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

项目总结/摘要 我是加州大学洛杉矶分校的胃肠病学家，我致力于研究肠道神经发生，以更好地 肠神经病如先天性巨结肠症、食管癌、 贲门失弛缓症和胃轻瘫。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的概念支持治疗方法 对于肠道神经病，主要寻求改善症状，但不纠正潜在的 病理生理学最近，这一概念受到了一种新的肠源性大肠杆菌的鉴定的挑战。 存在于肠外的神经元祖细胞，称为神经嵴干细胞（NCSC）。 这份建议书概述了一个5年的研究和职业发展计划，这将使我准备成为 从事高水平科学研究的独立的医生科学家。我的目的是阐明 发育中NCSC介导的肠神经发生的起源、迁移动力学和分子信号传导 和再生。我将采用斑马鱼作为我的模式生物，因为它克服了面临的几个限制， 其他模型，并适用于创新技术。目的1将调查胚胎后肠道， 使用原位杂交和单细胞RNA测序检测驻留ENS祖细胞。我的前期工作 支持肠内缺乏ENS祖细胞。目的2将采用诱导型Cre转基因株系 和延时共聚焦显微镜，以确定NCSC的起源和迁移动力学， 肠神经元这一目标将评估国家公务员合同委员会通过发展和 成年我的初步数据提供了NCSC介导的肠神经发生的证据，并支持 这一目标的基础。最后，目标3将探索5 HT 4受体激动剂在促进NCSC-1中的作用。 在发育和再生中介导肠神经发生。斑马鱼可以接受双光子激光 细胞消融，通过消融单个肠神经元允许ENS特异性损伤。我的前期工作 证实了用5 HT 4受体激动剂增加肠神经发生，支持了对该目的的追求。 沿着强有力的前期工作和精心构思的研究计划，本提案还包括 优秀和多学科的导师和顾问团队，成功过渡到一个关键方面， 独立我的导师布朗纳博士和波图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，他们是著名的导师，有着悠久的历史，成功地 培训年轻的调查员。我还将从具有广泛认可的专业知识的顾问那里获得重要指导 在ENS开发（Gershon博士），转录组学（Pachter博士），神经发生（Kornblum博士）和高级 显微镜（Collazo博士）。加上我非凡的研究环境，课程和研讨会， 以及我所在部门的机构支持，该计划将确保提供强有力的培训，使我能够 转型为神经胃肠病学的高影响力独立研究者。

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.