Zebrafish Based Model for Gastrointestinal Physiology

描述(由申请人提供)：这一新领域(R-15)研究提案的总体目标是开发、验证和利用基于斑马鱼的模型系统来阐明胃肠道(Gl)功能的细胞和分子细节，重点是运动。由于目前缺乏治疗Gl运动障碍的方法，因此需要一个新的模型系统来探索Gl生理学。基于斑马鱼的模型系统将提供几个重要的实验优势，例如在光学透明的幼虫中直接显示Gl运动，以及利用正向遗传方法来识别决定Gl运动的新的分子成分。受精后5天，当幼虫吞食、蠕动和吸收营养时，Gl区出现功能。在特定的目标1，调节哺乳动物Gl运动的细胞成分，Cajal间质细胞(ICC)，将用免疫组织化学方法在幼虫和成虫中表征。KIT基因的表达特异性地识别ICC，KIT的表达将识别斑马鱼中可能的ICC。与其他脊椎动物类似，ICC或起搏细胞的发育预计与自发收缩的发育相一致。ICC是肠道神经元和Gl平滑肌之间的中介，其解剖位置表明了人类运动的整合和协调的功能作用。假定的ICC细胞和网络的解剖特征将被确定。在具体目标2中，将使用野生型和KIT缺失突变斑马鱼来确定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。运动对起搏细胞的依赖性将使用具有失活KIT突变的斑马鱼来检验，该突变会损害其他脊椎动物模型中的ICC。基于斑马鱼的模型系统有可能发现调节Gl运动的新基因，这可能有助于治疗运动障碍。

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.