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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
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.
References
Freeman, M. R. Specification and morphogenesis of astrocytes. Science 330, 774–778 (2010).
Clarke, L. E. & Barres, B. A. Emerging roles of astrocytes in neural circuit development. Nat. Rev. Neurosci. 14, 311–321 (2013).
Ma, Z., Stork, T., Bergles, D. E. & Freeman, M. R. Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour. Nature 539, 428–432 (2016).
Nagai, J. et al. Hyperactivity with disrupted attention by activation of an astrocyte synaptogenic cue. Cell 177, 1280–1292 (2019).
Molofsky, A. V. et al. Astrocytes and disease: a neurodevelopmental perspective. Genes Dev. 26, 891–907 (2012).
Freeman, M. R. & Rowitch, D. H. Evolving concepts of gliogenesis: a look way back and ahead to the next 25 years. Neuron 80, 613–623 (2013).
Stork, T., Sheehan, A., Tasdemir-Yilmaz, O. E. & Freeman, M. R. Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes. Neuron 83, 388–403 (2014).
Lyons, D. A. & Talbot, W. S. Glial cell development and function in zebrafish. Cold Spring Harb. Perspect. Biol. 7, a020586 (2014).
Grupp, L., Wolburg, H. & Mack, A. F. Astroglial structures in the zebrafish brain. J. Comp. Neurol. 518, 4277–4287 (2010).
Kroehne, V., Freudenreich, D., Hans, S., Kaslin, J. & Brand, M. Regeneration of the adult zebrafish brain from neurogenic radial glia-type progenitors. Development 138, 4831–4841 (2011).
Kyritsis, N. et al. Acute inflammation initiates the regenerative response in the adult zebrafish brain. Science 338, 1353–1356 (2012).
Mu, Y. et al. Glia accumulate evidence that actions are futile and suppress unsuccessful behavior. Cell 178, 27–43 (2019).
Di Castro, M. A. et al. Local Ca2+ detection and modulation of synaptic release by astrocytes. Nat. Neurosci. 14, 1276–1284 (2011).
Shigetomi, E., Tong, X., Kwan, K. Y., Corey, D. P. & Khakh, B. S. TRPA1 channels regulate astrocyte resting calcium and inhibitory synapse efficacy through GAT-3. Nat. Neurosci. 15, 70–80 (2011).
Grosche, J. et al. Microdomains for neuron-glia interaction: parallel fiber signaling to Bergmann glial cells. Nat. Neurosci. 2, 139–143 (1999).
Nimmerjahn, A., Mukamel, E. A. & Schnitzer, M. J. Motor behavior activates Bergmann glial networks. Neuron 62, 400–412 (2009).
Ding, F. et al. ɑ1-Adrenergic receptors mediate coordinated Ca2+ signaling of cortical astrocytes in awake, behaving mice. Cell Calcium 54, 387–394 (2013).
Paukert, M. et al. Norepinephrine controls astroglial responsiveness to local circuit activity. Neuron 82, 1263–1270 (2014).
Pankratov, Y. & Lalo, U. Role for astroglial ɑ1-adrenoreceptors in gliotransmission and control of synaptic plasticity in the neocortex. Front. Cell Neurosci. 9, 230 (2015).
Cahoy, J. D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008).
Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014).
Turner, K. J., Bracewell, T. G. & Hawkins, T. A. Anatomical dissection of zebrafish brain development. Methods Mol. Biol. 1082, 197–214 (2014).
Stogsdill, J. A. et al. Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis. Nature 551, 192–197 (2017).
Regan, M. R. et al. Variations in promoter activity reveal a differential expression and physiology of glutamate transporters by glia in the developing and mature CNS. J. Neurosci. 27, 6607–6619 (2007).
Bellamy, T. C. Interactions between Purkinje neurones and Bergmann glia. Cerebellum 5, 116–126 (2006).
Lyons, D. A., Guy, A. T. & Clarke, J. D. Monitoring neural progenitor fate through multiple rounds of division in an intact vertebrate brain. Development 130, 3427–3436 (2003).
Norenberg, M. D. & Martinez-Hernandez, A. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161, 303–310 (1979).
Farnsworth, D. R., Saunders, L. M. & Miller, A. C. A single-cell transcriptome atlas for zebrafish development. Dev. Biol. 459, 100–108 (2020).
Christopherson, K. S. et al. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell 120, 421–433 (2005).
Bernardinelli, Y. et al. Activity-dependent structural plasticity of perisynaptic astrocytic domains promotes excitatory synapse stability. Curr. Biol. 24, 1679–1688 (2014).
Kucukdereli, H. et al. Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC. Proc. Natl Acad. Sci. USA 108, E440–E449 (2011).
Allen, N. J. et al. Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors. Nature 486, 410–414 (2012).
Bushong, E. A., Martone, M. E., Jones, Y. Z. & Ellisman, M. H. Protoplasmic astrocytes in CA1 stratum radiatum occupy separate anatomical domains. J. Neurosci. 22, 183–192 (2002).
Wang, Y. et al. Accurate quantification of astrocyte and neurotransmitter fluorescence dynamics for single-cell and population-level physiology. Nat. Neurosci. 22, 1936–1944 (2019).
Nett, W. J., Oloff, S. H. & McCarthy, K. D. Hippocampal astrocytes in situ exhibit calcium oscillations that occur independent of neuronal activity. J. Neurophysiol. 87, 528–537 (2002).
Araque, A. et al. Gliotransmitters travel in time and space. Neuron 81, 728–739 (2014).
Agarwal, A. et al. Transient opening of the mitochondrial permeability transition pore induces microdomain calcium transients in astrocyte processes. Neuron 93, 587–605 e587 (2017).
Kegel, L. et al. Disruption to NKCC1 impairs the response of myelinating Schwann cells to neuronal activity and leads to severe peripheral nerve pathology. Preprint at bioRxiv https://www.biorxiv.org/content/10.1101/757831v1.full (2019).
Ablain, J., Durand, E. M., Yang, S., Zhou, Y. & Zon, L. I. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Dev. Cell 32, 756–764 (2015).
Shah, A. N., Davey, C. F., Whitebirch, A. C., Miller, A. C. & Moens, C. B. Rapid reverse genetic screening using CRISPR in zebrafish. Nat. Methods 12, 535–540 (2015).
Rowitch, D. H. & Kriegstein, A. R. Developmental genetics of vertebrate glial-cell specification. Nature 468, 214–222 (2010).
Kriegstein, A. & Alvarez-Buylla, A. The glial nature of embryonic and adult neural stem cells. Annu. Rev. Neurosci. 32, 149–184 (2009).
Johnson, K. et al. Gfap-positive radial glial cells are an essential progenitor population for later-born neurons and glia in the zebrafish spinal cord. Glia 64, 1170–1189 (2016).
Goldshmit, Y. et al. Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J. Neurosci. 32, 7477–7492 (2012).
Eroglu, C. et al. Gabapentin receptor ɑ2Δ-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell 139, 380–392 (2009).
Zhou, Y. & Danbolt, N. C. GABA and glutamate transporters in brain. Front. Endocrinol. 4, 165 (2013).
Vaccarino, F. M. et al. Changes in cerebral cortex size are governed by fibroblast growth factor during embryogenesis. Nat. Neurosci. 2, 246–253 (1999).
Pringle, N. P. et al. Fgfr3 expression by astrocytes and their precursors: evidence that astrocytes and oligodendrocytes originate in distinct neuroepithelial domains. Development 130, 93–102 (2003).
Labun, K. et al. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 47, W171–W174 (2019).
Cunningham, R. L. & Monk, K. R. Whole mount in situ hybridization and immunohistochemistry for zebrafish larvae. Methods Mol. Biol. 1739, 371–384 (2018).
Kawakami, K., Shima, A. & Kawakami, N. Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage. Proc. Natl Acad. Sci. USA 97, 11403–11408 (2000).
Inoue, D. & Wittbrodt, J. One for all–a highly efficient and versatile method for fluorescent immunostaining in fish embryos. PLoS ONE 6, e19713 (2011).
Ackerman, S. D., Garcia, C., Piao, X., Gutmann, D. H. & Monk, K. R. The adhesion GPCR Gpr56 regulates oligodendrocyte development via interactions with Galpha12/13 and RhoA. Nat. Commun. 6, 6122 (2015).
Herbert, A. L. et al. Dynein/dynactin is necessary for anterograde transport of Mbp mRNA in oligodendrocytes and for myelination in vivo. Proc. Natl Acad. Sci. USA 114, E9153–E9162 (2017).
Baraban, M., Koudelka, S. & Lyons, D. A. Ca2+ activity signatures of myelin sheath formation and growth in vivo. Nat. Neurosci. 21, 19–23 (2018).
Acknowledgements
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.
Author information
Authors and Affiliations
Contributions
J.C., M.R.F. and K.R.M. conceived the project. J.C. carried out experiments and data analyses. K.E.P. provided support for the AQuA software analyses. All authors contributed to the final version of the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
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=1.07x10−7 (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.
Supplementary information
Supplementary Video 1
Time-lapse confocal imaging demonstrates that a slc1a3b:myrGFP-P2A-H2AmCherry+ cell elaborates dynamic processes at 2–3 dpf in the spinal cord.
Supplementary Video 2
z-stack movie showing one slc1a3b:myrGFP+ astrocyte closely tiling with a neighboring slc1a3b:mCD8mCherry+ astrocyte in the 6-dpf spinal cord.
Supplementary Video 3
Representative time-lapse Ca2+ imaging shows spontaneous microdomain Ca2+ transients in spinal cord astrocyte processes in Tg[slc1a3b:myrGCaMP6s] fish.
Supplementary Video 4
Representative time-lapse Ca2+ imaging and AQuA-detected Ca2+ events in DMSO-treated spinal cord astrocytes at 6 dpf.
Supplementary Video 5
Representative time-lapse Ca2+ imaging and AQuA-detected Ca2+ events in NE-treated spinal cord astrocytes at 6 dpf.
Supplementary Video 6
Representative time-lapse Ca2+ imaging and AQuA-detected Ca2+ events in DMSO-treated hindbrain radial astrocytes at 6 dpf.
Supplementary Video 7
Representative time-lapse Ca2+ imaging and AQuA-detected Ca2+ events in NE-treated hindbrain radial astrocytes at 6 dpf.
Source data
Source Data Extended Data Fig. 6
Unprocessed gels
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41593-020-0703-x
This article is cited by
-
Gastrointestinal and brain barriers: unlocking gates of communication across the microbiota–gut–brain axis
Nature Reviews Gastroenterology & Hepatology (2024)
-
Astroglial Hmgb1 regulates postnatal astrocyte morphogenesis and cerebrovascular maturation
Nature Communications (2023)
-
Functional roles of reactive astrocytes in neuroinflammation and neurodegeneration
Nature Reviews Neurology (2023)
-
Mitochondrial proteins encoded by the 22q11.2 neurodevelopmental locus regulate neural stem and progenitor cell proliferation
Molecular Psychiatry (2023)
-
Heterogeneity and Molecular Markers for CNS Glial Cells Revealed by Single-Cell Transcriptomics
Cellular and Molecular Neurobiology (2022)