application) Understanding the molecular mechanisms of intestinal epithelial differentiation will greatly impact our knowledge of the pathogenesis of diseases such as colo-rectal cancer, where the normal controls for proliferation and differentiation are not properly regulated. The goal of this applicant is to develop an independent research program aimed at understanding the molecular basis of epithelial differentiation using the mouse intestine as a model system. This research program will be conducted under the guidance of Dr. Jeffrey Gordon who has considerable experience with the study of intestinal epithelial biology using this in vivo system. Previous studies of chimeric-transgenic mice suggest that differentiation of this epithelium is regulated by instructions that are received and interpreted as cells migrate along crypt-villus units. To test this hypothesis, perturbations of Rae GTPases were made within in the 129/Sv embryonic stem cell‑derived component of the small intestinal epithelia of chimeric (C57Bl/6-R0SA26<->l29/Sv) mice. Forced expression of a constitutively active form of Rae induces precocious differentiation of members of the Paneth cell and enterocytic lineages in the fetal gut without suppressing proliferation. In contrast, forced expression of a dominant negative mutant of Rae 1 inhibits epithelial differentiation, again without affecting cell division. The results from these initial studies suggest that Rae GTPases are involved in the import and processing of extracellular signals that control differentiation of this epithelium. The hypothesis that stems from this work is that signal transduction cascades involved by Rae I mediate effects on gene transcription that control epithelial differentiation. To address this question the genetic alterations in response to inhibition of Rae GTPase function within the small intestinal epithelium of Rae IAsnl7 chimeric-transgenic mice will be determined in a systematic and quantitative fashion. A combination of laser capture microdissection and Affymetrix GeneChip analysis will be performed to specifically audit global gene expression within defined populations of epithelial cells. To generate a more robust phenotype of loss of Rae 1 function, newly developed experimental systems that allow both constitutive and inducible Cre-mediated gene knockouts that are limited to the adult small intestinal and colonic epithelium will be used. This approach will allow for a more comprehensive analysis of the effects of genetically ablating Rae I in the developing or fully formed crypt villus unit in the small intestine and crypt-surface cuff unit in the colon.
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