Identification and characterization of vitamin A dependent genes in the mouse embryo

Vitamin A (retinol) plays a key role in the regulation of vertebrate embryonic development. Over the years, an understanding of the function of retinoic acid (RA) has been achieved greatly via genetic disruption of the RARs and RXRs, however, the underlying mechanism remains unclear, owing in part to the difficulty in identifying downstream RA target genes in an intact embryo. In this study, I first describe a novel strategy allowing us to identify RA-dependent genes in mouse embryos. Utilizing a modified differential display technique, we isolated twenty-eight developmentally expressed genes from vitamin A deficient mouse embryos generated via RBP antisense oligodeoxynucleotide microinjection. Of these twenty-eight genes, we verified that the expression of twenty genes is truly dependent on endogenous RA in mouse embryos. While search the identities of the isolated genes, we discovered that sFRP-1, PP2A-Cα and cul-1 are part of the developmentally important Wnt signaling pathway and function in control of intracellular β-catenin stability. This discovery has led us to examine the possibility for an interaction between RA and Wnt signaling. Using whole mount in situ hybridization, we first verified that sFRP-1 and cul-1 transcripts are up-regulated whereas the expression of PP2A-Cα is downregulated in mouse embryos in response to 1µM RA. We also report for the first time the expression patterns of cul-1 and PP2A-Cα in the mouse embryos. In general, cul-1 and PP2A-Cα are not only co-expressed in but also reside proximally to vitamin A sensitive tissues. With respect to β-catenin regulation, we find that β-catenin protein abundance is increased in our VAD embryos whereas the protein abundance is decreased in RA exposed embryos. Consistent with the β-catenin result, expressions of two Wnt target genes, cyclin D1 and c-myc, also respond to RA. Taken together, our data suggest that vitamin A exerts an influence on Wnt signaling in the embryo. In our differential display study, we also isolated ten EST sequences. In the last part of my thesis, I report the cloning of mouse cul-4A full-length cDNA and verify that it is also a RA regulated gene in mouse embryo.