Identification of Ca2+ signaling components in neural stem/progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres
- 97 Downloads
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.
KeywordsCa2+ signaling neurospheres zebrafish neural stem/progenitor cells differentiation IP3 receptors ryanodine receptors STIM and Orai
Unable to display preview. Download preview PDF.
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).
- Alzayady, K.J., Sebé-Pedrós, A., Chandrasekhar, R., Wang, L., Ruiz-Trillo, I., and Yule, D.I. (2015). Tracing the evolutionary history of inositol 1,4,5-trisphosphate receptor: insights from analysis of Capsaspora owczarzaki Ca2+ release channel orthologues. Mol Biol Evol 32, 2236–2253.CrossRefPubMedPubMedCentralGoogle Scholar
- Azari, H., Rahman, M., Sharififar, S., and Reynolds, B.A. (2010). Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay. J Vis Exp in press doi: 10.3791/2393.Google Scholar
- Chan, H.Y.S., Cheung, M.C., Gao, Y., Miller, A.L., and Webb, S.E. (2016b). Expression and reconstitution of the bioluminescent Ca2+ reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes. Sci China Life Sci 59, 811–824.CrossRefPubMedGoogle Scholar
- Furlan, G., Cuccioli, V., Vuillemin, N., Dirian, L., Muntasell, A.J., Coolen, M., Dray, N., Bedu, S., Houart, C., Beaurepaire, E., et al. (2017). Lifelong neurogenic activity of individual neural stem cells and continuous growth establish an outside-in architecture in the teleost pallium. Curr Biol 27, 3288–3301.e3.CrossRefPubMedPubMedCentralGoogle Scholar
- Johnson, K., Barragan, J., Bashiruddin, S., Smith, C.J., Tyrrell, C., Parsons, M.J., Doris, R., Kucenas, S., Downes, G.B., Velez, C.M., et al. (2016). 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.CrossRefPubMedPubMedCentralGoogle Scholar
- Leclerc, C., Haeich, J., Aulestia, F.J., Kilhoffer, M.C., Miller, A.L., Néant, I., Webb, S.E., Schaeffer, E., Junier, M.P., Chneiweiss, H., et al. (2016). Calcium signaling orchestrates glioblastoma development: facts and conjunctures. Biochim Biophys Acta 1863, 1447–1459.CrossRefPubMedGoogle Scholar
- Lopez-Ramirez, M.A., Calvo, C.F., Ristori, E., Thomas, J.L., and Nicoli, S. (2016). Isolation and culture of adult zebrafish brain-derived neurospheres. J Vis Exp (108), 53617.Google Scholar
- Sharp, A.H., Nucifora, F.C., Blondel, O., Sheppard, C.A., Zhang, C., Snyder, S.H., Russell, J.T., Ryugoand, D.K., and Ross, C.A. (1999). Differential cellular expression of isoforms of inositol 1,4,5-triphosphate receptors in neurons and glia in brain. J Comp Neurol 406, 207–220.CrossRefPubMedGoogle Scholar
- Wie, M.B., Koh, J.Y., Won, M.H., Lee, J.C., Shin, T.K., Moon, C.J., Ha, H. J., Park, S.M., and Kim, H.C. (2001). BAPTA/AM, an intracellular calcium chelator, induces delayed necrosis by lipoxygenase-mediated free radicals in mouse cortical cultures. Prog Neuropsychopharmacol Biol Psychiatry 25, 1641–1659.CrossRefPubMedGoogle Scholar