Lineage analysis and identification of novel genes involved in differentiation processes in the zebrafish enteric nervous system

肠功能障碍是一个严重的健康和社会问题，可以使个人痛苦和昂贵的治疗。一些肠功能障碍是由于肠神经系统（ENS）的发育和功能不正常造成的，肠神经系统调节基本的肠道功能，如运动，血流和稳态。ENS由不同亚型的神经元和神经胶质组成。异常ENS发育引起了研究充分的人类疾病，如先天性巨结肠病，其中远端肠道无神经支配和非运动性，以及可能还有许多其他描述不太充分的人类肠道疾病。大多数使用脊椎动物遗传系统作为模型来了解人类ENS疾病的原因的研究都集中在ENS发育的早期事件上，例如ENS祖细胞如何导航到肠道。然而，对ENS发展的后期事件知之甚少。例如，祖细胞与分化的神经元和它们产生的神经胶质之间的谱系关系仍然难以捉摸，ENS分化过程的控制在很大程度上仍然未知。我建议利用斑马鱼的实验易处理性，1）调查导致ENS神经元和神经胶质正常发育的谱系，2）分析肠祖细胞不同亚群的潜力，3）通过表征和克隆影响ENS神经元生成的突变来分离参与这一过程的新基因。在我的项目中，我将使用肠祖细胞及其后代的遗传诱导命运作图来产生ENS的全面谱系分析。遗传标记肠祖细胞的个体亚群并随时间推移跟踪其后代将揭示不同肠祖细胞亚群的潜力差异。斑马鱼模型非常适合谱系追踪研究，因为其在发育过程中的光学清晰度允许在体内完整胚胎或幼虫中追踪前体及其衍生物。我对ENS突变体的表征和下一代高通量测序方法的克隆将揭示在ENS发展中发挥作用的新基因和途径。 了解ENS的谱系以及发现控制ENS中分化过程的新基因对于基础知识和开发治疗由异常ENS分化引起的肠道功能障碍的新疗法都至关重要。

Intestinal dysfunction is a serious health and societal problem that can make individuals miserable and be expensive to treat. Some intestinal dysfunction results from improper development and function of the enteric nervous system (ENS), which regulates essential gut functions such as motility, blood flow, and homeostasis. The ENS is composed of diverse subtypes of neurons and of glia. Abnormal ENS development causes well-studied human diseases such as Hirschsprung disease, in which the distal gut is uninnervated and nonmotile, and probably also many other human intestinal diseases that are less well-described. Most research using vertebrate genetic systems as models to understand the causes of human ENS diseases has focused on early events in ENS development, such as how ENS progenitors navigate to the gut. However, little is known about later events of ENS development. For example, lineage relationships between progenitors and the differentiated neurons and glia they generate remain elusive and control of the process of ENS differentiation remains largely unknown. I propose to exploit the experimental tractability of zebrafish to 1) investigate lineages that lead to normal development of ENS neurons and glia, 2) analyze the potential of different subpopulations of enteric progenitors, and 3) isolate novel genes involved in this process by characterizing and cloning mutations that affect the generation of ENS neurons. In my project, I will use genetic inducible fate mapping of enteric progenitors and their progeny to generate a comprehensive lineage analysis of the ENS. Genetically labeling the individual subpopulations of enteric progenitors and following their progeny over time will reveal differences in potential of different enteric progenitor subpopulations. The zebrafish model is exceptionally well-suited for lineage tracing studies due to its optical clarity during development which allows tracking of precursors and their derivatives in the intact embryo or larva in vivo. My characterization of ENS mutants and cloning with next-generation high throughput sequencing methods will reveal novel genes and pathways that play a role in ENS development. Understanding the lineages of the ENS as well as finding novel genes that control differentiation processes in the ENS is critical both for basic knowledge and for developing new therapies to treat gut dysfunction resulting from abnormal ENS differentiation.