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The Cerebellum

, Volume 18, Issue 1, pp 56–66 | Cite as

Zebrin II Is Ectopically Expressed in Microglia in the Cerebellum of Neurogenin 2 Null Mice

  • Maryam Rahimi-Balaei
  • Xiaodan Jiao
  • Shahin Shabanipour
  • Rajiv Dixit
  • Carol Schuurmans
  • Hassan MarzbanEmail author
Original Paper
  • 138 Downloads

Abstract

Zebrin II/aldolase C expression in the normal cerebellum is restricted to a Purkinje cell subset and is the canonical marker for stripes and zones. This spatial restriction has been confirmed in over 30 species of mammals, birds, fish, etc. In a transgenic mouse model in which the Neurogenin 2 gene has been disrupted (Neurog2−/−), the cerebellum is smaller than normal and Purkinje cell dendrites are disordered, but the basic zone and stripe architecture is preserved. Here, we show that in the Neurog2−/− mouse, in addition to the normal Purkinje cell expression, zebrin II is also expressed in a population of cells with a morphology characteristic of microglia. This identity was confirmed by double immunohistochemistry for zebrin II and the microglial marker, Iba1. The expression of zebrin II in cerebellar microglia is not restricted by zone or stripe or lamina. A second zone and stripe marker, PLCβ4, does not show the same ectopic expression. When microglia are compared in control vs. Neurog2−/− mice, no difference is seen in apparent number or distribution, suggesting that the ectopic zebrin II immunoreactivity in Neurog2−/− cerebellum reflects an ectopic expression rather than the invasion of a new population of microglia from the periphery. This ectopic expression of zebrin II in microglia is unique as it is not seen in numerous other models of cerebellar disruption, such as in Acp2−/− mice and in human pontocerebellar hypoplasia. The upregulation of zebrin II in microglia is thus specific to the disruption of Neurog2 downstream pathways, rather than a generic response to a cerebellar disruption.

Keywords

Cerebellum Microglia Neurogenin 2 mutant Zebrin II Acp2 mutant 

Notes

Acknowledgments

These studies were supported by grants from the Children Hospital Research Institute of Manitoba (HM). We are grateful to Carol Schuurmans for providing ngn2 knockout mice, and Marc Del Bigio for providing slides of pontocerebellar hypoplasia and normal control section samples.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12311_2018_944_Fig7_ESM.png (1 mb)
Fig. Suppl. 1

. A) Acp2−/− and wt sibling cerebellum at P5 and P7 (P5; n = 1 and P7; n = 2) were isolated and extracted total RNA was shipped to the McGill University and Genome Quebec Innovation Centre (MUGQIC). The raw RNA-sequencing data were trimmed and clipped with STAR (Spliced Transcripts Alignment to a Reference) program. RNA-sequencing data analysis indicates that the Neurog2 expression upregulated in Acp2−/− cerebella. B) In order to quantify microglia in cerebellum of the Neurog2−/− and wt, we counted microglia immunostained with Iba1 in six sections through the vermis of P15 animals, capturing 5 areas in each zone (each area = 0.6 mm*0.64 mm). We calculated microglia number to be: 59.69 ± 1.36 per 1mm2 in wild-type cerebella and 55.94 ± 1.28/1mm2 in Neurog2−/− cerebella (mean ± SE). There were no significant differences between wild-type and Neurog2−/− cerebella. (PNG 1.02 mb)

12311_2018_944_MOESM1_ESM.tif (11.5 mb)
High resolution image (TIF 11797 kb)

References

  1. 1.
    Brochu G, Maler L, Zebrin HR II. A polypeptide antigen expressed selectively by Purkinje cells reveals compartments in rat and fish cerebellum. J Comp Neurol. 1990;291(4):538–52.PubMedGoogle Scholar
  2. 2.
    Eisenman LM, Hawkes R. Antigenic compartmentation in the mouse cerebellar cortex: zebrin and HNK-1 reveal a complex, overlapping molecular topography. J Comp Neurol. 1993;335(4):586–605.PubMedGoogle Scholar
  3. 3.
    Sillitoe RV, Marzban H, Larouche M, Zahedi S, Affanni J, Hawkes R. Conservation of the architecture of the anterior lobe vermis of the cerebellum across mammalian species. Prog Brain Res. 2005;148:283–97.PubMedGoogle Scholar
  4. 4.
    Pakan JM, Iwaniuk AN, Wylie DR, Hawkes R, Marzban H. Purkinje cell compartmentation as revealed by zebrin II expression in the cerebellar cortex of pigeons (Columba livia). J Comp Neurol. 2007;501(4):619–30.PubMedGoogle Scholar
  5. 5.
    Marzban H, Hawkes R. On the architecture of the posterior zone of the cerebellum. Cerebellum. 2011;10(3):422–34.PubMedGoogle Scholar
  6. 6.
    Marzban H, Zahedi S, Sanchez M, Hawkes R. Antigenic compartmentation of the cerebellar cortex in the Syrian hamster Mesocricetus auratus. Brain Res. 2003;974(1):176–83.PubMedGoogle Scholar
  7. 7.
    Kim JY, Marzban H, Chung SH, Watanabe M, Eisenman LM, Hawkes R. Purkinje cell compartmentation of the cerebellum of microchiropteran bats. J Comp Neurol. 2009;517(2):193–209.PubMedGoogle Scholar
  8. 8.
    Sillitoe RV, Malz CR, Rockland K, Hawkes R. Antigenic compartmentation of the primate and tree shrew cerebellum: a common topography of zebrin II in Macaca mulatta and Tupaia belangeri. J Anat. 2004;204(4):257–69.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Staugaitis SM, Zerlin M, Hawkes R, Levine JM, Goldman JE. Aldolase C/zebrin II expression in the neonatal rat forebrain reveals cellular heterogeneity within the subventricular zone and early astrocyte differentiation. J Neurosci Off J Soc Neurosci. 2001;21(16):6195–205.Google Scholar
  10. 10.
    Caffe AR, Von Schantz M, Szel A, Voogd J, Van Veen T. Distribution of Purkinje cell-specific Zebrin-II/aldolase C immunoreactivity in the mouse, rat, rabbit, and human retina. J Comp Neurol. 1994;348(2):291–7.PubMedGoogle Scholar
  11. 11.
    Popovici T, Berwald-Netter Y, Vibert M, Kahn A, Skala H. Localization of aldolase C mRNA in brain cells. FEBS Lett. 1990;268(1):189–93.PubMedGoogle Scholar
  12. 12.
    Fode C, Ma Q, Casarosa S, Ang SL, Anderson DJ, Guillemot F. A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons. Genes Dev. 2000;14(1):67–80.PubMedPubMedCentralGoogle Scholar
  13. 13.
    Parras CM, Schuurmans C, Scardigli R, Kim J, Anderson DJ, Guillemot F. Divergent functions of the proneural genes Mash1 and Ngn2 in the specification of neuronal subtype identity. Genes Dev. 2002;16(3):324–38.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Schuurmans C, Armant O, Nieto M, Stenman JM, Britz O, Klenin N, et al. Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways. EMBO J. 2004;23(14):2892–902.PubMedPubMedCentralGoogle Scholar
  15. 15.
    Salsano E, Croci L, Maderna E, Lupo L, Pollo B, Giordana MT, et al. Expression of the neurogenic basic helix-loop-helix transcription factor NEUROG1 identifies a subgroup of medulloblastomas not expressing ATOH1. Neuro-oncology. 2007;9(3):298–307.PubMedPubMedCentralGoogle Scholar
  16. 16.
    Zordan P, Croci L, Hawkes R, Consalez GG. Comparative analysis of proneural gene expression in the embryonic cerebellum. Dev Dyn Off Publ Am Assoc Anatomists. 2008;237(6):1726–35.Google Scholar
  17. 17.
    Henke RM, Savage TK, Meredith DM, Glasgow SM, Hori K, Dumas J, et al. Neurog2 is a direct downstream target of the Ptf1a-Rbpj transcription complex in dorsal spinal cord. Development (Cambridge, England). 2009;136(17):2945–54.Google Scholar
  18. 18.
    Florio M, Leto K, Muzio L, Tinterri A, Badaloni A, Croci L, et al. Neurogenin 2 regulates progenitor cell-cycle progression and Purkinje cell dendritogenesis in cerebellar development. Development (Cambridge, England). 2012;139(13):2308–20.Google Scholar
  19. 19.
    Figueiredo C, Pais TF, Gomes JR, Chatterjee S. Neuron-microglia crosstalk up-regulates neuronal FGF-2 expression which mediates neuroprotection against excitotoxicity via JNK1/2. J Neurochem. 2008;107(1):73–85.PubMedGoogle Scholar
  20. 20.
    Parkhurst CN, Gan WB. Microglia dynamics and function in the CNS. Curr Opin Neurobiol. 2010;20(5):595–600.PubMedPubMedCentralGoogle Scholar
  21. 21.
    Cunningham C. Microglia and neurodegeneration: the role of systemic inflammation. Glia. 2013;61(1):71–90.PubMedGoogle Scholar
  22. 22.
    Mrak RE, Griffin WS. Glia and their cytokines in progression of neurodegeneration. Neurobiol Aging. 2005;26(3):349–54.PubMedGoogle Scholar
  23. 23.
    Rock RB, Gekker G, Hu S, Sheng WS, Cheeran M, Lokensgard JR, et al. Role of microglia in central nervous system infections. Clin Microbiol Rev. 2004;17(4):942–64. table of contentsPubMedPubMedCentralGoogle Scholar
  24. 24.
    Wong EL, Stowell RD, Majewska AK. What the Spectrum of microglial functions can teach us about fetal alcohol Spectrum disorder. Front Synaptic Neurosci. 2017;9:11.PubMedPubMedCentralGoogle Scholar
  25. 25.
    Marin-Teva JL, Dusart I, Colin C, Gervais A, van Rooijen N, Mallat M. Microglia promote the death of developing Purkinje cells. Neuron. 2004;41(4):535–47.PubMedGoogle Scholar
  26. 26.
    Polazzi E, Gianni T, Contestabile A. Microglial cells protect cerebellar granule neurons from apoptosis: evidence for reciprocal signaling. Glia. 2001;36(3):271–80.PubMedGoogle Scholar
  27. 27.
    Ma Q, Anderson DJ, Fritzsch B. Neurogenin 1 null mutant ears develop fewer, morphologically normal hair cells in smaller sensory epithelia devoid of innervation. J Assoc Res Otolaryngol. 2000;1(2):129–43.PubMedPubMedCentralGoogle Scholar
  28. 28.
    Ma Q, Fode C, Guillemot F, Anderson DJ. Neurogenin1 and neurogenin2 control two distinct waves of neurogenesis in developing dorsal root ganglia. Genes Dev. 1999;13(13):1717–28.PubMedPubMedCentralGoogle Scholar
  29. 29.
    Ma Q, Chen Z, del Barco Barrantes I, de la Pompa JL, Anderson DJ. neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron. 1998;20(3):469–82.PubMedGoogle Scholar
  30. 30.
    Mattar P, Britz O, Johannes C, Nieto M, Ma L, Rebeyka A, et al. A screen for downstream effectors of Neurogenin2 in the embryonic neocortex. Dev Biol. 2004;273(2):373–89.PubMedGoogle Scholar
  31. 31.
    Bailey K, Balaei MR, Mannan A, Del Bigio MR, Marzban H. Purkinje cell compartmentation in the cerebellum of the lysosomal acid phosphatase 2 mutant mouse (nax-naked-ataxia mutant mouse). PLoS One. 2014;9(4):e94327.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Bailey K, Balaei MR, Mehdizadeh M, Marzban H. Spatial and temporal expression of lysosomal acid phosphatase 2 (ACP2) reveals dynamic patterning of the mouse cerebellar cortex. Cerebellum. 2013;12(6):870–81.PubMedGoogle Scholar
  33. 33.
    Chung SH, Marzban H, Aldinger K, Dixit R, Millen K, Schuurmans C, et al. Zac1 plays a key role in the development of specific neuronal subsets in the mouse cerebellum. Neural Dev. 2011;6:25.PubMedPubMedCentralGoogle Scholar
  34. 34.
    Hazama GI, Yasuhara O, Morita H, Aimi Y, Tooyama I, Kimura H. Mouse brain IgG-like immunoreactivity: strain-specific occurrence in microglia and biochemical identification of IgG. J Comp Neurol. 2005;492(2):234–49.PubMedGoogle Scholar
  35. 35.
    Sillitoe RV, Hawkes R. Whole-mount immunohistochemistry: a high-throughput screen for patterning defects in the mouse cerebellum. J Histochem Cytochem Off J Histochem Soc. 2002;50(2):235–44.Google Scholar
  36. 36.
    Wilkinson G, Dennis D, Schuurmans C. Proneural genes in neocortical development. Neuroscience. 2013;253:256–73.PubMedGoogle Scholar
  37. 37.
    Sarna JR, Marzban H, Watanabe M, Hawkes R. Complementary stripes of phospholipase Cbeta3 and Cbeta4 expression by Purkinje cell subsets in the mouse cerebellum. J Comp Neurol. 2006;496(3):303–13.PubMedGoogle Scholar
  38. 38.
    Ito D, Imai Y, Ohsawa K, Nakajima K, Fukuuchi Y, Kohsaka S. Microglia-specific localisation of a novel calcium binding protein, Iba1. Mol Brain Res. 1998;57(1):1–9.PubMedGoogle Scholar
  39. 39.
    Vedeler C, Ulvestad E, Grundt I, Conti G, Nyland H, Matre R, et al. Fc receptor for IgG (FcR) on rat microglia. J Neuroimmunol. 1994;49(1):19–24.PubMedGoogle Scholar
  40. 40.
    Saftig P, Hartmann D, Lullmann-Rauch R, Wolff J, Evers M, Koster A, et al. Mice deficient in lysosomal acid phosphatase develop lysosomal storage in the kidney and central nervous system. J Biol Chem. 1997;272(30):18628–35.PubMedGoogle Scholar
  41. 41.
    Rahimi Balaei M, Jiao X, Ashtari N, Afsharinezhad P, Ghavami S, Marzban H. Cerebellar expression of the Neurotrophin receptor p75 in naked-ataxia mutant mouse. Int J Mol Sci. 2016;17(1).Google Scholar
  42. 42.
    Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330(6005):841–5.PubMedPubMedCentralGoogle Scholar
  43. 43.
    Eisenman LM, Gallagher E, Hawkes R. Regionalization defects in the weaver mouse cerebellum. J Comp Neurol. 1998;394(4):431–44.PubMedGoogle Scholar
  44. 44.
    Tano D, Napieralski JA, Eisenman LM, Messer A, Plummer J, Hawkes R. Novel developmental boundary in the cerebellum revealed by zebrin expression in the lurcher (Lc/+) mutant mouse. J Comp Neurol. 1992;323(1):128–36.PubMedGoogle Scholar
  45. 45.
    Hawkes R, Herrup K, Aldolase C. Zebrin II and the regionalization of the cerebellum. J Mol Neurosci. 1995;6(3):147–58.PubMedGoogle Scholar
  46. 46.
    Howell BW, Hawkes R, Soriano P, Cooper JA. Neuronal position in the developing brain is regulated by mouse disabled-1. Nature. 1997;389(6652):733–7.PubMedGoogle Scholar
  47. 47.
    Gallagher E, Howell BW, Soriano P, Cooper JA, Hawkes R. Cerebellar abnormalities in the disabled (mdab1–1) mouse. J Comp Neurol. 1998;402(2):238–51.PubMedGoogle Scholar
  48. 48.
    Edwards MA, Leclerc N, Crandall JE, Yamamoto M. Purkinje cell compartments in the reeler mutant mouse as revealed by Zebrin II and 90-acetylated glycolipid antigen expression. Anat Embryol. 1994;190(5):417–28.PubMedGoogle Scholar
  49. 49.
    Goldowitz D, Cushing RC, Laywell E, D’Arcangelo G, Sheldon M, Sweet HO, et al. Cerebellar disorganization characteristic of reeler in scrambler mutant mice despite presence of reelin. J Neurosci. 1997;17(22):8767–77.PubMedGoogle Scholar
  50. 50.
    Sarna J, Miranda SR, Schuchman EH, Hawkes R. Patterned cerebellar Purkinje cell death in a transgenic mouse model of Niemann pick type a/B disease. Eur J Neurosci. 2001;13(10):1873–80.PubMedGoogle Scholar
  51. 51.
    Sarna JR, Larouche M, Marzban H, Sillitoe RV, Rancourt DE, Hawkes R. Patterned Purkinje cell degeneration in mouse models of Niemann-pick type C disease. J Comp Neurol. 2003;456(3):279–91.PubMedGoogle Scholar
  52. 52.
    Ji Z, Hawkes R. Partial ablation of the neonatal external granular layer disrupts mossy fiber topography in the adult rat cerebellum. J Comp Neurol. 1996;371(4):578–88.PubMedGoogle Scholar
  53. 53.
    McCORMICK DA, Steinmetz JE, Thompson RF. Lesions of the inferior olivary complex cause extinction of the classically conditioned eyeblink response. Brain Res. 1985;359(1–2):120–30.PubMedGoogle Scholar
  54. 54.
    Ashwell K. Microglia and cell death in the developing mouse cerebellum. Dev Brain Res. 1990;55(2):219–30.Google Scholar
  55. 55.
    Kovach C, Dixit R, Li S, Mattar P, Wilkinson G, Elsen GE, et al. Neurog2 simultaneously activates and represses alternative gene expression programs in the developing neocortex. Cereb Cortex. 2013;23(8):1884–900.PubMedGoogle Scholar
  56. 56.
    Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua X, et al. Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell. 2001;104(3):365–76.PubMedGoogle Scholar
  57. 57.
    Fletcher CF, Lutz CM, O'Sullivan TN, Shaughnessy JD, Hawkes R, Frankel WN, et al. Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell. 1996;87(4):607–17.PubMedGoogle Scholar
  58. 58.
    Leclerc N, Schwarting GA, Herrup K, Hawkes R, Yamamoto M. Compartmentation in mammalian cerebellum: Zebrin II and P-path antibodies define three classes of sagittally organized bands of Purkinje cells. Proc Natl Acad Sci. 1992;89(11):5006–10.PubMedGoogle Scholar
  59. 59.
    Sawada K, Fukui Y. Expression of tyrosine hydroxylase in cerebellar Purkinje cells of ataxic mutant mice: its relation to the onset and/or development of ataxia. J Med Invest. 2001;48(1/2):5–10.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Maryam Rahimi-Balaei
    • 1
  • Xiaodan Jiao
    • 1
  • Shahin Shabanipour
    • 1
  • Rajiv Dixit
    • 2
  • Carol Schuurmans
    • 2
  • Hassan Marzban
    • 1
    Email author
  1. 1.Department of Human Anatomy and Cell Science, The Children’s Hospital Research Institute of Manitoba (CHRIM), Max Rady College of Medicine, Rady Faculty of Health SciencesUniversity of ManitobaWinnipegCanada
  2. 2.Biological Sciences Platform, Sunnybrook Research InstituteUniversity of TorontoTorontoCanada

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