Abstract
Adult neural stem cells (aNSCs) in zebrafish produce mature neurons throughout their entire life span in both the intact and regenerating brain. An understanding of the behavior of aNSCs in their intact niche and during regeneration in vivo should facilitate the identification of the molecular mechanisms controlling regeneration-specific cellular events. A greater understanding of the process in regeneration-competent species may enable regeneration to be achieved in regeneration-incompetent species, including humans. Here we describe a protocol for labeling and repetitive imaging of aNSCs in vivo. We label single aNSCs, allowing nonambiguous re-identification of single cells in repetitive imaging sessions using electroporation of a red-reporter plasmid in Tg(gfap:GFP)mi2001 transgenic fish expressing GFP in aNSCs. We image using two-photon microscopy through the thinned skull of anesthetized and immobilized fish. Our protocol allows imaging every 2 d for a period of up to 1 month. This methodology allowed the visualization of aNSC behavior in vivo in their natural niche, in contrast to previously available technologies, which rely on the imaging of either dissociated cells or tissue slices. We used this protocol to follow the mode of aNSC division, fate changes and cell death in both the intact and injured zebrafish telencephalon. This experimental setup can be widely used, with minimal prior experience, to assess key factors for processes that modulate aNSC behavior. A typical experiment with data analysis takes up to 1.5 months.
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Acknowledgements
We thank J. Fisher for critical reading of the manuscript. We also gratefully acknowledge funding to J.N. from the German Research Foundation (DFG) by the SFB 870 and SPP 'Integrative Analysis of Olfaction' grants; to M.G. from the DFG by the SFB 870, SYNERGY, SPP1757 'Functional Specializations of Neuroglia as Critical Determinants of Brain Activity', ICEMED Helmholtz Alliance and ERC ChroNeuroRepair: GA no. 340793 grants; and to J.S.B. from the Fundação para a Ciência e Tecnologia, Portugal (FCT). We obtained the plasmid ubi:Cherry (used in cytoplasmic localization) as a gift from Christian Mosimann (Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland); we obtained the plasmid pCS-H2B-mRFP (used in nuclear localization) from Sean Megason (Department of Systems Biology, Harvard Medical School, Boston, MA).
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J.S.B. designed and performed live imaging experiments and wrote the manuscript; R.D.G. performed FACS experiments; M.G. designed experiments and wrote the manuscript; J.N. designed and supervised experiments and wrote the manuscript.
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Supplementary Figure 1 Effect of the animal genetic background and the skull thinning procedure on aNSCs proliferation and microglia reaction.
(a) Quantification by fluorescence activated cell sorting (FACS) of the number of proliferating NSCs (gfap:GFP+ and PCNA+), performed according to Barbosa et al 2015, in fish with the brassy or the wild type AB/EK background. Data are shown as mean ± SEM with symbols representing single animals. Mann-Whitney test was used for the statistical evaluation (n=5). (b) Quantification of the number of proliferating NSCs in fish of the brassy background without skull thinning procedure and 24h or 48h after thinning the skull. Data are shown as mean ± SEM with symbols representing single animals. Kruskal–Wallis multiple comparison test was used for the statistical evaluation (n=5). (c) to (f) Coronal hemi-sections of the zebrafish telencephalon, stained for 4C4, which labels microglia cells, 19h after injury (as described in Baumgart et al) (c), removal of the skull (d) or thinning of the skull (e) in the area above the telencephalon. Note the strong reaction of microglia after injury (c) and skull removal (d), compared to the situation after thinning of the skull (e) and in the intact brain (f). All the pictures were acquired using the same laser settings, for an appropriate comparison of 4C4 levels. Scale bars 50µm. Dotted line outlines telencephalic hemisphere in (c) to (f).
Supplementary Figure 2 Immunostaining in the whole brain using the BABB clearing method.
(a) Example of a whole brain in the Tg(gfap:GFP) line, dissected, stained (see antibodies used in Table 2) and cleared after electroporation with TdTomatomem plasmid. Note the high amount of electroporated cells at the ventricle with a radial morphology typical of aNSCs in this system. (b) to (d) Examples of three types of plasmid used, in which the red fluorescent proteins are localized at the plasma membrane (b), nucleus (c) or the cytoplasm (d) of the cells. Abbreviation: dpe-days post electroporation. Scale bars b-d 20µm.
Supplementary Figure 3 Preparation of a sponge to hold the fish for injection and electroporation procedures.
(a) Using a normal sponge (the ones made for dish washing) make a longitudinal cut with a scalpel. (b) Place the fish within the cut.
Supplementary Figure 4 Skull thinning procedure.
(a)-(e) Dorsal view of the telencephalon before (a), at different steps during (b-d) the skull thinning procedure and after the procedure (e). Note that the skull is gradually more exposed, and the surface above the brain turns from a shiny skin (a) to a dull skull (e). Do not drill for more than 1min. Abbreviations: a-anterior, p-posterior.
Supplementary Figure 5 Orientation of the fish in different imaging sessions using pigment cells as landmarks.
(a)-(c) Dorsal view of the telencephalon at different imaging time-points, in which three pigments are visible and are stable throughout time (each colored arrow marks the same pigment over the different time-points). Abbreviations: a-anterior, p-posterior, dpe-days post electroporation.
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Barbosa, J., Di Giaimo, R., Götz, M. et al. Single-cell in vivo imaging of adult neural stem cells in the zebrafish telencephalon. Nat Protoc 11, 1360–1370 (2016). https://doi.org/10.1038/nprot.2016.077
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DOI: https://doi.org/10.1038/nprot.2016.077
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