Deciphering the role of STIM1 in nervous system development

The wiring of the nervous system during development requires each neuron to find and connect with a correct synaptic target in a process known as axon pathfinding. Defects in axon pathfinding cause significant neurodevelopmental disorders, and contribute to the inability of neural circuits to reconnect following injury or disease. Axon pathfinding is regulated by precise intracellular calcium signals produced by the influx of extracellular calcium and the release of calcium from internal stores, such as the endoplasmic reticulum (ER). Although the importance of discrete patterns of calcium signaling for determining axon pathfinding are well documented, the mechanisms that control such precise calcium signaling events are yet to be fully elucidated. Stromal interacting molecule 1 (STIM1) is an ER-resident calcium sensing protein that activates a specific mode of calcium influx termed store-operated calcium entry (SOCE). STIM1 is activated by the depletion of calcium from the ER, with activated STIM1 triggering calcium influx via SOCE. The calcium that enters the cell via SOCE refills the depleted ER calcium store, and sustains elevated intracellular calcium to potentiate calcium-dependent intracellular signaling pathways. STIM1 has been shown to be necessary for correct growth cone motility in vitro, and correct axon pathfinding in vivo. However, the cellular expression of STIM1 during nervous system development, and the function of STIM1 in axon pathfinding in vivo remain poorly understood. To this end, the central hypothesis examined by this thesis is that there are discrete patterns of STIM1 protein expression during embryonic development, and STIM1-mediated calcium signaling is necessary for correct nervous system development. The developmental expression of STIM1 was investigated in the mouse and zebrafish (Danio rerio) nervous systems. The pattern of STIM1 expression was highly conserved between mice and zebrafish nervous systems, suggesting that STIM1 has a conserved role in development. By examining the cellular expression of STIM1 in the embryonic mouse nervous system, it was confirmed that STIM1 is expressed by neurons during development, consistent with a function for STIM1 in the regulation of calcium signaling during nervous system development. The function of STIM1 during nervous system development was assessed by investigating the requirement of zebrafish STIM1a (zSTIM1a) expression for axon pathfinding in vivo. A reduced expression of zSTIM1a impacted the survival, growth and correct development of zebrafish embryos, consistent with STIM1 having important functions in development. zSTIM1a was shown to regulate calcium influx via SOCE in zebrafish spinal motor neurons, confirming that STIM1 regulates calcium signaling in zebrafish neurons. When axon pathfinding by spinal motor neurons was examined in zSTIM1a morphant embryos, the axons of caudal primary (CaP) motor neurons were observed to stall at intermediate targets, exhibit defects in extension from intermediate targets, express fewer filopodia, and were less likely to be branched. Defects in axon pathfinding correlated with perturb calcium signaling in navigating CaP axons when zSTIM1a expression was reduced, with bursting calcium spikes decreased in axons navigating via intermediate targets. Together, these data provide evidence that STIM1-mediated calcium signaling is required for correct axon pathfinding in vivo. These findings have significant implications for our understanding of the molecular mechanisms that contribute to the regulation of discrete patterns of calcium signaling during axon pathfinding in vivo and, hence, nervous system development.