TPC2-mediated Ca signaling is required for axon extension in caudal primary motor neurons in zebrafish embryos.

The role of two-pore channel type 2 (TPC2, encoded by )-mediated Ca release was recently characterized in zebrafish during establishment of the early spinal circuitry, one of the key events in the coordination of neuromuscular activity. Here, we extend our study to investigate the role of TPC2 in the regulation of caudal primary motor neuron (CaP) axon extension. We used a combination of TPC2 knockdown with a translation-blocking morpholino antisense oligonucleotide (MO), TPC2 knockout via the generation of a mutant line of zebrafish using CRISPR/Cas9 gene-editing and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H-ATPase inhibitor) or -ned-19 (an NAADP receptor antagonist), and showed that these treatments attenuated CaP Ca signaling and inhibited axon extension. We also characterized the expression of an transcript in CaPs grown in primary culture. MO-mediated knockdown of ARC1-like led to attenuation of the Ca transients in the CaP growth cones and an inhibition of axon extension. Together, our new data suggest a link between ARC1-like, TPC2 and Ca signaling during axon extension in zebrafish.