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Automated Immunofluorescence Staining for Analysis of Mitotic Stages and Division Orientation in Brain Sections

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Microcephaly

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2583))

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Abstract

Microcephaly often results from mitotic defects in neuronal progenitors, frequently by decreasing proliferation rates or shifting cell fates. During neurogenesis, oriented cell division—the molecular control of mitotic spindle positioning to control the axis of division—represents an important mechanism to balance expansion of the progenitor pool with generating cellular diversity. While mostly studied in the context of cortical development, more recently, spindle orientation has emerged as a key player in the formation of other brain regions such as the cerebellum. Here we describe methods to perform automated dual-color fluorescent immunohistochemistry on murine cerebellar sections using the mitotic markers phospho-Histone H3 and Survivin, and detail analytical and statistical approaches to display and compare division orientation datasets.

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References

  1. O’Neill RS, Schoborg TA, Rusan NM (2018) Same but different: pleiotropy in centrosome-related microcephaly. Mol Biol Cell 29:241–246

    Article  PubMed  PubMed Central  Google Scholar 

  2. Nano M, Basto R (2017) Consequences of centrosome dysfunction during brain development. Adv Exp Med Biol 1002:19–45

    Article  CAS  PubMed  Google Scholar 

  3. Capecchi MR, Pozner A (2015) ASPM regulates symmetric stem cell division by tuning Cyclin E ubiquitination. Nat Commun 6:8763

    Article  CAS  PubMed  Google Scholar 

  4. Fujimori A et al (2014) Disruption of Aspm causes microcephaly with abnormal neuronal differentiation. Brain Dev 36:661–669

    Article  PubMed  Google Scholar 

  5. Pulvers JN et al (2010) Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline. Proc Natl Acad Sci U S A 107:16595–16600

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jayaraman D et al (2016) Microcephaly proteins Wdr62 and Aspm define a mother centriole complex regulating centriole biogenesis, apical complex, and cell fate. Neuron 92:813–828

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Novorol C et al (2013) Microcephaly models in the developing zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression. Open Biol 3:130065

    Article  PubMed  PubMed Central  Google Scholar 

  8. Kim HT et al (2011) The microcephaly gene aspm is involved in brain development in zebrafish. Biochem Biophys Res Commun 409:640–644

    Article  CAS  PubMed  Google Scholar 

  9. Johnson MB et al (2018) Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size. Nature 556:370–375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Williams SE et al (2015) Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth. Development 142:3921–3932

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Gai M et al (2016) ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules. EMBO Rep 17:1396–1409

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Higgins J et al (2010) Human ASPM participates in spindle organisation, spindle orientation and cytokinesis. BMC Cell Biol 11:85

    Article  PubMed  PubMed Central  Google Scholar 

  13. Fish JL, Kosodo Y, Enard W, Paabo S, Huttner WB (2006) Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells. Proc Natl Acad Sci U S A 103:10438–10443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bergstralh DT, Dawney NS, Johnston DS (2017) Spindle orientation: a question of complex positioning. Development 144:1137–1145

    Article  CAS  PubMed  Google Scholar 

  15. Seldin L, Macara I (2017) Epithelial spindle orientation diversities and uncertainties: recent developments and lingering questions. F1000Res 6:984

    Article  PubMed  PubMed Central  Google Scholar 

  16. di Pietro F, Echard A, Morin X (2016) Regulation of mitotic spindle orientation: an integrated view. EMBO Rep 17:1106–1130

    Article  PubMed  PubMed Central  Google Scholar 

  17. Lu MS, Johnston CA (2013) Molecular pathways regulating mitotic spindle orientation in animal cells. Development 140:1843–1856

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Williams SE, Fuchs E (2013) Oriented divisions, fate decisions. Curr Opin Cell Biol 25:749–758

    Article  CAS  PubMed  Google Scholar 

  19. Morin X, Bellaiche Y (2011) Mitotic spindle orientation in asymmetric and symmetric cell divisions during animal development. Dev Cell 21:102–119

    Article  CAS  PubMed  Google Scholar 

  20. Gillies TE, Cabernard C (2011) Cell division orientation in animals. Curr Biol 21:R599–R609

    Article  CAS  PubMed  Google Scholar 

  21. Lancaster MA, Knoblich JA (2012) Spindle orientation in mammalian cerebral cortical development. Curr Opin Neurobiol 22:737–746

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wodarz A, Huttner WB (2003) Asymmetric cell division during neurogenesis in drosophila and vertebrates. Mech Dev 120:1297–1309

    Article  CAS  PubMed  Google Scholar 

  23. Li H et al (2017) Spindle Misorientation of cerebral and cerebellar progenitors is a mechanistic cause of megalencephaly. Stem Cell Rep 9:1071–1080

    Article  CAS  Google Scholar 

  24. Lejeune E, Dortdivanlioglu B, Kuhl E, Linder C (2019) Understanding the mechanical link between oriented cell division and cerebellar morphogenesis. Soft Matter 15:2204–2215

    Article  CAS  PubMed  Google Scholar 

  25. Feng Y, Walsh CA (2004) Mitotic spindle regulation by Nde1 controls cerebral cortical size. Neuron 44:279–293

    Article  CAS  PubMed  Google Scholar 

  26. Miyamoto T et al (2017) PLK1-mediated phosphorylation of WDR62/MCPH2 ensures proper mitotic spindle orientation. Hum Mol Genet 26:4429–4440

    Article  CAS  PubMed  Google Scholar 

  27. Chen JF et al (2014) Microcephaly disease gene Wdr62 regulates mitotic progression of embryonic neural stem cells and brain size. Nat Commun 5:3885

    Article  CAS  PubMed  Google Scholar 

  28. Hanafusa H et al (2015) PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2. Nat Cell Biol 17:1024–1035

    Article  CAS  PubMed  Google Scholar 

  29. Adachi Y, Mochida G, Walsh C, Barkovich J (2014) Posterior fossa in primary microcephaly: relationships between forebrain and mid-hindbrain size in 110 patients. Neuropediatrics 45:93–101

    PubMed  Google Scholar 

  30. Hibi M, Shimizu T (2012) Development of the cerebellum and cerebellar neural circuits. Dev Neurobiol 72:282–301

    Article  PubMed  Google Scholar 

  31. Roussel MF, Hatten ME (2011) Cerebellum development and medulloblastoma. Curr Top Dev Biol 94:235–282

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hatten ME, Rifkin DB, Furie MB, Mason CA, Liem RK (1982) Biochemistry of granule cell migration in developing mouse cerebellum. Prog Clin Biol Res 85 Pt B:509–519

    CAS  PubMed  Google Scholar 

  33. Espinosa JS, Luo L (2008) Timing neurogenesis and differentiation: insights from quantitative clonal analyses of cerebellar granule cells. J Neurosci 28:2301–2312

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Nakashima K, Umeshima H, Kengaku M (2015) Cerebellar granule cells are predominantly generated by terminal symmetric divisions of granule cell precursors. Dev Dyn 244:748–758

    Article  PubMed  Google Scholar 

  35. Zagon IS, McLaughlin PJ (1987) The location and orientation of mitotic figures during histogenesis of the rat cerebellar cortex. Brain Res Bull 18:325–336

    Article  CAS  PubMed  Google Scholar 

  36. Legue E, Riedel E, Joyner AL (2015) Clonal analysis reveals granule cell behaviors and compartmentalization that determine the folded morphology of the cerebellum. Development 142:1661–1671

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Miyashita S, Adachi T, Yamashita M, Sota T, Hoshino M (2017) Dynamics of the cell division orientation of granule cell precursors during cerebellar development. Mech Dev 147:1–7

    Article  CAS  PubMed  Google Scholar 

  38. Haldipur P, Sivaprakasam I, Periasamy V, Govindan S, Mani S (2015) Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum. Biol Open 4:865–872

    Article  PubMed  PubMed Central  Google Scholar 

  39. Knoblich JA (2010) Asymmetric cell division: recent developments and their implications for tumour biology. Nat Rev Mol Cell Biol 11:849–860

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lough KJ et al (2019) Telophase correction refines division orientation in stratified epithelia. Elife 8:e49249

    Google Scholar 

  41. Ying Z, Sandoval M, Beronja S (2018) Oncogenic activation of PI3K induces progenitor cell differentiation to suppress epidermal growth. Nat Cell Biol 20:1256–1266

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Poulson ND, Lechler T (2010) Robust control of mitotic spindle orientation in the developing epidermis. J Cell Biol 191:915–922

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Williams SE, Beronja S, Pasolli HA, Fuchs E (2011) Asymmetric cell divisions promote Notch-dependent epidermal differentiation. Nature 470:353–358

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hans F, Dimitrov S (2001) Histone H3 phosphorylation and cell division. Oncogene 20:3021–3027

    Article  CAS  PubMed  Google Scholar 

  45. Caldas H et al (2005) Survivin splice variants regulate the balance between proliferation and cell death. Oncogene 24:1994–2007

    Article  CAS  PubMed  Google Scholar 

  46. Beardmore VA, Ahonen LJ, Gorbsky GJ, Kallio MJ (2004) Survivin dynamics increases at centromeres during G2/M phase transition and is regulated by microtubule-attachment and Aurora B kinase activity. J Cell Sci 117:4033–4042

    Article  CAS  PubMed  Google Scholar 

  47. Byrd KM et al (2016) LGN plays distinct roles in oral epithelial stratification, filiform papilla morphogenesis and hair follicle development. Development 143:2803–2817

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Williams SE, Ratliff LA, Postiglione MP, Knoblich JA, Fuchs E (2014) Par3-mInsc and Galphai3 cooperate to promote oriented epidermal cell divisions through LGN. Nat Cell Biol 16:758–769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ichijo R et al (2017) Tbx3-dependent amplifying stem cell progeny drives interfollicular epidermal expansion during pregnancy and regeneration. Nat Commun 8:508

    Article  PubMed  PubMed Central  Google Scholar 

  50. Ding X et al (2016) mTORC1 and mTORC2 regulate skin morphogenesis and epidermal barrier formation. Nat Commun 7:13226

    Article  PubMed  PubMed Central  Google Scholar 

  51. Asrani K et al (2017) mTORC1 loss impairs epidermal adhesion via TGF-beta/rho kinase activation. J Clin Invest 127:4001–4017

    Article  PubMed  PubMed Central  Google Scholar 

  52. Aragona M et al (2017) Defining stem cell dynamics and migration during wound healing in mouse skin epidermis. Nat Commun 8:14684

    Article  PubMed  PubMed Central  Google Scholar 

  53. Dor-On E et al (2017) T-plastin is essential for basement membrane assembly and epidermal morphogenesis. Sci Signal 10:eaal3154

    Article  PubMed  Google Scholar 

  54. Cohen J et al (2019) The Wave complex controls epidermal morphogenesis and proliferation by suppressing Wnt-Sox9 signaling. J Cell Biol 218:1390–1406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Liu N et al (2019) Stem cell competition orchestrates skin homeostasis and ageing. Nature 568:344–350

    Article  CAS  PubMed  Google Scholar 

  56. Ellis SJ et al (2019) Distinct modes of cell competition shape mammalian tissue morphogenesis. Nature 569:497–502

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Jones KB et al (2019) Quantitative clonal analysis and single-cell transcriptomics reveal division kinetics, hierarchy, and fate of oral epithelial progenitor cells. Cell Stem Cell 24:183–192.e188

    Article  CAS  PubMed  Google Scholar 

  58. Wang X et al (2018) E-cadherin bridges cell polarity and spindle orientation to ensure prostate epithelial integrity and prevent carcinogenesis in vivo. PLoS Genet 14:e1007609

    Article  PubMed  PubMed Central  Google Scholar 

  59. Niessen MT et al (2013) aPKClambda controls epidermal homeostasis and stem cell fate through regulation of division orientation. J Cell Biol 202:887–900

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Juschke C, Xie Y, Postiglione MP, Knoblich JA (2014) Analysis and modeling of mitotic spindle orientations in three dimensions. Proc Natl Acad Sci U S A 111:1014–1019

    Article  PubMed  Google Scholar 

  61. Ségalen M et al (2010) The Fz-Dsh planar cell polarity pathway induces oriented cell division via Mud/NuMA in Drosophila and zebrafish. Dev Cell 19(5):740–752

    Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Pathology & Laboratory Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Gabriela De la Cruz, Nana Nikolaishvili Feinberg & Scott E. Williams

  2. Pathology Services Core, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Gabriela De la Cruz & Nana Nikolaishvili Feinberg

  3. Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Scott E. Williams

  4. Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Gabriela De la Cruz, Nana Nikolaishvili Feinberg & Scott E. Williams

Authors
  1. Gabriela De la Cruz
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  2. Nana Nikolaishvili Feinberg
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  3. Scott E. Williams
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Corresponding author

Correspondence to Scott E. Williams .

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Editors and Affiliations

  1. Department of Pediatrics, Children’s Center for Neuroscience Research, Emory University School of Medicine, Atlanta, GA, USA

    Timothy Gershon

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De la Cruz, G., Nikolaishvili Feinberg, N., Williams, S.E. (2023). Automated Immunofluorescence Staining for Analysis of Mitotic Stages and Division Orientation in Brain Sections. In: Gershon, T. (eds) Microcephaly. Methods in Molecular Biology, vol 2583. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2752-5_7

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  • DOI: https://doi.org/10.1007/978-1-0716-2752-5_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2751-8

  • Online ISBN: 978-1-0716-2752-5

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