Tissue-specific transgenesis with the mouse Villin gene: Exploring intestinal organogenesis.

摘要

The embryonic intestine begins as a simple tube composed of endoderm surrounded by mesenchyme. Further remodeling of this tube requires signaling crosstalk between these two tissue layers, resulting in the unique regional patterning characteristic of the adult esophagus, stomach, small intestine, and colon. In the intestine, the epithelium is sequentially remodeled to form finger-like villi, followed by flask-shaped crypts. Stem cells, anchored in the adult crypts, divide to constantly renew the intestinal epithelium. Early in vitro studies performed with isolated fractions of epithelium and mesenchyme revealed the importance of cell-cell crosstalk for acquisition of regional identity and region-specific morphological remodeling. Further identification of the specific cellular signals involved has been greatly limited by the lack of genetic tools for the manipulation of intestinal gene expression, in vivo.;In this dissertation, I describe a series of powerful tools developed using a promoter fragment from the mouse Villin gene that enable intestine-specific and temporal-controlled alteration of gene function in the intestinal epithelium. This promoter directs efficient expression of multiple transgenes in all cell lineages of the intestinal epithelium (including stem cells), permitting the examination of signaling pathways involved in intestinal development, physiology, and disease. Through the further dissection of this promoter, I have also discovered enhancers that drive expression in more restricted domains, such as the duodenum, cecum, and crypt compartments.;Using the Villin promoter, I have specifically examined the function of Indian (Ihh) and Sonic (Shh) hedgehog signaling in the developing mouse intestine by transgenic expression of the pan-hedgehog inhibitor, Hhip. Ihh and Shh each play multiple patterning roles during development, and the intestine is one of the few organs that express both molecules. Hhip-mediated attenuation of this combined signal reveals novel functions of the hedgehog signal during intestinal villus formation, smooth muscle development, and spatial restriction of the proliferative stem cell compartment in the intestinal epithelium. These developmental processes are mediated by hedgehog signals sent from epithelial cells to adjacent stromal tissues. This mouse model of hedgehog inhibition allows the identification of precise target genes through which the hedgehog pathway acts to mediate these events.