APC and Retinoids in Zebrafish Enterocyte Development

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DESCRIPTION (provided by applicant): Mutations in APC, or in its regulatory target, beta-catenin, are thought to cause colon neoplasms by promoting proliferation and preventing proper differentiation of colonocytes. However, our understanding of the mechanisms that control colonocyte differentiation is limited. Recent studies have shown that human colon adenomas and carcinomas show a profound deficiency of retinoic acid biosynthetic enzymes. Furthermore, re-introduction of wild type APC into an APC-deficient colon cancer cell line induced retinol dehydrogenase L and increased retinoic acid production. These observations suggest a novel model wherein APC promotes enterocyte differentiation by controlling retinoic acid biosynthesis. The studies outlined in this proposal will examine the genetic relationship between APC and retinoic acid biosynthesis in normal enterocytes using zebrafish as a model system. Preliminary data show that morpholino knockdown of either zAPC or zRDHB in zebrafish embryos results in defects in structures known to require retinoic acid, consistent with a role for these genes in RA-dependent pathways. In addition, APC or zRDHB morphant fish develop intestines that lack columnar epithelial cells and fail to express the differentiation marker intestinal fatty acid binding protein (i-FABP). Treatment of either APC or zRDHB morphant embryos with retinoic acid rescued the defective phenotypes, for the first time placing zAPC upstream of RA and implicating RA in intestinal differentiation. These preliminary data, therefore, strongly support a critical role for retinoic acid in zebrafish enterocyte development and provide genetic evidence placing retinoic acid and hox genes downstream of APC. The long term goal of this project is to facilitate the development of new preventive measures for colon adenoma formation by understanding the earliest cellular perturbations that follow APC mutation.