The role of TPC2-mediated Ca²⁺ release was recently characterized in zebrafish during the establishment of the early spinal circuitry, one of the key events in the coordination of neuromuscular activity. Here, I extended the study to investigate the role of TPC2-mediated Ca²⁺ release in the regulation of caudal primary motor neuron (CaP) axon extension from the spinal cord towards the trunk musculature. I used a combination of: TPC2 knock-down with a translational-blocking morpholino antisense oligonucleotide (MO); TPC2 knock-out via the generation of a SAIGFF213A:GFP;tpcn2^dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing; and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H⁺-ATPase inhibitor) or trans-ned-19 (an NAADP receptor antagonist...[ Read more ]

The role of TPC2-mediated Ca²⁺ release was recently characterized in zebrafish during the establishment of the early spinal circuitry, one of the key events in the coordination of neuromuscular activity. Here, I extended the study to investigate the role of TPC2-mediated Ca²⁺ release in the regulation of caudal primary motor neuron (CaP) axon extension from the spinal cord towards the trunk musculature. I used a combination of: TPC2 knock-down with a translational-blocking morpholino antisense oligonucleotide (MO); TPC2 knock-out via the generation of a SAIGFF213A:GFP;tpcn2^dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing; and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H⁺-ATPase inhibitor) or trans-ned-19 (an NAADP receptor antagonist). These treatments significantly attenuated Ca²⁺ signaling in CaP growth cones and inhibited axon extension. MO-mediated knockdown of ARC1-like, a key upstream component in generating NAADP (an endogenous agonist of TPC2) also led to a significant attenuation of the Ca²⁺ transients in the CaP growth cones, and an inhibition of axon extension. I also demonstrated that lysosomes, TPC2, ER, RyRs, and IP₃Rs are all present in the extending growth cones of CaP axons. This indicated that the components required for triggering inter-organellar Ca²⁺ release were present. Furthermore, I showed that AChR clusters are localized in the middle region of slow muscle pioneer cells, with a second distinct pattern of clustering occurring at the lateral ends of the slow muscle cells adjacent to the vertical myoseptae. In addition, attenuation of TPC2-mediated Ca²⁺ signaling appeared to have an indirect effect on reducing AChR clustering and the subsequent formation of NMJs in the middle part of the slow muscle pioneer cells due to the inhibition of CaP axon extension. Finally, Cre-LoxP gene editing techniques were applied to initiate the conditional knockout of tpcn2 in an inducible manner. I designed a gRNA, and a donor DNA (left homology arm-LoxP-tpcn2-venus-LoxP-right homology arm) plasmid. The latter was injected into embryos and confirmed from the expression of Venus fluorescent in the lens of the eye that successful integration had taken place. Together my data suggest a possible link between ARC1-like, TPC2 and Ca²⁺ signaling during axon extension in zebrafish.