CDX-1 AND CDX-2 IN DEVELOPMENT

指导肠道发育的分子机制尚未得到证实 完全阐明了。同源结构域转录因子已被证明 对许多生物体的发育具有核心重要性。初步 数据显示，MCDX-2可以激活肠道特异基因，因此 两种肠道特异性小鼠尾部相关同源结构域的研究 家族基因MCDX-1和MCDX-2可能提供有关 肠道分化发育的调控。目标是 这一提议的特点是同时刻画了时间和细胞 这些CDX基因的表达模式，并确定它们在 发育中的小鼠。为了实现这一目标，以下具体目标将 L)MCDX-L和MCDX-2在卵巢癌中的表达 小鼠的原位杂交和免疫组织化学研究 强调它们沿纵向和纵向的细胞定位 肠道的垂直(隐窝-绒毛轴)轴。2) 两种方法评价CDX基因在发育中的功能作用 功能获得和功能丧失研究。考察…的功能 MCDX-L和MCDX-2基因的功能获得实验 使用转基因小鼠进行实验。这些鼠标将被设计为使用 两种CDX基因在大肠杆菌中的异位表达 内皮源性组织以及性早熟表达。老鼠 将分析内胚层-肠道可能发生的变化 在发育过程中的隐窝-绒毛轴 组织学检查、原位杂交和免疫组织化学染色。 功能丧失实验也将使用转基因小鼠进行。 用MCDX-2的显性负性蛋白突变体选育，有效地 阻断天然蛋白质的结合。主导性的建构 阴性突变体将在绒毛蛋白启动子的控制下直接 表达于内皮源性组织。转基因小鼠将成为 对丢失后的组织学和生理学变化进行分析 MCDX-2功能。 这些实验旨在阐明一些机制， 直接的肠道发育和分化。该计划的完成 本建议书的第一个目标将提供描述性信息， 将用于分析增加或增加 转基因小鼠CDX基因功能降低。对.的使用 转基因动物允许进行体内研究，以放松对 基因，在这种情况下是转录因子MCDX-L和MCDX-2。这个 对转录调控的基本机制的理解 肠道基因，通过研究尾部相关的同源结构域基因，将 作为一种手段，加深我们对肠道发育和 可能对影响消化疾病的过程有深入的了解。

The molecular mechanisms that direct intestinal development have not been fully elucidated. The homeodomain transcription factors have been shown to be of central importance for development in many organisms. Preliminary data shows that mCdx-2 can activate intestinal specific genes, therefore the study of two intestine specific mouse caudal-related homeodomain family genes, mCdx-1 and mCdx-2, may give essential information regarding the control of intestinal differentiation and development. The objective of this proposal is to characterize both the temporal and cellular patterns of expression of these Cdx genes and to determine their role in developing mice. To reach this objective the following specific aims will be pursued: l) The characterization of mCdx-l and mCdx-2 expression in developing mice by in situ hybridization and immunohistochemistry with an emphasis on their cellular localization along the longitudinal and vertical (crypt-villus axis) axes of the intestinal tract. 2) The evaluation of the functional role of Cdx genes in development by both gain-of-function and loss-of-function studies. To examine the function of the mCdx-l and mCdx-2 genes, gain-of-function experiments will be performed using transgenic mice. These mice will be designed, using the villin promotor, to have both ectopic expression of the Cdx genes in endodermally derived tissue as well as precocious expression. The mice will be analyzed for changes that may occur at the endoderm-intestinal transition and at the crypt-villus axis during development by direct histologic inspection, in situ hybridization and immunohistochemistry. Loss-of-function experiments will also be performed using transgenic mice bred with a dominant negative protein mutant of mCdx-2 that effectively blocks the binding of the native protein. The construct of the dominant negative mutant will be under the control of the villin promotor to direct expression to endodermally derived tissue. The transgenic mice will be analyzed for histologic and physiologic changes that occur with the loss of mCdx-2 function. These experiments are designed to elucidate some of the mechanisms that direct intestinal development and differentiation. The completion of the first objective in this proposal will give descriptive information which will be used to analyze potential changes that occur with increased or decreased function of the Cdx genes in the transgenic mice. The use of transgenic animals allows for an in vivo study into the deregulation of genes, in this case the transcription factors mCdx-l and mCdx-2. The understanding of the basic mechanisms of transcriptional regulation of intestinal genes, by studying the caudal related homeodomain genes, will serve as a means to further our knowledge of intestinal development and may give insight into the processes influencing digestive diseases.