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The Cerebellum

, Volume 14, Issue 6, pp 688–698 | Cite as

MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2

  • Lena Constantin
  • Brandon J. Wainwright
Original Paper

Abstract

MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87 % in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

Keywords

Cerebellum Dicer1 protein Mouse Gli2 protein Mirn106 microRNA Mouse Growth and development 

Notes

Acknowledgments

The authors would like to thank Professor Witold Filipowicz for the kind gift of the Dicer 349 antibody, Professor Michael Waters for critical reading of the manuscript, and Mr. Hou Jiapeng for optimizing the Dicer immunofluorescence protocol. All imaging was performed in the Australian Cancer Research Foundation’s Imaging Facility at the Institute for Molecular Bioscience.

Funding

This research was financially supported by the National Health and Medical Research Council of Australia and The John Trivett Foundation. Lena Constantin is an Australian Postgraduate Award Scholar.

Conflict of Interest

The authors declare no conflict of interest.

References

  1. 1.
    Pascual-Castroviejo I, Gutierrez M, Morales C, Gonzalez-Mediero I, Martinez-Bermejo A, Pascual-Pascual SI. Primary degeneration of the granular layer of the cerebellum. A study of 14 patients and review of the literature. Neuropediatrics. 1994;25(4):183–90.CrossRefPubMedGoogle Scholar
  2. 2.
    Yang ZJ, Ellis T, Markant SL, Read TA, Kessler JD, Bourboulas M, et al. Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells. Cancer Cell. 2008;14(2):135–45.PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, et al. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007;114(2):97–109.PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Xenaki D, Martin IB, Yoshida L, Ohyama K, Gennarini G, Grumet M, et al. F3/contactin and TAG1 play antagonistic roles in the regulation of sonic hedgehog-induced cerebellar granule neuron progenitor proliferation. Development. 2011;138(3):519–29.PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Bai CB, Auerbach W, Lee JS, Stephen D, Joyner AL. Gli2, but not Gli1, is required for initial Shh signaling and ectopic activation of the Shh pathway. Development. 2002;129(20):4753–61.PubMedGoogle Scholar
  6. 6.
    Park HL, Bai C, Platt KA, Matise MP, Beeghly A, Hui CC, et al. Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation. Development. 2000;127(8):1593–605.PubMedGoogle Scholar
  7. 7.
    Bai CB, Stephen D, Joyner AL. All mouse ventral spinal cord patterning by hedgehog is Gli dependent and involves an activator function of Gli3. Dev Cell. 2004;6(1):103–15.CrossRefPubMedGoogle Scholar
  8. 8.
    Corrales JD, Rocco GL, Blaess S, Guo Q, Joyner AL. Spatial pattern of sonic hedgehog signaling through Gli genes during cerebellum development. Development. 2004;131(22):5581–90.CrossRefPubMedGoogle Scholar
  9. 9.
    Ozol K, Hayden JM, Oberdick J, Hawkes R. Transverse zones in the vermis of the mouse cerebellum. J Comp Neurol. 1999;412(1):95–111.CrossRefPubMedGoogle Scholar
  10. 10.
    Herrup K, Kuemerle B. The compartmentalization of the cerebellum. Annu Rev Neurosci. 1997;20:61–90.CrossRefPubMedGoogle Scholar
  11. 11.
    Millen KJ, Hui CC, Joyner AL. A role for En-2 and other murine homologues of Drosophila segment polarity genes in regulating positional information in the developing cerebellum. Development. 1995;121(12):3935–45.PubMedGoogle Scholar
  12. 12.
    Hollander WF, Waggie KS. Meander tail: a recessive mutant located in chromosome 4 of the mouse. J Hered. 1977;68(6):403–6.PubMedGoogle Scholar
  13. 13.
    Hawkes R, Beierbach E, Tan SS. Granule cell dispersion is restricted across transverse boundaries in mouse chimeras. Eur J Neurosci. 1999;11(11):3800–8.CrossRefPubMedGoogle Scholar
  14. 14.
    Goldowitz D, Hamre KM, Przyborski SA, Ackerman SL. Granule cells and cerebellar boundaries: analysis of Unc5h3 mutant chimeras. J Neurosci: Off J Soc Neurosci. 2000;20(11):4129–37.Google Scholar
  15. 15.
    Lorenz A, Deutschmann M, Ahlfeld J, Prix C, Koch A, Smits R, et al. Severe alterations of cerebellar cortical development after constitutive activation of Wnt signaling in granule neuron precursors. Mol Cell Biol. 2011;31(16):3326–38.PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Pan N, Jahan I, Lee JE, Fritzsch B. Defects in the cerebella of conditional Neurod1 null mice correlate with effective Tg(Atoh1-cre) recombination and granule cell requirements for Neurod1 for differentiation. Cell Tissue Res. 2009;337(3):407–28.PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Machold R, Fishell G. Math1 is expressed in temporally discrete pools of cerebellar rhombic-lip neural progenitors. Neuron. 2005;48(1):17–24.CrossRefPubMedGoogle Scholar
  18. 18.
    Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215–33.PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet. 2008;9(2):102–14.CrossRefPubMedGoogle Scholar
  20. 20.
    Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ. Processing of primary microRNAs by the Microprocessor complex. Nature. 2004;432(7014):231–5.CrossRefPubMedGoogle Scholar
  21. 21.
    Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003;425(6956):415–9.CrossRefPubMedGoogle Scholar
  22. 22.
    Hutvagner G, McLachlan J, Pasquinelli AE, Balint E, Tuschl T, Zamore PD. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science. 2001;293(5531):834–8.CrossRefPubMedGoogle Scholar
  23. 23.
    Schaefer A, O’Carroll D, Tan CL, Hillman D, Sugimori M, Llinas R, et al. Cerebellar neurodegeneration in the absence of microRNAs. J Exp Med. 2007;204(7):1553–8.PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Lee Y, Samaco RC, Gatchel JR, Thaller C, Orr HT, Zoghbi HY. miR-19, miR-101 and miR-130 co-regulate ATXN1 levels to potentially modulate SCA1 pathogenesis. Nat Neurosci. 2008;11(10):1137–9.PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Ferretti E, De Smaele E, Miele E, Laneve P, Po A, Pelloni M, et al. Concerted microRNA control of Hedgehog signalling in cerebellar neuronal progenitor and tumour cells. EMBO J. 2008;27(19):2616–27.PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Zindy F, Kawauchi D, Lee Y, Ayrault O, Ben Merzoug L, McKinnon PJ, et al. Role of the miR-17 approximately 92 cluster family in cerebellar and medulloblastoma development. Biol Open. 2014;3(7):597–605.PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Ferretti E, De Smaele E, Po A, Di Marcotullio L, Tosi E, Espinola MS, et al. MicroRNA profiling in human medulloblastoma. Int J Cancer. 2009;124(3):568–77.CrossRefPubMedGoogle Scholar
  28. 28.
    Northcott PA, Fernandez LA, Hagan JP, Ellison DW, Grajkowska W, Gillespie Y, et al. The miR-17/92 polycistron is up-regulated in sonic hedgehog-driven medulloblastomas and induced by N-myc in sonic hedgehog-treated cerebellar neural precursors. Cancer Res. 2009;69(8):3249–55.PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Kawauchi D, Saito T. Transcriptional cascade from Math1 to Mbh1 and Mbh2 is required for cerebellar granule cell differentiation. Dev Biol. 2008;322(2):345–54.CrossRefPubMedGoogle Scholar
  30. 30.
    Englund C, Kowalczyk T, Daza RA, Dagan A, Lau C, Rose MF, et al. Unipolar brush cells of the cerebellum are produced in the rhombic lip and migrate through developing white matter. J Neurosci : Off J Soc Neurosci. 2006;26(36):9184–95.CrossRefGoogle Scholar
  31. 31.
    Harfe BD, McManus MT, Mansfield JH, Hornstein E, Tabin CJ. The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb. Proc Natl Acad Sci U S A. 2005;102(31):10898–903.PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Schuller U, Zhao Q, Godinho SA, Heine VM, Medema RH, Pellman D, et al. Forkhead transcription factor FoxM1 regulates mitotic entry and prevents spindle defects in cerebellar granule neuron precursors. Mol Cell Biol. 2007;27(23):8259–70.PubMedCentralCrossRefPubMedGoogle Scholar
  33. 33.
    Luna LG, Armed Forces Institute of Pathology (U.S.), Armed Forces Institute of Pathology (U.S.). Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed. New York: Blakiston Division; 1968. xii, 258 p. p.Google Scholar
  34. 34.
    Adolphe C, Narang M, Ellis T, Wicking C, Kaur P, Wainwright B. An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis. Development. 2004;131(20):5009–19.CrossRefPubMedGoogle Scholar
  35. 35.
    Hui CC, Slusarski D, Platt KA, Holmgren R, Joyner AL. Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during postimplantation development. Dev Biol. 1994;162(2):402–13.CrossRefPubMedGoogle Scholar
  36. 36.
    Bremer J, O’Connor T, Tiberi C, Rehrauer H, Weis J, Aguzzi A. Ablation of Dicer from murine Schwann cells increases their proliferation while blocking myelination. PLoS One. 2010;5(8), e12450.PubMedCentralCrossRefPubMedGoogle Scholar
  37. 37.
    Mandelbaum AD, Melkman-Zehavi T, Oren R, Kredo-Russo S, Nir T, Dor Y, et al. Dysregulation of Dicer1 in beta cells impairs islet architecture and glucose metabolism. Exp Diabetes Res. 2012;2012:470302.PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Doughty ML, Lohof A, Campana A, Delhaye-Bouchaud N, Mariani J. Neurotrophin-3 promotes cerebellar granule cell exit from the EGL. Eur J Neurosci. 1998;10(9):3007–11.CrossRefPubMedGoogle Scholar
  39. 39.
    Flora A, Klisch TJ, Schuster G, Zoghbi HY. Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma. Science. 2009;326(5958):1424–7.PubMedCentralCrossRefPubMedGoogle Scholar
  40. 40.
    Corrales JD, Blaess S, Mahoney EM, Joyner AL. The level of sonic hedgehog signaling regulates the complexity of cerebellar foliation. Development. 2006;133(9):1811–21.CrossRefPubMedGoogle Scholar
  41. 41.
    Wechsler-Reya RJ, Scott MP. Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. Neuron. 1999;22(1):103–14.CrossRefPubMedGoogle Scholar
  42. 42.
    Dahmane N, Ruiz i Altaba A. Sonic hedgehog regulates the growth and patterning of the cerebellum. Development. 1999;126(14):3089–100.PubMedGoogle Scholar
  43. 43.
    Lewis PM, Gritli-Linde A, Smeyne R, Kottmann A, McMahon AP. Sonic hedgehog signaling is required for expansion of granule neuron precursors and patterning of the mouse cerebellum. Dev Biol. 2004;270(2):393–410.CrossRefPubMedGoogle Scholar
  44. 44.
    Zhang J, Zhang J, Zhou Y, Wu YJ, Ma L, Wang RJ, et al. Novel cerebellum-enriched miR-592 may play a role in neural progenitor cell differentiation and neuronal maturation through regulating Lrrc4c and Nfasc in rat. Curr Mol Med. 2013;13(9):1432–45.CrossRefPubMedGoogle Scholar
  45. 45.
    Berenguer J, Herrera A, Vuolo L, Torroba B, Llorens F, Sumoy L, et al. MicroRNA 22 regulates cell cycle length in cerebellar granular neuron precursors. Mol Cell Biol. 2013;33(14):2706–17.PubMedCentralCrossRefPubMedGoogle Scholar
  46. 46.
    Kuang Y, Liu Q, Shu X, Zhang C, Huang N, Li J, et al. Dicer1 and MiR-9 are required for proper Notch1 signaling and the Bergmann glial phenotype in the developing mouse cerebellum. Glia. 2012;60(11):1734–46.CrossRefPubMedGoogle Scholar
  47. 47.
    Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 2009;19(1):92–105.PubMedCentralCrossRefPubMedGoogle Scholar
  48. 48.
    Gibson P, Tong Y, Robinson G, Thompson MC, Currle DS, Eden C, et al. Subtypes of medulloblastoma have distinct developmental origins. Nature. 2010;468(7327):1095–9.PubMedCentralCrossRefPubMedGoogle Scholar
  49. 49.
    Murphy BL, Obad S, Bihannic L, Ayrault O, Zindy F, Kauppinen S, et al. Silencing of the miR-17~92 cluster family inhibits medulloblastoma progression. Cancer Res. 2013;73(23):7068–78.CrossRefPubMedGoogle Scholar
  50. 50.
    Kool M, Korshunov A, Remke M, Jones DT, Schlanstein M, Northcott PA, et al. Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas. Acta Neuropathol. 2012;123(4):473–84.PubMedCentralCrossRefPubMedGoogle Scholar
  51. 51.
    Northcott PA, Korshunov A, Witt H, Hielscher T, Eberhart CG, Mack S, et al. Medulloblastoma comprises four distinct molecular variants. J Clin Oncol : Off J Am Soc Clin Oncol. 2011;29(11):1408–14.CrossRefGoogle Scholar
  52. 52.
    Shih DJ, Northcott PA, Remke M, Korshunov A, Ramaswamy V, Kool M, et al. Cytogenetic prognostication within medulloblastoma subgroups. J Clin Oncol : Off J Am Soc Clin Oncol. 2014;32(9):886–96.CrossRefGoogle Scholar
  53. 53.
    Buczkowicz P, Ma J, Hawkins C. GLI2 is a potential therapeutic target in pediatric medulloblastoma. J Neuropathol Exp Neurol. 2011;70(6):430–7.CrossRefPubMedGoogle Scholar
  54. 54.
    Kool M, Jones DT, Jager N, Northcott PA, Pugh TJ, Hovestadt V, et al. Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition. Cancer Cell. 2014;25(3):393–405.PubMedCentralCrossRefPubMedGoogle Scholar
  55. 55.
    Low JA, de Sauvage FJ. Clinical experience with Hedgehog pathway inhibitors. J Clin Oncol : Off J Am Soc Clin Oncol. 2010;28(36):5321–6.CrossRefGoogle Scholar
  56. 56.
    Ng JM, Curran T. The Hedgehog’s tale: developing strategies for targeting cancer. Nat Rev Cancer. 2011;11(7):493–501.PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Institute for Molecular BioscienceThe University of QueenslandSt LuciaAustralia

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