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Nato3 Integrates with the Shh-Foxa2 Transcriptional Network Regulating the Differentiation of Midbrain Dopaminergic Neurons

Abstract

Mesencephalic dopaminergic (mesDA) neurons originate from the floor plate of the midbrain, a transient embryonic organizing center located at the ventral-most midline. Since the loss of mesDA leads to Parkinson’s disease, the molecular mechanisms controlling the genesis and differentiation of dopaminergic progenitors are extensively studied and the identification and characterization of new genes is of interest. Here, we show that the expression of the basic helix-loop-helix transcription factor Nato3 (Ferd3l) increases in parallel to the differentiation of SN4741 dopaminergic cells in vitro. Nato3 transcription is directly regulated by the transcription factor Foxa2, a target and effector of the Sonic hedgehog (Shh) signaling cascade. Moreover, pharmacological inhibition of Shh signaling downregulated the expression of Nato3, thus defining Nato3 as a novel component of one of the major pathways controlling cell patterning and generation of mesDA. Furthermore, we show that Nato3 regulated Shh and Foxa2 through a novel feed-backward loop. Up- and downregulation of Nato3 further affected the transcription of Nurr1, implicated in the genesis of mesDA, but not of TH. Taken together, these data shed new light on the transcriptional networks controlling the generation of mesDA and may be utilized in the efforts to direct stem cells towards a dopaminergic fate.

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References

  1. Abeliovich A, Hammond R (2007) Midbrain dopamine neuron differentiation: factors and fates. Dev Biol 304:447–454

    PubMed  Article  CAS  Google Scholar 

  2. Ang SL, Rossant J (1994) HNF-3 beta is essential for node and notochord formation in mouse development. Cell 78:561–574

    PubMed  Article  CAS  Google Scholar 

  3. Ang SL, Wierda A, Wong D, Stevens KA, Cascio S, Rossant J et al (1993) The formation and maintenance of the definitive endoderm lineage in the mouse: involvement of HNF3/forkhead proteins. Development 119:1301–1315

    PubMed  CAS  Google Scholar 

  4. Baffi JS, Palkovits M, Castillo SO, Mezey E, Nikodem VM (1999) Differential expression of tyrosine hydroxylase in catecholaminergic neurons of neonatal wild-type and Nurr1-deficient mice. Neuroscience 93:631–642

    PubMed  Article  CAS  Google Scholar 

  5. Baker H, Liu N, Chun HS, Saino S, Berlin R, Volpe B et al (2001) Phenotypic differentiation during migration of dopaminergic progenitor cells to the olfactory bulb. J Neurosci 21:8505–8513

    PubMed  CAS  Google Scholar 

  6. Bayly RD, Brown CY, Agarwala S (2012) A novel role for FOXA2 and SHH in organizing midbrain signaling centers. Dev Biol 369:32–42

    PubMed  Article  CAS  Google Scholar 

  7. Ben-Shushan E, Marshak S, Shoshkes M, Cerasi E, Melloul D (2001) A pancreatic beta-cell-specific enhancer in the human PDX-1 gene is regulated by hepatocyte nuclear factor 3beta (HNF-3beta ), HNF-1alpha, and SPs transcription factors. J Biol Chem 276:17533–17540

    PubMed  Article  CAS  Google Scholar 

  8. Bryja V, Cajanek L, Grahn A, Schulte G (2007a) Inhibition of endocytosis blocks Wnt signalling to beta-catenin by promoting dishevelled degradation. Acta Physiol (Oxf) 190:55–61

    Article  CAS  Google Scholar 

  9. Bryja V, Schulte G, Arenas E (2007b) Wnt-3a utilizes a novel low dose and rapid pathway that does not require casein kinase 1-mediated phosphorylation of Dvl to activate beta-catenin. Cell Signal 19:610–616

    PubMed  Article  CAS  Google Scholar 

  10. Bryja V, Schulte G, Rawal N, Grahn A, Arenas E (2007c) Wnt-5a induces dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism. Journal of Cell Science 120:586–595

    PubMed  Article  CAS  Google Scholar 

  11. Castillo SO, Baffi JS, Palkovits M, Goldstein DS, Kopin IJ, Witta J et al (1998) Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in mice with targeted disruption of the Nurr1 gene. Mol Cell Neurosci 11:36–46

    PubMed  Article  CAS  Google Scholar 

  12. Chakrabarti SK, James JC, Mirmira RG (2002) Quantitative assessment of gene targeting in vitro and in vivo by the pancreatic transcription factor, Pdx1. Importance of chromatin structure in directing promoter binding. J Biol Chem 277:13286–13293

    PubMed  Article  CAS  Google Scholar 

  13. Chen JK, Taipale J, Cooper MK, Beachy PA (2002) Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev 16:2743–2748

    PubMed  Article  CAS  Google Scholar 

  14. Choi K-C, Kim S-H, Ha J-Y, Kim S-T, Son JH (2010) A novel mTOR activating protein protects dopamine neurons against oxidative stress by repressing autophagy related cell death. J Neurochem 112:366–376

    PubMed  Article  CAS  Google Scholar 

  15. Chung S, Leung A, Han BS, Chang MY, Moon JI, Kim CH et al (2009) Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway. Cell Stem Cell 5:646–658

    PubMed  Article  CAS  Google Scholar 

  16. Echelard Y, Epstein DJ, St-Jacques B, Shen L, Mohler J, McMahon JA et al (1993) Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75:1417–1430

    PubMed  Article  CAS  Google Scholar 

  17. Epstein DJ, Martinu L, Michaud JL, Losos KM, Fan C, Joyner AL (2000) Members of the bHLH-PAS family regulate Shh transcription in forebrain regions of the mouse CNS. Development 127:4701–4709

    PubMed  CAS  Google Scholar 

  18. Ferri AL, Lin W, Mavromatakis YE, Wang JC, Sasaki H, Whitsett JA et al (2007) Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner. Development 134:2761–2769

    PubMed  Article  CAS  Google Scholar 

  19. Fishman-Jacob T, Youdim MBH, Mandel SA (2010) Silencing/overexpressing selected genes as a model of sporadic Parkinson’s disease. Neurodegener Dis 7:108–111

    PubMed  Article  CAS  Google Scholar 

  20. Gale E, Li M (2008) Midbrain dopaminergic neuron fate specification: of mice and embryonic stem cells. Mol Brain 1:8

    PubMed  Article  Google Scholar 

  21. Hemsley A, Arnheim N, Toney MD, Cortopassi G, Galas DJ (1989) A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res 17:6545–6551

    PubMed  Article  CAS  Google Scholar 

  22. Hwang CK, Chun HS (2012) Isoliquiritigenin isolated from licorice Glycyrrhiza uralensis prevents 6-hydroxydopamine-induced apoptosis in dopaminergic neurons. Biosci Biotechnol Biochem 76:536–543

    PubMed  Article  CAS  Google Scholar 

  23. Hynes M, Porter JA, Chiang C, Chang D, Tessier-Lavigne M, Beachy PA et al (1995a) Induction of midbrain dopaminergic neurons by Sonic hedgehog. Neuron 15:35–44

    PubMed  Article  CAS  Google Scholar 

  24. Hynes M, Poulsen K, Tessier-Lavigne M, Rosenthal A (1995b) Control of neuronal diversity by the floor plate: contact-mediated induction of midbrain dopaminergic neurons. Cell 80:95–101

    PubMed  Article  CAS  Google Scholar 

  25. Hynes M, Rosenthal A (1999) Specification of dopaminergic and serotonergic neurons in the vertebrate CNS. Curr Opin Neurobiol 9:26–36

    PubMed  Article  CAS  Google Scholar 

  26. Hynes M, Stone DM, Dowd M, Pitts-Meek S, Goddard A, Gurney A et al (1997) Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1. Neuron 19:15–26

    PubMed  Article  CAS  Google Scholar 

  27. Iwawaki T, Kohno K, Kobayashi K (2000) Identification of a potential nurr1 response element that activates the tyrosine hydroxylase gene promoter in cultured cells. Biochem Biophys Res Commun 274:590–595

    PubMed  Article  CAS  Google Scholar 

  28. Joksimovic M, Anderegg A, Roy A, Campochiaro L, Yun B, Kittappa R et al (2009a) Spatiotemporally separable Shh domains in the midbrain define distinct dopaminergic progenitor pools. Proc Natl Acad Sci U S A 106:19185–19190

    PubMed  Article  CAS  Google Scholar 

  29. Joksimovic M, Yun BA, Kittappa R, Anderegg AM, Chang WW, Taketo MM et al (2009b) Wnt antagonism of Shh facilitates midbrain floor plate neurogenesis. Nat Neurosci 12:125–131

    PubMed  Article  CAS  Google Scholar 

  30. Kim HJ (2011) Stem cell potential in Parkinson’s disease and molecular factors for the generation of dopamine neurons. Biochim Biophys Acta 1812:1–11

    PubMed  Article  CAS  Google Scholar 

  31. Kim HJ, Sugimori M, Nakafuku M, Svendsen CN (2007) Control of neurogenesis and tyrosine hydroxylase expression in neural progenitor cells through bHLH proteins and Nurr1. Exp Neurol 203:394–405

    PubMed  Article  CAS  Google Scholar 

  32. Kim KS, Kim CH, Hwang DY, Seo H, Chung S, Hong SJ et al (2003) Orphan nuclear receptor Nurr1 directly transactivates the promoter activity of the tyrosine hydroxylase gene in a cell-specific manner. J Neurochem 85:622–634

    PubMed  Article  CAS  Google Scholar 

  33. Kittappa R, Chang WW, Awatramani RB, McKay RD (2007) The foxa2 gene controls the birth and spontaneous degeneration of dopamine neurons in old age. PLoS Biol 5:e325

    PubMed  Article  Google Scholar 

  34. Kuo MH, Allis CD (1999) In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in a chromatin environment. Methods 19:425–433

    PubMed  Article  CAS  Google Scholar 

  35. Lin W, Metzakopian E, Mavromatakis YE, Gao N, Balaskas N, Sasaki H et al (2009) Foxa1 and Foxa2 function both upstream of and cooperatively with Lmx1a and Lmx1b in a feedforward loop promoting mesodiencephalic dopaminergic neuron development. Dev Biol 333:386–396

    PubMed  Article  CAS  Google Scholar 

  36. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 25:402–408

    PubMed  Article  CAS  Google Scholar 

  37. Mandel SA, Fishman-Jacob T, Youdim MB (2012) Genetic reduction of the E3 ubiquitin ligase element, SKP1A and environmental manipulation to emulate cardinal features of Parkinson’s disease. Parkinsonism Relat Disord 18(Suppl 1):S177–S179

    PubMed  Article  Google Scholar 

  38. Mansour AA, Nissim-Eliraz E, Zisman S, Golan-Lev T, Schatz O, Klar A et al (2011) Foxa2 regulates the expression of Nato3 in the floor plate by a novel evolutionarily conserved promoter. Mol Cell Neurosci 46:187–199

    PubMed  Article  CAS  Google Scholar 

  39. Marti E, Takada R, Bumcrot DA, Sasaki H, McMahon AP (1995) Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo. Development 121:2537–2547

    PubMed  CAS  Google Scholar 

  40. Matise MP, Epstein DJ, Park HL, Platt KA, Joyner AL (1998) Gli2 is required for induction of floor plate and adjacent cells, but not most ventral neurons in the mouse central nervous system. Development 125:2759–2770

    PubMed  CAS  Google Scholar 

  41. Monaghan AP, Kaestner KH, Grau E, Schutz G (1993) Postimplantation expression patterns indicate a role for the mouse forkhead/HNF-3 alpha, beta and gamma genes in determination of the definitive endoderm, chordamesoderm and neuroectoderm. Development 119:567–578

    PubMed  CAS  Google Scholar 

  42. Nunes I, Tovmasian LT, Silva RM, Burke RE, Goff SP (2003) Pitx3 is required for development of substantia nigra dopaminergic neurons. Proc Natl Acad Sci U S A 100:4245–4250

    PubMed  Article  CAS  Google Scholar 

  43. Ono Y, Nakatani T, Minaki Y, Kumai M (2010) The basic helix-loop-helix transcription factor Nato3 controls neurogenic activity in mesencephalic floor plate cells. Development 137:1897–1906

    PubMed  Article  CAS  Google Scholar 

  44. Orlando V, Strutt H, Paro R (1997) Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11:205–214

    PubMed  Article  CAS  Google Scholar 

  45. Placzek M, Briscoe J (2005) The floor plate: multiple cells, multiple signals. Nat Rev Neurosci 6:230–240

    PubMed  Article  CAS  Google Scholar 

  46. Prakash N, Wurst W (2006) Genetic networks controlling the development of midbrain dopaminergic neurons. J Physiol 575:403–410

    PubMed  Article  CAS  Google Scholar 

  47. Ruiz i Altaba A (1998) Combinatorial Gli gene function in floor plate and neuronal inductions by Sonic hedgehog. Development 125:2203–2212

    PubMed  Google Scholar 

  48. Ruiz i Altaba A, Placzek M, Baldassare M, Dodd J, Jessell TM (1995) Early stages of notochord and floor plate development in the chick embryo defined by normal and induced expression of HNF-3 beta. Dev Biol 170:299–313

    PubMed  Article  Google Scholar 

  49. Sakurada K, Ohshima-Sakurada M, Palmer TD, Gage FH (1999) Nurr1, an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain. Development 126:4017–4026

    PubMed  CAS  Google Scholar 

  50. Sasaki H, Hogan BL (1993) Differential expression of multiple fork head related genes during gastrulation and axial pattern formation in the mouse embryo. Development 118:47–59

    PubMed  CAS  Google Scholar 

  51. Sasaki H, Hui C, Nakafuku M, Kondoh H (1997) A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 124:1313–1322

    PubMed  CAS  Google Scholar 

  52. Saucedo-Cardenas O, Quintana-Hau JD, Le WD, Smidt MP, Cox JJ, De Mayo F et al (1998) Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Natl Acad Sci U S A 95:4013–4018

    PubMed  Article  CAS  Google Scholar 

  53. Segev E, Halachmi N, Salzberg A, Ben-Arie N (2001) Nato3 is an evolutionarily conserved bHLH transcription factor expressed in the CNS of Drosophila and mouse. Mech Dev 106:197–202

    PubMed  Article  CAS  Google Scholar 

  54. She H, Yang Q, Mao Z (2012) Neurotoxin-induced selective ubiquitination and regulation of MEF2A isoform in neuronal stress response. J Neurochem 122:1203–1210

    PubMed  Article  CAS  Google Scholar 

  55. Silva JP, von Meyenn F, Howell J, Thorens B, Wolfrum C, Stoffel M (2009) Regulation of adaptive behaviour during fasting by hypothalamic Foxa2. Nature 462:646–650

    PubMed  Article  CAS  Google Scholar 

  56. Smidt MP, Asbreuk CH, Cox JJ, Chen H, Johnson RL, Burbach JP (2000) A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b. Nat Neurosci 3:337–341

    PubMed  Article  CAS  Google Scholar 

  57. Son JH, Chun HS, Joh TH, Cho S, Conti B, Lee JW (1999) Neuroprotection and neuronal differentiation studies using substantia nigra dopaminergic cells derived from transgenic mouse embryos. J Neurosci 19:10–20

    PubMed  CAS  Google Scholar 

  58. Sousa KM, Villaescusa JC, Cajanek L, Ondr JK, Castelo-Branco G, Hofstra W et al (2010) Wnt2 regulates progenitor proliferation in the developing ventral midbrain. J Biol Chem 285:7246–7253

    PubMed  Article  CAS  Google Scholar 

  59. Verzi MP, Anderson JP, Dodou E, Kelly KK, Greene SB, North BJ et al (2002) N-twist, an evolutionarily conserved bHLH protein expressed in the developing CNS, functions as a transcriptional inhibitor. Dev Biol 249:174–190

    PubMed  Article  CAS  Google Scholar 

  60. Wallen AA, Castro DS, Zetterstrom RH, Karlen M, Olson L, Ericson J et al (2001) Orphan nuclear receptor Nurr1 is essential for Ret expression in midbrain dopamine neurons and in the brain stem. Mol Cell Neurosci 18:649–663

    Article  CAS  Google Scholar 

  61. Wang MZ, Jin P, Bumcrot DA, Marigo V, McMahon AP, Wang EA et al (1995) Induction of dopaminergic neuron phenotype in the midbrain by Sonic hedgehog protein. Nat Med 1:1184–1188

    PubMed  Article  CAS  Google Scholar 

  62. Weinstein DC, Ruiz i Altaba A, Chen WS, Hoodless P, Prezioso VR, Jessell TM et al (1994) The winged-helix transcription factor HNF-3 beta is required for notochord development in the mouse embryo. Cell 78:575–588

    PubMed  Article  CAS  Google Scholar 

  63. Wolfrum C, Besser D, Luca E, Stoffel M (2003) Insulin regulates the activity of forkhead transcription factor Hnf-3beta/Foxa-2 by Akt-mediated phosphorylation and nuclear/cytosolic localization. Proc Natl Acad Sci U S A 100:11624–11629

    PubMed  Article  CAS  Google Scholar 

  64. Yan CH, Levesque M, Claxton S, Johnson RL, Ang SL (2011) Lmx1a and lmx1b function cooperatively to regulate proliferation, specification, and differentiation of midbrain dopaminergic progenitors. J Neurosci 31:12413–12425

    PubMed  Article  CAS  Google Scholar 

  65. Yao L, Li W, She H, Dou J, Jia L, He Y et al (2012) Activation of transcription factor MEF2D by Bis(3)-cognitin protects dopaminergic neurons and ameliorates parkinsonian motor defects. J Biol Chem 287:34246–34255

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

The research was supported by grants from the Israel Science Foundation (431/07) and the Legacy-Heritage Biomedical Program of the Israel Science Foundation (1914/08). We thank Eti Golenser and Theodora Bar-El for carefully reading and correcting the manuscript. We are grateful to Dr. Naomi Melamed-Book for expert assistance with confocal imaging.

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Correspondence to Nissim Ben-Arie.

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Nissim-Eliraz, E., Zisman, S., Schatz, O. et al. Nato3 Integrates with the Shh-Foxa2 Transcriptional Network Regulating the Differentiation of Midbrain Dopaminergic Neurons. J Mol Neurosci 51, 13–27 (2013). https://doi.org/10.1007/s12031-012-9939-6

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Keywords

  • Nato3
  • Midbrain dopaminergic neuron
  • Differentiation
  • Transcriptional regulation
  • SN4741 cell line