CDX-1 AND CDX-2 IN DEVELOPMENT

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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.