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Performing Single-Cell Clonal Analysis in the Mouse Brain Using Mosaic Analysis with Double Markers (MADM)

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Neuronal Cell Death

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2515))

Abstract

A central question in neuroscience is how 100 billion neurons come together to build the human brain. The wiring, morphology, survival, and death of each neuron are controlled by genes that encode intrinsic and extrinsic factors. Determining the function of these genes at a high spatiotemporal resolution is a critical step toward understanding brain development and function. Moreover, an increasing number of somatic mutations are being discovered in many brain disorders. However, neurons are embedded in complex networks, making it difficult to distinguish cell-autonomous from non–cell-autonomous function of any given gene in the brain. Here, I describe MADM (mosaic analysis with double markers), a genetic method that allows for labeling and manipulating gene function at the single-cell level within the mouse brain. I present mouse breeding schemes to employ MADM analysis and important considerations for experimental design. This powerful system can be adapted to make fundamental neuroscience discoveries by targeting genetically defined cell types in the mouse brain with high spatiotemporal resolution.

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References

  1. Sudhof TC (2017) Molecular neuroscience in the 21(st) century: a personal perspective. Neuron 96(3):536–541. https://doi.org/10.1016/j.neuron.2017.10.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Okawa H, Hoon M, Yoshimatsu T, Della Santina L, Wong ROL (2014) Illuminating the multifaceted roles of neurotransmission in shaping neuronal circuitry. Neuron 83(6):1303–1318. https://doi.org/10.1016/j.neuron.2014.08.029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kolodkin AL, Tessier-Lavigne M (2011) Mechanisms and molecules of neuronal wiring: a primer. Cold Spring Harb Perspect Biol 3(6):a001727. https://doi.org/10.1101/cshperspect.a001727

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. McAllister AK (2002) Conserved cues for axon and dendrite growth in the developing cortex. Neuron 33(1):2–4. https://doi.org/10.1016/s0896-6273(01)00577-3

    Article  CAS  PubMed  Google Scholar 

  5. Jan YN, Jan LY (2010) Branching out: mechanisms of dendritic arborization. Nat Rev Neurosci 11(5):316–328. https://doi.org/10.1038/nrn2836

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dekkers MP, Nikoletopoulou V, Barde YA (2013) Cell biology in neuroscience: death of developing neurons: new insights and implications for connectivity. J Cell Biol 203(3):385–393. https://doi.org/10.1083/jcb.201306136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sudhof TC (2018) Towards an understanding of synapse formation. Neuron 100(2):276–293. https://doi.org/10.1016/j.neuron.2018.09.040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lewandoski M (2001) Conditional control of gene expression in the mouse. Nat Rev Genet 2(10):743–755. https://doi.org/10.1038/35093537

    Article  CAS  PubMed  Google Scholar 

  9. D'Gama AM, Walsh CA (2018) Somatic mosaicism and neurodevelopmental disease. Nat Neurosci 21(11):1504–1514. https://doi.org/10.1038/s41593-018-0257-3

    Article  CAS  PubMed  Google Scholar 

  10. Joo W, Hippenmeyer S, Luo L (2014) Neurodevelopment. Dendrite morphogenesis depends on relative levels of NT-3/TrkC signaling. Science 346(6209):626–629. https://doi.org/10.1126/science.1258996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hippenmeyer S, Youn YH, Moon HM, Miyamichi K, Zong H, Wynshaw-Boris A, Luo L (2010) Genetic mosaic dissection of Lis1 and Ndel1 in neuronal migration. Neuron 68(4):695–709. https://doi.org/10.1016/j.neuron.2010.09.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zong H, Espinosa JS, Su HH, Muzumdar MD, Luo L (2005) Mosaic analysis with double markers in mice. Cell 121(3):479–492. https://doi.org/10.1016/j.cell.2005.02.012

    Article  CAS  PubMed  Google Scholar 

  13. Contreras X, Amberg N, Davaatseren A, Hansen AH, Sonntag J, Andersen L, Bernthaler T, Streicher C, Heger A, Johnson RL, Schwarz LA, Luo L, Rulicke T, Hippenmeyer S (2021) A genome-wide library of MADM mice for single-cell genetic mosaic analysis. Cell Rep 35(12):109274. https://doi.org/10.1016/j.celrep.2021.109274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Espinosa JS, Luo L (2008) Timing neurogenesis and differentiation: insights from quantitative clonal analyses of cerebellar granule cells. J Neurosci 28(10):2301–2312. https://doi.org/10.1523/JNEUROSCI.5157-07.2008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Gao P, Postiglione MP, Krieger TG, Hernandez L, Wang C, Han Z, Streicher C, Papusheva E, Insolera R, Chugh K, Kodish O, Huang K, Simons BD, Luo L, Hippenmeyer S, Shi SH (2014) Deterministic progenitor behavior and unitary production of neurons in the neocortex. Cell 159(4):775–788. https://doi.org/10.1016/j.cell.2014.10.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Espinosa JS, Wheeler DG, Tsien RW, Luo L (2009) Uncoupling dendrite growth and patterning: single-cell knockout analysis of NMDA receptor 2B. Neuron 62(2):205–217. https://doi.org/10.1016/j.neuron.2009.03.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Takeo YH, Shuster SA, Jiang L, Hu MC, Luginbuhl DJ, Rulicke T, Contreras X, Hippenmeyer S, Wagner MJ, Ganguli S, Luo L (2020) GluD2- and Cbln1-mediated competitive interactions shape the dendritic arbors of cerebellar Purkinje cells. Neuron 109(4):629–644.e8. https://doi.org/10.1016/j.neuron.2020.11.028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ortiz-Alvarez G, Daclin M, Shihavuddin A, Lansade P, Fortoul A, Faucourt M, Clavreul S, Lalioti ME, Taraviras S, Hippenmeyer S, Livet J, Meunier A, Genovesio A, Spassky N (2019) Adult neural stem cells and multiciliated ependymal cells share a common lineage regulated by the geminin family members. Neuron 102(1):159–172.e7. https://doi.org/10.1016/j.neuron.2019.01.051

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Shi W, Xianyu A, Han Z, Tang X, Li Z, Zhong H, Mao T, Huang K, Shi SH (2017) Ontogenetic establishment of order-specific nuclear organization in the mammalian thalamus. Nat Neurosci 20(4):516–528. https://doi.org/10.1038/nn.4519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hippenmeyer S, Johnson RL, Luo L (2013) Mosaic analysis with double markers reveals cell-type-specific paternal growth dominance. Cell Rep 3(3):960–967. https://doi.org/10.1016/j.celrep.2013.02.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Laukoter S, Pauler FM, Beattie R, Amberg N, Hansen AH, Streicher C, Penz T, Bock C, Hippenmeyer S (2020) Cell-type specificity of genomic imprinting in cerebral cortex. Neuron 107(6):1160–1179.e9. https://doi.org/10.1016/j.neuron.2020.06.031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Muzumdar MD, Luo L, Zong H (2007) Modeling sporadic loss of heterozygosity in mice by using mosaic analysis with double markers (MADM). Proc Natl Acad Sci U S A 104(11):4495–4500. https://doi.org/10.1073/pnas.0606491104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Knudson AG Jr (1971) Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci U S A 68(4):820–823. https://doi.org/10.1073/pnas.68.4.820

    Article  PubMed  PubMed Central  Google Scholar 

  24. Yao M, Ventura PB, Jiang Y, Rodriguez FJ, Wang L, Perry JSA, Yang Y, Wahl K, Crittenden RB, Bennett ML, Qi L, Gong CC, Li XN, Barres BA, Bender TP, Ravichandran KS, Janes KA, Eberhart CG, Zong H (2020) Astrocytic trans-differentiation completes a multicellular paracrine feedback loop required for medulloblastoma tumor growth. Cell 180(3):502–520.e19. https://doi.org/10.1016/j.cell.2019.12.024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Liu C, Sage JC, Miller MR, Verhaak RG, Hippenmeyer S, Vogel H, Foreman O, Bronson RT, Nishiyama A, Luo L, Zong H (2011) Mosaic analysis with double markers reveals tumor cell of origin in glioma. Cell 146(2):209–221. https://doi.org/10.1016/j.cell.2011.06.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Crino PB, Aronica E, Baltuch G, Nathanson KL (2010) Biallelic TSC gene inactivation in tuberous sclerosis complex. Neurology 74(21):1716–1723. https://doi.org/10.1212/WNL.0b013e3181e04325

    Article  PubMed  PubMed Central  Google Scholar 

  27. Lindhurst MJ, Sapp JC, Teer JK, Johnston JJ, Finn EM, Peters K, Turner J, Cannons JL, Bick D, Blakemore L, Blumhorst C, Brockmann K, Calder P, Cherman N, Deardorff MA, Everman DB, Golas G, Greenstein RM, Kato BM, Keppler-Noreuil KM, Kuznetsov SA, Miyamoto RT, Newman K, Ng D, O'Brien K, Rothenberg S, Schwartzentruber DJ, Singhal V, Tirabosco R, Upton J, Wientroub S, Zackai EH, Hoag K, Whitewood-Neal T, Robey PG, Schwartzberg PL, Darling TN, Tosi LL, Mullikin JC, Biesecker LG (2011) A mosaic activating mutation in AKT1 associated with the Proteus syndrome. N Engl J Med 365(7):611–619. https://doi.org/10.1056/NEJMoa1104017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pangalos C, Avramopoulos D, Blouin JL, Raoul O, deBlois MC, Prieur M, Schinzel AA, Gika M, Abazis D, Antonarakis SE (1994) Understanding the mechanism(s) of mosaic trisomy 21 by using DNA polymorphism analysis. Am J Hum Genet 54(3):473–481

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Lim ET, Uddin M, De Rubeis S, Chan Y, Kamumbu AS, Zhang X, D'Gama AM, Kim SN, Hill RS, Goldberg AP, Poultney C, Minshew NJ, Kushima I, Aleksic B, Ozaki N, Parellada M, Arango C, Penzol MJ, Carracedo A, Kolevzon A, Hultman CM, Weiss LA, Fromer M, Chiocchetti AG, Freitag CM, Autism Sequencing Consortium, Church GM, Scherer SW, Buxbaum JD, Walsh CA (2017) Rates, distribution and implications of postzygotic mosaic mutations in autism spectrum disorder. Nat Neurosci 20(9):1217–1224. https://doi.org/10.1038/nn.4598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Javed S, Selliah T, Lee YJ, Huang WH (2020) Dosage-sensitive genes in autism spectrum disorders: from neurobiology to therapy. Neurosci Biobehav Rev 118:538–567. https://doi.org/10.1016/j.neubiorev.2020.08.009

    Article  CAS  PubMed  Google Scholar 

  31. Park JS, Lee J, Jung ES, Kim MH, Kim IB, Son H, Kim S, Kim S, Park YM, Mook-Jung I, Yu SJ, Lee JH (2019) Brain somatic mutations observed in Alzheimer's disease associated with aging and dysregulation of tau phosphorylation. Nat Commun 10(1):3090. https://doi.org/10.1038/s41467-019-11000-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Veeriah S, Taylor BS, Meng S, Fang F, Yilmaz E, Vivanco I, Janakiraman M, Schultz N, Hanrahan AJ, Pao W, Ladanyi M, Sander C, Heguy A, Holland EC, Paty PB, Mischel PS, Liau L, Cloughesy TF, Mellinghoff IK, Solit DB, Chan TA (2010) Somatic mutations of the Parkinson’s disease-associated gene PARK2 in glioblastoma and other human malignancies. Nat Genet 42(1):77–82. https://doi.org/10.1038/ng.491

    Article  CAS  PubMed  Google Scholar 

  33. Lim JS, Kim WI, Kang HC, Kim SH, Park AH, Park EK, Cho YW, Kim S, Kim HM, Kim JA, Kim J, Rhee H, Kang SG, Kim HD, Kim D, Kim DS, Lee JH (2015) Brain somatic mutations in MTOR cause focal cortical dysplasia type II leading to intractable epilepsy. Nat Med 21(4):395–400. https://doi.org/10.1038/nm.3824

    Article  CAS  PubMed  Google Scholar 

  34. Shirley MD, Tang H, Gallione CJ, Baugher JD, Frelin LP, Cohen B, North PE, Marchuk DA, Comi AM, Pevsner J (2013) Sturge-Weber syndrome and port-wine stains caused by somatic mutation in GNAQ. N Engl J Med 368(21):1971–1979. https://doi.org/10.1056/NEJMoa1213507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lim JS, Gopalappa R, Kim SH, Ramakrishna S, Lee M, Kim WI, Kim J, Park SM, Lee J, Oh JH, Kim HD, Park CH, Lee JS, Kim S, Kim DS, Han JM, Kang HC, Kim HH, Lee JH (2017) Somatic mutations in TSC1 and TSC2 cause focal cortical dysplasia. Am J Hum Genet 100(3):454–472. https://doi.org/10.1016/j.ajhg.2017.01.030

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tang SH, Silva FJ, Tsark WM, Mann JR (2002) A Cre/loxP-deleter transgenic line in mouse strain 129S1/SvImJ. Genesis 32(3):199–202

    Article  CAS  Google Scholar 

  37. Petersen PH, Zou K, Hwang JK, Jan YN, Zhong W (2002) Progenitor cell maintenance requires numb and numblike during mouse neurogenesis. Nature 419(6910):929–934. https://doi.org/10.1038/nature01124

    Article  CAS  PubMed  Google Scholar 

  38. Gorski JA, Talley T, Qiu M, Puelles L, Rubenstein JL, Jones KR (2002) Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage. J Neurosci 22(15):6309–6314. https://doi.org/10.1523/JNEUROSCI.22-15-06309.2002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Xu Q, Tam M, Anderson SA (2008) Fate mapping Nkx2.1-lineage cells in the mouse telencephalon. J Comp Neurol 506(1):16–29. https://doi.org/10.1002/cne.21529

    Article  CAS  PubMed  Google Scholar 

  40. Luo L, Ambrozkiewicz MC, Benseler F, Chen C, Dumontier E, Falkner S, Furlanis E, Gomez AM, Hoshina N, Huang WH, Hutchison MA, Itoh-Maruoka Y, Lavery LA, Li W, Maruo T, Motohashi J, Pai EL, Pelkey KA, Pereira A, Philips T, Sinclair JL, Stogsdill JA, Traunmüller L, Wang J, Wortel J, You W, Abumaria N, Beier KT, Brose N, Burgess HA, Cepko CL, Cloutier JF, Eroglu C, Goebbels S, Kaeser PS, Kay JN, Lu W, Mandai K, McBain CJ, Nave KA, Prado MAM, Prado VF, Rothstein J, Rubenstein JLR, Saher G, Sakimura K, Sanes JR, Scheiffele P, Takai Y, Umemori H, Verhage M, Yuzaki M, Zoghbi HY, Kawabe H, Craig AM (2020) Optimizing nervous system-specific gene targeting with Cre driver lines: prevalence of germline recombination and influencing factors. Neuron 106(1):37–65.e5. https://doi.org/10.1016/j.neuron.2020.01.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kessaris N, Fogarty M, Iannarelli P, Grist M, Wegner M, Richardson WD (2006) Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage. Nat Neurosci 9(2):173–179. https://doi.org/10.1038/nn1620

    Article  CAS  PubMed  Google Scholar 

  42. Taniguchi H, He M, Wu P, Kim S, Paik R, Sugino K, Kvitsiani D, Kvitsani D, Fu Y, Lu J, Lin Y, Miyoshi G, Shima Y, Fishell G, Nelson SB, Huang ZJ (2011) A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71(6):995–1013. https://doi.org/10.1016/j.neuron.2011.07.026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work is supported by a 2021 NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation and Fonds de recherche du Québec—Santé to Wei-Hsiang Huang. Wei-Hsiang Huang is also an Azrieli Centre for Autism Research (ACAR) Researcher.

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  1. Department of Neurology and Neurosurgery, Centre for Research in Neuroscience, McGill University, Montréal, QC, Canada

    Wei-Hsiang Huang

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Correspondence to Wei-Hsiang Huang .

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  1. Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada

    Arezu Jahani-Asl

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Huang, WH. (2022). Performing Single-Cell Clonal Analysis in the Mouse Brain Using Mosaic Analysis with Double Markers (MADM). In: Jahani-Asl, A. (eds) Neuronal Cell Death. Methods in Molecular Biology, vol 2515. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2409-8_4

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  • DOI: https://doi.org/10.1007/978-1-0716-2409-8_4

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  • Publisher Name: Humana, New York, NY

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  • Online ISBN: 978-1-0716-2409-8

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