Journal of Molecular Neuroscience

, Volume 55, Issue 3, pp 684–705 | Cite as

Transcriptional Regulatory Events Initiated by Ascl1 and Neurog2 During Neuronal Differentiation of P19 Embryonic Carcinoma Cells

  • Holly S. Huang
  • Tanya M. Redmond
  • Ginger M. Kubish
  • Shweta Gupta
  • Robert C. Thompson
  • David L. Turner
  • Michael D. Uhler


As members of the proneural basic-helix-loop-helix (bHLH) family of transcription factors, Ascl1 and Neurog2 direct the differentiation of specific populations of neurons at various times and locations within the developing nervous system. In order to characterize the mechanisms employed by these two bHLH factors, we generated stable, doxycycline-inducible lines of P19 embryonic carcinoma cells that express comparable levels of Ascl1 and Neurog2. Upon induction, both Ascl1 and Neurog2 directed morphological and immunocytochemical changes consistent with initiation of neuronal differentiation. Comparison of Ascl1- and Neurog2-regulated genes by microarray analyses showed both shared and distinct transcriptional changes for each bHLH protein. In both Ascl1- and Neurog2-differentiating cells, repression of Oct4 mRNA levels was accompanied by increased Oct4 promoter methylation. However, DNA demethylation was not detected for genes induced by either bHLH protein. Neurog2-induced genes included glutamatergic marker genes while Ascl1-induced genes included GABAergic marker genes. The Neurog2-specific induction of a gene encoding a protein phosphatase inhibitor, Ppp1r14a, was dependent on distinct, canonical E-box sequences within the Ppp1r14a promoter and the nucleotide sequences within these E-boxes were partially responsible for Neurog2-specific regulation. Our results illustrate multiple novel mechanisms by which Ascl1 and Neurog2 regulate gene repression during neuronal differentiation in P19 cells.


Ascl1 Differentiation Neurog2 Neuron Transcription 



Basic helix-loop-helix


Ca2+/calmodulin-dependent protein kinase


Central nervous system


Delta/Notch-like EGF-related receptor


DNA methyltransferases




Embryonic carcinoma


Enhanced green fluorescent protein


ES cell-expressed Ras


Embryonic stem


Growth arrest and DNA damage-inducible gamma


Integrin-linked kinase


Internal ribosome entry site


Mitogen-activated protein kinase


p21-activated protein kinase


cAMP-dependent protein kinase


Protein kinase C


Protein kinase N


Protein phosphatase 1


Quantitative real-time PCR


Rho-associated coiled-coil kinase


Reverse transcriptional activator protein



The authors would like to thank Dr. Fan Meng for helpful discussions of the microarray data. This work was supported by NIH/NINDSR01NS051472 (MDU), the Medical School of the University of Michigan, and the Pritzker Neuropsychiatric Disorders Research Fund.

Supplementary material

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Supplementary Fig. 1

Gene expression of Ascl1, Neurog2, EGFP, and Mtap2 in A6 and N3 cells following Dox treatment. a qRT-PCR analysis of gene expression changes over the course of 8 days in response to 0.5 μg/ml Dox. Treating A6 and N3 cells with 0.5 μg/ml Dox resulted in a transient increase in Ascl1 or Neurog2 expression, respectively. Bicistronic Egfp expression is also transiently induced, with kinetics mirroring bHLH expression for each cell line. Mtap2 expression significantly, but transiently, increases in both cell lines, albeit to a greater extent in response to Neurog2. b qRT-PCR analysis of gene expression in response to varying concentrations of Dox at 48 h. A6 and N3 cells express Ascl1, Neurog2, and Egfp in a dose-dependent manner, with significant increases in gene expression in as low as 100 ng/ml of Dox. While Ascl1 and Neurog2 both induce expression of general neuronal differentiation marker, Mtap2, N3 cells appear to be more sensitive to lower concentrations of Dox (GIF 62 kb)

12031_2014_408_MOESM1_ESM.tif (15.2 mb)
High-Resolution Image (TIFF 15,577 kb)
12031_2014_408_Fig10_ESM.gif (42 kb)
Supplementary Fig. 2

Characterization of Isl1 gene regulation by Ascl1 and Neurog2. a qRT-PCR analysis of Isl1 gene expression changes over the course of 8 days in response to 0.5 μg/ml Dox. Isl1 mRNA transiently increases in response to both Ascl1 and Neurog2, with expression peaking 2 days after treatment with Dox. The induction of Isl1 appears to be 2.8-fold higher in response to Ascl1 after 2 days of treatment with Dox. b Western blot for Isl1 protein expression changes over the course of 8 days shows a significant increase in Isl1 protein expression after 2 days of Dox treatment, with elevated levels in response to Ascl1. c qRT-PCR analysis of Isl1 gene expression in response to varying concentrations of Dox again shows higher induction by Ascl1 (GIF 42 kb)

12031_2014_408_MOESM2_ESM.tif (3.6 mb)
High-Resolution Image (TIFF 3,652 kb)
12031_2014_408_Fig11_ESM.gif (42 kb)
Supplementary Fig. 3

Analysis of Cldn5 gene expression changes. a qRT-PCR analysis of Cldn5 gene expression changes over the course of 8 days in response to 0.5 μg/ml Dox. Cldn5 exhibits induction by only Ascl1, and the expression is undetectable in response to Neurog2. b Western blot for Cldn5 protein expression changes over the course of 8 days shows a substantial increase in Cldn5 protein expression in response to Ascl1, but not to Neurog2. c qRT-PCR analysis of Cldn5 gene expression in response to varying concentrations of Dox again shows induction by only Ascl1 and not Neurog2 (GIF 42 kb)

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12031_2014_408_MOESM4_ESM.docx (15 kb)
Supplementary Table 1 (DOCX 15 kb)
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Supplementary Table 2 (DOCX 34 kb)
12031_2014_408_MOESM6_ESM.xlsx (13 kb)
Supplementary Table 3 (XLSX 12 kb)
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Supplementary Table 4 (XLSX 28 kb)


  1. Azam N, Vairapandi M, Zhang W, Hoffman B, Liebermann DA (2001) Interaction of CR6 (GADD45gamma) with proliferating cell nuclear antigen impedes negative growth control. J Biol Chem 276(4):2766–2774PubMedCrossRefGoogle Scholar
  2. Azmi S, Sun H, Ozog A, Taneja R (2003) mSharp-1/DEC2, a basic helix-loop-helix protein functions as a transcriptional repressor of E box activity and Stra13 expression. J Biol Chem 278(22):20098–20109PubMedCrossRefGoogle Scholar
  3. Bain G, Ray WJ, Yao M, Gottlieb DI (1994) From embryonal carcinoma cells to neurons: the P19 pathway. Bioessays 16(5):343–348PubMedCrossRefGoogle Scholar
  4. Bertrand N, Castro DS, Guillemot F (2002) Proneural genes and the specification of neural cell types. Nat Rev Neurosci 3(7):517–530PubMedCrossRefGoogle Scholar
  5. Breslin MB, Zhu M, Lan MS (2003) NeuroD1/E47 regulates the E-box element of a novel zinc finger transcription factor, IA-1, in developing nervous system. J Biol Chem 278(40):38991–38997PubMedCentralPubMedCrossRefGoogle Scholar
  6. Bruniquel D, Schwartz RH (2003) Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nat Immunol 4(3):235–240PubMedCrossRefGoogle Scholar
  7. Brzezinski JA, Kim EJ, Johnson JE, Reh TA (2011) Ascl1 expression defines a subpopulation of lineage-restricted progenitors in the mammalian retina. Development 138(16):3519–3531PubMedCentralPubMedCrossRefGoogle Scholar
  8. Castro DS, Skowronska-Krawczyk D, Armant O, Donaldson IJ, Parras C, Hunt C, Critchley JA, Nguyen L, Gossler A, Gottgens B et al (2006) Proneural bHLH and Brn proteins coregulate a neurogenic program through cooperative binding to a conserved DNA motif. Dev Cell 11(6):831–844PubMedCrossRefGoogle Scholar
  9. Castro DS, Martynoga B, Parras C, Ramesh V, Pacary E, Johnston C, Drechsel D, Lebel-Potter M, Garcia LG, Hunt C et al (2011) A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets. Genes Dev 25(9):930–945PubMedCentralPubMedCrossRefGoogle Scholar
  10. Cau E, Casarosa S, Guillemot F (2002) Mash1 and Ngn1 control distinct steps of determination and differentiation in the olfactory sensory neuron lineage. Development 129(8):1871–1880PubMedGoogle Scholar
  11. Chambers I, Silva J, Colby D, Nichols J, Nijmeijer B, Robertson M, Vrana J, Jones K, Grotewold L, Smith A (2007) Nanog safeguards pluripotency and mediates germline development. Nature 450(7173):1230–1234PubMedCrossRefGoogle Scholar
  12. Dalgard CL, Zhou Q, Lundell TG, Doughty ML (2011) Altered gene expression in the emerging cerebellar primordium of Neurog1−/− mice. Brain Res 1388:12–21PubMedCrossRefGoogle Scholar
  13. Dalton GD, Dewey WL (2006) Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function. Neuropeptides 40(1):23–34PubMedCrossRefGoogle Scholar
  14. Deaton AM, Bird A (2011) CpG islands and the regulation of transcription. Genes Dev 25(10):1010–1022PubMedCentralPubMedCrossRefGoogle Scholar
  15. Deb-Rinker P, Ly D, Jezierski A, Sikorska M, Walker PR (2005) Sequential DNA methylation of the Nanog and Oct-4 upstream regions in human NT2 cells during neuronal differentiation. J Biol Chem 280(8):6257–6260PubMedCrossRefGoogle Scholar
  16. Deng JT, Sutherland C, Brautigan DL, Eto M, Walsh MP (2002) Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase. Biochem J 367(Pt 2):517–524PubMedCentralPubMedCrossRefGoogle Scholar
  17. Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z (2009) GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinforma 10:48CrossRefGoogle Scholar
  18. Eiraku M, Tohgo A, Ono K, Kaneko M, Fujishima K, Hirano T, Kengaku M (2005) DNER acts as a neuron-specific Notch ligand during Bergmann glial development. Nat Neurosci 8(7):873–880PubMedCrossRefGoogle Scholar
  19. Eto M (2009) Regulation of cellular protein phosphatase-1 (PP1) by phosphorylation of the CPI-17 family, C-kinase-activated PP1 inhibitors. J Biol Chem 284(51):35273–35277PubMedCentralPubMedCrossRefGoogle Scholar
  20. Eto M, Wong L, Yazawa M, Brautigan DL (2000) Inhibition of myosin/moesin phosphatase by expression of the phosphoinhibitor protein CPI-17 alters microfilament organization and retards cell spreading. Cell Motil Cytoskeleton 46(3):222–234PubMedCrossRefGoogle Scholar
  21. Farah MH, Olson JM, Sucic HB, Hume RI, Tapscott SJ, Turner DL (2000) Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. Development 127(4):693–702PubMedGoogle Scholar
  22. Flames N, Hobert O (2009) Gene regulatory logic of dopamine neuron differentiation. Nature 458(7240):885–889PubMedCentralPubMedCrossRefGoogle Scholar
  23. Fode C, Ma Q, Casarosa S, Ang SL, Anderson DJ, Guillemot F (2000) A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons. Genes Dev 14(1):67–80PubMedCentralPubMedGoogle Scholar
  24. Francius C, Clotman F (2010) Dynamic expression of the one cut transcription factors HNF-6, OC-2 and OC-3 during spinal motor neuron development. Neuroscience 165(1):116–129PubMedCrossRefGoogle Scholar
  25. Galichet C, Guillemot F, Parras CM (2008) Neurogenin 2 has an essential role in development of the dentate gyrus. Development 135(11):2031–2041PubMedCrossRefGoogle Scholar
  26. Gidekel S, Bergman Y (2002) A unique developmental pattern of Oct-3/4 DNA methylation is controlled by a cis-demodification element. J Biol Chem 277(37):34521–34530PubMedCrossRefGoogle Scholar
  27. Gohlke JM, Armant O, Parham FM, Smith MV, Zimmer C, Castro DS, Nguyen L, Parker JS, Gradwohl G, Portier CJ et al (2008) Characterization of the proneural gene regulatory network during mouse telencephalon development. BMC Biol 6:15PubMedCentralPubMedCrossRefGoogle Scholar
  28. Gu P, Xu X, Le Menuet D, Chung AC, Cooney AJ (2011) Differential recruitment of methyl CpG-binding domain factors and DNA methyltransferases by the orphan receptor germ cell nuclear factor initiates the repression and silencing of Oct4. Stem Cells 29(7):1041–1051PubMedCentralPubMedCrossRefGoogle Scholar
  29. Guillemot F (2007) Spatial and temporal specification of neural fates by transcription factor codes. Development 134(21):3771–3780PubMedCrossRefGoogle Scholar
  30. Guillemot F, Lo LC, Johnson JE, Auerbach A, Anderson DJ, Joyner AL (1993) Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell 75(3):463–476PubMedCrossRefGoogle Scholar
  31. Han DW, Do JT, Arauzo-Bravo MJ, Lee SH, Meissner A, Lee HT, Jaenisch R, Scholer HR (2009) Epigenetic hierarchy governing Nestin expression. Stem Cells 27(5):1088–1097PubMedCrossRefGoogle Scholar
  32. Hand R, Polleux F (2011) Neurogenin2 regulates the initial axon guidance of cortical pyramidal neurons projecting medially to the corpus callosum. Neural Dev 6:30PubMedCentralPubMedCrossRefGoogle Scholar
  33. Hand R, Bortone D, Mattar P, Nguyen L, Heng JI, Guerrier S, Boutt E, Peters E, Barnes AP, Parras C et al (2005) Phosphorylation of neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex. Neuron 48(1):45–62PubMedCrossRefGoogle Scholar
  34. Hatada I, Morita S, Kimura M, Horii T, Yamashita R, Nakai K (2008) Genome-wide demethylation during neural differentiation of P19 embryonal carcinoma cells. J Hum Genet 53(2):185–191PubMedCrossRefGoogle Scholar
  35. Henke RM, Meredith DM, Borromeo MD, Savage TK, Johnson JE (2009) Ascl1 and Neurog2 form novel complexes and regulate delta-like3 (Dll3) expression in the neural tube. Dev Biol 328(2):529–540PubMedCentralPubMedCrossRefGoogle Scholar
  36. Hirabayashi Y, Gotoh Y (2010) Epigenetic control of neural precursor cell fate during development. Nat Rev Neurosci 11(6):377–388PubMedCrossRefGoogle Scholar
  37. Huang HS, Kubish GM, Redmond TM, Turner DL, Thompson RC, Murphy GG, Uhler MD (2010) Direct transcriptional induction of Gadd45gamma by Ascl1 during neuronal differentiation. Mol Cell Neurosci 44(3):282–296PubMedCentralPubMedCrossRefGoogle Scholar
  38. Huang HS, Turner DL, Thompson RC, Uhler MD (2012) Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cyclic AMP-dependent protein kinase. J Neurochem 120(5):667–683PubMedCentralPubMedCrossRefGoogle Scholar
  39. Jang SK, Pestova TV, Hellen CU, Witherell GW, Wimmer E (1990) Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site. Enzyme 44(1–4):292–309PubMedGoogle Scholar
  40. Kageyama R, Ohtsuka T, Hatakeyama J, Ohsawa R (2005) Roles of bHLH genes in neural stem cell differentiation. Exp Cell Res 306(2):343–348PubMedCrossRefGoogle Scholar
  41. Kaufmann LT, Niehrs C (2011) Gadd45a and Gadd45g regulate neural development and exit from pluripotency in Xenopus. Mech Dev 128(7–10):401–411PubMedCrossRefGoogle Scholar
  42. Kaufmann LT, Gierl MS, Niehrs C (2011) Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development. Gene Expr Patterns 11(8):465–470PubMedCrossRefGoogle Scholar
  43. Kearsey JM, Coates PJ, Prescott AR, Warbrick E, Hall PA (1995) Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1. Oncogene 11(9):1675–1683PubMedGoogle Scholar
  44. Kellner S, Kikyo N (2010) Transcriptional regulation of the Oct4 gene, a master gene for pluripotency. Histol Histopathol 25(3):405–412PubMedCentralPubMedGoogle Scholar
  45. Koyama M, Ito M, Feng J, Seko T, Shiraki K, Takase K, Hartshorne DJ, Nakano T (2000) Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase. FEBS Lett 475(3):197–200PubMedCrossRefGoogle Scholar
  46. Lee S, Lee B, Joshi K, Pfaff SL, Lee JW, Lee SK (2008) A regulatory network to segregate the identity of neuronal subtypes. Dev Cell 14(6):877–889PubMedCentralPubMedCrossRefGoogle Scholar
  47. Lee MY, Lee SH, Park JH, Han HJ (2009) Interaction of galectin-1 with caveolae induces mouse embryonic stem cell proliferation through the Src, ERas, Akt and mTOR signaling pathways. Cell Mol Life Sci 66(8):1467–1478PubMedCrossRefGoogle Scholar
  48. Li JY, Pu MT, Hirasawa R, Li BZ, Huang YN, Zeng R, Jing NH, Chen T, Li E, Sasaki H et al (2007) Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog. Mol Cell Biol 27(24):8748–8759PubMedCentralPubMedCrossRefGoogle Scholar
  49. Liang X, Song MR, Xu Z, Lanuza GM, Liu Y, Zhuang T, Chen Y, Pfaff SL, Evans SM, Sun Y (2011) Isl1 is required for multiple aspects of motor neuron development. Mol Cell Neurosci 47(3):215–222PubMedCentralPubMedCrossRefGoogle Scholar
  50. Liu ML, Zang T, Zou Y, Chang JC, Gibson JR, Huber KM, Zhang CL (2013) Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. Nat Commun 4:2183PubMedGoogle Scholar
  51. Lyden D, Young AZ, Zagzag D, Yan W, Gerald W, O’Reilly R, Bader BL, Hynes RO, Zhuang Y, Manova K et al (1999) Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature 401(6754):670–677PubMedCrossRefGoogle Scholar
  52. Ma Q, Fode C, Guillemot F, Anderson DJ (1999) Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev 13(13):1717–1728PubMedCentralPubMedCrossRefGoogle Scholar
  53. Ma YC, Song MR, Park JP, Henry Ho HY, Hu L, Kurtev MV, Zieg J, Ma Q, Pfaff SL, Greenberg ME (2008) Regulation of motor neuron specification by phosphorylation of neurogenin 2. Neuron 58(1):65–77PubMedCentralPubMedCrossRefGoogle Scholar
  54. Ma DK, Guo JU, Ming GL, Song H (2009) DNA excision repair proteins and Gadd45 as molecular players for active DNA demethylation. Cell Cycle 8(10):1526–1531PubMedCentralPubMedCrossRefGoogle Scholar
  55. McBurney MW (1993) P19 embryonal carcinoma cells. Int J Dev Biol 37(1):135–140PubMedGoogle Scholar
  56. McBurney MW, Rogers BJ (1982) Isolation of male embryonal carcinoma cells and their chromosome replication patterns. Dev Biol 89(2):503–508PubMedCrossRefGoogle Scholar
  57. Mellor J, Dudek P, Clynes D (2008) A glimpse into the epigenetic landscape of gene regulation. Curr Opin Genet Dev 18(2):116–122PubMedCrossRefGoogle Scholar
  58. Messmer K, Shen WB, Remington M, Fishman PS (2012) Induction of neural differentiation by the transcription factor NeuroD2. Int J Dev Neurosci 30(2):105–112PubMedCrossRefGoogle Scholar
  59. Nam HS, Benezra R (2009) High levels of Id1 expression define B1 type adult neural stem cells. Cell Stem Cell 5(5):515–526PubMedCentralPubMedCrossRefGoogle Scholar
  60. Narumi O, Mori S, Boku S, Tsuji Y, Hashimoto N, Nishikawa S, Yokota Y (2000) OUT, a novel basic helix-loop-helix transcription factor with an Id-like inhibitory activity. J Biol Chem 275(5):3510–3521PubMedCrossRefGoogle Scholar
  61. Nefzger CM, Haynes JM, Pouton CW (2011) Directed expression of Gata2, Mash1, and Foxa2 synergize to induce the serotonergic neuron phenotype during in vitro differentiation of embryonic stem cells. Stem Cells 29(6):928–939PubMedCrossRefGoogle Scholar
  62. Neilson KM, Klein SL, Mhaske P, Mood K, Daar IO, Moody SA (2012) Specific domains of FoxD4/5 activate and repress neural transcription factor genes to control the progression of immature neural ectoderm to differentiating neural plate. Dev Biol 365(2):363–375PubMedCentralPubMedCrossRefGoogle Scholar
  63. Niehrs C, Schafer A (2012) Active DNA demethylation by Gadd45 and DNA repair. Trends Cell Biol 22(4):220–227PubMedCrossRefGoogle Scholar
  64. Nieto M, Schuurmans C, Britz O, Guillemot F (2001) Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors. Neuron 29(2):401–413PubMedCrossRefGoogle Scholar
  65. Oda H, Fushimi F, Kato M, Kitagawa M, Araki K, Seki N, Ohkubo H (2005) Microarray analysis of the genes induced by tetracycline-regulated expression of NDRF/NeuroD2 in P19 cells. Biochem Biophys Res Commun 335(2):458–468PubMedCrossRefGoogle Scholar
  66. Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99(3):247–257PubMedCrossRefGoogle Scholar
  67. Oropez D, Horb M (2012) Transient expression of Ngn3 in Xenopus endoderm promotes early and ectopic development of pancreatic beta and delta cells. Genesis 50(3):271–285PubMedCrossRefGoogle Scholar
  68. Osorio J, Mueller T, Retaux S, Vernier P, Wullimann MF (2010) Phylotypic expression of the bHLH genes Neurogenin2, Neurod, and Mash1 in the mouse embryonic forebrain. J Comp Neurol 518(6):851–871PubMedCrossRefGoogle Scholar
  69. Oswald J, Engemann S, Lane N, Mayer W, Olek A, Fundele R, Dean W, Reik W, Walter J (2000) Active demethylation of the paternal genome in the mouse zygote. Curr Biol 10(8):475–478PubMedCrossRefGoogle Scholar
  70. Paling NR, Wheadon H, Bone HK, Welham MJ (2004) Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J Biol Chem 279(46):48063–48070PubMedCrossRefGoogle Scholar
  71. Parras CM, Schuurmans C, Scardigli R, Kim J, Anderson DJ, Guillemot F (2002) Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity. Genes Dev 16(3):324–338PubMedCentralPubMedCrossRefGoogle Scholar
  72. Peltopuro P, Kala K, Partanen J (2010) Distinct requirements for Ascl1 in subpopulations of midbrain GABAergic neurons. Dev Biol 343(1–2):63–70PubMedCrossRefGoogle Scholar
  73. Pesce M, Scholer HR (2001) Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19(4):271–278PubMedCrossRefGoogle Scholar
  74. Powell LM, Zur Lage PI, Prentice DR, Senthinathan B, Jarman AP (2004) The proneural proteins Atonal and Scute regulate neural target genes through different E-box binding sites. Mol Cell Biol 24(21):9517–9526PubMedCentralPubMedCrossRefGoogle Scholar
  75. Razin A, Webb C, Szyf M, Yisraeli J, Rosenthal A, Naveh-Many T, Sciaky-Gallili N, Cedar H (1984) Variations in DNA methylation during mouse cell differentiation in vivo and in vitro. Proc Natl Acad Sci U S A 81(8):2275–2279PubMedCentralPubMedCrossRefGoogle Scholar
  76. Rigbolt KT, Prokhorova TA, Akimov V, Henningsen J, Johansen PT, Kratchmarova I, Kassem M, Mann M, Olsen JV, Blagoev B (2011) System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation. Sci Signal 4(164):rs3PubMedCrossRefGoogle Scholar
  77. Ross SE, Greenberg ME, Stiles CD (2003) Basic helix-loop-helix factors in cortical development. Neuron 39(1):13–25PubMedCrossRefGoogle Scholar
  78. Roybon L, Hjalt T, Stott S, Guillemot F, Li JY, Brundin P (2009) Neurogenin2 directs granule neuroblast production and amplification while NeuroD1 specifies neuronal fate during hippocampal neurogenesis. PLoS One 4(3):e4779PubMedCentralPubMedCrossRefGoogle Scholar
  79. Ruzinova MB, Benezra R (2003) Id proteins in development, cell cycle and cancer. Trends Cell Biol 13(8):410–418PubMedCrossRefGoogle Scholar
  80. Schuurmans C, Guillemot F (2002) Molecular mechanisms underlying cell fate specification in the developing telencephalon. Curr Opin Neurobiol 12(1):26–34PubMedCrossRefGoogle Scholar
  81. Seo S, Lim JW, Yellajoshyula D, Chang LW, Kroll KL (2007) Neurogenin and NeuroD direct transcriptional targets and their regulatory enhancers. EMBO J 26(24):5093–5108PubMedCentralPubMedCrossRefGoogle Scholar
  82. Sheng N, Xie Z, Wang C, Bai G, Zhang K, Zhu Q, Song J, Guillemot F, Chen YG, Lin A et al (2010) Retinoic acid regulates bone morphogenic protein signal duration by promoting the degradation of phosphorylated Smad1. Proc Natl Acad Sci U S A 107(44):18886–18891PubMedCentralPubMedCrossRefGoogle Scholar
  83. Shimozaki K, Nakashima K, Niwa H, Taga T (2003) Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures. Development 130(11):2505–2512PubMedCrossRefGoogle Scholar
  84. Spada F, Haemmer A, Kuch D, Rothbauer U, Schermelleh L, Kremmer E, Carell T, Langst G, Leonhardt H (2007) DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells. J Cell Biol 176(5):565–571PubMedCentralPubMedCrossRefGoogle Scholar
  85. Sukhanova MJ, Deb DK, Gordon GM, Matakatsu MT, Du W (2007) Proneural basic helix-loop-helix proteins and epidermal growth factor receptor signaling coordinately regulate cell type specification and cdk inhibitor expression during development. Mol Cell Biol 27(8):2987–2996PubMedCentralPubMedCrossRefGoogle Scholar
  86. Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9(6):465–476PubMedCrossRefGoogle Scholar
  87. Szyf M, Theberge J, Bozovic V (1995) Ras induces a general DNA demethylation activity in mouse embryonal P19 cells. J Biol Chem 270(21):12690–12696PubMedCrossRefGoogle Scholar
  88. Takahashi K, Mitsui K, Yamanaka S (2003) Role of ERas in promoting tumour-like properties in mouse embryonic stem cells. Nature 423(6939):541–545PubMedCrossRefGoogle Scholar
  89. Takizawa N, Koga Y, Ikebe M (2002) Phosphorylation of CPI17 and myosin binding subunit of type 1 protein phosphatase by p21-activated kinase. Biochem Biophys Res Commun 297(4):773–778PubMedCrossRefGoogle Scholar
  90. Thoma EC, Wischmeyer E, Offen N, Maurus K, Siren AL, Schartl M, Wagner TU (2012) Ectopic expression of neurogenin 2 alone is sufficient to induce differentiation of embryonic stem cells into mature neurons. PLoS One 7(6):e38651PubMedCentralPubMedCrossRefGoogle Scholar
  91. Uemura O, Okada Y, Ando H, Guedj M, Higashijima S, Shimazaki T, Chino N, Okano H, Okamoto H (2005) Comparative functional genomics revealed conservation and diversification of three enhancers of the isl1 gene for motor and sensory neuron-specific expression. Dev Biol 278(2):587–606PubMedCrossRefGoogle Scholar
  92. Vairapandi M, Balliet AG, Hoffman B, Liebermann DA (2002) GADD45b and GADD45g are cdc2/cyclinB1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints induced by genotoxic stress. J Cell Physiol 192(3):327–338PubMedCrossRefGoogle Scholar
  93. Yan B, Neilson KM, Moody SA (2010) Microarray identification of novel downstream targets of FoxD4L1/D5, a critical component of the neural ectodermal transcriptional network. Dev Dyn 239(12):3467–3480PubMedCentralPubMedCrossRefGoogle Scholar
  94. Yang Z, Song L, Huang C (2009) Gadd45 proteins as critical signal transducers linking NF-kappaB to MAPK cascades. Curr Cancer Drug Targets 9(8):915–930PubMedCentralPubMedCrossRefGoogle Scholar
  95. Ying J, Srivastava G, Hsieh WS, Gao Z, Murray P, Liao SK, Ambinder R, Tao Q (2005) The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors. Clin Cancer Res 11(18):6442–6449PubMedCrossRefGoogle Scholar
  96. Zemlickova E, Johannes FJ, Aitken A, Dubois T (2004) Association of CPI-17 with protein kinase C and casein kinase I. Biochem Biophys Res Commun 316(1):39–47PubMedCrossRefGoogle Scholar
  97. Zhang J, Piontek J, Wolburg H, Piehl C, Liss M, Otten C, Christ A, Willnow TE, Blasig IE, Abdelilah-Seyfried S (2010) Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion. Proc Natl Acad Sci U S A 107(4):1425–1430PubMedCentralPubMedCrossRefGoogle Scholar
  98. Zhang H, Deo M, Thompson RC, Uhler MD, Turner DL (2012) Negative regulation of Yap during neuronal differentiation. Dev Biol 361(1):103–115PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Holly S. Huang
    • 1
  • Tanya M. Redmond
    • 1
    • 2
  • Ginger M. Kubish
    • 3
  • Shweta Gupta
    • 1
  • Robert C. Thompson
    • 1
    • 4
  • David L. Turner
    • 1
    • 2
  • Michael D. Uhler
    • 1
    • 2
  1. 1.Molecular and Behavioral Neuroscience InstituteUniversity of MichiganAnn ArborUSA
  2. 2.Department of Biological ChemistryUniversity of MichiganAnn ArborUSA
  3. 3.Life Sciences InstituteUniversity of MichiganAnn ArborUSA
  4. 4.Department of PsychiatryUniversity of MichiganAnn ArborUSA

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