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.


CONCLUSIONS
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.
Delporte
François M
FM
Unit of Molecular Biology and Genetic Engineering, University of Liège, Giga-R, B34, Avenue de l'hôpital, 1, B-4000 Liège, Belgium. fdelporte@ulg.ac.be
Pasque
Vincent
V
Devos
Nathalie
N
Manfroid
Isabelle
I
Voz
Marianne L
ML
Motte
Patrick
P
Biemar
Frédéric
F
Martial
Joseph A
JA
Peers
Bernard
B
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005136:Eye Proteins / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000235:genetics*; D010179:Pancreas / Q000196:embryology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D012160:Retina / Q000196:embryology / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013687:Telencephalon / Q000196:embryology / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D046912:Multiprotein Complexes; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D029961:Zebrafish Proteins; C084010:pancreatic and duodenal homeobox 1 protein; D049452:Green Fluorescent Proteins