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In vivo pulse labeling of isochronic cohorts of cells in the central nervous system using FlashTag

Nature Protocols volume 13, pages 2297–2311 (2018)Cite this article

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Abstract

The tracing of neuronal cell lineages is critical to our understanding of cellular diversity in the CNS. This protocol describes a fluorescence birth-dating technique to label, track and isolate isochronic cohorts of newborn cells in the CNS in vivo in mouse embryos. Injection of carboxyfluorescein esters (CFSEs) into the cerebral ventricle allows pulse labeling of mitotic (M phase) ventricular zone (VZ) progenitors and their progeny across the CNS, a procedure we termed FlashTag. Specificity for M-phase apical progenitors is a result of the somata of these cells transiently contacting the ventricular wall during this cell-cycle phase, exposing them to CFSE injected into the cerebrospinal fluid. Using this approach, the developmental trajectory of progenitors and their daughter neurons can be tracked. Labeled cells can be imaged ex vivo or in fixed tissue, targeted for electrophysiological experiments or isolated using FACS for cell culture or (single-cell) RNA sequencing. Multiple embryos can be labeled within 30 min. The dye is retained for several weeks, allowing labeled cells to be identified postnatally. This protocol describes the labeling procedure using in utero injection, the isolation of live cells using FACS and the processing of labeled tissue for immunohistochemistry.

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Fig. 1: FlashTag specifically labels M-phase VZ progenitors.
Fig. 2: Brightly labeled FT neurons (FT+) are directly born from VZ progenitors.
Fig. 3: Comparison of BrdU, FT and in utero electroporation pulse labeling.
Fig. 4: Labeling of remotely migrating cells.
Fig. 5: Detection of FT labeling in fixed tissue.
Fig. 6: Pipette preparation and surgical procedure.
Fig. 7: Detection of FT labeling in live tissue.

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Acknowledgements

We thank the members of our laboratory for helpful discussions. We also thank A. Benoit and M. Lanzillo for technical assistance, L. Telley for his contribution to initial discussions and R. Wagener for providing the photomicrograph in Fig. 4. Work in the Jabaudon laboratory is supported by the Swiss National Science Foundation, the Fondation des Hôpitaux Universitaires de Genève and the Brain and Behavior Foundation. S.G. was supported in part by a grant from iGE3.

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  1. Subashika Govindan

    Present address: Interfaculty Institute of Bioengineering, EPFL, Lausanne, Switzerland

Authors and Affiliations

  1. Department of Basic Neurosciences, University of Geneva, Geneva, Switzerland

    Subashika Govindan, Polina Oberst & Denis Jabaudon

  2. Clinic of Neurology, Geneva University Hospital, Geneva, Switzerland

    Denis Jabaudon

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  1. Subashika Govindan
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  2. Polina Oberst
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Contributions

S.G. and D.J. developed the initial protocol, which was later updated by members of the laboratory, including P.O. S.G., P.O. and D.J. wrote the manuscript. S.G. performed the experiments.

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Correspondence to Denis Jabaudon.

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The authors declare no competing interests.

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Reference describing the development of the approach

1. Telley L. et al. Science 351, 1443–1446 (2016): https://doi.org/10.1126/science.aad8361

Integrated supplementary information

Supplementary Figure 1 Gating strategies for FAC-sorting of FT-labeled cells.

(a) Side scatter (SSC) and forward scatter (FSC) are used to identify singlet cells. Debris and doublets are excluded based on their granularity and size. (b) Draq7 dye is added to the cell suspension to identify live cells. Dead and dying cells are excluded based on strong Draq7 signal intensity. (c) CFSE intensity of cells 6h after labeling. Only the top 5% of cells are collected. Cells with low intensity likely correspond to cells that were labeled through diffusion of CFSE into the tissue and are therefore excluded. (d) CFSE intensity of cells 12h after labeling. Only the top 5% of cells are collected. (e) CFSE intensity of cells 24h after labeling. Note that the overall signal intensity is decreased compared to earlier collection time-points. Only cells with high signal intensity (top 5%) are collected. Cutoff values are determined based on pre-validation with chronic BrdU co-perfusion (see main text and Fig. 2). For all experiments involving animals appropriate institutional regulatory board permission was obtained.

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Govindan, S., Oberst, P. & Jabaudon, D. In vivo pulse labeling of isochronic cohorts of cells in the central nervous system using FlashTag. Nat Protoc 13, 2297–2311 (2018). https://doi.org/10.1038/s41596-018-0038-1

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