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

BMC Developmental Biology Expression of zebrafish pax6b in pancreas is regulated by two enhancers containing highly conserved cis-elements bound by PDX1, PBX and PREP factors Franois M Delporte 2 Vincent Pasque 2 3 Nathalie Devos 2 Isabelle Manfroid 2 Marianne L Voz 2 Patrick Motte 1 Frdric Biemar 0 2 Joseph A Martial 2 Bernard Peers 2 0 Department of Biology, Temple University , Philadelphia, PA 19122 , USA 1 Laboratory of Plant Cell and Molecular Biology, University of Liege, Department of Life Sciences, Institute of Botany , B-4000 Liege , Belgium 2 Unit of Molecular Biology and Genetic Engineering, University of Liege , Giga-R, B34, Avenue de l'hopital, 1, B-4000 Liege , Belgium 3 Wellcome/Cancer Research UK Gurdon Institute, University of Cambridge Tennis Court Road, Cambridge CB2 1QN, U.K 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. - Background The pancreas has two major functions fulfilled by distinct tissues: i) production of digestive enzymes by the exocrine cells and ii) release of various hormones by distinct endocrine cell types (i.e. secretion of glucagon, insulin, somatostatin, pancreatic polypeptide and ghrelin by , , , PP and cells, respectively). These mature pancreatic endocrine and exocrine cells derive from a pool of endodermal progenitor cells located in the embryonic gut. Differentiation of these cells is controlled by a regulatory cascade involving a battery of pancreatic transcription factors (reviewed in [1,2]). The HOX-like homeoprotein PDX1 is expressed in pancreatic progenitor cells and plays a crucial role in pancreas development. Its absence results in an early arrest of pancreatic bud growth and blocks both exocrine and endocrine cell differentiation [3-5]. PDX1 regulates the expression of downstream target genes by acting in concert with regulatory proteins, including other homeodomain proteins such as PBX and MEIS/PREP [39]. Subsequent commitment of PDX1+ pancreatic progenitors to the endocrine lineage is controlled by a set of other transcription factors such as NGN3, NEUROD, ISL1 and INSM1/IA1 [10-13]. Specification of the various endocrine cell subtypes involves the action of downstream regulators such as the homeodomain-containing proteins PAX6, PAX4, ARX, NKX2.2 and NKX6.1 [14-18]. In mouse, Pax6 is expressed in all pancreatic endocrine cells and its disruption leads to a reduction of , and cells and an increase of cells [17,19,20]. Pax6 plays crucial functions in the development of several organs/tissues besides the endocrine pancreas, such as the eyes, olfactory system, brain, spinal cord, enteroendocrine cells and the pituitary (reviewed in [21-24]). Expression of the Pax6 gene is controlled by a highly complex system of regulatory elements comprising at least three distinct promoters (P0, P1 and P) and many cisacting elements or enhancers, located along the gene [2527]. Long-range control elements have been identified in the human PAX6 locus located more than 150 kb downstream from the transcription unit [28]. Sequence comparisons of different vertebrate Pax6 genes have revealed that most of these cis-acting elements are evolutionarily conserved. Reports published so far indicate that expression in a specific tissue can be driven by several distinct enhancers. Experiments focusing on the zebrafish, quail, mouse, and human Pax6 genes have highlighted several distinct enhancers targeting expression to the neuroretina. In vitro experiments with the quail Pax6 gene have revealed a region located 7.5 kb downstream from the quail P0 promoter, acting as an enhancer in neural retina cells [29]. Several in vivo transgenic studies have led to identification of at least five other retinal enhancers, located respectively at 2 kb upstream from the murine P0 promoter [30,31], just upstream from the promoter P [30,32-34], within intron 7 [35], and even 70 kb and 100 kb downstream from the Pax6 gene [35,36]. Two enhancers have been identified as participating in the control of Pax6 expression in the lens. The first, driving expression in the lens placode and corneal ectoderm, lies about 3.5 kb upstream from the P0 murine promoter [31,37-40] and is recognized by a multi-protein complex composed of the homeoproteins MEIS1 and MEIS2 [40]. This enhancer is also bound by a complex containing the SRY-like HMG box SOX2 transcription factor and the OCT-1 factor, which are essential for Pax6 expression in the lens placode and its derivatives [41]. The second lens enhancer consists of the EI/SIMO elements located 80 kb downstream the last Pax6 exon [36]. Regulatory regions involved in pancreatic expression have also been identified within the Pax6 locus. Two different groups have shown that in the mouse, the pancreatic regulatory elements are located upstream from the P0 promoter [31,32,42]. These groups disagree, however, as to the precise location of these elements: Kammandel and coworkers observed disrupted pancreatic expression of the LacZ reporter gene after deletion of sequences located 4 kb upstream from exon 0, i (...truncated)