[en] In mice, the Nkx6 genes are crucial to alpha- and beta-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in beta-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of alpha-cells but has no effect on beta-, delta-, or epsilon-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of beta-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both beta- and alpha-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for alpha-cell than for beta-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control alpha- and beta-cell differentiation.
Research Center/Unit :
Giga-Development and Stem Cells - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Binot, Anne-Catherine
Manfroid, Isabelle ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Flasse, Lydie ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Winandy, Marie ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-Management : Plate-forme zebrafish
Motte, Patrick ; Université de Liège - ULiège > Département des sciences de la vie > Génomique fonctionnelle et imagerie moléculaire végétale
Martial, Joseph ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Peers, Bernard ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Voz, Marianne ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Language :
English
Title :
Nkx6.1 and nkx6.2 regulate alpha- and beta-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells.
Ahlgren U., Pfaff S.L., Jessell T.M., Edlund T., Edlund H. Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells. Nature 1997, 385:257-260.
Ahnfelt-Ronne J., Hald J., Bodker A., Yassin H., Serup P., Hecksher-Sorensen J. Preservation of proliferating pancreatic progenitor cells by Delta-Notch signaling in the embryonic chicken pancreas. BMC Dev. Biol. 2007, 7:63.
Argenton F., Zecchin E., Bortolussi M. Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo. Mech. Dev. 1999, 87:217-221.
Awatramani R., Beesley J., Yang H., Jiang H., Cambi F., Grinspan J., Garbern J., Kamholz J. Gtx, an oligodendrocyte-specific homeodomain protein, has repressor activity. J. Neurosci. Res. 2000, 61:376-387.
Biemar F., Argenton F., Schmidtke R., Epperlein S., Peers B., Driever W. Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet. Dev. Biol. 2001, 230:189-203.
Cheesman S.E., Layden M.J., Von Ohlen T., Doe C.Q., Eisen J.S. Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation. Development 2004, 131:5221-5232.
Delporte F.M., Pasque V., Devos N., Manfroid I., Voz M.L., Motte P., Biemar F., Martial J.A., Peers B. Expression of zebrafish pax6b in pancreas is regulated by two enhancers containing highly conserved cis-elements bound by PDX1, PBX and PREP factors. BMC Dev. Biol. 2008, 8:53.
Field H.A., Dong P.D., Beis D., Stainier D.Y. Formation of the digestive system in zebrafish. II. Pancreas morphogenesis. Dev. Biol. 2003, 261:197-208.
Hauptmann G., Gerster T. Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet. 1994, 10:266.
Henseleit K.D., Nelson S.B., Kuhlbrodt K., Hennings J.C., Ericson J., Sander M. NKX6 transcription factor activity is required for alpha- and beta-cell development in the pancreas. Development 2005, 132:3139-3149.
Huang H.P., Liu M., El-Hodiri H.M., Chu K., Jamrich M., Tsai M.J. Regulation of the pancreatic islet-specific gene BETA2 (neuroD) by neurogenin 3. Mol. Cell. Biol. 2000, 20:3292-3307.
Hutchinson S.A., Cheesman S.E., Hale L.A., Boone J.Q., Eisen J.S. Nkx6 proteins specify one zebrafish primary motoneuron subtype by regulating late islet1 expression. Development 2007, 134:1671-1677.
Johansson K.A., Dursun U., Jordan N., Gu G., Beermann F., Gradwohl G., Grapin-Botton A. Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types. Dev. Cell 2007, 12:457-465.
Jorgensen M.C., Vestergard Petersen H., Ericson J., Madsen O.D., Serup P. Cloning and DNA-binding properties of the rat pancreatic beta-cell-specific factor Nkx6.1. FEBS Lett. 1999, 461:287-294.
Kimmel C.B., Ballard W.W., Kimmel S.R., Ullmann B., Schilling T.F. Stages of embryonic development of the zebrafish. Dev. Dyn. 1995, 203:253-310.
Korzh V., Edlund T., Thor S. Zebrafish primary neurons initiate expression of the LIM homeodomain protein Isl-1 at the end of gastrulation. Development 1993, 118:417-425.
Korzh V., Sleptsova I., Liao J., He J., Gong Z. Expression of zebrafish bHLH genes ngn1 and nrd defines distinct stages of neural differentiation. Dev. Dyn. 1998, 213:92-104.
Li Z., Korzh V., Gong Z. DTA-mediated targeted ablation revealed differential interdependence of endocrine cell lineages in early development of zebrafish pancreas. Differentiation 2009, 78:241-452.
Mavropoulos A., Devos N., Biemar F., Zecchin E., Argenton F., Edlund H., Motte P., Martial J.A., Peers B. sox4b is a key player of pancreatic alpha cell differentiation in zebrafish. Dev. Biol. 2005, 285:211-223.
Milewski W.M., Duguay S.J., Chan S.J., Steiner D.F. Conservation of PDX-1 structure, function, and expression in zebrafish. Endocrinology 1998, 139:1440-1449.
Mirmira R.G., Watada H., German M.S. Beta-cell differentiation factor Nkx6.1 contains distinct DNA binding interference and transcriptional repression domains. J. Biol. Chem. 2000, 275:14743-14751.
Nelson S.B., Schaffer A.E., Sander M. The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate specification in Pdx1+ pancreatic progenitor cells. Development 2007, 134:2491-2500.
Nornes S., Clarkson M., Mikkola I., Pedersen M., Bardsley A., Martinez J.P., Krauss S., Johansen T. Zebrafish contains two pax6 genes involved in eye development. Mech. Dev. 1998, 77:185-196.
Odenthal J., Nusslein-Volhard C. fork head domain genes in zebrafish. Dev. Genes Evol. 1998, 208:245-258.
Pauls S., Zecchin E., Tiso N., Bortolussi M., Argenton F. Function and regulation of zebrafish nkx2.2a during development of pancreatic islet and ducts. Dev. Biol. 2007, 304:875-890.
Robu M.E., Larson J.D., Nasevicius A., Beiraghi S., Brenner C., Farber S.A., Ekker S.C. p53 activation by knockdown technologies. PLoS Genet. 2007, 3:e78.
Sander M., Sussel L., Conners J., Scheel D., Kalamaras J., Dela Cruz F., Schwitzgebel V., Hayes-Jordan A., German M. Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. Development 2000, 127:5533-5540.
Soyer J., Flasse L., Raffelsberger W., Beucher A., Orvain C., Peers B., Ravassard P., Vermot J., Voz M.L., Mellitzer G., Gradwohl G. Rfx6 as an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development. Development 2010, 137:203-212.
Vallstedt A., Muhr J., Pattyn A., Pierani A., Mendelsohn M., Sander M., Jessell T.M., Ericson J. Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification. Neuron 2001, 31:743-755.
Weinberg E.S., Allende M.L., Kelly C.S., Abdelhamid A., Murakami T., Andermann P., Doerre O.G., Grunwald D.J., Riggleman B. Developmental regulation of zebrafish MyoD in wild-type, no tail and spadetail embryos. Development 1996, 122:271-280.
Westerfield M. The Zebrafish Book: Guide for the Laboratory Use of Zebrafish (Danio Rerio) 1995, University of Oregon Press, Eugene, Oregon. third ed.