[en] 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.
Research Center/Unit :
Giga-Development and Stem Cells - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Delporte, F. M.
Pasque, Vincent; University of Cambridge > Wellcome/Cancer Research UK Gurdon Institute
Gene regulatory factors in pancreatic development. J Jensen, Dev Dyn 2004 229 1 176 200 10.1002/dvdy.10460 14699589
Pancreas and beta-cell development: from the actual to the possible. LC Murtaugh, Development 2007 134 3 427 438 10.1242/dev.02770 17185316
Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells. U Ahlgren SL Pfaff TM Jessell T Edlund H Edlund, Nature 1997 385 6613 257 260 10.1038/385257a0 9000074
Insulin-promoter-factor 1 is required for pancreas development in mice. J Jonsson L Carlsson T Edlund H Edlund, Nature 1994 371 6498 606 609 10.1038/371606a0 7935793
PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. MF Offield TL Jetton PA Labosky M Ray RW Stein MA Magnuson BL Hogan CV Wright, Development 1996 122 3 983 995 8631275
PDX:PBX complexes are required for normal proliferation of pancreatic cells during development. S Dutta M Gannon B Peers C Wright S Bonner-Weir M Montminy, Proc Natl Acad Sci U S A 2001 98 3 1065 1070 11158595 10.1073/pnas.031561298
Functional and cooperative interactions between the homeodomain PDX1, Pbx, and Prep1 factors on the somatostatin promoter. G Goudet S Delhalle F Biemar JA Martial B Peers, J Biol Chem 1999 274 7 4067 4073 10.1074/jbc.274.7. 4067 9933599
The pancreatic islet factor STF-1 binds cooperatively with Pbx to a regulatory element in the somatostatin promoter: importance of the FPWMK motif and of the homeodomain. B Peers S Sharma T Johnson M Kamps M Montminy, Mol Cell Biol 1995 15 12 7091 7097 8524276
An endocrine-exocrine switch in the activity of the pancreatic homeodomain protein PDX1 through formation of a trimeric complex with PBX1b and MRG1 (MEIS2). GH Swift Y Liu SD Rose LJ Bischof S Steelman AM Buchberg CV Wright RJ MacDonald, Mol Cell Biol 1998 18 9 5109 5120 9710595
Pancreatic organogenesis - developmental mechanisms and implications for therapy. H Edlund, Nat Rev Genet 2002 3 7 524 532 10.1038/nrg841 12094230
neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. G Gradwohl A Dierich M LeMeur F Guillemot, Proc Natl Acad Sci U S A 2000 97 4 1607 1611 10677506 10.1073/pnas.97.4.1607
IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas. G Mellitzer S Bonne RF Luco M Van De Casteele N Lenne-Samuel P Collombat A Mansouri J Lee M Lan D Pipeleers FC Nielsen J Ferrer G Gradwohl H Heimberg, Embo J 2006 25 6 1344 1352 16511571 10.1038/sj.emboj.7601011
Opposing actions of Arx and Pax4 in endocrine pancreas development. P Collombat A Mansouri J Hecksher-Sorensen P Serup J Krull G Gradwohl P Gruss, Genes Dev 2003 17 20 2591 2603 14561778 10.1101/gad.269003
Isolation, characterization, and chromosomal mapping of the human Nkx6.1 gene (NKX6A), a new pancreatic islet homeobox gene. H Inoue A Rudnick MS German R Veile H Donis-Keller MA Permutt, Genomics 1997 40 2 367 370 10.1006/geno.1996.4568 9119408
The Pax4 gene is essential for differentiation of insulin-producing beta cells in the mammalian pancreas. B Sosa-Pineda K Chowdhury M Torres G Oliver P Gruss, Nature 1997 386 6623 399 402 10.1038/386399a0 9121556
Pax6 is required for differentiation of glucagon-producing alpha-cells in mouse pancreas. L St-Onge B Sosa-Pineda K Chowdhury A Mansouri P Gruss, Nature 1997 387 6631 406 409 10.1038/387406a0 9163426
Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells. L Sussel J Kalamaras DJ Hartigan-O'Connor JJ Meneses RA Pedersen JL Rubenstein MS German, Development 1998 125 12 2213 2221 9584121
The role of Brn4/Pou3f4 and Pax6 in forming the pancreatic glucagon cell identity. RS Heller DA Stoffers A Liu A Schedl EB Crenshaw 3rd OD Madsen P Serup, Dev Biol 2004 268 1 123 134 10.1016/j.ydbio.2003.12.008 15031110
Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. M Sander L Sussel J Conners D Scheel J Kalamaras F Dela Cruz V Schwitzgebel A Hayes-Jordan M German, Development 2000 127 24 5533 5540 11076772
PAX-6 in development and evolution. P Callaerts G Halder WJ Gehring, Annu Rev Neurosci 1997 20 483 532 10.1146/annurev.neuro.20.1.483 9056723
Differential induction of Pax genes by NGF and BDNF in cerebellar primary cultures. C Kioussi P Gruss, J Cell Biol 1994 125 2 417 425 8163557 10.1083/jcb.125.2.417
The role of Pax3 and Pax7 in development and cancer. A Mansouri, Crit Rev Oncog 1998 9 2 141 149 9973247
Pax-6, a murine paired box gene, is expressed in the developing CNS. C Walther P Gruss, Development 1991 113 4 1435 1449 1687460
Subfunctionalization of Duplicated Zebrafish pax6 Genes by cis-Regulatory Divergence. DA Kleinjan RM Bancewicz P Gautier R Dahm HB Schonthaler G Damante A Seawright AM Hever PL Yeyati V van Heyningen P Coutinho, PLoS Genet 2008 4 2 e29 18282108 10.1371/journal.pgen.0040029
Mechanisms controlling Pax6 isoform expression in the retina have been conserved between teleosts and mammals. J Lakowski A Majumder JD Lauderdale, Dev Biol 2007 307 2 498 520 10.1016/j.ydbio.2007.04.015 17509554
Pax6 is a direct, positively regulated target of the circadian gene Clock. R Morgan, Dev Dyn 2004 230 4 643 650 10.1002/dvdy.20097 15254898
Long-range downstream enhancers are essential for Pax6 expression. DA Kleinjan A Seawright S Mella CB Carr DA Tyas TI Simpson JO Mason DJ Price V van Heyningen, Dev Biol 2006 299 2 563 581 17014839 10.1016/j.ydbio.2006.08.060
High conservation of cis-regulatory elements between quail and human for the Pax-6 gene. S Plaza S Saule C Dozier, Dev Genes Evol 1999 209 3 165 173 10.1007/s004270050240 10079359
Highly conserved non-coding sequences are associated with vertebrate development. A Woolfe M Goodson DK Goode P Snell GK McEwen T Vavouri SF Smith P North H Callaway K Kelly K Walter I Abnizova W Gilks YJ Edwards JE Cooke G Elgar, PLoS Biol 2005 3 1 e7 15630479 10.1371/journal.pbio.0030007
Regulation of Pax6 expression is conserved between mice and flies. PX Xu X Zhang S Heaney A Yoon AM Michelson RL Maas, Development 1999 126 2 383 395 9847251
Distinct cis-essential modules direct the time-space pattern of the Pax6 gene activity. B Kammandel K Chowdhury A Stoykova S Aparicio S Brenner P Gruss, Dev Biol 1999 205 1 79 97 10.1006/dbio.1998.9128 9882499
Identification and characterization of a neuroretina-specific enhancer element in the quail Pax-6 (Pax-QNR) gene. S Plaza C Dozier MC Langlois S Saule, Mol Cell Biol 1995 15 2 892 903 7529875
C-Myb acts as transcriptional activator of the quail PAX6 (PAX-QNR) promoter through two different mechanisms. S Plaza N Turque C Dozier M Bailly S Saule, Oncogene 1995 10 2 329 340 7838532
New 3' elements control Pax6 expression in the developing pretectum, neural retina and olfactory region. C Griffin DA Kleinjan B Doe V van Heyningen, Mech Dev 2002 112 1-2 89 100 10.1016/S0925-4773(01)00646-3 11850181
Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6. DA Kleinjan A Seawright A Schedl RA Quinlan S Danes V van Heyningen, Hum Mol Genet 2001 10 19 2049 2059 10.1093/hmg/10.19.2049 11590122
The upstream ectoderm enhancer in Pax6 has an important role in lens induction. PV Dimanlig SC Faber W Auerbach HP Makarenkova RA Lang, Development 2001 128 22 4415 4424 11714668
Pax6-dependence of Six3, Eya1 and Dach1 expression during lens and nasal placode induction. P Purcell G Oliver G Mardon AL Donner RL Maas, Gene Expr Patterns 2005 6 1 110 118 10.1016/j.modgep.2005.04.010 16024294
A highly conserved lens transcriptional control element from the Pax-6 gene. SC Williams CR Altmann RL Chow A Hemmati-Brivanlou RA Lang, Mech Dev 1998 73 2 225 229 10.1016/S0925-4773(98)00057-4 9622640
Meis homeoproteins directly regulate Pax6 during vertebrate lens morphogenesis. X Zhang A Friedman S Heaney P Purcell RL Maas, Genes Dev 2002 16 16 2097 2107 12183364 10.1101/gad.1007602
Sox2 and Pou2f1 interact to control lens and olfactory placode development. AL Donner V Episkopou RL Maas, Dev Biol 2007 303 2 784 799 10.1016/j.ydbio.2006.10.047 17140559
Cre-loxp fate-mapping of Pax6 enhancer active retinal and pancreatic progenitors. X Zhang S Heaney RL Maas, Genesis 2003 35 1 22 30 10.1002/gene.10160 12481295
Zebrafish contains two pax6 genes involved in eye development. S Nornes M Clarkson I Mikkola M Pedersen A Bardsley JP Martinez S Krauss T Johansen, Mech Dev 1998 77 2 185 196 10.1016/S0925-4773(98)00156-7 9831649
Zebrafish pax[zf-a]: a paired box-containing gene expressed in the neural tube. S Krauss T Johansen V Korzh U Moens JU Ericson A Fjose, Embo J 1991 10 12 3609 3619 1718739
Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice. AW Puschel P Gruss M Westerfield, Development 1992 114 3 643 651 1352238
Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet. F Biemar F Argenton R Schmidtke S Epperlein B Peers W Driever, Dev Biol 2001 230 2 189 203 10.1006/dbio.2000.0103 11161572
Expression pattern of zebrafish pax genes suggests a role in early brain regionalization. S Krauss T Johansen V Korzh A Fjose, Nature 1991 353 6341 267 270 10.1038/353267a0 1680220
Positional cloning and characterization of a paired box- and homeobox-containing gene from the aniridia region. CC Ton H Hirvonen H Miwa MM Weil P Monaghan T Jordan V van Heyningen ND Hastie H Meijers-Heijboer M Drechsler, Cell 1991 67 6 1059 1074 10.1016/0092-8674(91)90284-6 1684738
Cell fate decisions and patterning in the vertebrate retina: the importance of timing, asymmetry, polarity and waves. J Malicki, Curr Opin Neurobiol 2004 14 1 15 21 10.1016/j.conb.2004.01.015 15018933
I-SceI meganuclease mediates highly efficient transgenesis in fish. V Thermes C Grabher F Ristoratore F Bourrat A Choulika J Wittbrodt JS Joly, Mech Dev 2002 118 1-2 91 98 10.1016/S0925-4773(02)00218-6 12351173
Transposon tools and methods in zebrafish. K Kawakami, Dev Dyn 2005 234 2 244 254 10.1002/dvdy.20516 16110506
Fusion with E2A alters the transcriptional properties of the homeodomain protein PBX1 in t(1;19) leukemias. DP LeBrun ML Cleary, Oncogene 1994 9 6 1641 1647 8183558
Fusion with E2A converts the Pbx1 homeodomain protein into a constitutive transcriptional activator in human leukemias carrying the t(1;19) translocation. Q Lu DD Wright MP Kamps, Mol Cell Biol 1994 14 6 3938 3948 7910944
Hox gene products modulate the DNA binding activity of Pbx1 and Pbx2. MA van Dijk LT Peltenburg C Murre, Mech Dev 1995 52 1 99 108 10.1016/0925-4773(95) 00394-G 7577680
Control of the nuclear localization of Extradenticle by competing nuclear import and export signals. M Abu-Shaar HD Ryoo RS Mann, Genes Dev 1999 13 8 935 945 10215621 10.1101/gad.13.8.935
The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH. J Berthelsen C Kilstrup-Nielsen F Blasi F Mavilio V Zappavigna, Genes Dev 1999 13 8 946 953 10215622 10.1101/gad.13.8.946
Conserved regulation of proximodistal limb axis development by Meis1/Hth. N Mercader E Leonardo N Azpiazu A Serrano G Morata C Martinez M Torres, Nature 1999 402 6760 425 429 10.1038/46580 10586884
Nuclear translocation of extradenticle requires homothorax, which encodes an extradenticle-related homeodomain protein. GE Rieckhof F Casares HD Ryoo M Abu-Shaar RS Mann, Cell 1997 91 2 171 183 10.1016/S0092-8674(00)80400-6 9346235
Identification of a new family of Pbx-related homeobox genes. T Nakamura NA Jenkins NG Copeland, Oncogene 1996 13 10 2235 2242 8950991
Pax6 is regulated by Meis and Pbx homeoproteins during pancreatic development. X Zhang S Rowan Y Yue S Heaney Y Pan A Brendolan L Selleri RL Maas, Dev Biol 2006 300 2 748 757 10.1016/j.ydbio.2006.06.030 17049510
IPF1, a homeodomain-containing transactivator of the insulin gene. H Ohlsson K Karlsson T Edlund, Embo J 1993 12 11 4251 4259 7901001
Gene regulation and large-scale chromatin organization in the nucleus. N Dillon, Chromosome Res 2006 14 1 117 126 10.1007/s10577-006-1027-8 16506101
Pax-QNR/Pax-6, a paired box- and homeobox-containing gene expressed in neurons, is also expressed in pancreatic endocrine cells. N Turque S Plaza F Radvanyi C Carriere S Saule, Mol Endocrinol 1994 8 7 929 938 10.1210/me.8.7.929 7984154
Conserved elements in Pax6 intron 7 involved in (auto)regulation and alternative transcription. DA Kleinjan A Seawright AJ Childs V van Heyningen, Dev Biol 2004 265 2 462 477 10.1016/j.ydbio.2003.09.011 14732405
http://vega.sanger.ac.uk/Danio_rerio/. Vega
http://www.imagenes-bio.de/. Imagenes
The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. KM Kwan E Fujimoto C Grabher BD Mangum ME Hardy DS Campbell JM Parant HJ Yost JP Kanki CB Chien, Dev Dyn 2007 236 11 3088 3099 10.1002/dvdy.21343 17937395
Conservation of RET regulatory function from human to zebrafish without sequence similarity. S Fisher EA Grice RM Vinton SL Bessling AS McCallion, Science 2006 312 5771 276 279 10.1126/science.1124070 16556802
Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish. S Fisher EA Grice RM Vinton SL Bessling A Urasaki K Kawakami AS McCallion, Nat Protoc 2006 1 3 1297 1305 10.1038/nprot.2006.230 17406414
Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. G Hauptmann T Gerster, Trends Genet 1994 10 8 266 10.1016/0168-9525(90) 90008-T 7940754
sox4b is a key player of pancreatic alpha cell differentiation in zebrafish. A Mavropoulos N Devos F Biemar E Zecchin F Argenton H Edlund P Motte JA Martial B Peers, Dev Biol 2005 285 1 211 223 10.1016/j.ydbio.2005.06.024 16055112
Conservation of PDX-1 structure, function, and expression in zebrafish. WM Milewski SJ Duguay SJ Chan DF Steiner, Endocrinology 1998 139 3 1440 1449 10.1210/en.139.3.1440 9492081
Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo. F Argenton E Zecchin M Bortolussi, Mech Dev 1999 87 1-2 217 221 10.1016/S0925-4773(99)00151-3 10495291
Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation. V Korzh I Sleptsova J Liao J He Z Gong, Dev Dyn 1998 213 1 92 104 10.1002/(SICI)1097-0177(199809)213:1<92::AID-AJA9>3.0.CO;2-T 9733104
Differential expression of two somatostatin genes during zebrafish embryonic development. N Devos G Deflorian F Biemar M Bortolussi JA Martial B Peers F Argenton, Mech Dev 2002 115 1-2 133 137 10.1016/S0925-4773(02)00082-5 12049777
Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric kidney tubules. X Zhou PD Vize, Dev Biol 2004 271 2 322 338 10.1016/j.ydbio.2004.03.036 15223337
Insulin expression in pancreatic islet cells relies on cooperative interactions between the helix loop helix factor E47 and the homeobox factor STF-1. B Peers J Leonard S Sharma G Teitelman MR Montminy, Mol Endocrinol 1994 8 12 1798 1806 10.1210/me.8.12.1798 7708065
Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. E Schreiber P Matthias MM Muller W Schaffner, Nucleic Acids Res 1989 17 15 6419 2771659 10.1093/nar/17.15.6419
Heat shock stimulation of a tilapia heat shock protein 70 promoter is mediated by a distal element. A Molina E Di Martino JA Martial M Muller, Biochem J 2001 356 Pt 2 353 359 11368761 10.1042/0264-6021:3560353