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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Slc1a3b/Glast+ cells in zebrafish exhibit complex cellular morphologies.
Fig. 2: Zebrafish spinal cord astrocytes show dynamic cellular process elaboration and establish individual cell territories between 2 and 4 dpf.
Fig. 3: Spinal cord astrocytes express GS, closely associate with synapses and tile with one another.
Fig. 4: Zebrafish astrocytes exhibit spontaneous microdomain Ca2+ transients in the fine processes and respond to NE activation.
Fig. 5: Cell-specific inactivation of fgfr3 and fgfr4 disturbs spinal cord astrocyte morphogenesis.

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

  1. 1.

    Freeman, M. R. Specification and morphogenesis of astrocytes. Science 330, 774–778 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Clarke, L. E. & Barres, B. A. Emerging roles of astrocytes in neural circuit development. Nat. Rev. Neurosci. 14, 311–321 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Nagai, J. et al. Hyperactivity with disrupted attention by activation of an astrocyte synaptogenic cue. Cell 177, 1280–1292 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Molofsky, A. V. et al. Astrocytes and disease: a neurodevelopmental perspective. Genes Dev. 26, 891–907 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Lyons, D. A. & Talbot, W. S. Glial cell development and function in zebrafish. Cold Spring Harb. Perspect. Biol. 7, a020586 (2014).

    PubMed  Google Scholar 

  9. 9.

    Grupp, L., Wolburg, H. & Mack, A. F. Astroglial structures in the zebrafish brain. J. Comp. Neurol. 518, 4277–4287 (2010).

    CAS  PubMed  Google Scholar 

  10. 10.

    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).

    CAS  PubMed  Google Scholar 

  11. 11.

    Kyritsis, N. et al. Acute inflammation initiates the regenerative response in the adult zebrafish brain. Science 338, 1353–1356 (2012).

    CAS  PubMed  Google Scholar 

  12. 12.

    Mu, Y. et al. Glia accumulate evidence that actions are futile and suppress unsuccessful behavior. Cell 178, 27–43 (2019).

    CAS  PubMed  Google Scholar 

  13. 13.

    Di Castro, M. A. et al. Local Ca2+ detection and modulation of synaptic release by astrocytes. Nat. Neurosci. 14, 1276–1284 (2011).

    PubMed  Google Scholar 

  14. 14.

    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).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Grosche, J. et al. Microdomains for neuron-glia interaction: parallel fiber signaling to Bergmann glial cells. Nat. Neurosci. 2, 139–143 (1999).

    CAS  PubMed  Google Scholar 

  16. 16.

    Nimmerjahn, A., Mukamel, E. A. & Schnitzer, M. J. Motor behavior activates Bergmann glial networks. Neuron 62, 400–412 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Ding, F. et al. ɑ1-Adrenergic receptors mediate coordinated Ca2+ signaling of cortical astrocytes in awake, behaving mice. Cell Calcium 54, 387–394 (2013).

    CAS  Google Scholar 

  18. 18.

    Paukert, M. et al. Norepinephrine controls astroglial responsiveness to local circuit activity. Neuron 82, 1263–1270 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    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).

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Turner, K. J., Bracewell, T. G. & Hawkins, T. A. Anatomical dissection of zebrafish brain development. Methods Mol. Biol. 1082, 197–214 (2014).

    PubMed  Google Scholar 

  23. 23.

    Stogsdill, J. A. et al. Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis. Nature 551, 192–197 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Bellamy, T. C. Interactions between Purkinje neurones and Bergmann glia. Cerebellum 5, 116–126 (2006).

    PubMed  Google Scholar 

  26. 26.

    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).

    CAS  PubMed  Google Scholar 

  27. 27.

    Norenberg, M. D. & Martinez-Hernandez, A. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161, 303–310 (1979).

    CAS  PubMed  Google Scholar 

  28. 28.

    Farnsworth, D. R., Saunders, L. M. & Miller, A. C. A single-cell transcriptome atlas for zebrafish development. Dev. Biol. 459, 100–108 (2020).

    CAS  PubMed  Google Scholar 

  29. 29.

    Christopherson, K. S. et al. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell 120, 421–433 (2005).

    CAS  PubMed  Google Scholar 

  30. 30.

    Bernardinelli, Y. et al. Activity-dependent structural plasticity of perisynaptic astrocytic domains promotes excitatory synapse stability. Curr. Biol. 24, 1679–1688 (2014).

    CAS  PubMed  Google Scholar 

  31. 31.

    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).

    CAS  PubMed  Google Scholar 

  32. 32.

    Allen, N. J. et al. Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors. Nature 486, 410–414 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    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).

    PubMed  Google Scholar 

  36. 36.

    Araque, A. et al. Gliotransmitters travel in time and space. Neuron 81, 728–739 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    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).

  39. 39.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Rowitch, D. H. & Kriegstein, A. R. Developmental genetics of vertebrate glial-cell specification. Nature 468, 214–222 (2010).

    CAS  PubMed  Google Scholar 

  42. 42.

    Kriegstein, A. & Alvarez-Buylla, A. The glial nature of embryonic and adult neural stem cells. Annu. Rev. Neurosci. 32, 149–184 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    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).

    PubMed  PubMed Central  Google Scholar 

  44. 44.

    Goldshmit, Y. et al. Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J. Neurosci. 32, 7477–7492 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Eroglu, C. et al. Gabapentin receptor ɑ2Δ-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell 139, 380–392 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Zhou, Y. & Danbolt, N. C. GABA and glutamate transporters in brain. Front. Endocrinol. 4, 165 (2013).

    Google Scholar 

  47. 47.

    Vaccarino, F. M. et al. Changes in cerebral cortex size are governed by fibroblast growth factor during embryogenesis. Nat. Neurosci. 2, 246–253 (1999).

    CAS  PubMed  Google Scholar 

  48. 48.

    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).

    CAS  PubMed  Google Scholar 

  49. 49.

    Labun, K. et al. CHOPCHOP v3: expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res. 47, W171–W174 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Cunningham, R. L. & Monk, K. R. Whole mount in situ hybridization and immunohistochemistry for zebrafish larvae. Methods Mol. Biol. 1739, 371–384 (2018).

    CAS  PubMed  Google Scholar 

  51. 51.

    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).

    CAS  PubMed  Google Scholar 

  52. 52.

    Inoue, D. & Wittbrodt, J. One for all–a highly efficient and versatile method for fluorescent immunostaining in fish embryos. PLoS ONE 6, e19713 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    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).

    CAS  PubMed  Google Scholar 

  55. 55.

    Baraban, M., Koudelka, S. & Lyons, D. A. Ca2+ activity signatures of myelin sheath formation and growth in vivo. Nat. Neurosci. 21, 19–23 (2018).

    CAS  PubMed  Google Scholar 

Download references

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

Affiliations

Authors

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

Correspondence to Jiakun Chen or Kelly R. Monk.

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. Source data

Supplementary information

Reporting Summary

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

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

Download citation

Search

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing