Zebrafish Based Model for Gastrointestinal Physiology

描述（由申请人提供）：这个新的区域（R-15）研究计划的总体目标是开发，验证和利用基于斑马鱼的模型系统来阐明胃肠（Gl）功能的细胞和分子细节，重点是运动性。由于目前缺乏针对Gl运动障碍的治疗方法，因此需要一种探索Gl生理学的新模型系统。基于斑马鱼的模型系统将提供几个重要的实验优势，例如直接可视化透明幼虫的Gl运动，并有机会利用前向遗传方法识别决定Gl运动的新分子成分。受精后5天，当幼虫吞咽食物，表现出蠕动和吸收营养物质时，消化道开始发挥功能。在Specific Aim 1中，将使用免疫组织化学方法对哺乳动物幼虫和成虫体内调节Gl运动的细胞成分——Cajal间质细胞（ICC）进行表征。kit基因的表达特异性地识别ICC， kit表达将识别斑马鱼中假定的ICC。ICC或起搏器细胞的发育预计与自发收缩的发育相吻合，与其他脊椎动物相似。ICC位于肠神经元和Gl平滑肌之间，其解剖位置表明其在人体运动整合和协调中的功能作用。假定的ICC细胞和网络的解剖特征将被确定。在Specific Aim 2中，将使用野生型和零kit突变斑马鱼来确定kit阳性细胞的功能意义。将确定正常的运动模式，包括自发收缩的频率和食物清除胃肠道的时间。运动对起搏器细胞的依赖性将通过失活试剂盒突变的斑马鱼进行检查，这种突变在其他脊椎动物模型中损害ICC。基于斑马鱼的模型系统具有鉴定调节Gl运动的新基因的潜力，这可能有利于治疗无组织运动。

DESCRIPTION (provided by applicant): The overall goal of this new AREA (R-15) research proposal is to develop, validate, and utilize a zebrafish-based model system to elucidate the cellular and molecular details of gastrointestinal (Gl) function with an emphasis on motility. A new model system exploring Gl physiology is needed because current therapies for Gl motility disorders are lacking. A zebrafish-based model system will provide several important experimental advantages, such as the direct visualization of Gl motility in optically transparent larvae and the opportunity to utilize a forward genetic approach to identify novel molecular components determining Gl motility. The Gl tract appears functional by 5 days post-fertilization when larvae swallow food, exhibit peristalsis, and absorb nutrients. In Specific Aim 1 the cellular components that regulate Gl motility in mammals, interstitial cells of Cajal (ICC), will be characterized in larvae and adults, using immunohistochemical procedures. Expression of the kit gene specifically identifies ICC, and kit expression will identify putative ICC in the zebrafish. Development of ICC, or pacemaker cells, is expected to coincide with development of spontaneous contractions, similar to other vertebrates. The anatomical position of ICC, intermediary between enteric neurons and Gl smooth muscles, indicates the functional role of integration and coordination of motility in humans. Anatomical features of putative ICC cells and networks will be determined. In Specific Aim 2 the functional significance of kit-positive cells will be determined using wild type and null kit mutant zebrafish. Normal motility patterns, including the frequency of spontaneous contractions and the time for food to clear the Gl tract, will be determined. The dependence of motility on the pacemaker cell will be examined using zebrafish with inactivating kit mutations, which lesion ICC in other vertebrate models. A zebrafish based model system has the potential for the identification of novel genes regulating Gl motility, which may benefit treatment for disorganized motility.