The Journal of Membrane Biology

, Volume 245, Issue 5–6, pp 263–273 | Cite as

Cx36 Is a Target of Beta2/NeuroD1, Which Associates with Prenatal Differentiation of Insulin-producing β Cells

  • Rachel Nlend Nlend
  • Aouatef Aït-Lounis
  • Florent Allagnat
  • Valentina Cigliola
  • Anne Charollais
  • Walter Reith
  • Jacques-Antoine Haefliger
  • Paolo Meda


The insulin-producing β cells of pancreatic islets are coupled by connexin36 (Cx36) channels. To investigate what controls the expression of this connexin, we have investigated its pattern during mouse pancreas development, and the influence of three transcription factors that are critical for β-cell development and differentiation. We show that (1) the Cx36 gene (Gjd2) is activated early in pancreas development and is markedly induced at the time of the surge of the transcription factors that determine β-cell differentiation; (2) the cognate protein is detected about a week later and is selectively expressed by β cells throughout the prenatal development of mouse pancreas; (3) a 2-kbp fragment of the Gjd2 promoter, which contains three E boxes for the binding of the bHLH factor Beta2/NeuroD1, ensures the expression of Cx36 by β cells; and (4) Beta2/NeuroD1 binds to these E boxes and, in the presence of the E47 ubiquitous cofactor, transactivates the Gjd2 promoter. The data identify Cx36 as a novel early marker of β cells and as a target of Beta2/NeuroD1, which is essential for β-cell development and differentiation.


Beta2/neurod1 Connexin Gap junctions Gene regulation Pancreas Promoter Transcription factor 



Our team is supported by Grants from the Swiss National Science Foundation (310000-141162, IZ73Z0_127935, CR32I3_129987), the Juvenile Diabetes Research Foundation (40-2011-11, 5-2012-281), and the European Union (BETAIMAGE 222980; IMIDIA C2008-T7, BETATRAIN 289932). We are pleased to dedicate this study to Ross G. Johnson on occasion of the starting of his new life. Ross has been a pioneer in our field, a passionate and influential scholar, and a much appreciated host while Paolo was striving in snowy Minneapolis. We wish him all the best for a new exciting adventure.


  1. Aramata S, Han SI, Yasuda K, Kataoka K (2005) Synergistic activation of the insulin gene promoter by the beta-cell enriched transcription factors MafA, Beta2, and Pdx1. Biochim Biophys Acta 1730:41–46PubMedCrossRefGoogle Scholar
  2. Berthoud VM, Singh R, Minogue PJ, Ragsdale CW, Beyer EC (2004) Highly restricted pattern of connexin36 expression in chick somite development. Anat Embryol 209:11–18PubMedCrossRefGoogle Scholar
  3. Bosco D, Haefliger JA, Meda P (2011) Connexins: key mediators of endocrine function. Physiol Rev 91:1393–1445PubMedCrossRefGoogle Scholar
  4. Carvalho CP, Barbosa HC, Britan A, Santos-Silva JC, Boschero AC, Meda P, Collares-Buzato CB (2010) Beta cell coupling and connexin expression change during the functional maturation of rat pancreatic islets. Diabetologia 53:1428–1437PubMedCrossRefGoogle Scholar
  5. Carvalho CP, Oliveira RB, Britan A, Silva-Santos JC, Boschero AC, Meda P, Collares-Buzato CB (2012) Impaired beta-to-beta cell coupling mediated by Cx36 gap junctions in pre-diabetic mice. Am J Physiol Endocrinol Metab 302 (in press)Google Scholar
  6. Cho JH, Tsai MJ (2004) The role of BETA2/NeuroD1 in the development of the nervous system. Mol Neurobiol 30:35–47PubMedCrossRefGoogle Scholar
  7. Cina C, Bechberger JF, Ozog MA, Naus CC (2007) Expression of connexins in embryonic mouse neocortical development. J Comp Neurol 504:298–313PubMedCrossRefGoogle Scholar
  8. Cummings DM, Yamazaki I, Cepeda C, Paul DL, Levine MS (2008) Neuronal coupling via connexin36 contributes to spontaneous synaptic currents of striatal medium-sized spiny neurons. J Neurosci Res 86:2147–2158PubMedCrossRefGoogle Scholar
  9. Degen J, Meier C, Van Der Giessen RS, Sohl G, Petrasch-Parwez E, Urschel S, Dermietzel R, Schilling K, De Zeeuw CI, Willecke K (2004) Expression pattern of lacZ reporter gene representing connexin36 in transgenic mice. J Comp Neurol 473:511–525PubMedCrossRefGoogle Scholar
  10. Docherty HM, Hay CW, Ferguson LA, Barrow J, Durward E, Docherty K (2005) Relative contribution of PDX-1, MafA and E47/beta2 to the regulation of the human insulin promoter. Biochem J 389:813–820PubMedCrossRefGoogle Scholar
  11. Gulisano M, Parenti R, Spinella F, Cicirata F (2000) Cx36 is dynamically expressed during early development of mouse brain and nervous system. Neuroreport 11:3823–3828PubMedCrossRefGoogle Scholar
  12. Habener JF, Kemp DM, Thomas MK (2005) Minireview: transcriptional regulation in pancreatic development. Endocrinology 146:1025–1034PubMedCrossRefGoogle Scholar
  13. Head WS, Orseth ML, Nunemaker CS, Satin LS, Piston DW, Benninger RK (2012) Connexin-36 gap junctions regulate in vivo first- and second-phase insulin secretion dynamics and glucose tolerance in the conscious mouse. Diabetes 61 (in press)Google Scholar
  14. Henderson E, Stein R (1994) c-jun inhibits transcriptional activation by the insulin enhancer, and the insulin control element is the target of control. Mol Cell Biol 14:655–662PubMedGoogle Scholar
  15. Herrera PL (2000) Adult insulin- and glucagon-producing cells differentiate from two independent cell lineages. Development 127:2317–2322PubMedGoogle Scholar
  16. Hohl M, Thiel G (2005) Cell type-specific regulation of RE-1 silencing transcription factor (REST) target genes. Eur J Neurosci 22:2216–2230PubMedCrossRefGoogle Scholar
  17. Itkin-Ansari P, Marcora E, Geron I, Tyrberg B, Demeterco C, Hao E, Padilla C, Ratineau C, Leiter A, Lee JE, Levine F (2005) NeuroD1 in the endocrine pancreas: localization and dual function as an activator and repressor. Dev Dyn 233:946–953PubMedCrossRefGoogle Scholar
  18. Iwata I, Nagafuchi S, Nakashima H, Kondo S, Koga T, Yokogawa Y, Akashi T, Shibuya T, Umeno Y, Okeda T, Shibata S, Kono S, Yasunami M, Ohkubo H, Niho Y (1999) Association of polymorphism in the NeuroD/BETA2 gene with type 1 diabetes in the Japanese. Diabetes 48:416–419PubMedCrossRefGoogle Scholar
  19. Jonsson J, Carlsson L, Edlund T, Edlund H (1994) Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371:606–609PubMedCrossRefGoogle Scholar
  20. Kim SK, MacDonald RJ (2002) Signaling and transcriptional control of pancreatic organogenesis. Curr Opin Genet Dev 12:540–547PubMedCrossRefGoogle Scholar
  21. Kim JW, Seghers V, Cho JH, Kang Y, Kim S, Ryu Y, Baek K, Aguilar-Bryan L, Lee YD, Bryan J, Suh-Kim H (2002) Transactivation of the mouse sulfonylurea receptor I gene by BETA2/NeuroD. Mol Endocrinol 16:1097–1107PubMedCrossRefGoogle Scholar
  22. Klee P, Allagnat F, Pontes H, Cederroth M, Charollais A, Caille D, Britan A, Haefliger JA, Meda P (2011) Connexins protect mouse pancreatic β cells against apoptosis. J Clin Invest 121:4870–4879PubMedCrossRefGoogle Scholar
  23. Le Gurun S, Martin D, Formenton A, Maechler P, Caille D, Waeber G, Meda P, Haefliger JA (2003) Connexin-36 contributes to control function of insulin-producing cells. J Biol Chem 278:37690–37697PubMedCrossRefGoogle Scholar
  24. Lee JE (1997) NeuroD and neurogenesis. Dev Neurosci 19:27–32PubMedCrossRefGoogle Scholar
  25. Lee JE, Hollenberg SM, Snider L, Turner DL, Lipnick N, Weintraub H (1995) Conversion of Xenopus ectoderm into neurons by NeuroD, a basic helix-loop-helix protein. Science 268:836–884PubMedCrossRefGoogle Scholar
  26. Liu M, Pereira FA, Price SD, Chu MJ, Shope C, Himes D, Eatock RA, Brownell WE, Lysakowski A, Tsai MJ (2000) Essential role of Beta2/NeuroD1 in development of the vestibular and auditory systems. Genes Dev 14:2839–2854PubMedCrossRefGoogle Scholar
  27. MacDonald MJ (2007) Synergistic potent insulin release by combinations of weak secretagogues in pancreatic islets and INS-1 cells. J Biol Chem 282:6043–6052PubMedCrossRefGoogle Scholar
  28. MacDonald PE, Rorsman P (2007) The ins and outs of secretion from pancreatic beta-cells: control of single-vesicle exo- and endocytosis. Physiology (Bethesda) 22:113–121CrossRefGoogle Scholar
  29. Malecki MT, Jhala US, Antonellis A, Fields L, Doria A, Orban T, Saad M, Warram JH, Montminy M, Krolewski AS (1999) Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus. Nat Genet 23:323–328PubMedCrossRefGoogle Scholar
  30. Malecki MT, Cyganek K, Klupa T, Sieradzki J (2003) The Ala45Thr polymorphism of Beta2/NeuroD1 gene and susceptibility to type 2 diabetes mellitus in a Polish population. Acta Diabetol 40:109–111PubMedGoogle Scholar
  31. Martin D, Tawadros T, Meylan L, Abderrahmani A, Condorelli DF, Waeber G, Haefliger JA (2003) Critical role of the transcriptional repressor neuron-restrictive silencer factor in the specific control of connexin36 in insulin-producing cell lines. J Biol Chem 278:53082–53089PubMedCrossRefGoogle Scholar
  32. Masternak K, Peyraud N, Krawczyk M, Barras E, Reith W (2003) Chromatin remodeling and extragenic transcription at the MHC class II locus control region. Nat Immunol 4:132–137PubMedCrossRefGoogle Scholar
  33. Meda P (2012) The in vivo β-to-β-cell chat room: connexin connections matter. Diabetes 61 (in press)Google Scholar
  34. Mirasierra M, Vallejo M (2006) The homeoprotein Alx3 expressed in pancreatic beta-cells regulates insulin gene transcription by interacting with the basic helix-loop-helix protein E47. Mol Endocrinol 20:2876–2889PubMedCrossRefGoogle Scholar
  35. Miyata T, Maeda T, Lee JE (1999) NeuroD is required for differentiation of the granule cells in the cerebellum and hippocampus. Genes Dev 13:1647–1652PubMedCrossRefGoogle Scholar
  36. Murtaugh LC (2007) Pancreas and beta-cell development: from the actual to the possible. Development 134:427–438PubMedCrossRefGoogle Scholar
  37. Naya FJ, Stellrecht CM, Tsai MJ (1994) Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor. Genes Dev 9:1009–1919CrossRefGoogle Scholar
  38. Naya FJ, Stellrecht CM, Tsai MJ (1995) Tissue-specific regulation of the insulin gene by a novel basic helix-loop-helix transcription factor. Genes Dev 9:1009–1019PubMedCrossRefGoogle Scholar
  39. Naya FJ, Huang HP, Qiu Y, Mutoh H, DeMayo FJ, Leiter AB, Tsai MJ (1997) Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD-deficient mice. Genes Dev 11:2323–2334PubMedCrossRefGoogle Scholar
  40. Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BL, Wright CV (1996) PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122:983–995PubMedGoogle Scholar
  41. Pérez-Armendariz EM, Cruz-Miguel L, Coronel-Cruz C, Esparza-Aguilar M, Pinzon-Estrada E, Rancaño-Camacho E, Zacarias-Climaco G, Olivares PF, Espinosa AM, Becker I, Sáez JC, Berumen J, Pérez-Palacios G (2012) Connexin 36 is expressed in beta and connexins 26 and 32 in acinar cells at the end of the secondary transition of mouse pancreatic development and increase during fetal and perinatal life. Anat Rec 295:980–990CrossRefGoogle Scholar
  42. Pictet RL, Clark WR, Williams RH, Rutter WJ (1972) An ultrastructural analysis of the developing embryonic pancreas. Dev Biol 29:436–467PubMedCrossRefGoogle Scholar
  43. Potolicchio I, Cigliola V, Velazquez-Garcia S, Klee P, Valjevac A, Kapic D, Cosovic E, Lepara O, Hadzovic-Dzuvo A, Mornjacovic Z, Meda P (2012) Connexin-dependent signaling in neuro-hormonal systems. Biochim Biophys Acta 1818:1919–1936Google Scholar
  44. Poulin G, Turgeon B, Drouin J (1997) NeuroD1/beta2 contributes to cell-specific transcription of the proopiomelanocortin gene. Mol Cell Biol 17:6673–6682PubMedGoogle Scholar
  45. Qiu Y, Guo M, Huang S, Stein R (2002) Insulin gene transcription is mediated by interactions between the p300 coactivator and PDX-1, BETA2, and E47. Mol Cell Biol 22:412–420PubMedCrossRefGoogle Scholar
  46. Ravier MA, Guldenagel M, Charollais A, Gjinovci A, Caille D, Sohl G, Wollheim CB, Willecke K, Henquin JC, Meda P (2005) Loss of connexin36 channels alters beta-cell coupling, islet synchronization of glucose-induced Ca2+ and insulin oscillations, and basal insulin release. Diabetes 54:1798–1807PubMedCrossRefGoogle Scholar
  47. Robinson KA, Koepke JI, Kharodawala M, Lopes JM (2000) A network of yeast basic helix-loop-helix interactions. Nucleic Acids Res 28:4460–4466PubMedCrossRefGoogle Scholar
  48. Serre-Beinier V, Le Gurun S, Belluardo N, Trovato-Salinaro A, Charollais A, Haefliger JA, Condorelli DF, Meda P (2000) Cx36 preferentially connects beta-cells within pancreatic islets. Diabetes 49:727–734PubMedCrossRefGoogle Scholar
  49. Serre-Beinier V, Bosco D, Zulianello L, Charollais A, Caille D, Charpantier E, Gauthier BR, Diaferia GR, Giepmans BN, Lupi R, Marchetti P, Deng S, Buhler L, Berney T, Cirulli V, Meda P (2009) Cx36 makes channels coupling human pancreatic beta-cells, and correlates with insulin expression. Hum Mol Genet 18:428–439PubMedCrossRefGoogle Scholar
  50. Sommer L, Ma Q, Anderson DJ (1996) Neurogenins, a novel family of atonal-related bHLH transcription factors, are putative mammalian neuronal determination genes that reveal progenitor cell heterogeneity in the developing CNS and PNS. Mol Cell Neurosci 8:221–241PubMedCrossRefGoogle Scholar
  51. Speier S, Gjinovci A, Charollais A, Meda P, Rupnik M (2007) Cx36-mediated coupling reduces beta-cell heterogeneity, confines the stimulating glucose concentration range, and affects insulin release kinetics. Diabetes 56:1078–1086PubMedCrossRefGoogle Scholar
  52. Theis M, Mas C, Doring B, Degen J, Brink C, Caille D, Charollais A, Kruger O, Plum A, Nepote V, Herrera P, Meda P, Willecke K (2004) Replacement by a lacZ reporter gene assigns mouse connexin36, 45 and 43 to distinct cell types in pancreatic islets. Exp Cell Res 294:18–29PubMedCrossRefGoogle Scholar
  53. Wellershaus K, Degen J, Deuchars J, Theis M, Charollais A, Caille D, Gauthier B, Janssen-Bienhold U, Sonntag S, Herrera P, Meda P, Willecke K (2008) A new conditional mouse mutant reveals specific expression and functions of connexin36 in neurons and pancreatic beta-cells. Exp Cell Res 314:997–1012PubMedCrossRefGoogle Scholar
  54. Yamada S, Motohashi Y, Yanagawa T, Maruyama T, Kasuga A, Hirose H, Matsubara K, Shimada A, Saruta T (2001) NeuroD/beta2 gene G→A polymorphism may affect onset pattern of type 1 diabetes in Japanese. Diabetes Care 24:1438–1441PubMedCrossRefGoogle Scholar
  55. Zhang C, Moriguchi T, Kajihara M, Esaki R, Harada A, Shimohata H, Oishi H, Hamada M, Morito N, Hasegawa K, Kudo T, Engel JD, Yamamoto M, Takahashi S (2005) MafA is a key regulator of glucose-stimulated insulin secretion. Mol Cell Biol 25:4969–4976PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Rachel Nlend Nlend
    • 1
  • Aouatef Aït-Lounis
    • 4
  • Florent Allagnat
    • 3
  • Valentina Cigliola
    • 1
  • Anne Charollais
    • 1
  • Walter Reith
    • 2
  • Jacques-Antoine Haefliger
    • 3
  • Paolo Meda
    • 1
  1. 1.Department of Cell Physiology and MetabolismUniversity of GenevaGeneva 4Switzerland
  2. 2.Department of Pathology and ImmunologyUniversity of GenevaGeneva 4Switzerland
  3. 3.Department of Internal MedicineUniversity of LausanneLausanneSwitzerland
  4. 4.Laboratory of Cellular and Molecular BiologyUniversity of Algiers, USTHBAlgiersAlgeria

Personalised recommendations