Optogenetic investigation of mechanical force propagation across scales in the developing zebrafish neuroepithelium

Abstract

During embryonic development, morphogenetic processes orchestrate the formation of tissues and organs with complex three-dimensional architecture. Morphogenesis involves changes in tissue shape, that in turn are driven by coordinated cell behaviours, such as cell shape changes and movements. These behaviours are themselves driven by intracellular forces, generated via actomyosin-mediated cell contractility. Mechanical forces are also transmitted to the wider tissue, forming the basis of mechanical signalling – an important regulator of development and disease. This thesis addresses the fundamental question of how mechanical forces propagate from individual cells to coordinate tissue-scale morphogenesis during neural development. To do this, I investigated how cell-scale forces generated at the lateral edges of neuroepithelial progenitor cells propagate on the tissue scale during zebrafish neural rod morphogenesis. It has been technically challenging to manipulate mechanical forces in deep three-dimensional tissues. To address this issue, I developed an in vivo optogenetic approach to reversibly manipulate endogenous actomyosin contractility via RhoA activation in the zebrafish neural rod. This method is non-invasive, reversible, has high spatiotemporal specificity and can be performed at depth. Manipulation of endogenous actomyosin contractility also ensures that forces are within a physiological range. The response of tissue to optogenetic induction of actomyosin contractility is measured using particle image velocimetry analysis. This experimental system enabled the investigation of cellular force propagation to tissues during zebrafish neural rod morphogenesis. Optogenetic activation of actomyosin contractility at the lateral edges of neuroepithelial progenitor cells induced mediolateral cell shortening. At the tissue-scale, these local changes in cell shape led to long-ranging and elastic tissue displacement along the tangential anterior-posterior axis. Surprisingly, this displacement was asymmetric in the anterior-posterior axis, occurring in either the anterior or the posterior directions, correlating with local gradients in optogenetic activation. Interestingly, this response did not significantly change as development progressed, suggesting that the state of epithelial transition did not significantly impact the extent of force propagation from lateral cortices. Tissue movement was attenuated by rhombomere boundaries (hindbrain segments), indicating pre-existing mechanical patterning along the anterior-posterior axis. I also discovered oscillations in expansion and contraction along the anterior-posterior axis, on the scale of rhombomere segments, which may emerge from mechanical coupling between individual cells. Together this work helps explain how precise morphogenetic patterns occur across distances in response to small local changes in cellular contractility. This work suggests long-range mechanical coupling exists between cells, even before the neuroepithelial precursor tissue has fully polarised. In summary, this thesis presents a method enabling the reversible control of cellular force generation in an in vivo vertebrate model and provides the basis for a mechanical explanation for signal amplification and patterning on the tissue scale, both fundamental motifs in morphogenesis.