We recently demonstrated, via the introduction of a translational-blocking morpholino antisense oligonucleotide (MO), a critical role for two-pore channel type 2 (TPC2)-mediated Ca2+ release during the differentiation of slow (skeletal) muscle cells (SMCs) in intact zebrafish embryos. Following this initial report, we extended our study and demonstrated that knockdown of TPC2 (with a non-overlapping splice-blocking MO); knockout of TPC2 (via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing); or the pharmacological inhibition of TPC2 action (with bafilomycin A1 or trans-ned-19), also led to a significant attenuation of SMC differentiation, characterized by a disruption of SMC myofibrillogenesis and gross morphological changes in the trunk musculature. STED super-resolution microscopy revealed a close physical relationship between clusters of ryanodine receptors (RyR) in the terminal cisternae of the sarcoplasmic reticulum (SR), and TPC2 in lysosomes, with a mean estimated separation of ~52-87 nm. Our data therefore add to the increasing body of evidence, which indicate that localized Ca2+ release via TPC2 might trigger the generation of more global Ca2+ release from the SR via Ca2+-induced Ca2+ release. Furthermore, in zebrafish, one of the first observed behavioral activities is the SMC-mediated spontaneous coiling contractions of the trunk. This behavior begins at ~17 hours post-fertilization (hpf) and coincides with the spontaneous activity of the primary neurons in the spinal cord as well as Ca2+ transients generated in the SMCs in the trunk. Here, we report that TPC2-mediated Ca2+-release is also required for the establishment of highly synchronized connectivity within the zebrafish embryonic spinal circuitry. Using the SAIGFF213A;UAS:GCaMP7a double-transgenic line of fish (a kind gift from Prof. Koichi Kawakami), which expresses GCaMP7a in the caudal primary motor neurons (CaPs), Ca2+ transients were visualized starting from the early stages of spontaneous activity at ~18 hpf. We report that TPC2 inhibition resulted in a decrease in the frequency and amplitude, as well as the ipsilateral and contralateral correlation, of the CaP Ca2+ transients, indicating a significant disruption of the maturing spinal circuitry. Together, our new data suggest a novel function for TPC2-mediated Ca2+ signalling in the development, coordination, and maturation of the early neuromuscular activity of zebrafish embryos. This work was supported by: HK RGC-GRF awards 16101714 & 16100115; ANR/RGC award A-HKUST601/13, and HK-ITC (ITCPD/17-9).