•RESEARCH PAPER• ................... .......................... November 2018 Vol.61 No.11: 1352–1368 ............................................................................... https://doi.org/10.1007/s11427-018-9315-6 Identification of Ca 2+ signaling components in neural stem/ progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres Man Kit Tse 1 , Ting Shing Hung 1 , Ching Man Chan 1 , Tiffany Wong 1 , Mike Dorothea 1 , Catherine Leclerc 2 , Marc Moreau 2 , Andrew L. Miller 1 & Sarah E. Webb 1* 1 Division of Life Science & State Key Laboratory of Molecular Neuroscience, HKUST, Clear Water Bay, Hong Kong, China; 2 Centre de Biologie du Développement (CBD), Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse F-31062, France Received February 20, 2018; accepted May 3, 2018; published online June 20, 2018 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 (IP 3 R) type-1 and ryanodine receptor (RyR) type-2 are differentially expressed in cells with neuron- or radial glial-like prop- erties. Furthermore, ATP but not caffeine (IP 3 R and RyR agonists, respectively), induced the generation of Ca 2+ transients in cells exhibiting neuron- or glial-like morphology. These results indicate the differential expression of components of the Ca 2+ -sig- naling 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 Ca 2+ signaling as a read-out. Ca 2+ signaling, neurospheres, zebrafish, neural stem/progenitor cells, differentiation, IP 3 receptors, ryanodine receptors, STIM and Orai Citation: Tse, M.K., Hung, T.S., Chan, C.M., Wong, T., Dorothea, M., Leclerc, C., Moreau, M., Miller, A.L., and Webb, S.E. (2018). Identification of Ca 2+ 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. https://doi.org/10.1007/s11427-018-9315-6 INTRODUCTION Neurogenesis and gliogenesis take place in the developing central nervous system (CNS) and involve the induction and proliferation of neural stem cells (NSCs) and neural pro- genitor cells (NPCs) followed by their differentiation into mature and functional neurons and glia (including astrocytes and oligodendrocytes), respectively (Schmidt et al., 2013; Urbán and Guillemot, 2014). While neurogenesis and glio- genesis begin (and are most dynamic) in vertebrates during embryogenesis, both continue to occur (to a greater or lesser extent) in adults (Schmidt et al., 2013; Urbán and Guillemot, 2014). In mammals, only a few neurogenic regions persist in the adult stage (Urbán and Guillemot, 2014). In the adult mouse, for example, the main neurogenic areas are restricted © Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018 ....................... life.scichina.com link.springer.com SCIENCE CHINA Life Sciences *Corresponding author (email: barnie@ust.hk)