DEVELOPMENT OF NEURONS IN GUT--GROWTH FACTORS

肠道需要内源性神经元的存在才能进行运动和吸收/分泌。内源性神经元或肠神经细胞是从神经管迁移到肠道的神经脊细胞发展而来的。在肠道内，这些脊细胞迁移、聚集、增殖和分化为神经元，神经元相互作用形成肠道的神经回路。在肠道内，这些脊细胞迁移、聚集、增殖和分化为神经元，神经元相互作用形成肠道的神经回路。本研究的长期目标是阐明调节肠道神经系统形成的机制。肠道神经系统的形成和维持对肠道的正常功能至关重要。胶质源性神经营养因子(GDNF)及其受体Ret的基因敲除导致了相似的表型。这些小鼠在前肠显示少量神经元，但在小肠和大肠中没有。这项建议的总体目标是发现GDNF在肠道神经系统发育中所起的关键作用。我们的第一个目标是确定GDNF在肠道中何时何地产生。我们将使用原位杂交、免疫细胞化学、ELISA法和显微外科技术来定位和定量GDNF的表达。我们的第二个目标是确定独立细胞何时何地能够在肠道中定植。免疫细胞化学和原位杂交将被用来检测Ret的外观。为了确定Ret非依赖细胞(不需要GDNF生存)是否能够在肠道定植，GDNF基因缺失的小鼠的肠道将被移植入QUAL和小鼠的躯干神经脊细胞。这项实验将揭示Ret依赖是在神经峰严重依赖GDNF之前还是之后确定的。肠道神经系统的形成分为3个阶段：神经峰迁移、神经峰进入肠道并分化、神经峰为神经元和胶质细胞。我们将评估GDNF对这些阶段的影响。我们将培养每个阶段的细胞，并对它们进行免疫染色，以评估在GDNF存在的情况下，细胞增殖、分化和存活的变化。我们还将通过培养成人神经元来确定GDNF是否对维持成人肠道神经系统是必要的。这项工作的完成将对GDNF在肠道神经系统的形成和可能的维持中的作用提供深入的认识。这些信息将促进我们了解GDNF或Ret在发育过程中或成人中的表达缺陷如何影响肠道神经系统的结构和功能。

The gut requires the presence of intrinsic neurons for motility and absorption/secretion to occur. The intrinsic or enteric neurons develop from neural crest cells that migrate from the neural tube and enter the gut. Within the gut these crest cells migrate, aggregate, proliferate, and differentiate into neurons which interact to form the neural circuitry of the gut. Within the gut these crest cells migrate, aggregate, proliferate, and differentiate into neurons which interact to form the neural circuitry of the gut. The long-term objective of this study is to elucidate the mechanisms which regulate the formation of the enteric nervous system. The formation and maintenance of the enteric nervous system are essential to the formal function of the gut. A knockout of the gene for Glial Derived Neurotrophic Factor (GDNF) and for its receptor Ret results in similar phenotypes. These mice show a small number of neurons in the foregut but none in the small and large intestine. The overall objective of this proposal is to discover the critical role GDNF plays in the development of enteric nervous system. Our first aim is to determine when and where GDNF is made in the gut. We will use in situ hybridization, immunocytochemistry, ELISA, and microsurgery to localize and quantitate GDNF expression. Our second goal is to determine when and where independent cells are able to colonize the intestine. Immunocytochemistry and in situ hybridization will be used to examine the appearance of Ret. To determine whether Ret independent cells (which do not require GDNF for survival) are able to colonize the gut, gut from GDNF null mice will be explanted with qual and mouse truncal neural crest cells. This experiment will reveal whether Ret dependence is determined before or after neural crest are critically dependent on GDNF. The enteric nervous system is formed in 3 stages: neural crest migrate, neural crest enter the gut and differentiate, neural crest as neurons and glia. We will evaluate the effects of GDNF on these stages. We will culture cells from each of these stages and immunostain them to assess changes in proliferation, differentiation, and survival in the presence of GDNF. We will also determine whether GDNF is necessary for the maintenance of the adult enteric nervous system by culturing adult neurons. Completion of this work will provide insight into the role of GDNF in the formation and possibly the maintenance of the enteric nervous system. This information will advance our knowledge of how the defects in GDNF or Ret expression in the development or in the adult might affect the structure and function of the enteric nervous system.