Foxa2 and Hif1ab regulate maturation of intestinal goblet cells by modulating agr2 expression in zebrafish embryos

Mammalian anterior gradient 2 (AGR2), an endoplasmic reticulum (ER) protein disulfide-isomerase (PDI), is involved in cancer cell growth and metastasis, asthma and inflammatory bowel disease (IBD). Mice lacking Agr2 exhibit decreased Muc2 protein in intestinal goblet cells, abnormal Paneth cell development, ileitis and colitis. Despite its importance in cancer biology and inflammatory diseases, the mechanisms regulating agr2 expression in the gastrointestinal tract remain unclear. In the present study, we investigated the mechanisms that control agr2 expression in the pharynx and intestine of zebrafish by transient/stable transgenesis, coupled with motif mutation, morpholino knockdown, mRNA rescue and ChIP. A 350 bp DNA sequence with a hypoxia-inducible response element (HRE) and forkhead-response element (FHRE) within a region -4.5 to -4.2 kbp upstream of agr2 directed EGFP expression specifically in the pharynx and intestine. No EGFP expression was detected in the intestinal goblet cells of Tg(HREM:EGFP) or Tg(FHREM:EGFP) embryos with mutated HRE or FHRE, whereas EGFP was expressed in the pharynx of Tg(HREM:EGFP), but not Tg(FHREM:EGFP), embryos. Morpholino knockdown of foxa1 (forkhead box A1) reduced agr2 levels in the pharynx, whereas knockdown of foxa2 or hif1ab decreased intestinal agr2 expression and affected the differentiation and maturation of intestinal goblet cells. These results demonstrate that Foxa1 regulates agr2 expression in the pharynx, whereas both Foxa2 and Hif1ab control agr2 expression in intestinal goblet cells to regulate maturation of these cells.