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Microinjection of membrane-impermeable molecules into single neural stem cells in brain tissue

Abstract

This microinjection protocol allows the manipulation and tracking of neural stem and progenitor cells in tissue at single-cell resolution. We demonstrate how to apply microinjection to organotypic brain slices obtained from mice and ferrets; however, our technique is not limited to mouse and ferret embryos, but provides a means of introducing a wide variety of membrane-impermeable molecules (e.g., nucleic acids, proteins, hydrophilic compounds) into neural stem and progenitor cells of any developing mammalian brain. Microinjection experiments are conducted by using a phase-contrast microscope equipped with epifluorescence, a transjector and a micromanipulator. The procedure normally takes 2 h for an experienced researcher, and the entire protocol, including tissue processing, can be performed within 1 week. Thus, microinjection is a unique and versatile method for changing and tracking the fate of a cell in organotypic slice culture.

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Figure 1: Timeline of the microinjection protocol.
Figure 2: Flow scheme of the microinjection procedure of organotypic slices of embryonic telencephalon followed by slice culture.
Figure 3: Microinjection of organotypic brain slices: setup.
Figure 4: Characterization of microinjected cells and their progeny in organotypic brain slices at 0 h and 24 h after microinjection.
Figure 5: Acute manipulation via microinjection.
Figure 6: Microinjection of organotypic ferret brain slices.

References

  1. 1

    Costa, M.R. et al. Continuous live imaging of adult neural stem cell division and lineage progression in vitro. Development 138, 1057–1068 (2011).

    CAS  Article  Google Scholar 

  2. 2

    Saito, K. et al. Morphological asymmetry in dividing retinal progenitor cells. Dev. Growth Differ. 45, 219–229 (2003).

    Article  Google Scholar 

  3. 3

    Miyata, T. et al. Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells. Development 131, 3133–3145 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Takahashi, M., Sato, K., Nomura, T. & Osumi, N. Manipulating gene expressions by electroporation in the developing brain of mammalian embryos. Differentiation 70, 155–162 (2002).

    CAS  Article  Google Scholar 

  5. 5

    Calegari, F., Haubensak, W., Yang, D., Huttner, W.B. & Buchholz, F. Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA. Proc. Natl. Acad. Sci. USA 99, 14236–14240 (2002).

    CAS  Article  Google Scholar 

  6. 6

    Molotkov, D.A., Yukin, A.Y., Afzalov, R.A. & Khiroug, L.S. Gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation. J. Vis. Exp. 43, 2244 (2010).

    Google Scholar 

  7. 7

    Borrell, V. In vivo gene delivery to the postnatal ferret cerebral cortex by DNA electroporation. J. Neurosci. Methods 186, 186–195 (2010).

    CAS  Article  Google Scholar 

  8. 8

    Kawasaki, H., Toda, T. & Tanno, K. In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation. Biol. Open 2, 95–100 (2013).

    Article  Google Scholar 

  9. 9

    Kawasaki, H., Iwai, L. & Tanno, K. Rapid and efficient genetic manipulation of gyrencephalic carnivores using in utero electroporation. Mol. Brain 5, 24 (2012).

    Article  Google Scholar 

  10. 10

    Sessa, A., Mao, C.A., Hadjantonakis, A.K., Klein, W.H. & Broccoli, V. Tbr2 directs conversion of radial glia into basal precursors and guides neuronal amplification by indirect neurogenesis in the developing neocortex. Neuron 60, 56–69 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Farkas, L.M. et al. Insulinoma-associated 1 has a panneurogenic role and promotes the generation and expansion of basal progenitors in the developing mouse neocortex. Neuron 60, 40–55 (2008).

    CAS  Article  Google Scholar 

  12. 12

    Fietz, S.A. & Huttner, W.B. Cortical progenitor expansion, self-renewal and neurogenesis—a polarized perspective. Curr. Opin. Neurobiol. 21, 23–35 (2011).

    CAS  Article  Google Scholar 

  13. 13

    Lui, J.H., Hansen, D.V. & Kriegstein, A.R. Development and evolution of the human neocortex. Cell 146, 18–36 (2011).

    CAS  Article  Google Scholar 

  14. 14

    Borrell, V. & Reillo, I. Emerging roles of neural stem cells in cerebral cortex development and evolution. Dev. Neurobiol. 72, 955–971 (2012).

    Article  Google Scholar 

  15. 15

    Rosa, P. et al. An antibody against secretogranin I (chromogranin B) is packaged into secretory granules. J. Cell Biol. 109, 17–34 (1989).

    CAS  Article  Google Scholar 

  16. 16

    Pepperkok, R., Saffrich, R. & Ansorge, W. Computer-automated capillary microinjection of macromolecules into living cells. In Cell Biology: A Laboratory Handbook, Vol. 3 (ed. Celis, J.E.) 22–30 (Academic Press, 1994).

  17. 17

    Taverna, E., Haffner, C., Pepperkok, R. & Huttner, W.B. A new approach to manipulate the fate of single neural stem cells in tissue. Nat. Neurosci. 15, 329–337 (2012).

    CAS  Article  Google Scholar 

  18. 18

    Götz, M. & Huttner, W.B. The cell biology of neurogenesis. Nat. Rev. Mol. Cell Biol. 6, 777–788 (2005).

    Article  Google Scholar 

  19. 19

    Echeverri, K., Clarke, J.D. & Tanaka, E.M. In vivo imaging indicates muscle fiber dedifferentiation is a major contributor to the regenerating tail blastema. Dev. Biol. 236, 151–164 (2001).

    CAS  Article  Google Scholar 

  20. 20

    Echeverri, K. & Tanaka, E.M. Electroporation as a tool to study in vivo spinal cord regeneration. Dev. Dyn. 226, 418–425 (2003).

    CAS  Article  Google Scholar 

  21. 21

    Arai, Y. et al. Neural stem and progenitor cells shorten S-phase on commitment to neuron production. Nat. Commun. 2, 154 (2011).

    Article  Google Scholar 

  22. 22

    Takahashi, T., Nowakowski, R.S. & Caviness, V.S. Jr. The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall. J. Neurosci. 15, 6046–6057 (1995).

    CAS  Article  Google Scholar 

  23. 23

    Reillo, I. & Borrell, V. Germinal zones in the developing cerebral cortex of ferret: ontogeny, cell cycle kinetics, and diversity of progenitors. Cereb. Cortex 22, 2039–2054 (2012).

    Article  Google Scholar 

  24. 24

    Lukaszewicz, A. et al. G1 phase regulation, area-specific cell cycle control, and cytoarchitectonics in the primate cortex. Neuron 47, 353–364 (2005).

    CAS  Article  Google Scholar 

  25. 25

    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. Proc. Natl. Acad. Sci. USA 101, 3196–3201 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Miyata, T., Kawaguchi, A., Saito, K., Kuramochi, H. & Ogawa, M. Visualization of cell cycling by an improvement in slice culture methods. J. Neurosci. Res. 69, 861–868 (2002).

    CAS  Article  Google Scholar 

  27. 27

    Ansorge, W. & Pepperkok, R. Performance of an automated system for capillary microinjection into living cells. J. Biochem. Biophys. Meth. 16, 283–292 (1988).

    CAS  Article  Google Scholar 

  28. 28

    Pulvers, J.N. & Huttner, W.B. Brca1 is required for embryonic development of the mouse cerebral cortex to normal size by preventing apoptosis of early neural progenitors. Development 136, 1859–1868 (2009).

    CAS  Article  Google Scholar 

  29. 29

    Minobe, S. et al. Rac is involved in the interkinetic nuclear migration of cortical progenitor cells. Neurosci. Res. 63, 294–301 (2009).

    CAS  Article  Google Scholar 

  30. 30

    Chen, L., Melendez, J., Campbell, K., Kuan, C.Y. & Zheng, Y. Rac1 deficiency in the forebrain results in neural progenitor reduction and microcephaly. Dev. Biol. 325, 162–170 (2009).

    CAS  Article  Google Scholar 

  31. 31

    Leone, D.P., Srinivasan, K., Brakebusch, C. & McConnell, S.K. The rho GTPase Rac1 is required for proliferation and survival of progenitors in the developing forebrain. Dev. Neurobiol. 70, 659–678 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Miyata, T., Kawaguchi, A., Okano, H. & Ogawa, M. Asymmetric inheritance of radial glial fibers by cortical neurons. Neuron 31, 727–741 (2001).

    CAS  Article  Google Scholar 

  33. 33

    Konno, D. et al. Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis. Nat. Cell Biol. 10, 93–101 (2008).

    CAS  Article  Google Scholar 

  34. 34

    Kosodo, Y. et al. Cytokinesis of neuroepithelial cells can divide their basal process before anaphase. EMBO J. 27, 3151–3163 (2008).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank J. Helppi and other members of the animal facility as well as H. Wolf of the workshop and K. Margitudis of the photo laboratory of the Max Planck Institute of Molecular Cell Biology and Genetics for excellent support; the staff of BioCrea, especially B. Langen, for ferret care and housing; M. Turrero-García for advice with ferret slice culture; F. Mora-Bermúdez for acquiring and processing the photographs of the microinjection equipment (Fig. 3a and Supplementary Fig. 1a,b); and Y.J. Chang, E. Lewitus and M. Wilsch-Bräuninger for helpful comments on the manuscript. W.B.H. was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (SFB 655, A2; TRR 83, Tp6) and the European Research Council (250197), by the DFG-funded Center for Regenerative Therapies Dresden, and by the Fonds der Chemischen Industrie.

Author information

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Authors

Contributions

F.K.W. and E.T. designed and performed all microinjections and most other experimental work, analyzed the data and wrote the manuscript; C.H. performed experiments; E.T. and W.B.H. supervised the project and wrote the manuscript.

Corresponding authors

Correspondence to Wieland B Huttner or Elena Taverna.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Microinjection pipette optimization.

Comparison between bad and good microinjection pipette. A good microinjection pipette has longer taper (a, b, arrows) and smaller tip (c, note the smaller angle), as compared to a bad one.

Supplementary Figure 2 Collagen embedding.

Comparison between optimal and suboptimal collagen embedding. Top row: photographs of glass-bottom Petri dishes containing organotypic slices embedded with too little, an optimal amount, or too much collagen. Bottom row: diagram illustrating optimal versus suboptimal collagen embedding (side view of Petri dish). Note the difference in the amount of collagen solution. All animal studies were conducted in accordance with the German animal welfare legislation.

Supplementary information

Supplementary Figure 1

Microinjection pipette optimization. (PDF 3869 kb)

Supplementary Figure 2

Collagen embedding. (PDF 38091 kb)

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Wong, F., Haffner, C., Huttner, W. et al. Microinjection of membrane-impermeable molecules into single neural stem cells in brain tissue. Nat Protoc 9, 1170–1182 (2014). https://doi.org/10.1038/nprot.2014.074

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