前期工作 证明了5HT4受体激动剂增加了肠神经发生，支持了这一目标的追求。 除了扎实的前期工作和精心设计的研究计划外，这项提案还包括 卓越的多学科导师和顾问团队，这是成功过渡到 独立。我的导师，布朗纳博士和波特拉基斯博士，是各自领域的领导者 发育生物学和细胞生物学，他们都是著名的导师，有着悠久的成功历史 培养年轻的调查员。我还将从具有广泛认可的专业知识的顾问那里获得批判性指导 ENS发育(Gershon博士)、转录学(Pachter博士)、神经发生(Kornblum博士)和高级 显微镜(科拉佐博士)。加上我非凡的研究环境、课程和研讨会， 和我部门的机构支持，这项计划将确保我得到强有力的培训，使我做好准备 转变为神经胃肠病学领域的高影响力独立研究员。

PROJECT SUMMARY/ABSTRACT I am a gastroenterologist at UCLA, and I am committed to studying enteric neurogenesis to better inform development of therapies for enteric neuropathies such as Hirschsprung disease, esophageal achalasia, and gastroparesis. Historically, the enteric nervous system (ENS) was thought to be composed of a finite pool of terminally differentiated neurons. This conception of the ENS supported therapeutic approaches for enteric neuropathies that largely seek to ameliorate symptoms but do not correct the underlying pathophysiology. Recently, this concept has been challenged by the identification of a novel source of enteric neuronal progenitors that reside outside of the intestine, termed neural crest stem cells (NCSCs). This proposal outlines a 5-year research and career development plan that will prepare me to become an independent physician-scientist engaged in high-level scientific research. My aims are to elucidate the origin, migration dynamics, and molecular signaling of NCSC-mediated enteric neurogenesis in development and regeneration. I will employ the zebrafish as my model organism as it overcomes several limitations faced by other models and is amenable to innovative techniques. Aim 1 will survey the post-embryonic intestine for resident ENS progenitors using in situ hybridization and single cell RNA sequencing. My preliminary work supports the absence of ENS progenitors in the intestine. Aim 2 will employ an inducible Cre transgenic line and time-lapse confocal microscopy to determine the origin and migration dynamics of NCSCs that give rise to enteric neurons. This Aim will assess persistence of NCSC contributions to the ENS through development and adulthood. My preliminary data provides evidence of NCSC-mediated enteric neurogenesis and supports the basis of this Aim. Lastly, Aim 3 will explore the role of 5HT4 receptor agonists in the promotion of NCSC- mediated enteric neurogenesis in development and regeneration. Zebrafish are amenable to two-photon laser cell ablation, allowing ENS-specific injury by ablating individual enteric neurons. My preliminary work demonstrates increased enteric neurogenesis with 5HT4 receptor agonists, supporting the pursuit of this Aim. Along with strong preliminary work and a well-conceived research plan, this proposal also includes an outstanding and multidisciplinary team of mentors and advisors, a crucial aspect to a successful transition to independence. My mentors, Dr. Bronner and Dr. Pothoulakis, are leaders in their respective fields of developmental biology and cellular biology, and they are renowned mentors with a long history of successfully training young investigators. I will also receive critical guidance from advisors with widely recognized expertise in ENS development (Dr. Gershon), transcriptomics (Dr. Pachter), neurogenesis (Dr. Kornblum), and advanced microscopy (Dr. Collazo). Together, with my extraordinary research environment, coursework and seminars, and the institutional support from my Division, this plan will ensure a robust training that will prepare me to transition into a high-impact independent investigator in neurogastroenterology.