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In Utero Electroporation to Study Mouse Brain Development

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Brain Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2047))

Abstract

In utero electroporation is a rapid and powerful technique to study the development of many brain regions. This approach presents several advantages over other methods to study specific steps of brain development in vivo, from proliferation to synaptic integration. Here, we describe in detail the individual steps necessary to carry out the technique. We also highlight the variations that can be implemented to target different cerebral structures and to study specific steps of development.

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References

  1. Baumgart J, Baumgart N (2016) Cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the C57BL/6 mouse. J Vis Exp 107:e53303. https://doi.org/10.3791/53303

    Article  CAS  Google Scholar 

  2. Matsui A, Yoshida AC, Kubota M, Ogawa M, Shimogori T (2011) Mouse in utero electroporation: controlled spatiotemporal gene transfection. J Vis Exp (54). https://doi.org/10.3791/3024

  3. Szczurkowska J, Cwetsch AW, dal Maschio M, Ghezzi D, Ratto GM, Cancedda L (2016) Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe. Nat Protoc 11(3):399–412. https://doi.org/10.1038/nprot.2016.014

    Article  CAS  PubMed  Google Scholar 

  4. Matsuda T, Cepko CL (2007) Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci U S A 104(3):1027–1032. https://doi.org/10.1073/pnas.0610155104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kalebic N, Taverna E, Tavano S, Wong FK, Suchold D, Winkler S, Huttner WB, Sarov M (2016) CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo. EMBO Rep 17(3):338–348. https://doi.org/10.15252/embr.201541715

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shinmyo Y, Tanaka S, Tsunoda S, Hosomichi K, Tajima A, Kawasaki H (2016) CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation. Sci Rep 6:20611. https://doi.org/10.1038/srep20611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Cheng M, Jin X, Mu L, Wang F, Li W, Zhong X, Liu X, Shen W, Liu Y, Zhou Y (2016) Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development. J Neurosci Res 94(9):814–824. https://doi.org/10.1002/jnr.23776

    Article  CAS  PubMed  Google Scholar 

  8. Yoshida A, Yamaguchi Y, Nonomura K, Kawakami K, Takahashi Y, Miura M (2010) Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system. Genes Cells 15(5):501–512. https://doi.org/10.1111/j.1365-2443.2010.01397.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gee JM, Gibbons MB, Taheri M, Palumbos S, Morris SC, Smeal RM, Flynn KF, Economo MN, Cizek CG, Capecchi MR, Tvrdik P, Wilcox KS, White JA (2015) Imaging activity in astrocytes and neurons with genetically encoded calcium indicators following in utero electroporation. Front Mol Neurosci 8:10. https://doi.org/10.3389/fnmol.2015.00010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bocchi R, Egervari K, Carol-Perdiguer L, Viale B, Quairiaux C, De Roo M, Boitard M, Oskouie S, Salmon P, Kiss JZ (2017) Perturbed Wnt signaling leads to neuronal migration delay, altered interhemispheric connections and impaired social behavior. Nat Commun 8(1):1158. https://doi.org/10.1038/s41467-017-01046-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ishii K, Kubo K, Endo T, Yoshida K, Benner S, Ito Y, Aizawa H, Aramaki M, Yamanaka A, Tanaka K, Takata N, Tanaka KF, Mimura M, Tohyama C, Kakeyama M, Nakajima K (2015) Neuronal heterotopias affect the activities of distant brain areas and lead to behavioral deficits. J Neurosci 35(36):12432–12445. https://doi.org/10.1523/JNEUROSCI.3648-14.2015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bitzenhofer SH, Ahlbeck J, Wolff A, Wiegert JS, Gee CE, Oertner TG, Hanganu-Opatz IL (2017) Layer-specific optogenetic activation of pyramidal neurons causes beta-gamma entrainment of neonatal networks. Nat Commun 8:14563. https://doi.org/10.1038/ncomms14563

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Niwa M, Kamiya A, Murai R, Kubo K, Gruber AJ, Tomita K, Lu L, Tomisato S, Jaaro-Peled H, Seshadri S, Hiyama H, Huang B, Kohda K, Noda Y, O’Donnell P, Nakajima K, Sawa A, Nabeshima T (2010) Knockdown of DISC1 by in utero gene transfer disturbs postnatal dopaminergic maturation in the frontal cortex and leads to adult behavioral deficits. Neuron 65(4):480–489

    Article  CAS  Google Scholar 

  14. Kawasaki H, Iwai L, Tanno K (2012) Rapid and efficient genetic manipulation of gyrencephalic carnivores using in utero electroporation. Mol Brain 5:24. https://doi.org/10.1186/1756-6606-5-24

    Article  PubMed  PubMed Central  Google Scholar 

  15. Kawasaki H, Toda T, Tanno K (2013) In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation. Biol Open 2(1):95–100. https://doi.org/10.1242/bio.20123160

    Article  PubMed  Google Scholar 

  16. Liu Y, Fu S, Niu R, Yang C, Lin J (2014) Transcriptional activity assessment of three different promoters for mouse in utero electroporation system. Plasmid 74:52–58. https://doi.org/10.1016/j.plasmid.2014.06.003

    Article  CAS  PubMed  Google Scholar 

  17. Pacary E, Haas MA, Wildner H, Azzarelli R, Bell DM, Abrous DN, Guillemot F (2012) Visualization and genetic manipulation of dendrites and spines in the mouse cerebral cortex and hippocampus using in utero electroporation. J Vis Exp 65:pii: 4163. https://doi.org/10.3791/4163

    Article  CAS  Google Scholar 

  18. Saito T, Nakatsuji N (2001) Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 240(1):237–246

    Article  CAS  Google Scholar 

  19. Pacary E, Guillemot F (2016) Cerebral cortex electroporation to study projection neuron migration. Curr Protoc Neurosci 77:2-26-21-22-26 18. https://doi.org/10.1002/cpns.13

    Article  Google Scholar 

  20. dal Maschio M, Ghezzi D, Bony G, Alabastri A, Deidda G, Brondi M, Sato SS, Zaccaria RP, Di Fabrizio E, Ratto GM, Cancedda L (2012) High-performance and site-directed in utero electroporation by a triple-electrode probe. Nat Commun 3:960. https://doi.org/10.1038/ncomms1961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Bai J, Ramos RL, Paramasivam M, Siddiqi F, Ackman JB, LoTurco JJ (2008) The role of DCX and LIS1 in migration through the lateral cortical stream of developing forebrain. Dev Neurosci 30(1-3):144–156

    Article  CAS  Google Scholar 

  22. Soma M, Aizawa H, Ito Y, Maekawa M, Osumi N, Nakahira E, Okamoto H, Tanaka K, Yuasa S (2009) Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation. J Comp Neurol 513(1):113–128

    Article  CAS  Google Scholar 

  23. Nakahira E, Yuasa S (2005) Neuronal generation, migration, and differentiation in the mouse hippocampal primordium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation. J Comp Neurol 483(3):329–340

    Article  Google Scholar 

  24. Navarro-Quiroga I, Chittajallu R, Gallo V, Haydar TF (2007) Long-term, selective gene expression in developing and adult hippocampal pyramidal neurons using focal in utero electroporation. J Neurosci 27(19):5007–5011

    Article  CAS  Google Scholar 

  25. Garcia-Frigola C, Carreres MI, Vegar C, Herrera E (2007) Gene delivery into mouse retinal ganglion cells by in utero electroporation. BMC Dev Biol 7:103

    Article  Google Scholar 

  26. Petros TJ, Rebsam A, Mason CA (2009) In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells. J Vis Exp (31). https://doi.org/10.3791/1333

  27. Borrell V, Yoshimura Y, Callaway EM (2005) Targeted gene delivery to telencephalic inhibitory neurons by directional in utero electroporation. J Neurosci Methods 143(2):151–158

    Article  CAS  Google Scholar 

  28. Castro DS, Martynoga B, Parras C, Ramesh V, Pacary E, Johnston C, Drechsel D, Lebel-Potter M, Garcia LG, Hunt C, Dolle D, Bithell A, Ettwiller L, Buckley N, Guillemot F (2011) A novel function of the proneural factor Ascl1 in progenitor proliferation identified by genome-wide characterization of its targets. Genes Dev 25(9):930–945

    Article  CAS  Google Scholar 

  29. Rouaux C, Arlotta P (2010) Fezf2 directs the differentiation of corticofugal neurons from striatal progenitors in vivo. Nat Neurosci 13(11):1345–1347

    Article  CAS  Google Scholar 

  30. Gelman DM, Martini FJ, Nobrega-Pereira S, Pierani A, Kessaris N, Marin O (2009) The embryonic preoptic area is a novel source of cortical GABAergic interneurons. J Neurosci 29(29):9380–9389

    Article  CAS  Google Scholar 

  31. Bonnin A, Torii M, Wang L, Rakic P, Levitt P (2007) Serotonin modulates the response of embryonic thalamocortical axons to netrin-1. Nat Neurosci 10(5):588–597

    Article  CAS  Google Scholar 

  32. Kataoka A, Shimogori T (2008) Fgf8 controls regional identity in the developing thalamus. Development 135(17):2873–2881. https://doi.org/10.1242/dev.021618

    Article  CAS  PubMed  Google Scholar 

  33. Haddad-Tovolli R, Szabo NE, Zhou X, Alvarez-Bolado G (2013) Genetic manipulation of the mouse developing hypothalamus through in utero electroporation. J Vis Exp (77). https://doi.org/10.3791/50412

  34. Willett RT, Greene LA (2011) Gata2 is required for migration and differentiation of retinorecipient neurons in the superior colliculus. J Neurosci 31(12):4444–4455

    Article  CAS  Google Scholar 

  35. Kawauchi D, Taniguchi H, Watanabe H, Saito T, Murakami F (2006) Direct visualization of nucleogenesis by precerebellar neurons: involvement of ventricle-directed, radial fibre-associated migration. Development 133(6):1113–1123

    Article  CAS  Google Scholar 

  36. Nishiyama J, Hayashi Y, Nomura T, Miura E, Kakegawa W, Yuzaki M (2012) Selective and regulated gene expression in murine Purkinje cells by in utero electroporation. Eur J Neurosci 36(7):2867–2876

    Article  Google Scholar 

  37. Okada T, Keino-Masu K, Masu M (2007) Migration and nucleogenesis of mouse precerebellar neurons visualized by in utero electroporation of a green fluorescent protein gene. Neurosci Res 57(1):40–49

    Article  CAS  Google Scholar 

  38. Fukuchi-Shimogori T, Grove EA (2001) Neocortex patterning by the secreted signaling molecule FGF8. Science 294(5544):1071–1074

    Article  CAS  Google Scholar 

  39. Pacary E, Azzarelli R, Guillemot F (2013) Rnd3 coordinates early steps of cortical neurogenesis through actin-dependent and -independent mechanisms. Nat Commun 4:1635. https://doi.org/10.1038/ncomms2614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Quan XJ, Yuan L, Tiberi L, Claeys A, De Geest N, Yan J, van der Kant R, Xie WR, Klisch TJ, Shymkowitz J, Rousseau F, Bollen M, Beullens M, Zoghbi HY, Vanderhaeghen P, Hassan BA (2016) Post-translational control of the temporal dynamics of transcription factor activity regulates neurogenesis. Cell 164(3):460–475. https://doi.org/10.1016/j.cell.2015.12.048

    Article  CAS  PubMed  Google Scholar 

  41. Kitazawa A, Kubo K, Hayashi K, Matsunaga Y, Ishii K, Nakajima K (2014) Hippocampal pyramidal neurons switch from a multipolar migration mode to a novel “climbing” migration mode during development. J Neurosci 34(4):1115–1126. https://doi.org/10.1523/JNEUROSCI.2254-13.2014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kobayashi H, Saragai S, Naito A, Ichio K, Kawauchi D, Murakami F (2015) Calm1 signaling pathway is essential for the migration of mouse precerebellar neurons. Development 142(2):375–384. https://doi.org/10.1242/dev.112680

    Article  CAS  PubMed  Google Scholar 

  43. Cubelos B, Sebastian-Serrano A, Beccari L, Calcagnotto ME, Cisneros E, Kim S, Dopazo A, Alvarez-Dolado M, Redondo JM, Bovolenta P, Walsh CA, Nieto M (2010) Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex. Neuron 66(4):523–535

    Article  CAS  Google Scholar 

  44. Mire E, Hocine M, Bazellieres E, Jungas T, Davy A, Chauvet S, Mann F (2018) Developmental upregulation of ephrin-B1 silences Sema3C/neuropilin-1 signaling during post-crossing navigation of corpus callosum axons. Curr Biol 28(11):1768–1782. https://doi.org/10.1016/j.cub.2018.04.026

    Article  CAS  PubMed  Google Scholar 

  45. Mire E, Mezzera C, Leyva-Diaz E, Paternain AV, Squarzoni P, Bluy L, Castillo-Paterna M, Lopez MJ, Peregrin S, Tessier-Lavigne M, Garel S, Galceran J, Lerma J, Lopez-Bendito G (2012) Spontaneous activity regulates Robo1 transcription to mediate a switch in thalamocortical axon growth. Nat Neurosci 15(8):1134–1143. https://doi.org/10.1038/nn.3160

    Article  CAS  PubMed  Google Scholar 

  46. Namba T, Kibe Y, Funahashi Y, Nakamuta S, Takano T, Ueno T, Shimada A, Kozawa S, Okamoto M, Shimoda Y, Oda K, Wada Y, Masuda T, Sakakibara A, Igarashi M, Miyata T, Faivre-Sarrailh C, Takeuchi K, Kaibuchi K (2014) Pioneering axons regulate neuronal polarization in the developing cerebral cortex. Neuron 81(4):814–829. https://doi.org/10.1016/j.neuron.2013.12.015

    Article  CAS  PubMed  Google Scholar 

  47. Kwon HB, Kozorovitskiy Y, Oh WJ, Peixoto RT, Akhtar N, Saulnier JL, Gu C, Sabatini BL (2012) Neuroligin-1-dependent competition regulates cortical synaptogenesis and synapse number. Nat Neurosci 15(12):1667–1674. https://doi.org/10.1038/nn.3256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Garcia-Marques J, Lopez-Mascaraque L (2013) Clonal identity determines astrocyte cortical heterogeneity. Cereb Cortex 23(6):1463–1472. https://doi.org/10.1093/cercor/bhs134

    Article  PubMed  Google Scholar 

  49. Subramanian L, Sarkar A, Shetty AS, Muralidharan B, Padmanabhan H, Piper M, Monuki ES, Bach I, Gronostajski RM, Richards LJ, Tole S (2011) Transcription factor Lhx2 is necessary and sufficient to suppress astrogliogenesis and promote neurogenesis in the developing hippocampus. Proc Natl Acad Sci U S A 108(27):E265–E274

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. INSERM U1215, Neurocentre Magendie, Bordeaux, France

    Emilie Pacary

  2. Université de Bordeaux, Bordeaux, France

    Emilie Pacary

  3. The Francis Crick Institute, London, UK

    François Guillemot

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  1. Emilie Pacary
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  2. François Guillemot
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Correspondence to Emilie Pacary .

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  1. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Simon G. Sprecher

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Pacary, E., Guillemot, F. (2020). In Utero Electroporation to Study Mouse Brain Development. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 2047. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9732-9_29

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  • DOI: https://doi.org/10.1007/978-1-4939-9732-9_29

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9731-2

  • Online ISBN: 978-1-4939-9732-9

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