The mechanical regulation of Semaphorin3A signalling in the developing Xenopus laevis brain

Abstract

Establishing neuronal connectivity accurately during development is vital for the functioning of the mature brain. Failure of axons to connect to their correct downstream targets is linked to a host of neurodevelopmental disorders. Axons extending through the developing brain interpret gradients of both chemical and mechanical cues for navigation. In this thesis, I investigated how these two signalling modalities interact to achieve robust axon guidance, using the Xenopus retinotectal system as a model. Specifically, I studied how the response of retinal ganglion cell axons to the classical repulsive chemical guidance cue Semaphorin3A is regulated by substrate stiffness. The strength of axonal turning responses to Semaphorin3A can be quantified using a turning assay, whereby axons change their trajectory in response to a gradient of a chemical cue. I developed two alternative turning assay setups with the potential to improve the stability of these gradients, and so the consistency of axon responses. I first used the Fluicellâ Biopen, a free-standing microfluidic device that enables controlled chemical stimulation within subcellular regions. The second assay was based on functionalised beads designed to bind and release chemical guidance cues. I designed novel assay protocols for both approaches, characterised them and conducted preliminary turning assays. However, each turning assay design had significant limitations that prevented demonstrations of reliable turning responses. I then investigated the mechanisms by which substrate stiffness regulates axon responses to Semaphorin3A. I focused on the mechanosensitive ion channel Piezo1 due to data supporting its in vivo requirement for Xenopus retinal ganglion cell axon pathfinding. To replicate increased axon outgrowth on stiff gels, I refined the protocols for compliant gel fabrication and Xenopus eye primordium dissection. I re-established optical membrane potential recording of Xenopus axons, replicating the finding that axons on soft gels are more depolarised than on stiff gels. To develop a Piezo1 knockdown model complimentary to the established translation-blocking morpholino, I introduced the use of both CRISPR-Cas13 and CRISPR-Cas9 to the Xenopus model in the lab. CRISPR-Cas9 proved more promising; therefore, I designed, characterised and tested several guide RNAs. I evaluated the crispant embryos using their morphology, DNA sequencing, protein levels, retinal ganglion cell axon pathfinding and chemical guidance cue expression in the brain. My results indicated that, despite successfully editing the targeted DNA, the CRISPR-generated phenotypes were not distinguishable from control embryos. This unexpected discrepancy warrants further investigation. The study of how Semaphorin3A signalling is regulated by substrate stiffness bears relevance not only in development, but also in homeostasis, disease and injury across multiple tissue types. An improved understanding of chemo-mechanical interplay will thus be of great benefit in many research fields.