[en] BACKGROUND: In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors. RESULTS: Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells. CONCLUSIONS: We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In contrast to the mouse, pancreatic progenitor markers nkx6.1 and pdx1 continue to be expressed in adult ductal cells, a subset of which we show are still able to proliferate and undergo ductal and endocrine differentiation, providing robust evidence of the existence of pancreatic progenitor/stem cells in the adult zebrafish. Our findings support the hypothesis that nkx6.1+ pancreatic progenitors contribute to beta cell regeneration. Further characterization of these cells will open up new perspectives for anti-diabetic therapies.
Pisharath H, Rhee JM, Swanson MA, Leach SD, Parsons MJ. Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase. Mech Dev. 2007;124:218-29.
Moss JB, Koustubhan P, Greenman M, Parsons MJ, Walter I, Moss LG. Regeneration of the pancreas in adult zebrafish. Diabetes. 2009;58:1844-51.
Curado S, Anderson RM, Jungblut B, Mumm J, Schroeter E, Stainier DY. Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies. Dev Dyn. 2007;236:1025-35.
Field HA, Dong PD, Beis D, Stainier DY. Formation of the digestive system in zebrafish. II. Pancreas morphogenesis. Dev Biol. 2003;261:197-208.
Kumar M, Melton D. Pancreas specification: a budding question. Curr Opin Genet Dev. 2003;13:401-7.
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.
Wang Y, Rovira M, Yusuff S, Parsons MJ. Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing beta-cells. Development. 2011;138:609-17.
Hesselson D, Anderson RM, Beinat M, Stainier DY. Distinct populations of quiescent and proliferative pancreatic beta-cells identified by HOTcre mediated labeling. Proc Natl Acad Sci U S A. 2009;106:14896-901.
Kimmel RA, Onder L, Wilfinger A, Ellertsdottir E, Meyer D. Requirement for Pdx1 in specification of latent endocrine progenitors in zebrafish. BMC Biol. 2011;9:75.
Chung WS, Andersson O, Row R, Kimelman D, Stainier DY. Suppression of Alk8-mediated Bmp signaling cell-autonomously induces pancreatic beta-cells in zebrafish. Proc Natl Acad Sci U S A. 2010;107:1142-7.
Dong PD, Munson CA, Norton W, Crosnier C, Pan X, Gong Z, et al. Fgf10 regulates hepatopancreatic ductal system patterning and differentiation. Nat Genet. 2007;39:397-402.
Parsons MJ, Pisharath H, Yusuff S, Moore JC, Siekmann AF, Lawson N, et al. Notch-responsive cells initiate the secondary transition in larval zebrafish pancreas. Mech Dev. 2009;126:898-912.
Afelik S, Jensen J. Notch signaling in the pancreas: patterning and cell fate specification. Wiley Interdiscip Rev Dev Biol. 2013;2:531-44.
Apelqvist A, Li H, Sommer L, Beatus P, Anderson DJ, Honjo T, et al. Notch signalling controls pancreatic cell differentiation. Nature. 1999;400:877-81.
Flasse LC, Pirson JL, Stern DG, Von Berg V, Manfroid I, Peers B, et al. Ascl1b and Neurod1, instead of Neurog3, control pancreatic endocrine cell fate in zebrafish. BMC Biol. 2013;11:78.
Ninov N, Borius M, Stainier DY. Different levels of Notch signaling regulate quiescence, renewal and differentiation in pancreatic endocrine progenitors. Development. 2012;139:1557-67.
Zhou Q, Law AC, Rajagopal J, Anderson WJ, Gray PA, Melton DA. A multipotent progenitor domain guides pancreatic organogenesis. Dev Cell. 2007;13:103-14.
Binot AC, Manfroid I, Flasse L, Winandy M, Motte P, Martial JA, et al. Nkx6.1 and nkx6.2 regulate alpha- and beta-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells. Dev Biol. 2010;340:397-407.
Schaffer AE, Freude KK, Nelson SB, Sander M. Nkx6 transcription factors and Ptf1a function as antagonistic lineage determinants in multipotent pancreatic progenitors. Dev Cell. 2010;18:1022-9.
Hesselson D, Anderson RM, Stainier DY. Suppression of Ptf1a activity induces acinar-to-endocrine conversion. Curr Biol. 2011;21:712-17.
Dong PD, Provost E, Leach SD, Stainier DY. Graded levels of Ptf1a differentially regulate endocrine and exocrine fates in the developing pancreas. Genes Dev. 2008;22:1445-50.
Manfroid I, Ghaye A, Naye F, Detry N, Palm S, Pan L, et al. Zebrafish sox9b is crucial for hepatopancreatic duct development and pancreatic endocrine cell regeneration. Dev Biol. 2012;366:268-78.
Sander M, Sussel L, Conners J, Scheel D, Kalamaras J, Dela Cruz F, et al. Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas. Development. 2000;127:5533-40.
Suster ML, Sumiyama K, Kawakami K. Transposon-mediated BAC transgenesis in zebrafish and mice. BMC Genomics. 2009;10:477.
Suster ML, Abe G, Schouw A, Kawakami K. Transposon-mediated BAC transgenesis in zebrafish. Nat Protoc. 2011;6:1998-2021.
Corish P, Tyler-Smith C. Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 1999;12:1035-40.
Zhou Y, Cashman TJ, Nevis KR, Obregon P, Carney SA, Liu Y, et al. Latent TGF-beta binding protein 3 identifies a second heart field in zebrafish. Nature. 2011;474:645-8.
Mosimann C, Kaufman CK, Li P, Pugach EK, Tamplin OJ, Zon LI. Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish. Development. 2011;138:169-77.
Chung WS, Shin CH, Stainier DY. Bmp2 signaling regulates the hepatic versus pancreatic fate decision. Dev Cell. 2008;15:738-48.
Chung WS, Stainier DY. Intra-endodermal interactions are required for pancreatic beta cell induction. Dev Cell. 2008;14:582-93.
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013;31:227-9.
Itoh M, Kim CH, Palardy G, Oda T, Jiang YJ, Maust D, et al. Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta. Dev Cell. 2003;4:67-82.
Scheer N, Groth A, Hans S, Campos-Ortega JA. An instructive function for Notch in promoting gliogenesis in the zebrafish retina. Development. 2001;128:1099-107.
Lorent K, Moore JC, Siekmann AF, Lawson N, Pack M. Reiterative use of the notch signal during zebrafish intrahepatic biliary development. Dev Dyn. 2010;239:855-64.
Ninov N, Hesselson D, Gut P, Zhou A, Fidelin K, Stainier DY. Metabolic regulation of cellular plasticity in the pancreas. Curr Biol. 2013;23:1242-50.
Delaspre F, Beer RL, Rovira M, Huang W, Wang G, Gee S, et al. Centroacinar cells are progenitors that contribute to endocrine pancreas regeneration. Diabetes. 2015. Epub ahead of print.
European Nucleotide Archive. RNAseq from mature ductal cells from nkx6.1:GFP zebrafish lines. http://www.ebi.ac.uk/ena/data/view/PRJEB10137
Shih HP, Kopp JL, Sandhu M, Dubois CL, Seymour PA, Grapin-Botton A, et al. A Notch-dependent molecular circuitry initiates pancreatic endocrine and ductal cell differentiation. Development. 2012;139:2488-99.
Zecchin E, Mavropoulos A, Devos N, Filippi A, Tiso N, Meyer D, et al. Evolutionary conserved role of ptf1a in the specification of exocrine pancreatic fates. Dev Biol. 2004;268:174-84.
Briscoe J, Pierani A, Jessell TM, Ericson J. A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell. 2000;101:435-45.
Olguin HC, Yang Z, Tapscott SJ, Olwin BB. Reciprocal inhibition between Pax7 and muscle regulatory factors modulates myogenic cell fate determination. J Cell Biol. 2007;177:769-79.
Laslo P, Spooner CJ, Warmflash A, Lancki DW, Lee HJ, Sciammas R, et al. Multilineage transcriptional priming and determination of alternate hematopoietic cell fates. Cell. 2006;126:755-66.
Afelik S, Qu X, Hasrouni E, Bukys MA, Deering T, Nieuwoudt S, et al. Notch-mediated patterning and cell fate allocation of pancreatic progenitor cells. Development. 2012;139:1744-53.
Nelson SB, Schaffer AE, 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;134:2491-500.
Rovira M, Scott SG, Liss AS, Jensen J, Thayer SP, Leach SD. Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas. Proc Natl Acad Sci U S A. 2010;107:75-80.
Kopinke D, Brailsford M, Shea JE, Leavitt R, Scaife CL, Murtaugh LC. Lineage tracing reveals the dynamic contribution of Hes1+ cells to the developing and adult pancreas. Development. 2011;138:431-41.
Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature. 2008;455:627-32.
Ferber S, Halkin A, Cohen H, Ber I, Einav Y, Goldberg I, et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nat Med. 2000;6:568-72.
Shternhall-Ron K, Quintana FJ, Perl S, Meivar-Levy I, Barshack I, Cohen IR, et al. Ectopic PDX-1 expression in liver ameliorates type 1 diabetes. J Autoimmun. 2007;28:134-42.
Ber I, Shternhall K, Perl S, Ohanuna Z, Goldberg I, Barshack I, et al. Functional, persistent, and extended liver to pancreas transdifferentiation. J Biol Chem. 2003;278:31950-7.
Miyashita K, Miyatsuka T, Matsuoka TA, Sasaki S, Takebe S, Yasuda T, et al. Sequential introduction and dosage balance of defined transcription factors affect reprogramming efficiency from pancreatic duct cells into insulin-producing cells. Biochem Biophys Res Commun. 2014;444:514-9.
Gefen-Halevi S, Rachmut IH, Molakandov K, Berneman D, Mor E, Meivar-Levy I, et al. NKX6.1 promotes PDX-1-induced liver to pancreatic beta-cells reprogramming. Cell Reprogram. 2010;12:655-64.
Ye L, Robertson MA, Hesselson D, Stainier DY, Anderson RM. Glucagon is essential for alpha cell transdifferentiation and beta cell neogenesis. Development. 2015;142:1407-17.
Miyamoto Y, Maitra A, Ghosh B, Zechner U, Argani P, Iacobuzio-Donahue CA, et al. Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis. Cancer Cell. 2003;3:565-76.
Strobel O. In vivo lineage tracing defines the role of acinar-to-ductal transdifferentiation in inflammatory ductal metaplasia. Gastroenterology. 2007;133:1999-2009.
Stanger BZ, Stiles B, Lauwers GY, Bardeesy N, Mendoza M, Wang Y, et al. Pten constrains centroacinar cell expansion and malignant transformation in the pancreas. Cancer Cell. 2005;8:185-95.
Hua H, Zhang YQ, Dabernat S, Kritzik M, Dietz D, Sterling L, et al. BMP4 regulates pancreatic progenitor cell expansion through Id2. J Biol Chem. 2006;281:13574-80.
Collu GM, Hidalgo-Sastre A, Brennan K. Wnt-Notch signalling crosstalk in development and disease. Cell Mol Life Sci. 2014;71:3553-67.
Baumgartner BK, Cash G, Hansen H, Ostler S, Murtaugh LC. Distinct requirements for beta-catenin in pancreatic epithelial growth and patterning. Dev Biol. 2014;391:89-98.
Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish (Danio rerio). 3rd ed. Eugene, OR: University of Oregon Press; 1995.
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Dev Dyn. 1995;203:253-310.
Haddon C, Jiang YJ, Smithers L, Lewis J. Delta-Notch signalling and the patterning of sensory cell differentiation in the zebrafish ear: evidence from the mind bomb mutant. Development. 1998;125:4637-44.
Warming S, Costantino N, Court DL, Jenkins NA, Copeland NG. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 2005;33, e36.
Bussmann J, Schulte-Merker S. Rapid BAC selection for tol2-mediated transgenesis in zebrafish. Development. 2011;138:4327-32.
Jao LE, Wente SR, Chen W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci U S A. 2013;110:13904-9.
ZiFiT Targeter Version 4.2. http://zifit.partners.org/ZiFiT/
Ota S, Hisano Y, Muraki M, Hoshijima K, Dahlem TJ, Grunwald DJ, et al. Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays. Genes Cells. 2013;18:450-8.
Mavropoulos A, Devos N, Biemar F, Zecchin E, Argenton F, Edlund H, et al. sox4b is a key player of pancreatic alpha cell differentiation in zebrafish. Dev Biol. 2005;285:211-23.
Hauptmann G, Gerster T. Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet. 1994;10:266.
Allende ML, Weinberg ES. The expression pattern of two zebrafish achaete-scute homolog (ash) genes is altered in the embryonic brain of the cyclops mutant. Dev Biol. 1994;166:509-30.
Cheesman SE, Layden MJ, Von Ohlen T, Doe CQ, Eisen JS. Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation. Development. 2004;131:5221-32.
Argenton F, Zecchin E, Bortolussi M. Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo. Mech Dev. 1999;87:217-21.
Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562-78.
Anders S, Pyl PT, Huber W. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics. 2014;31:166-9.
Leng N, Dawson JA, Thomson JA, Ruotti V, Rissman AI, Smits BM, et al. EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments. Bioinformatics. 2013;29:1035-43.
Nikolaou N, Watanabe-Asaka T, Gerety S, Distel M, Koster RW, Wilkinson DG. Lunatic fringe promotes the lateral inhibition of neurogenesis. Development. 2009;136:2523-33.
Amoyel M, Cheng YC, Jiang YJ, Wilkinson DG. Wnt1 regulates neurogenesis and mediates lateral inhibition of boundary cell specification in the zebrafish hindbrain. Development. 2005;132:775-85.
Zhang D, Golubkov VS, Han W, Correa RG, Zhou Y, Lee S, et al. Identification of Annexin A4 as a hepatopancreas factor involved in liver cell survival. Dev Biol. 2014;395:96-110.
