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

Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts and functions to maintain cortical myosin during the flow phase. The subsequent direct myosin recruitment, however, is Dunk-independent but requires Slam. The Slam-dependent direct recruitment of myosin is sufficient to drive cleavage in the dunk mutant, and the subsequent development of the mutant is normal. In the dunk mutant, cortical myosin loss triggers misdirected flow and disrupts the hexagonal packing of the ingressing furrows. Computer simulation coupled with laser ablation suggests that Dunk-dependent maintenance of cortical myosin enables mechanical tension build-up, thereby providing a mechanism to guide myosin flow and define the hexagonal symmetry of the furrows.

Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity.

The Toll-Dorsal signaling pathway controls dorsal-ventral (DV) patterning in early Drosophila embryos, which defines specific cell fates along the DV axis and controls morphogenetic behavior of cells during gastrulation and beyond. The extent by which DV patterning information regulates subcellular organization in pre-gastrulation embryos remains unclear. We find that during Drosophila cleavage, the late endosome marker Rab7 is increasingly recruited to the yolk granules and promotes the formation of dynamic membrane tubules. The biogenesis of yolk granule tubules is positively regulated by active Rab7 and its effector complex HOPS, but negatively regulated by the Rab7 effector retromer. The occurrence of tubules is strongly biased towards the ventral side of the embryo, which we show is controlled by the Toll-Dorsal signaling pathway. Our work provides the first evidence for the formation and regulation of yolk granule tubulation in oviparous embryos and elucidates an unexpected role of Toll-Dorsal signaling in regulating this process.

Toll-Dorsal signaling regulates the spatiotemporal dynamics of yolk granule tubulation during Drosophila cleavage.

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.

Epithelial folding mediated by apical constriction serves as a fundamental mechanism to convert flat epithelial sheets into multilayered structures. It remains unknown whether additional mechanical inputs are required for apical constriction-mediated folding. Using Drosophila mesoderm invagination as a model, we identified an important role for the non-constricting, lateral mesodermal cells adjacent to the constriction domain ('flanking cells') in facilitating epithelial folding. We found that depletion of the basolateral determinant Dlg1 disrupts the transition between apical constriction and invagination without affecting the rate of apical constriction. Strikingly, the observed delay in invagination is associated with ineffective apical myosin contractions in the flanking cells that lead to overstretching of their apical domain. The defects in the flanking cells impede ventral-directed movement of the lateral ectoderm, suggesting reduced mechanical coupling between tissues. Specifically disrupting the flanking cells in wild-type embryos by laser ablation or optogenetic depletion of cortical actin is sufficient to delay the apical constriction-to-invagination transition. Our findings indicate that effective mesoderm invagination requires intact flanking cells and suggest a role for tissue-scale mechanical coupling during epithelial folding.

The cell polarity determinant Dlg1 facilitates epithelial invagination by promoting tissue-scale mechanical coordination.

During tissue morphogenesis, the changes in cell shape, resulting from cell-generated forces, often require active regulation of intracellular trafficking. How mechanical stimuli influence intracellular trafficking and how such regulation impacts tissue mechanics are not fully understood. In this study, we identify an actomyosin-dependent mechanism involving Rab11-mediated trafficking in regulating apical constriction in the Drosophila embryo. During Drosophila mesoderm invagination, apical actin and Myosin II (actomyosin) contractility induces apical accumulation of Rab11-marked vesicle-like structures ("Rab11 vesicles") by promoting a directional bias in dynein-mediated vesicle transport. At the apical domain, Rab11 vesicles are enriched near the adherens junctions (AJs). The apical accumulation of Rab11 vesicles is essential to prevent fragmented apical AJs, breaks in the supracellular actomyosin network, and a reduction in the apical constriction rate. This Rab11 function is separate from its role in promoting apical Myosin II accumulation. These findings suggest a feedback mechanism between actomyosin activity and Rab11-mediated intracellular trafficking that regulates the force generation machinery during tissue folding.

Actomyosin activity-dependent apical targeting of Rab11 vesicles reinforces apical constriction.

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)..

Bing He

Department of Biological Sciences

Dartmouth College

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

Bing He

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biological Sciences

Dartmouth College

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

Department of Biological Sciences