optogenetic-inhibition-rho1-mediated-actomyosin-contractility-coupled

Optogenetic Inhibition of Rho1-Mediated Actomyosin Contractility Coupled with Measurement of Epithelial Tension in <em>Drosophila</em> Embryos

Contractile forces generated by actin and non-muscle myosin II ("actomyosin contractility") are critical for morphological changes of cells and tissues at ...

Dartmouth College

Westlake University

Apical constriction driven by actin and non-muscle myosin II (actomyosin) provides a well-conserved mechanism to mediate epithelial folding. It remains unclear how contractile forces near the apical surface of a cell sheet drive out-of-the-plane bending of the sheet and whether myosin contractility is required throughout folding. By optogenetic-mediated acute inhibition of actomyosin, we find that during  mesoderm invagination, actomyosin contractility is critical to prevent tissue relaxation during the early, 'priming' stage of folding but is dispensable for the actual folding step after the tissue passes through a stereotyped transitional configuration. This binary response suggests that  mesoderm is mechanically bistable during gastrulation. Computer modeling analysis demonstrates that the binary tissue response to actomyosin inhibition can be recapitulated in the simulated epithelium that undergoes buckling-like deformation jointly mediated by apical constriction in the mesoderm and in-plane compression generated by apicobasal shrinkage of the surrounding ectoderm. Interestingly, comparison between wild-type and  mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. We propose that  mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling.

Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during  mesoderm invagination.

The folding of two-dimensional epithelial sheets into specific three-dimensional structures is a fundamental tissue construction mechanism in animal development. A common mechanism that mediates epithelial folding is apical constriction, the active shrinking of cell apices driven by actomyosin contractions. It remains unclear whether cells outside of the constriction domain also contribute to folding. During  mesoderm invagination, ventrally localized mesoderm epithelium undergoes apical constriction and subsequently folds into a furrow. While the critical role of apical constriction in ventral furrow formation has been well demonstrated, it remains unclear whether, and if so, how the laterally localized ectodermal tissue adjacent to the mesoderm contributes to furrow invagination. In this study, we combine experimental and computational approaches to test the potential function of the ectoderm in mesoderm invagination. Through laser-mediated, targeted disruption of cell formation prior to gastrulation, we found that the presence of intact lateral ectoderm is important for the effective transition between apical constriction and furrow invagination in the mesoderm. In addition, using a laser-ablation approach widely used for probing tissue tension, we found that the lateral ectodermal tissues exhibit signatures of tissue compression when ablation was performed shortly before the onset of mesoderm invagination. These observations led to the hypothesis that in-plane compression from the surrounding ectoderm facilitates mesoderm invagination by triggering buckling of the mesoderm epithelium. In support of this notion, we show that the dynamics of tissue flow during mesoderm invagination displays characteristic of elastic buckling, and this tissue dynamics can be recapitulated by combining local apical constriction and global compression in a simulated elastic monolayer. We propose that  mesoderm invagination is achieved through epithelial buckling jointly mediated by apical constriction in the mesoderm and compression from the neighboring ectoderm.

Evidence for a Role of the Lateral Ectoderm in  Mesoderm Invagination.

We describe a protocol for optogenetic inhibition of the small GTPase Rho1 (RhoA) in Drosophila embryos, which allows rapid and spatially confined inactivation of Rho1 and Rho1-mediated actomyosin contractility. We provide step-by-step instruction for optogenetic manipulations of Drosophila embryos using confocal and multiphoton imaging systems. This tool is useful for determining the site- and stage-specific function of Rho1 in Drosophila embryos and for studying the immediate tissue response to acute elimination of cellular contractility. For complete details on the use and execution of this protocol, please refer to Guo et al. (2022)..

Article	Year
Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during mesoderm invagination. eLife\| PubMed ID: 35195065	2022
Evidence for a Role of the Lateral Ectoderm in Mesoderm Invagination. Frontiers in cell and developmental biology\| PubMed ID: 35547820	2022
An optogenetic tool to inhibit RhoA in Drosophila embryos. STAR protocols\| PubMed ID: 36598852	2023

Hanqing Guo

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