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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
O’Neill RS, Schoborg TA, Rusan NM (2018) Same but different: pleiotropy in centrosome-related microcephaly. Mol Biol Cell 29:241–246
Nano M, Basto R (2017) Consequences of centrosome dysfunction during brain development. Adv Exp Med Biol 1002:19–45
Capecchi MR, Pozner A (2015) ASPM regulates symmetric stem cell division by tuning Cyclin E ubiquitination. Nat Commun 6:8763
Fujimori A et al (2014) Disruption of Aspm causes microcephaly with abnormal neuronal differentiation. Brain Dev 36:661–669
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
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
Novorol C et al (2013) Microcephaly models in the develo** zebrafish retinal neuroepithelium point to an underlying defect in metaphase progression. Open Biol 3:130065
Kim HT et al (2011) The microcephaly gene aspm is involved in brain development in zebrafish. Biochem Biophys Res Commun 409:640–644
Johnson MB et al (2018) Aspm knockout ferret reveals an evolutionary mechanism governing cerebral cortical size. Nature 556:370–375
Williams SE et al (2015) Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth. Development 142:3921–3932
Gai M et al (2016) ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules. EMBO Rep 17:1396–1409
Higgins J et al (2010) Human ASPM participates in spindle organisation, spindle orientation and cytokinesis. BMC Cell Biol 11:85
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
Bergstralh DT, Dawney NS, Johnston DS (2017) Spindle orientation: a question of complex positioning. Development 144:1137–1145
Seldin L, Macara I (2017) Epithelial spindle orientation diversities and uncertainties: recent developments and lingering questions. F1000Res 6:984
di Pietro F, Echard A, Morin X (2016) Regulation of mitotic spindle orientation: an integrated view. EMBO Rep 17:1106–1130
Lu MS, Johnston CA (2013) Molecular pathways regulating mitotic spindle orientation in animal cells. Development 140:1843–1856
Williams SE, Fuchs E (2013) Oriented divisions, fate decisions. Curr Opin Cell Biol 25:749–758
Morin X, Bellaiche Y (2011) Mitotic spindle orientation in asymmetric and symmetric cell divisions during animal development. Dev Cell 21:102–119
Gillies TE, Cabernard C (2011) Cell division orientation in animals. Curr Biol 21:R599–R609
Lancaster MA, Knoblich JA (2012) Spindle orientation in mammalian cerebral cortical development. Curr Opin Neurobiol 22:737–746
Wodarz A, Huttner WB (2003) Asymmetric cell division during neurogenesis in drosophila and vertebrates. Mech Dev 120:1297–1309
Li H et al (2017) Spindle Misorientation of cerebral and cerebellar progenitors is a mechanistic cause of megalencephaly. Stem Cell Rep 9:1071–1080
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
Feng Y, Walsh CA (2004) Mitotic spindle regulation by Nde1 controls cerebral cortical size. Neuron 44:279–293
Miyamoto T et al (2017) PLK1-mediated phosphorylation of WDR62/MCPH2 ensures proper mitotic spindle orientation. Hum Mol Genet 26:4429–4440
Chen JF et al (2014) Microcephaly disease gene Wdr62 regulates mitotic progression of embryonic neural stem cells and brain size. Nat Commun 5:3885
Hanafusa H et al (2015) PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2. Nat Cell Biol 17:1024–1035
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
Hibi M, Shimizu T (2012) Development of the cerebellum and cerebellar neural circuits. Dev Neurobiol 72:282–301
Roussel MF, Hatten ME (2011) Cerebellum development and medulloblastoma. Curr Top Dev Biol 94:235–282
Hatten ME, Rifkin DB, Furie MB, Mason CA, Liem RK (1982) Biochemistry of granule cell migration in develo** mouse cerebellum. Prog Clin Biol Res 85 Pt B:509–519
Espinosa JS, Luo L (2008) Timing neurogenesis and differentiation: insights from quantitative clonal analyses of cerebellar granule cells. J Neurosci 28:2301–2312
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
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
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
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
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
Knoblich JA (2010) Asymmetric cell division: recent developments and their implications for tumour biology. Nat Rev Mol Cell Biol 11:849–860
Lough KJ et al (2019) Telophase correction refines division orientation in stratified epithelia. Elife 8:e49249
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
Poulson ND, Lechler T (2010) Robust control of mitotic spindle orientation in the develo** epidermis. J Cell Biol 191:915–922
Williams SE, Beronja S, Pasolli HA, Fuchs E (2011) Asymmetric cell divisions promote Notch-dependent epidermal differentiation. Nature 470:353–358
Hans F, Dimitrov S (2001) Histone H3 phosphorylation and cell division. Oncogene 20:3021–3027
Caldas H et al (2005) Survivin splice variants regulate the balance between proliferation and cell death. Oncogene 24:1994–2007
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
Byrd KM et al (2016) LGN plays distinct roles in oral epithelial stratification, filiform papilla morphogenesis and hair follicle development. Development 143:2803–2817
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
Ichijo R et al (2017) Tbx3-dependent amplifying stem cell progeny drives interfollicular epidermal expansion during pregnancy and regeneration. Nat Commun 8:508
Ding X et al (2016) mTORC1 and mTORC2 regulate skin morphogenesis and epidermal barrier formation. Nat Commun 7:13226
Asrani K et al (2017) mTORC1 loss impairs epidermal adhesion via TGF-beta/rho kinase activation. J Clin Invest 127:4001–4017
Aragona M et al (2017) Defining stem cell dynamics and migration during wound healing in mouse skin epidermis. Nat Commun 8:14684
Dor-On E et al (2017) T-plastin is essential for basement membrane assembly and epidermal morphogenesis. Sci Signal 10:eaal3154
Cohen J et al (2019) The Wave complex controls epidermal morphogenesis and proliferation by suppressing Wnt-Sox9 signaling. J Cell Biol 218:1390–1406
Liu N et al (2019) Stem cell competition orchestrates skin homeostasis and ageing. Nature 568:344–350
Ellis SJ et al (2019) Distinct modes of cell competition shape mammalian tissue morphogenesis. Nature 569:497–502
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
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
Niessen MT et al (2013) aPKClambda controls epidermal homeostasis and stem cell fate through regulation of division orientation. J Cell Biol 202:887–900
Juschke C, **e 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
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
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2752-5_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2751-8
Online ISBN: 978-1-0716-2752-5
eBook Packages: Springer Protocols