Conserved roles for Hnf4 family transcription factors in zebrafish development and intestinal function

Abstract

Transcription factors play important roles in development of the intestinal epithelium and its ability to respond to endocrine, nutritional, and microbial signals. Hepatocyte Nuclear Factor 4 (Hnf4) family nuclear receptors are liganded transcription factors that are critical for the development and function of multiple digestive organs in vertebrates, including the intestinal epithelium. Zebrafish have three Hnf4 homologs, of which, hnf4a was previously shown to mediate intestinal responses to microbiota in zebrafish larvae. To discern the functions of other Hnf4 family members in zebrafish development and intestinal function, we created and characterized mutations in hnf4g and hnf4b. We addressed the possibility of genetic redundancy amongst these factors by creating double and triple mutants which showed different rates of survival, including apparent early lethality in hnf4a;hnf4b double mutants and triple mutants. RNA sequencing performed on digestive tracts from single and double mutant larvae revealed extensive changes in intestinal gene expression in hnf4a mutants that were amplified in hnf4a;hnf4g mutants, but limited in hnf4g mutants. Changes in hnf4a and hnf4a;hnf4g mutants were reminiscent of those seen in mice including decreased expression of genes involved in intestinal function and increased expression of cell proliferation genes, and were validated using transgenic reporters and EdU labeling in the intestinal epithelium. Gnotobiotics combined with RNA sequencing also showed hnf4g has subtler roles than hnf4a in host responses to microbiota. Overall, phenotypic changes in hnf4a single mutants were strongly enhanced in hnf4a;hnf4g double mutants, suggesting a conserved partial genetic redundancy between hnf4a and hnf4g in the vertebrate intestine.

Multiple roles and regulatory mechanisms of the transcription factor HNF4 in the intestine

“…Germline loss of hnf4a alone has a stronger impact on intestinal processes than loss of hnf4g and these changes are further exacerbated in a hnf4a/hnf4g double mutant model. The double mutant ultimately results in larval lethality between 6 to 14 days post fertilization ( 97 ). Moreover, zebrafish and other lower vertebrates like Xenopus laevis express a third paralog, hnf4b ( 98 , 99 ).…”

“…hnf4a and hnf4b are reported to redundantly regulate genes involved in yolk lipid mobilization to the embryonic body during early zebrafish development; a function which might be conserved in other egg-laying vertebrates but lost in higher vertebrates due to the lack of hnf4b . Double mutants of hnf4a and hnf4b or triple mutants of hnf4a , hnf4g and hnf4b in zebrafish are not viable, further pointing to multiple layers of redundancy between paralogs ( 97 ).…”