Investigation of the role of two-pore channel type 2 (TPC2) in calcium signalling during slow muscle cell and primary motor neuron development in zebrafish embryos

Abstract

The calcium (Ca²⁺) signals that help to regulate neuro-muscular development consist of a complex interaction between signal transduction pathways and gene transcription. The challenge when exploring, modulating, and visualizing these Ca²⁺ signals, lies in their spatial and temporal nature, as well as in the variety of Ca²⁺ mobilizing agents and receptors that come into play. The most recently identified agonist/ receptor pair that has been reported to be involved in skeletal muscle differentiation and nervous system development is nicotinic acid adenine dinucleotide phosphate (NAADP) and the two-pore channel (TPC), respectively, where NAADP has been shown to be the most potent intracellular Ca²⁺ mobilizing agent described to date. Here, I present new data that describe what appears to be a complex relationship between Ca²⁺ released via TPC2, and that released via the well-known ryanodine receptor (RyR) and inositol trisphosphate receptor (IP₃R) during zebrafish neuro-muscular development. I show that their differential couplings and interactions, which were deduced from: 1) Various molecular, genetic, and pharmacological manipulations; in conjunction with 2) Ca²⁺ imaging; and 3) immunolabelling followed by either confocal or stimulated emission depletion super-resolution microscopy, are necessary for myogenesis, motor axonogenesis, and spinal circuitry maturation during the segmentation period of zebrafish development. In addition, I suggest that the neuromuscular junctions that form the functional link between the motor network and muscle are also regulated by TPC2-mediated Ca²⁺ signalling. Furthermore, I identify the putative enzyme responsible for NAADP synthesis (i.e., ADP-ribosyl cyclase; ARC), in zebrafish embryos in silico. I then confirm the expression of the ARC homolog at the transcript and protein levels across the segmentation stages in embryos. My study, therefore, demonstrates the robust requirement of NAADP/TPC2/Ca²⁺ signalling throughout the formation and onset of function of the motor neurons and muscle cells in zebrafish embryos, which results in the development of their motility.

Date of Award	2017
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology