Live-imaging of astrocyte morphogenesis and function in zebrafish neural circuits

Abstract

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.

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.

Extended Data Fig. 3. In situ hybridization of slc1a3b, kcnj10a/Kir4.1, and gfap in 3 dpf larvae.

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=1.07×10⁻⁷ (c), p<1.0×10⁻¹⁵ (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=4.43×10⁻⁹ (d), p=6.55×10⁻⁶ (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.

Figure 1.. Slc1a3b/Glast⁺ cells in zebrafish exhibit complex cellular morphologies.

a, Schematic of slc1a3b:myrGFP-P2A-H2AmCherry Tol2 DNA construct used for generating stable transgenic line. b, MAX projection from the lateral and dorsal view of a representative Tg[slc1a3b:myrGFP-P2A-H2AmCherry] larva showing the expression of myrGFP and H2AmCherry at 6 dpf. Scale bar, 200 μm. c, MAX projection of Tg[slc1a3b:myrGFP-P2A-H2AmCherry] larval brain at 6 dpf from dorsal view. Small dashed boxes indicate the regions showed in panel e and f. Scale bar, 200 μm. d-d”, Representative images of Slc1a3b-expressing nuclear H2AmCherry and membrane myrGFP in 6 dpf spinal cord. Small dashed box indicates the region showed in panel g. d, MAX projection; d’, a single z-plane from dorsal view; d”, a single z-plane from lateral view. Scale bar, 20 μm. e-g’, Mosaic labeling single-cell clones using slc1a3b:myrGFP-P2A-H2AmCherry DNA construct in different CNS regions at 6 dpf. e, posterior hindbrain (dorsal view); f, cerebellum (lateral view); g, spinal cord (lateral view); g’, 3D reconstruction of the spinal cord clone (g) from dorsal view. Scale bar, 20 μm. b-d”, independently repeated three or four times; e-g’, representative images from N=6 (e), N=3 (f), N=5 (g, g’) fish larvae.

Figure 2.. Zebrafish spinal cord astrocytes show dynamic cellular process elaboration and establish individual cell territories between 2–4 dpf.

a, Time-lapse still images of a slc1a3b:myrGFP-P2A-H2AmCherry-expressing astrocyte in the spinal cord between 2 and 3 dpf. Scale bar, 20 μm. See also Supplementary Video 1. b, Representative images show the same astrocyte at different developmental stages in the spinal cord. MAX projection, lateral view. Scale bar, 20 μm. c, Quantification of individual spinal cord astrocyte cell territory between 2 and 9 dpf. ****, p<0.0001; ns, not significant; One-way ANOVA with Tukey’s post hoc test. N, number of fish analyzed. Error bars represent mean values +/− standard deviations (SD). d, Schematic showing the developmental stages important for spinal cord astrocyte growth.

Figure 3.. Spinal cord astrocytes express Glutamine synthetase, closely associate with synapses, and tile with one another.

a, Immunostaining of Glutamine synthetase (GS) in Tg[slc1a3b:myrGFP-P2A-H2AmCherry] fish spinal cord at 6 dpf. Single z-plane, dorsal view. Representative images from N=8 fish larvae. b, Immunostaining of synaptic vesicle glycoprotein 2A (SV2) in Tg[slc1a3b:myrGFP-P2A-H2AmCherry] fish spinal cord at 2–6 dpf. Single z-plane, dorsal view. Representative images from N=7 fish larvae, respectively. c, Higher resolution imaging shows the close apposition of myrGFP-labeled astrocyte membranes with synapses (α-SV2, red) in the spinal cord neuropil at 6 dpf (Lateral view). Scale bar, 20 μm. Representative images from four independent repeats. d, Representative images show two tiling astrocytes labeled with slc1a3b:myrGFP (Green) and slc1a3b:mCD8mCherry (Red), respectively, in the 6 dpf larval spinal cord. Cartoon to the right depicts how the experiments were performed to produce two individually labeled cells. Scale bar, 20 μm. Representative images from N=5 fish larvae. See also Supplementary Video 2.

Figure 4.. Zebrafish astrocytes exhibit spontaneous microdomain Ca²⁺ transients in the fine processes and respond to NE activation.

a, Representative image showing 5 minute time-overlay of Ca²⁺ events in a single z-plane in Tg[slc1a3b:myrGCaMP6s] fish cellular process-enriched lateral region (lateral view) at 6 dpf. Scale bar, 20 μm. See also Supplementary Video 3. b, AQuA-detected Ca²⁺ events with individual events pseudo-colored. c, Representative ΔF/F traces of 20 individual microdomain Ca²⁺ transients. d-f, Quantifications of astrocyte microdomain Ca²⁺ event area size (d), amplitude (e), and duration (f) in the spinal cord. Error bars represent mean values +/− standard error of the mean (SEM). g, AQuA-detected Ca²⁺ events in DMSO control versus NE-treated Tg[slc1a3b:myrGCaMP6s] fish spinal cord astrocytes, and corresponding 20 individual ΔF/F traces in 5 minutes. Scale bar, 20 μm. See also Supplementary Videos 4 and 5. h-k, Comparisons of average microdomain Ca²⁺ events frequencies (h), area sizes (i), amplitudes (j), and durations (k) in DMSO control and NE-treated fish. Error bars represent mean values +/− SD.**, p<0.01; ns, not significant; ****, p<0.0001. p=0.0023 (h); p=0.0837 (i); p<1.0×10–15 for j and k. Two-tailed unpaired t test. N, number of fish analyzed.

Figure 5.. Cell-specific inactivation of fgfr3/4 disturbs spinal cord astrocyte morphogenesis.

a, Schematic of the slc1a3b:nlsCas9nls;U6-sgRNA DNA construct used for gene inactivation and the experimental design. b, Representative images showing the three main classes of labeled clones observed at 6 dpf following injections, and the quantifications of each fgfr gene-inactivated cells in comparison with control sgRNA group. Scale bar, 20 μm. c, Representative images and Imaris-generated 3D models of control, fgfr3, and fgfr4-disrupted astrocytes. Scale bar, 20 μm. d, Quantification of individual astrocyte volumes in control, fgfr3, and fgfr4-disrupted cells in the 6 dpf spinal cord. Error bars represent mean values +/− SD. Control, n=33 cells/18 fish; fgfr3, n=68 cells/33 fish; fgfr4, n=21 cells/12 fish analyzed. *, p<0.05. For control vs. fgfr3, p=0.0131; For control vs. fgfr4, p=0.0379. Two-tailed unpaired t test.