Epithelia, such as those lining the gastrointestinal tract, are able to undergo continuous self-renewal because they contain a population of undifferentiated stem cells that have a large capacity for self renewal and can also give rise to daughter cells which are able to differentiate into all of the mature cell types needed for normal function of the epithelial layer. The nature of the signals that mediate the establishment of this hierarchical organization of the epithelium during normal gastrointestinal development has not yet been defined. Fibroblast growth factors (FGFs) are a family of at least 23 related mesenchymally derived peptide growth factors that modulate a wide array of morphogenic and differentiation events occurring during normal ontogeny of a variety of tissues. We have recently found that expression of one FGF receptor gene, FGFR- 3, is restricted to undifferentiated cells in the lower two-thirds of the intestinal crypt epithelium and is maximally expressed during crypt morphogenesis. Additionally, preliminary studies of intestinal development in FGFR3-/- mice, demonstrate that FGFR-3 regulates both the rate of nascent crypt formation and the number of replicating crypt transit cells in the suckling mouse intestine. The central hypothesis of this proposal is that signaling through FGFR-3 regulates the fate and/or proliferation of the multipotent epithelial stem cells during normal intestinal ontogeny.
Aim 1 is to determine whether FGFR-3-mediated signaling directly regulates expansion of the epithelial stem cell population during normal intestinal development through effects on stem cell proliferation and/or programmed cell death. Mice with targeted mutations in the FGFR3 receptor gene will be used to determine the effects of FGFR3 mediated signaling on the number of clonogenic stem cells and on apoptosis at various developmental time points.
Aim 2 will examine whether the effects of FGFR3 on crypt morphogenesis are mediated through a beta-catenin/TCF-4 dependent mechanism. Evidence in the literature suggests that the HMG transcription factors TCF-4 and Lef-1 are important downstream regulatory mediators of proliferative and apoptotic events in the crypt epithelium. Both cell culture and animal models will be used to determine whether signaling through FGFR3 can modulate TCF-4 activity. The goal of aim 3 is to define the intermediate signaling cascades that FGFR3 uses to regulate morphogenic events during intestinal development. The operant FGFR3 signaling pathways in intestinal epithelial cell lines, especially those impinging on TCF-4, will be investigated using a combination of biochemical and molecular approaches.
