Adenomatous Polyposis Coli Control of Retinoic Acid Biosynthesis Is Critical for Zebrafish Intestinal Development and Differentiation

Abstract

Mutations in the APC (adenomatous polyposis coli) tumor suppressor gene cause uncontrolled proliferation and impaired differentiation of intestinal epithelial cells. Recent studies indicate that human colon adenomas and carcinomas lack retinol dehydrogenases (RDHs) and that APC regulates the expression of human RDHL. These data suggest a model wherein APC controls enterocyte differentiation by controlling retinoic acid production. However, the importance of APC and retinoic acid in mediating control of normal enterocyte development and differentiation remains unclear. To examine the relationship between APC and retinoic acid biosynthesis in normal enterocytes, we have identified two novel zebrafish retinol dehydrogenases, termed zRDHA and zRDHB, that show strong expression within the gut of developing zebrafish embryos. Morpholino knockdown of either APC or zRDHB in zebrafish embryos resulted in defects in structures known to require retinoic acid. These defects included cardiac abnormalities, pericardial edema, failed jaw and pectoral fin development, and the absence of differentiated endocrine and exocrine pancreas. In addition, APC or zRDHB morphant fish developed intestines that lacked columnar epithelial cells and failed to express the differentiation marker intestinal fatty acid-binding protein. Treatment of either APC or zRDHB morphant embryos with retinoic acid rescued the defective phenotypes. Downstream of retinoic acid production, we identified hoxc8 as a retinoic acid-induced gene that, when ectopically expressed, rescued phenotypes of APC-and zRDHB-deficient zebrafish. Our data establish a genetic link supporting a critical role for retinoic acid downstream of APC and confirm the importance of retinoic acid in enterocyte differentiation.

The Retinol Dehydrogenase Rdh10 Localizes to Lipid Droplets during Acyl Ester Biosynthesis

“…not a high fat diet) weight 37% more than WT, if dietary vitamin A is not copious (7). In contrast, mouse Dhrs9 has not been knocked out, but the zebrafish ortholog of Dhrs9 contributes to atRA production that supports gut development (8). In mammalian cells, Dhrs9 seems to function as a tumor suppressor, consistent with its poor expression in colon cancer cell lines accompanied by insubstantial atRA biosynthesis, relative to normal colon cells (9).…”

Dual roles for adenomatous polyposis coli in regulating retinoic acid biosynthesis and Wnt during ocular development

“…PCR primers used were as follows: rdh5 forward, 5Ј-GACCGGTTGTGACTCTGGTT-3Ј; rdh5 reverse, 5Ј-TGCTTTCGAAGTCCTCGATT-3Ј; prrdh1 forward, 5Ј-CAGCGTCAAAGACCGACATA-3Ј; prrdh1 reverse, 5Ј-ATCGACATGGTGACGTTGAA-3Ј; prrdh2 forward, 5Ј-ACTC-TGCTGCCTCTGGACAT-3Ј; prrdh2 reverse, 5Ј-GACGC-TGCTCATGACGATAA-3Ј; rdh10 forward, 5Ј-GAATATCGC-CACCGAGTTGT-3Ј; rdh10 reverse, 5Ј-GGGCTTCAATT-GTCGGTAGA-3Ј; rdh12 forward, 5Ј-GCTGGTGTCATGATGT-GTCC-3Ј; rdh12 reverse, 5Ј-CTCTGGCAGTAGGCAAGTCC-3Ј. ␤-Actin primers were as described previously (26).…”

“…The riboprobe for GFP was a generous gift from R. Dorsky (University of Utah). Whole-mount in situ hybridizations were carried out as described (26). Embryos were cleared in 70% glycerol in PBS and photographed by using an Olympus DP12 digital camera.…”

“…HCT116 cells were transfected with a vector (pDEST40) containing rdh5 cDNA. Cells were then treated with retinol, and ATRA production was quantified as previously described (26).…”

Retinoic Acid and the Development of the Endoderm

“…The requirement for both is explained by the observation that APC is required for the expression of rdh1l and the loss of markers of intestinal differentiation seen when either are suppressed can be rescued by exogenous RA treatment [138]. Another retinol dehydrogenase, rdh1, is also required for intestinal differentiation in zebrafish but its requirement is restricted to the anterior gut and this corresponds to its graded expression pattern with highest levels of expression in the anterior gut [139]. The link to the Hox genes is reinforced by this study as over expression of hoxc8, normally regulated by RA, was able to partially rescue the differentiation of the intestine.…”