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Abstract

Introduction

Protocol

Representative Results

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Materials

References

Neuroscience

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

Published: March 8th, 2024

DOI:

10.3791/66631

1Department of Anatomy, Keio University School of Medicine, 2Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

During the development of the cerebral cortex, (apical) radial glia in the pallial ventricular zone (VZ) lining the lateral ventricle produce first neurons and then glial progenitors with some overlapping period1. Neurons are also generated from intermediate progenitors or basal radial glia in the subventricular zone (SVZ) adjacent to the VZ, both of which originate from the (apical) radial glia2,3. In mice, radial glial cells produce only neurons on embryonic day (E) 12-14, both neurons and glial progenitors on E15-16, and glial progenitors from E17 onward4. The....

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The present study was performed with the approval of and following the guidelines of the Animal Care and Use Committee of the Institute for Developmental Research, Aichi Developmental Disability Center (#2019-013), and Keio University (A2021-030). Timed pregnant ICR (wild-type) mice were obtained commercially (see Table of Materials). To observe the relationship between migrating cells and blood vessels, Flt1-DsRed mice, in which the endothelial cells express DsRed22, were us.......

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Radial glial cells in the pallial VZ produce only neurons until E14, and both neurons and glial cells at E15 and E16. To observe the migratory behaviors of neurons and glial cells simultaneously, we labeled them with enhanced GFP (EGFP) and RFP, respectively, by using a neuron-specific promoter, Tα1 promoter27, and human glial fibrillary acidic protein (hGFAP) promoter28, which is preferentially activated in astrocytes. Astrocyte progenitors repeat cell divisi.......

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This protocol introduced a method for the time-lapse observation of cells derived from the pallial (cortical) VZ. To label the migrating cells from the VZ, we used in utero electroporation, in which individual cells were clearly labeled with a higher signal-to-noise ratio than in viral vector-mediated labeling. Using in utero electroporation, any type of vector in any combination can be easily introduced into the radial glial cells (neural stem cells) in living embryos. Neurons and glial progenitors can.......

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Tα1 promoter is a gift from P. Barker and F.D. Miller. Dcx promoter is a gift from Q. Lu. hGFAP-Cre was a gift from Albee Messing. The PiggyBac transposon vector system was provided by the Sanger Institute. Flt1-DsRed mice were provided by M. Ema (Shiga University). This work was supported by JSPS KAKENHI (Grant Number JP21K07309 to H. Tabata, JP20H05688 and JP22K19365 to K. Nakajima) and Takeda Science Foundation, Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research, Keio Gijuku Academic Development Funds to K. Nakajima.

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NameCompanyCatalog NumberComments
Aspirator tube assemblyDrummond2-040-000
Atipamezole (5 mg/mL)MeijiMepatia
AutoclipBecton Dickinson4276309 mm
B27 supplementGibco17504-044
Butorphanol (5 mg/mL)MeijiVetorphale
Cell culture insertMilliporePICM ORG 50
Confocal microscopeNikonA1RHD25Equipped with a long working distance lens (S Plan Fluor ELWD 20XC)
CryomoldTissue-Tek4566
Culture chamberTokkenTK-NBCMPCustom-made
ElectroporatorNEPA GeneNEPA21
Fast GreenSigma-AldrichF7258
Gas mixerTokkenTK-MIGM01-02
Glass base dishIwaki3910-035Diameter of glass base is 27 mm
Glass capillariesNarishigeGD-1
HBS (2x)Sigma-Aldrich51558
HBSS(-)Wako084-08345
Heater UnitTokkenTK-0003HU20Custom-made, including hood and heater
hGFAP-CreAddgene#40591A gift from Albee Messing
ImageJhttps://imagej.net/ij/
L-glutamine (200 mM)Gibco25030
Low melting temperature agaroseLonza50100
Medetomidine (1 mg/mL)MeijiMedetomin
MicroinjectorNarishigeIM-300
Midazolam (5 mg/mL)SandozMidazolam
MTrackJhttps://imagescience.org/meijering/software/mtrackj/
Neurobasal mediumGibco21103-049
pCAG-hyPBaseThe hyPBase cDNA from pCMV-hyPBase (a gift from Sanger Institute) was inserted into the downstream of the CAG promoter of pCAGGS (a gift from J. Miyazaki).
pDcx-DreThe Dcx promoter from Dcx4kbEGFP70 (a gift from Q. Lu) was exchanged with CAG promoter of pCAG-NLS-HA-Dre34 (a gift from Pawel Pelczar, Addgene #51272).
Penicillin + StreptomycinGibco15140122
Plasmid purification kitInvitrogenPureLink HiPure plasmid midiprep kit (K210005)
pPB-CAG-LNL-RFPCAG-LNL cassette from pCALNL-DsRed (a gift from Connie Cepko, Addgene #13769), and TurboRFP cDNA (Evrogen, FP232) were inserted into the cloning site of pPB-CAG.EBNXN (a gift from Sanger Institute).
pPB-CAG-rDIO-EGFPThe sequence containning synthetic rox sites, synthetic DIO cassette, and EGFP cDNA from pEGFP-N1 (Clontech, U55762) in reverse direction  were inserted into the cloning site of pPB-CAG.EBNXN (a gift from Sanger Institute). The sequence is provided in the Supplementary File.
PullerNarishigePN-31
StackReda plugin for ImageJhttp://bigwww.epfl.ch/thevenaz/stackreg/
Suture needleNazmeC-24-521-R No.11/2 circle, length 14 mm
Suture threadNazmeC-23-B2Silk, size 5-0
Timed pregnant ICR (wild-type) miceJapan SLCICR mouse
TrackMatehttps://imagej.net/plugins/trackmate/index
Tweezer-type electrodeBEX or NEPA GeneCUY650P5 
Tα1-EGFPEGFP cDNA from pEGFP-N1 (Clontech, U55762) was inserted into the downstream of the Tα1 promoter in plasmid 253 (a gift from P. Barker and F.D.Miller)
Vibrating microtomeLeica or ZeissVibrating blade microtome VT1000S or Hyrax V50.

  1. Kriegstein, A., Alvarez-Buylla, A. The glial nature of embryonic and adult neural stem cells. Annu Rev Neurosci. 32 (1), 149-184 (2009).
  2. Noctor, S. C., Martínez-Cerdeño, V., Ivic, L., Kriegstein, A. R. Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases. Nat Neurosci. 7 (2), 136-144 (2004).
  3. Haubensak, W., Attardo, A., Denk, W., Huttner, W. B. Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: A major site of neurogenesis. P Natl Acad Sci USA. 101 (9), 3196-3201 (2004).
  4. Yoshida, A., Yamaguchi, Y., Nonomura, K., Kawakami, K., Takahashi, Y., Miura, M. 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 (2010).
  5. Tabata, H., et al. Erratic and blood vessel-guided migration of astrocyte progenitors in the cerebral cortex. Nat Commun. 13 (1), 6571 (2022).
  6. Tabata, H., Nakajima, K. Multipolar Migration: The third mode of radial neuronal migration in the developing cerebral cortex. J Neurosci. 23 (31), 9996-10001 (2003).
  7. Tabata, H., Kanatani, S., Nakajima, K. Differences of migratory behavior between direct progeny of apical progenitors and basal progenitors in the developing cerebral cortex. Cereb Cortex. 19 (9), 2092-2105 (2009).
  8. Rakic, P. Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol. 145 (1), 61-83 (1972).
  9. Sekine, K., Honda, T., Kawauchi, T., Kubo, K., Nakajima, K. The outermost region of the developing cortical plate is crucial for both the switch of the radial migration mode and the Dab1-dependent "inside-out" lamination in the neocortex. J Neurosci. 31 (25), 9426-9439 (2011).
  10. Shin, M., et al. Both excitatory and inhibitory neurons transiently form clusters at the outermost region of the developing mammalian cerebral neocortex. J Comp Neurol. 527 (10), 1577-1597 (2019).
  11. Sekine, K., et al. Reelin controls neuronal positioning by promoting cell-matrix adhesion via inside-out activation of integrin α5β1. Neuron. 76 (2), 353-369 (2012).
  12. Morimoto, K., Tabata, H., Takahashi, R., Nakajima, K. Interactions between neural cells and blood vessels in central nervous system development. BioEssays. 230091, (2023).
  13. Tabata, H., Nagata, K. Decoding the molecular mechanisms of neuronal migration using in utero electroporation. Med Mol Morphol. 49 (2), 63-75 (2016).
  14. Ishii, K., Kubo, K., Nakajima, K. Reelin and neuropsychiatric disorders. Front Cell Neurosci. 10, 229 (2016).
  15. Bosworth, A. P., Allen, N. J. The diverse actions of astrocytes during synaptic development. Curr Opin Neurobiol. 47, 38-43 (2017).
  16. Tabata, H. Crosstalk between blood vessels and glia during the central nervous system development. Life. 12 (11), 1761 (2022).
  17. Wiegreffe, C., Feldmann, S., Gaessler, S., Britsch, S. Time-lapse confocal imaging of migrating neurons in organotypic slice culture of embryonic mouse brain using in utero electroporation. J Vis Exp. (125), e55886 (2017).
  18. Saito, T., Nakatsuji, N. Efficient Gene Transfer into the Embryonic Mouse Brain Using in Vivo Electroporation. Dev Biol. 240 (1), 237-246 (2001).
  19. Tabata, H., Nakajima, K. Labeling embryonic mouse central nervous system cells by in utero electroporation. Dev Growth Differ. 50 (6), 507-511 (2008).
  20. Tabata, H., Nakajima, K. Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex. Neuroscience. 103 (4), 865-872 (2001).
  21. Fukuchi-Shimogori, T. Neocortex patterning by the secreted signaling molecule FGF8. Science. 294 (5544), 1071-1074 (2001).
  22. Matsumoto, K., et al. Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice. Genesis. 50 (7), 561-571 (2012).
  23. Kawai, S., Takagi, Y., Kaneko, S., Kurosawa, T. Effect of three types of mixed anesthetic agents alternate to ketamine in mice. Exp Anim. 60 (5), 481-487 (2011).
  24. Meijering, E., Dzyubachyk, O., Smal, I. Methods for cell and particle tracking. Methods Enzymol. 504, 183-200 (2012).
  25. Ershov, D., et al. TrackMate 7: Integrating state-of-the-art segmentation algorithms into tracking pipelines. Nat Methods. 19 (7), 829-832 (2022).
  26. Tinevez, J. -. Y., et al. TrackMate: An open and extensible platform for single-particle tracking. Methods. 115, 80-90 (2017).
  27. Gloster, A., et al. The T alpha 1 alpha-tubulin promoter specifies gene expression as a function of neuronal growth and regeneration in transgenic mice. J Neurosci. 14 (12), 7319-7330 (1994).
  28. Zhuo, L., et al. hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo. Genesis. 31 (2), 85-94 (2001).
  29. Yusa, K., Zhou, L., Li, M. A., Bradley, A., Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. P Natl Acad Sci USA. 108 (4), 1531-1536 (2011).
  30. Chen, F., LoTurco, J. A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition. J Neurosci Meth. 207 (2), 172-180 (2012).
  31. Wang, X., Qiu, R., Tsark, W., Lu, Q. Rapid promoter analysis in developing mouse brain and genetic labeling of young neurons by doublecortin-DsRed-express. J Neurosci Res. 85 (16), 3567-3573 (2007).
  32. Sauer, B. DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages. Nucleic Acids Res. 32 (20), 6086-6095 (2004).
  33. Hermann, M., et al. Binary recombinase systems for high-resolution conditional mutagenesis. Nucleic Acids Res. 42 (6), 3894-3907 (2014).
  34. Kanatani, S., et al. The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain. P Natl Acad Sci USA. 112 (36), E4985-E4994 (2015).
  35. Yozu, M., Tabata, H., Nakajima, K. The caudal migratory stream: A novel migratory stream of interneurons derived from the caudal ganglionic eminence in the developing mouse forebrain. J Neurosci. 25 (31), 7268-7277 (2005).
  36. Kanatani, S., Yozu, M., Tabata, H., Nakajima, K. COUP-TFII is preferentially expressed in the caudal ganglionic eminence and is involved in the caudal migratory stream. J Neurosci. 28 (50), 13582-13591 (2008).
  37. Kitazawa, A., et al. Hippocampal pyramidal neurons switch from a multipolar migration mode to a novel "climbing" migration mode during development. J Neurosci. 34 (4), 1115-1126 (2014).

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