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A new approach to manipulate the fate of single neural stem cells in tissue

Nature Neuroscience volume 15, pages 329–337 (2012)Cite this article

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Abstract

A challenge in the field of neural stem cell biology is the mechanistic dissection of single stem cell behavior in tissue. Although such behavior can be tracked by sophisticated imaging techniques, current methods of genetic manipulation do not allow researchers to change the level of a defined gene product on a truly acute time scale and are limited to very few genes at a time. To overcome these limitations, we established microinjection of neuroepithelial/radial glial cells (apical progenitors) in organotypic slice culture of embryonic mouse brain. Microinjected apical progenitors showed cell cycle parameters that were indistinguishable to apical progenitors in utero, underwent self-renewing divisions and generated neurons. Microinjection of single genes, recombinant proteins or complex mixtures of RNA was found to elicit acute and defined changes in apical progenitor behavior and progeny fate. Thus, apical progenitor microinjection provides a new approach to acutely manipulating single neural stem and progenitor cells in tissue.

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Figure 1: Microinjection of apical progenitors in brain slices.
Figure 2: Microinjection of a tracer into E13.5 hindbrain apical progenitors reveals their asymmetric division.
Figure 3: Microinjection of apical progenitors does not alter their cell cycle parameters.
Figure 4: Microinjection of apical progenitors with a plasmid encoding a transcription factor causes a change in gene expression.
Figure 5: Detection of a single translation product in apical progenitors microinjected with a poly-A+ mRNA pool.
Figure 6: Microinjection of poly-A+ mRNA from neural stem cells alters apical progenitor fate.
Figure 7: Microinjection of cdc42T17N into apical progenitors alters centrosome localization and induces apical progenitor delamination.

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Acknowledgements

We are grateful to R.F. Hevner for kindly providing the Tbr2-GFP mouse line, T. Miyata for pCS2-Ngn2, E. Tanaka for pCAGGS-Cherry and C. Eckmann for pTNT and helpful discussion. We thank J. Helppi and other members of the animal facility, as well as H. Wolf of the workshop, of the Max Planck Institute of Molecular Cell Biology and Genetics for excellent support, A. Ettinger for advice with NS-5 cells, K. Saito for advice with retina slice culture, J.F. Fei and Y.J. Chang for experimental advice, Y. Arai and J. Pulvers for discussion, and A.-M. Marzesco, F. Mora-Bermudéz, E. Paluch and F.K. Wong 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.

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Authors and Affiliations

  1. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Elena Taverna, Christiane Haffner & Wieland B Huttner

  2. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Rainer Pepperkok

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  1. Elena Taverna
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Contributions

E.T. designed and performed all of the microinjections and most of the other experimental work, analyzed the data and wrote the manuscript. C.H. performed experiments. R.P. taught the microinjection technique to E.T. W.B.H. supervised the project, analyzed the data and wrote the manuscript.

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Correspondence to Wieland B Huttner.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 1 (PDF 3775 kb)

Supplementary Video 1

The microinjection procedure. (AVI 5426 kb)

Supplementary Video 2

Microinjected hindbrain APs maintain contact with the ventricular surface and the basal lamina. (AVI 2397 kb)

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Taverna, E., Haffner, C., Pepperkok, R. et al. A new approach to manipulate the fate of single neural stem cells in tissue. Nat Neurosci 15, 329–337 (2012). https://doi.org/10.1038/nn.3008

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