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Piezo1-dependent mechanical regulation of chemical signalling in the developing Xenopus laevis brain https://doi.org/10.17863/CAM.116962 Mukherjee, Sudipta Franze, kristian Adams, Richard During brain development, neurons extend axons to distant regions. These axons are guided towards their correct targets by various chemo-mechanical signals. Major chemical signals include semaphorins, slits, netrins and ephrins/Eph, whereas mechanical signals include tissue stiffness, viscosity and cellular forces. In the developing embryo, chemical and mechanical signals are not isolated. However, how cells integrate these two different types of signals remains poorly understood. Here, I investigated how tissue stiffness and longrange chemical guidance cues interact, using the developing Xenopus laevis forebrain as a model system. First, I adapted hybridisation chain reaction RNA fluorescence in situ hybridisation (HCR RNA-FISH) to Xenopus laevis brains. I then probed the expression of different chemical cues upon knockdown of the mechanosensitive ion channel Piezo1, which transduces mechanical signals into intracellular chemical signals. Piezo1 downregulation led to a decrease in both semaphorin 3A (sema3A) and slit1 expression. Piezo1 depletion not only altered the chemical landscape of the developing brain, but also its mechanical properties. To understand how Piezo1 regulates tissue stiffness, I investigated microtubule acetylation, single cell stiffness and cell adhesions. Microtubule acetylation and single cell stiffness were Piezo1-independent. However, the expression of adhesion molecules NCAM1 and N-cadherin were highly decreased upon Piezo1 downregulation. Knocking down both NCAM1 and N-cadherin decreased tissue stiffness and reduced both sema3A and Piezo1 expression, suggesting mutual regulation of cell-cell adhesions and Piezo1. To investigate if mechanical cues are sufficient to alter chemical signalling cues, I modulated stiffness in vitro and in vivo. Culturing soft parts of the brain, which normally do not produce sema3A or slit1, in stiff hydrogel substrates increased sema3A and slit1 expression, demonstrating that the expression of chemical cues is regulated by substrate stiffness. Compression stiffening soft parts of the brain in vivo increased sema3A but not slit1 expression. Stiffening Piezo1 knockdown brains, however, did not increase sema3A expression, suggesting compression stiffened sema3A upregulation is Piezo1-dependent. My findings suggest that cell-cell adhesions and tissue mechanics mutually regulate each other and tissue mechanics in turn regulates the transcription of chemical cues. Due to the conserved nature of the molecules involved, this dynamic and complex crosstalk between chemical and mechanical signalling might underlie many developmental and disease-related phenomena across various species. University of Cambridge 2024-09-29 Thesis Doctoral Doctor of Philosophy (PhD) eng Wellcome trust PhD Studentship (222280/Z/20/Z) and Cambridge Trust. https://www.repository.cam.ac.uk/handle/1810/381956 embargo 2031-03-26 https://www.repository.cam.ac.uk/bitstreams/cd37db5c-ab2a-47c5-bdb4-42387f39570d/download 87eda9de84448d1f82354d60eee3eb5f https://www.repository.cam.ac.uk/bitstreams/689f31d1-89b5-4a80-974d-ec7b4674dab1/download f1e6c1f40221628bececea7ccf3a94d9 http://purl.org/NET/rdflicense/allrightsreserved axon pathfinding mechanobiology mechanotransduction neuronal guidance