How astrocytes grow and integrate into neural circuits remains poorly defined. Zebrafish are well suited for such investigations, but bona fide astrocytes have not been described in this system. Here we characterize a zebrafish cell type that is remarkably similar to mammalian astrocytes that derive from radial glial cells and elaborate processes to establish their territories at early larval stages. Zebrafish astrocytes associate closely with synapses, tile with one another and express markers, including Glast and glutamine synthetase. Once integrated into circuits, they exhibit whole-cell and microdomain Ca2+ transients, which are sensitive to norepinephrine. Finally, using a cell-specific CRISPR–Cas9 approach, we demonstrate that fgfr3 and fgfr4 are required for vertebrate astrocyte morphogenesis. This work provides the first visualization of astrocyte morphogenesis from stem cell to post-mitotic astrocyte in vivo, identifies a role for Fgf receptors in vertebrate astrocytes and establishes zebrafish as a valuable new model system to study astrocyte biology in vivo.
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The data that support the findings of this study are available in the manuscript or the Supplementary Information. All reagents and additional data from this study are available upon reasonable request. Source data are provided with this paper.
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We thank S. Ackerman, D. Lyons and members of the Freeman and Monk labs for helpful discussions and comments on the manuscript. We are indebted to M. Cahill, S. Pittolo and M. Reitman of the Poskanzer lab for assistance with the AQuA software platform and to K. Cole of the Lyons lab for TTX experiment advice. We thank A. Forbes and G. Halsell-Vore for excellent zebrafish care and L. Vaskalis for graphics. This work was supported by R01NS099254 to K.E.P., R37NS053538 to M.R.F. and R21NS115437 to K.R.M.
The authors declare no competing interests.
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Extended Data Fig. 1 Whole mount in situ hybridization of mammalian astrocyte markers in different stage zebrafish larvae.
a, 6 dpf expression patterns of slc1a2a/EAAT2b, slc1a2b/EAAT2a, slc1a3a/Glasta, slc1a3b/Glastb, slc6a11a/GAT-3a, slc6a11b/GAT-3b, aldh1l1, and aqp4. b, Expression patterns of slc1a2b/EAAT2a, slc1a3b/Glastb, and slc6a11b/GAT-3b at 1 dpf, 3 dpf, and 6 dpf in lateral and dorsal view. c, Expression patterns of slc1a2b/EAAT2a, slc1a3b/Glastb, slc6a11b/GAT-3b, aldh1l1, and aqp4 in 14 dpf dissected brains. Scale bar, 200 μm. All images are representative of three or four independent repeats.
Extended Data Fig. 2 slc1a3b:myrGFP-P2A-H2AmCherry-labeled RGCs and ependymal cells in 6 dpf zebrafish larvae.
a, b, Representative images show RGCs (a) and ependymal cells (b) in zebrafish spinal cord labeled by the slc1a3b:myrGFP-P2A-H2AmCherry DNA construct. Scale bar, 20 μm. Representative images from three independent repeats.
a, Representative images show the comparison of slc1a3b, kcnj10a/Kir4.1, and gfap in the spinal cord. Dash lines mark the outline of spinal cord. Images are representative of N=3-4 fish larvae. Scale bars represent 500 μm for left panel, 200 μm for middle panel, and 20 μm for right panel, respectively. b, RNAScope in situ hybridization of kcnj10a in Tg[slc1a3b:myrGFP-P2A-H2AmCherry] fish spinal cord at 3 dpf. Single z-plane, dorsal view. Scale bar, 20 μm. Representative images from N=3 fish larvae. c, Double staining in situ hybridization of slc1a3b (purple) and gfap (red) at 3 dpf. Scale bar, 500 μm. Representative images from N=6 fish larvae.
Extended Data Fig. 4 NE-induced Microdomain Ca2+ transients in spinal cord astrocytes are not driven by neuronal activity.
a, Schematic overview of the TTX injection experiments. b-e, Comparisons of average microdomain Ca2+ events frequencies (b), area sizes (c), amplitudes (d), and durations (e) in DMSO control and NE-treated fish following TTX injections. Error bars represent Mean values +/- SD. **, p<0.01; ****, p<0.0001. p=0.0094 (b), p=1.07x10−7 (c), p<1.0x10−15 (d and e). Two-tailed unpaired t test. N, number of fish analyzed.
Extended Data Fig. 5 Microdomain Ca2+ transients in the hindbrain radial astrocytes are sensitive to NE treatment.
a, b, AQuA-detected Ca2+ events in DMSO control versus NE-treated Tg[slc1a3b:myrGCaMP6s] fish hindbrain radial astrocytes, and corresponding 20 individual ΔF/F traces. Scale bar, 20 μm. Dashed lines mark the regions representing the fine cellular processes of radial astrocytes that were analyzed. See also Supplementary Videos 6 and 7. c-f, Quantifications of average microdomain Ca2+ events frequency, area size, amplitude, and duration in DMSO control and NE-treated fish hindbrain regions. Error bars represent mean values +/- SD. *, p<0.05; **, p<0.01; ****, p<0.0001. p=0.0136 (c), p=4.43x10−9 (d), p=6.55x10-6 (e), p=0.0058 (f). Two-tailed unpaired t test. N, number of fish analyzed.
Extended Data Fig. 6 Designed sgRNAs targeting fgfr1-4 are effective in disrupting corresponding genes.
a, Genotyping PCR results show that the co-injections of individual sgRNAs together with Cas9 protein led to the disruptions of endogenous restriction enzyme sites in contrast to uninjected controls. Two independent sgRNAs were tested except for fgfr4. NA, not available due to high toxicity. Source data
Time-lapse confocal imaging demonstrates that a slc1a3b:myrGFP-P2A-H2AmCherry+ cell elaborates dynamic processes at 2–3 dpf in the spinal cord.
z-stack movie showing one slc1a3b:myrGFP+ astrocyte closely tiling with a neighboring slc1a3b:mCD8mCherry+ astrocyte in the 6-dpf spinal cord.
Representative time-lapse Ca2+ imaging shows spontaneous microdomain Ca2+ transients in spinal cord astrocyte processes in Tg[slc1a3b:myrGCaMP6s] fish.
Representative time-lapse Ca2+ imaging and AQuA-detected Ca2+ events in DMSO-treated spinal cord astrocytes at 6 dpf.
Representative time-lapse Ca2+ imaging and AQuA-detected Ca2+ events in NE-treated spinal cord astrocytes at 6 dpf.
Representative time-lapse Ca2+ imaging and AQuA-detected Ca2+ events in DMSO-treated hindbrain radial astrocytes at 6 dpf.
Representative time-lapse Ca2+ imaging and AQuA-detected Ca2+ events in NE-treated hindbrain radial astrocytes at 6 dpf.
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Chen, J., Poskanzer, K.E., Freeman, M.R. et al. Live-imaging of astrocyte morphogenesis and function in zebrafish neural circuits. Nat Neurosci 23, 1297–1306 (2020). https://doi.org/10.1038/s41593-020-0703-x