Geometric control of myosin II orientation during axis elongation
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eLife assessment
This paper should be of broad interest to developmental biologists who seek to understand spatiotemporal control of myosin-based force generation during tissue morphogenesis during early development. The central conclusions are well-grounded in rigorous quantitative data analysis and modeling. The results challenge current views of how gene expression patterns control myosin II anisotropies and provide new testable hypotheses on the role and importance of tissue geometry.
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Abstract
The actomyosin cytoskeleton is a crucial driver of morphogenesis. Yet how the behavior of large-scale cytoskeletal patterns in deforming tissues emerges from the interplay of geometry, genetics, and mechanics remains incompletely understood. Convergent extension in Drosophila melanogaster embryos provides the opportunity to establish a quantitative understanding of the dynamics of anisotropic non-muscle myosin II. Cell-scale analysis of protein localization in fixed embryos suggests that gene expression patterns govern myosin anisotropy via complex rules. However, technical limitations have impeded quantitative and dynamic studies of this process at the whole embryo level, leaving the role of geometry open. Here, we combine in toto live imaging with quantitative analysis of molecular dynamics to characterize the distribution of myosin anisotropy and the corresponding genetic patterning. We found pair rule gene expression continuously deformed, flowing with the tissue frame. In contrast, myosin anisotropy orientation remained approximately static and was only weakly deflected from the stationary dorsal-ventral axis of the embryo. We propose that myosin is recruited by a geometrically defined static source, potentially related to the embryo-scale epithelial tension, and account for transient deflections by cytoskeletal turnover and junction reorientation by flow. With only one parameter, this model quantitatively accounts for the time course of myosin anisotropy orientation in wild-type, twist , and even-skipped embryos, as well as embryos with perturbed egg geometry. Geometric patterning of the cytoskeleton suggests a simple physical strategy to ensure a robust flow and formation of shape.
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eLife assessment
This paper should be of broad interest to developmental biologists who seek to understand spatiotemporal control of myosin-based force generation during tissue morphogenesis during early development. The central conclusions are well-grounded in rigorous quantitative data analysis and modeling. The results challenge current views of how gene expression patterns control myosin II anisotropies and provide new testable hypotheses on the role and importance of tissue geometry.
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Reviewer #1 (Public Review):
In this study, Lefebvre et al. investigate the interplay between tissue geometry and the expression patterns of Runt and Tartan in establishing anisotropic myosin localization during germband extension in the Drosophila embryo. Using live and fixed light sheet imaging, computational analysis, and modeling, the authors establish a global time-resolved map of Runt expression and myosin localization during germband extension. They show that a posterior Runt stripe increasingly deviates from the dorsoventral (DV) axis during elongation, while myosin anisotropy in this region transiently deviates from the DV axis and then realigns with this axis after a delay. The authors attribute this delay to the timescale of myosin turnover and the realignment to an unidentified geometric cue. The authors develop a model that …
Reviewer #1 (Public Review):
In this study, Lefebvre et al. investigate the interplay between tissue geometry and the expression patterns of Runt and Tartan in establishing anisotropic myosin localization during germband extension in the Drosophila embryo. Using live and fixed light sheet imaging, computational analysis, and modeling, the authors establish a global time-resolved map of Runt expression and myosin localization during germband extension. They show that a posterior Runt stripe increasingly deviates from the dorsoventral (DV) axis during elongation, while myosin anisotropy in this region transiently deviates from the DV axis and then realigns with this axis after a delay. The authors attribute this delay to the timescale of myosin turnover and the realignment to an unidentified geometric cue. The authors develop a model that can largely account for myosin localization in wild-type, eve mutant, and twist mutant embryos using a myosin lifetime parameter representing myosin turnover. These results provide evidence for a static signal that aligns myosin anisotropy with the DV axis during elongation.
The strengths of this paper are the combination of modeling and quantitative measurements. Powerful in toto measurements show that myosin anisotropy becomes increasingly misaligned with Runt, an essential regulator of myosin planar polarity, at later stages of elongation in posterior regions of the embryo. In addition, the authors present a simple model in which changes in one parameter representing the myosin lifetime can recapitulate the relationship between myosin and edge orientation in wild-type, eve mutant, and twist mutant embryos.
The main weakness of the paper is that the authors do not directly test if their model correctly predicts the myosin lifetime in eve mutants, twist mutants, or in Fat2-RNAi embryos with altered geometry. As myosin turnover is the key parameter in their model, measuring myosin dynamics in these backgrounds would provide an important first test of their model. In addition, the authors should attempt to relate their measurements of myosin dynamics in wild-type embryos to the myosin lifetime value predicted by their model, and they should consider alternative explanations that could account for their observations in wild-type and mutant embryos.
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Reviewer #2 (Public Review):
The manuscript by Lefebvre et al. investigates how the tissue-scale spatial organization of protein evolves during germ band extension. The key question is whether changes in the localization of important features such as pair-rule gene (PRG) stripes and apical myosin orientation can be explained purely via passive advection without the need for additional regulatory mechanisms. In the case of the PRG, as well as TLRs, their data strongly suggests the answer is yes: the authors show that the deformation of the characteristic stripe pattern closely matches that predicted by advecting the initial pattern in a velocity field extracted from the observed tissue flow. By contrast, the authors find that anisotropic myosin orientation cannot be explained purely in terms of the local velocity field, in particular the …
Reviewer #2 (Public Review):
The manuscript by Lefebvre et al. investigates how the tissue-scale spatial organization of protein evolves during germ band extension. The key question is whether changes in the localization of important features such as pair-rule gene (PRG) stripes and apical myosin orientation can be explained purely via passive advection without the need for additional regulatory mechanisms. In the case of the PRG, as well as TLRs, their data strongly suggests the answer is yes: the authors show that the deformation of the characteristic stripe pattern closely matches that predicted by advecting the initial pattern in a velocity field extracted from the observed tissue flow. By contrast, the authors find that anisotropic myosin orientation cannot be explained purely in terms of the local velocity field, in particular the fact that myosin remains robustly oriented with the DV axis. This leads the authors to postulate that myosin orientation is continually re-established via a static source aligned with said axis, which dominates over re-orientation due to advection. A simple model of myosin reorientation is developed from this hypothesis, which produces qualitatively similar relationships between orientation and local vorticity to that seen both in WT and in several mutants.
The strongest feature of this paper is illustrated by the results in Figure 2. The result it presents, which the authors summarize as "PRGs flow with tissue while myosin does not," is a very nice application of recent advances in using toto microscopy for embryonic systems to extract and quantify whole embryo expression patterns and flow fields, which are needed information for this kind of result. Tissue flow is a complicated, active process, and identifying which parts of the dynamics can be sufficiently explained by passive transport can tremendously simplify the conceptual challenges of germ band extension and related tissue movements found during neurulation or organogenesis. The resistance the authors found that myosin exhibits to re-orientation is likewise very interesting because it implies that information about global geometry (the direction of the DV axis) is somehow maintained at the cellular level throughout the convergent extension.
The principle weakness in this manuscript is the vagueness of the proposed static source mechanism and the lack of direct evidence for it in experiments. The FRAP experiments performed here suggest that binding/unbinding happens on the right timescale to play a role in anisotropy maintenance, but if the principle question is 'how does myosin remain oriented along the DV axis' then the static source hypothesis just kicks the can down the road to ask 'how does the static source remain oriented along the DV axis'? The minimal model the authors employ has the benefit that it lets them relate angular deviation to vorticity, at the cost that it is agnostic to the form and nature of the source term, so it cannot be used to extract useful constraints. This said the evidence provided regarding the connection between vorticity and binding rates to myosin deflection is sufficient indirect evidence of the hypothesized mechanism that I suspect it will be of interest to a good number of people interested in epithelial morphogenesis.
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Reviewer #3 (Public Review):
Lefevbre et al combine in toto imaging with "tissue cartography" to investigate the respective roles of pair-rule (PR) and toll-like receptor (TLR) gene expression, and embryo geometry, in shaping anisotropic distributions of myosin II during germband elongation (GBE) in Drosophila embryos. The authors find that the simple dependence of Myosin II on PR and TLR expression gradients cannot explain observed global patterns of myosin II. PR and TLR expression patterns evolve continuously as expressing cells are advected by tissue flow during GBE, while myosin II anisotropies remain roughly stationary even as myosin-rich junctions are advected and reoriented by tissue flows. The authors show that the observed spatiotemporal evolution of myosin II anisotropies in wild-type and certain mutant embryos can instead be …
Reviewer #3 (Public Review):
Lefevbre et al combine in toto imaging with "tissue cartography" to investigate the respective roles of pair-rule (PR) and toll-like receptor (TLR) gene expression, and embryo geometry, in shaping anisotropic distributions of myosin II during germband elongation (GBE) in Drosophila embryos. The authors find that the simple dependence of Myosin II on PR and TLR expression gradients cannot explain observed global patterns of myosin II. PR and TLR expression patterns evolve continuously as expressing cells are advected by tissue flow during GBE, while myosin II anisotropies remain roughly stationary even as myosin-rich junctions are advected and reoriented by tissue flows. The authors show that the observed spatiotemporal evolution of myosin II anisotropies in wild-type and certain mutant embryos can instead be explained by a simple model in which a geometric cue promotes myosin II accumulation of vertically oriented junctions, flows advect myosin-rich junctions, and myosin II turns over on a ~5-minute timescale.
The core findings are well-supported by rigorous quantitative analysis and modeling; they provide a fresh perspective on the role of geometry in the dynamic control of myosin II anisotropies. Thus they are likely to stimulate further experimental work to identify and characterize the underlying basis for this geometric control.
Key strengths
A key strength is the use of in toto light sheet imaging and tissue cartography, plus the high stereotypy of early Drosophila development, which allows the authors to assimilate data across multiple embryos to extract robust quantitative signatures of gene expression, protein localization, and tissue flows that allows robust analysis of relationships between these different factors in the wild type and across different mutants.
A second strength is the introduction of a very simple model for the evolution of myosin II anisotropy driven by local tissue rotation and myosin turnover which allows decomposing of their respective contributions.
Weaknesses
The power of the model is tested only by its sufficiency to reproduce observed features of myosin II anisotropy over time. There is no direct test/verification of a core model assumption - that the local binding of myosin II is biased with respect to a static geometric signal. Similarly, the inference from the model fits that myosin binding times are reduced in eve mutants has not been confirmed (e.g. by FRAP experiments).
There are a number of (clearly fixable) issues with the clarity of presentation - especially if the authors wish to make their work accessible to a broad audience. The comparison of model predictions and experimental observations is presented in a somewhat confusing way. Ditto for the analysis of mutant phenotypes and the conclusions drawn from this analysis. Some key information about the choices made to justify a very simple model (i.e. why alternative hypotheses and/or additional complexity in the junctional dynamics can be ignored) is presented only in the Supplementary text and should be summarized in the main text.
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