Interplay between Piezo1-mediated mechanosensing and the chemical guidance cue Semaphorin3A during development of the embryonic nervous system

During nervous system development, growing neurons extend axons over long distances to defined targets. Despite decades of research dedicated to understanding how neurons are guided along the correct pathways, we still do not fully understand the mechanisms underlying axon pathfinding. In this project, I used the embryonic Xenopus laevis optic pathway as a model to study how axons navigate so precisely to their destination. Here, retinal ganglion cells (RGCs) send their axons out of the retina, to cross with axons of the opposite eye in the optic chiasm and continue their journey through the diencephalon as an optic tract, before reaching their target, the optic tectum. During formation of the optic tract, RGC axons respond to both chemical and mechanical signals in the environment. A key player in sensing mechanical cues is the mechanosensitive ion channel (MSC) Piezo1. Perturbations of Piezo1 resulted in aberrant axon guidance in vivo. When I studied Piezo1 downregulation further, I found that it regulates not only axon pathfinding but also mechanical tissue properties. Tissue-specific downregulation of Piezo1 in either optic tract axons or in the surrounding neuroepithelia both resulted in optic pathway defects; suggesting that cell-autonomous and non-cell autonomous processes are mechanosensitive. Since Piezo1 is thought to respond exclusively to mechanical signals, I therefore set out to determine if tissue stiffness, cellular mechanosensing, or both were affected by Piezo1 downregulation. Knocking down Piezo1 softened brain tissue, suggesting that the axons were encountering an environment with altered mechanical properties, and therefore mechanical signalling was affected. In addition to tissue softening, the downregulation of Piezo1 dramatically altered the expression of semaphorin3A mRNA (sema3A), a chemical guidance cue known to be critical in axon pathfinding. Thus, both chemical and mechanical signals encountered by the axons were altered when Piezo1 was less abundant in the brain. To determine if tissue softening was caused by decreased Sema3A expression, I downregulated Sema3A and measured brain mechanics. However, I found that tissue stiffness actually increased, suggesting that the tissue softening observed in Piezo1-depleted brains was not the result of decreased Sema3A. While decreasing or increasing tissue stiffness did not significantly alter sema3A levels in vivo, increasing substrate stiffness in vitro resulted in an increase in sema3A levels in culture neuroepithelia, suggesting that expression and availability of this signalling molecule may still be modulated by tissue mechanics. My results suggest that Piezo1 plays critical cell autonomous and non-cell autonomous roles in axon guidance, and that perturbing this MSC results in alterations in both mechanical and chemical signalling. Furthermore, preliminary results suggest that mechanical signalling potentially regulates the transcription of chemical signalling molecules during development, implying that mechanical and chemical signalling are inextricably linked. This could have major implications for understanding the regulation of key biological processes in development, physiology, and pathophysiology.Malaysian Commonwealth Studies Centr