Gata6 and Hnf1ba are the major downstream effectors of retinoic acid for the specification of zebrafish pancreas

Retinoic acid (RA) is a key signaling molecule required for the specification of the pancreatic field within the endodermal germ layer. Still, the gene regulatory cascade triggered by RA in endoderm remains poorly characterized. In this study, we investigated the gene regulatory network induced by RA signaling in zebrafish endodermal cells by a combination of RNA-seq, RAR ChIP-seq and ATAC-seq experiments. By analysing the effect of RA and BMS439 on the transcriptome and on the chromatin accessibility of endodermal cells, we identified a large set of genes and regulatory regions regulated by RA signaling. Localization of RAR binding sites in the zebrafish genome by ChIP-seq highlighted the putative direct RAR target genes. Among them, Hnf1ba and Gata6, two known pancreatic regulatory factors activated by RA treatment, play a crucial role in opening chromatin at many genomic loci as revealed by the strong enrichment of their sequence binding motifs in RA-induced nucleosome-free regions. Furthermore, comparison of RAR ChIP-seq data obtained in zebrafish and in mice highlights the evolutionary-conserved direct targets, comprising the well-known Cyp26a or Hox genes but also Hnf1b and Gata6. Some RAR binding sites are located in highly conserved noncoding regions revealing the strong evolutionary constraint to maintain the function of such regulatory sequences. Among them, we identify a novel RA-induced enhancer located far upstream from the Hoxb Locus. In conclusion, our data reveal the central role of HNF1ba and Gata6 as pioneer transcription factors for the RA-dependent specification of the pancreatic field and highlight the RAR sites conserved from fish to mammals.