Abstract :
[en] The pancreas is developing from the endodermal germ layer. Differential analysis of gene expression and open regions of the chromatin between endodermal and non-endodermal cells, through RNA-seq and ATAC-seq, highlighted 820 genes with enriched expression in endoderm and 10601 regions of the chromatin specifically open in these cells. We found that half of the genes enriched in endoderm are located around at least one of these specific open chromatin regions; moreover, motif enrichment analysis showed
that these regions are enriched in binding sites corresponding to endodermal specific transcription factors, such as Gata, Sox, and Forkhead.
Furthermore, it is known that Retinoic Acid (RA) is required during gastrulation for the proper anterior-posterior regionalization of the embryo and for pancreas specification in vertebrates, however, the mechanisms by which RA induces pancreas in the endoderm are poorly understood. In this study, we identified the genes regulated by RA through RNA-seq. From the 371 genes up-regulated by RA, 26 of them were also down-regulated by BMS493 (an antagonist of RA). Besides, 94 of these up-regulated genes have RA receptor (RAR) binding sites, found by ChIP-seq analysis using a Myc-tagged RAR protein, around their transcription starting site (TSS), suggesting that RA might directly regulate them. Among these 94 genes, we found cyp26a1, cyp26b1 and dhrs3a, genes involved in the synthesis pathway of RA. Moreover, two known pancreatic markers, gata6 and hnf1ba, are also among these 94 genes, indicating that these two genes might act as mediators of RA to induce pancreas formation. Open chromatin analysis performed through ATAC-seq, showed that RA has also an effect on the chromatin structure, the motifs present in the regions of the chromatin induced by RA are enriched in RA response elements (RARE), and in binding sites for Gata6 and Hnf1b proteins. All these data together suggest that the RAR might act as pioneer factors modifying the chromatin structure and that gata6 and/or hnf1ba might be the key mediators of RA action to induce pancreas in the endoderm.
Finally, transcriptomic analyses of the pancreatic progenitor cells from the ventral pancreas highlighted genes with enriched expression in these cells. In situ hybridization analysis for some of these genes showed specific expression of olfm4 in the pancreatic progenitor cells. Besides, nupr1, elf3 and
cebpa showed an expression not only in the developing pancreas but also in other gut tissues.
