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 ...

Princeton University

During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction.

Passive mechanical forces control cell-shape change during Drosophila ventral furrow formation.

Many models of morphogenesis are forced to assume specific mechanical properties of cells, because the actual mechanical properties of living tissues are largely unknown. Here, we measure the rheology of epithelial cells in the cellularizing Drosophila embryo by injecting magnetic particles and studying their response to external actuation. We establish that, on timescales relevant to epithelial morphogenesis, the cytoplasm is predominantly viscous, whereas the cellular cortex is elastic. The timescale of elastic stress relaxation has a lower bound of 4 min, which is comparable to the time required for internalization of the ventral furrow during gastrulation. The cytoplasm was measured to be ∼10-fold as viscous as water. We show that elasticity depends on the actin cytoskeleton and conclude by discussing how these results relate to existing mechanical models of morphogenesis.

Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids.

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.

Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal the kinetics of gene expression downstream of a developmental transcription factor in vivo. We engineer light-controlled versions of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent transcription of giant and hunchback and delayed repression of Krüppel. In addition, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a noncanonical role for Bicoid in directly suppressing knirps transcription. Acute modulation of transcription factor concentration while recording output gene activity represents a powerful approach for studying developmental gene networks in vivo.

Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation.

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
Passive mechanical forces control cell-shape change during Drosophila ventral furrow formation. Biophysical journal\| PubMed ID: 25140436	2014
Measurement of cortical elasticity in Drosophila melanogaster embryos using ferrofluids. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 28096360	2017
Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during mesoderm invagination. eLife\| PubMed ID: 35195065	2022
Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation. Cell reports\| PubMed ID: 35320726	2022
An optogenetic tool to inhibit RhoA in Drosophila embryos. STAR protocols\| PubMed ID: 36598852	2023

Michael Swan

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