DEVELOPMENT OF NEURONS IN GUT--GROWTH FACTORS

肠道的运动和吸收/分泌需要内在神经元的存在。内源性或肠性神经元由神经嵴细胞发育而来，这些细胞从神经管迁移进入肠道。在肠道内，这些嵴细胞迁移、聚集、增殖并分化成神经元，这些神经元相互作用形成肠道的神经回路。在肠道内，这些嵴细胞迁移、聚集、增殖并分化成神经元，这些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神经系统形成的机制。肠神经系统的形成和维持对肠道的正常功能至关重要。神经胶质源性神经营养因子（GDNF）及其受体Ret基因的敲除导致相似的表型。这些小鼠在前肠中显示出少量的神经元，但在小肠和大肠中没有。本提案的总体目标是发现GDNF在肠神经系统发育中的关键作用。我们的第一个目标是确定GDNF在何时何地在肠道中产生。我们将使用原位杂交、免疫细胞化学、ELISA和显微手术来定位和定量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用于检查Ret的外观。为了确定Ret独立细胞（不需要GDNF存活）是否能够定植肠道，将来自GDNF缺失小鼠的肠道与相同的小鼠截断神经嵴细胞一起外植。本实验将揭示神经嵴对GDNF的依赖性是在神经嵴之前还是之后决定的。肠道神经系统的形成经历3个阶段：神经嵴迁移、神经嵴进入肠道分化、神经嵴成为神经元和神经胶质。我们将评估GDNF在这些阶段的作用。我们将培养这些阶段的细胞，并对它们进行免疫染色，以评估GDNF存在下增殖、分化和存活的变化。我们还将通过培养成体神经元来确定GDNF对维持成体肠神经系统是否必要。这项工作的完成将有助于深入了解GDNF在肠神经系统形成和可能维持中的作用。这一信息将促进我们对发育或成人中GDNF或Ret表达缺陷如何影响肠神经系统结构和功能的认识。

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.