A conserved retinoic acid responsive element in the murine Hoxb-1 gene is required for expression in the developing gut

Abstract

The murine Hoxb-1 gene contains a homeobox sequence and is expressed in a spatiotemporal specific pattern in neuroectoderm, mesoderm and gut endoderm during development. We previously identified a conserved retinoic acid (RA)-inducible enhancer, named the RAIDR5, which contains a DR5 RARE; this RAIDR5 enhancer is located 3' of the Hoxb-1-coding region in both the mouse and chick. In the F9 murine teratocarcinoma cell line, this DR5 RARE is required for the RA response of the Hoxb-1 gene, suggesting a functional role of the DR5 RARE in Hoxb-1 gene expression during embryogenesis. From the analysis of Hoxb-1/lacZ reporter genes in transgenic mice, we have shown that a wild-type (WT) transgene with 15 kb of Hoxb-1 genomic DNA, including this Hoxb-1 3' RAIDR5, is expressed in the same tissues and at the same times as the endogenous Hoxb-1 gene. However, a transgene construct with point mutations in the DR5 RARE (DR5mu) was not expressed in the developing foregut, which gives rise to organs such as the esophagus, lung, stomach, liver and pancreas. Like the wild-type transgene, this DR5 RARE mutated transgene was expressed in rhombomere 4 in 9.5 day postcoitum (d.p.c.) embryos. Similarly, transgene staining in the foregut of animals carrying a deletion of the entire Hox-b1 RAIDR5 enhancer (3'-del) was greatly reduced relative to that seen with the WT transgene. We also demonstrated that expression of the WT transgene in the gut increases in response to exogenous RA, resulting in anterior expansion of the expression in the gut. These observations that the Hoxb-1 gene is expressed in the developing gut and that this expression is regulated through a DR5 RARE strongly suggest a role for Hoxb-1 in the anteroposterior axis patterning of the gut and a critical role for endogenous retinoids in early gut development.