The Pax6b homeodomain is dispensable for pancreatic endocrine cell differentiation in zebrafish.

Pax6 is a well conserved transcription factor that contains two DNA-binding domains, a paired domain and a homeodomain, and plays a key role in the development of eye, brain, and pancreas in vertebrates. The recent identification of the zebrafish sunrise mutant, harboring a mutation in the pax6b homeobox and presenting eye abnormalities but no obvious pancreatic defects, raised a question about the role of pax6b in zebrafish pancreas. We show here that pax6b does play an essential role in pancreatic endocrine cell differentiation, as revealed by the phenotype of a novel zebrafish pax6b null mutant and of embryos injected with pax6b morpholinos. Pax6b-depleted embryos have almost no beta cells, a strongly reduced number of delta cells, and a significant increase of epsilon cells. Through the use of various morpholinos targeting intron-exon junctions, pax6b RNA splicing was perturbed at several sites, leading either to retention of intronic sequences or to deletion of exonic sequences in the pax6b transcript. By this strategy, we show that deletion of the Pax6b homeodomain in zebrafish embryos does not disturb pancreas development, whereas lens formation is strongly affected. These data thus provide the explanation for the lack of pancreatic defects in the sunrise pax6b mutants. In addition, partial reduction of Pax6b function in zebrafish embryos performed by injection of small amounts of pax6b morpholinos caused a clear rise in alpha cell number and in glucagon expression, emphasizing the importance of the fine tuning of the Pax6b level to its biological activity.

Expression of zebrafish pax6b in pancreas is regulated by two enhancers containing highly conserved cis-elements bound by PDX1, PBX and PREP factors.

BACKGROUND: PAX6 is a transcription factor playing a crucial role in the development of the eye and in the differentiation of the pancreatic endocrine cells as well as of enteroendocrine cells. Studies on the mouse Pax6 gene have shown that sequences upstream from the P0 promoter are required for expression in the lens and the pancreas; but there remain discrepancies regarding the precise location of the pancreatic regulatory elements. RESULTS: Due to genome duplication in the evolution of ray-finned fishes, zebrafish has two pax6 genes, pax6a and pax6b. While both zebrafish pax6 genes are expressed in the developing eye and nervous system, only pax6b is expressed in the endocrine cells of the pancreas. To investigate the cause of this differential expression, we used a combination of in silico, in vivo and in vitro approaches. We show that the pax6b P0 promoter targets expression to endocrine pancreatic cells and also to enteroendocrine cells, retinal neurons and the telencephalon of transgenic zebrafish. Deletion analyses indicate that strong pancreatic expression of the pax6b gene relies on the combined action of two conserved regulatory enhancers, called regions A and C. By means of gel shift assays, we detected binding of the homeoproteins PDX1, PBX and PREP to several cis-elements of these regions. In constrast, regions A and C of the zebrafish pax6a gene are not active in the pancreas, this difference being attributable to sequence divergences within two cis-elements binding the pancreatic homeoprotein PDX1. CONCLUSION: Our data indicate a conserved role of enhancers A and C in the pancreatic expression of pax6b and emphasize the importance of the homeoproteins PBX and PREP cooperating with PDX1, in activating pax6b expression in endocrine pancreatic cells. This study also provides a striking example of how adaptative evolution of gene regulatory sequences upon gene duplication progressively leads to subfunctionalization of the paralogous gene pair.

Reciprocal endoderm-mesoderm interactions mediated by fgf24 and fgf10 govern pancreas development.

In amniotes, the pancreatic mesenchyme plays a crucial role in pancreatic epithelium growth, notably through the secretion of fibroblast growth factors. However, the factors involved in the formation of the pancreatic mesenchyme are still largely unknown. In this study, we characterize, in zebrafish embryos, the pancreatic lateral plate mesoderm, which is located adjacent to the ventral pancreatic bud and is essential for its specification and growth. We firstly show that the endoderm, by expressing the fgf24 gene at early stages, triggers the patterning of the pancreatic lateral plate mesoderm. Based on the expression of isl1, fgf10 and meis genes, this tissue is analogous to the murine pancreatic mesenchyme. Secondly, Fgf10 acts redundantly with Fgf24 in the pancreatic lateral plate mesoderm and they are both required to specify the ventral pancreas. Our results unveil sequential signaling between the endoderm and mesoderm that is critical for the specification and growth of the ventral pancreas, and explain why the zebrafish ventral pancreatic bud generates the whole exocrine tissue.