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Identification of Ca2+ signaling components in neural stem/progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres

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Abstract

The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells (NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor (IP3R) type-1 and ryanodine receptor (RyR) type-2 are differentially expressed in cells with neuron- or radial glial-like properties. Furthermore, ATP but not caffeine (IP3R and RyR agonists, respectively), induced the generation of Ca2+ transients in cells exhibiting neuron- or glial-like morphology. These results indicate the differential expression of components of the Ca2+-signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca2+ signaling as a read-out.

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Acknowledgements

We would like to thank Dr. Jeffrey J. Kelu (Division of Life Science, HKUST) for helping us with the statistics. This work was supported by the ANR/RGC Joint Research Scheme Award (A-HKUST601/13), the HK RGC General Research Fund awards (662113, 16101714, 16100115) and Funding from the HKITC (ITCPD/17-9).

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  1. Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, China

    Man Kit Tse, Ting Shing Hung, Ching Man Chan, Tiffany Wong, Mike Dorothea, Andrew L. Miller & Sarah E. Webb

  2. Centre de Biologie du Développement (CBD), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, F-31062, France

    Catherine Leclerc & Marc Moreau

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Tse, M.K., Hung, T.S., Chan, C.M. et al. Identification of Ca2+ signaling components in neural stem/progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres. Sci. China Life Sci. 61, 1352–1368 (2018). https://doi.org/10.1007/s11427-018-9315-6

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