Author response: Optogenetic inhibition of actomyosin reveals mechanical bistability of the mesoderm epithelium during Drosophila mesoderm invagination
Year: 2021
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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 Drosophila 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 Drosophila 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 snail mutants that fail to specify the mesoderm demonstrates that the lateral ectoderm undergoes apicobasal shrinkage during gastrulation independently of mesoderm invagination. We propose that Drosophila mesoderm invagination is achieved through an interplay between local apical constriction and mechanical bistability of the epithelium that facilitates epithelial buckling. Editor's evaluation The authors examine the process of mesoderm invagination in the Drosphila embryo and found that while myosin contractility is critical to prevent tissue relaxation during the early phase of the process, it is dispensable for the subsequent folding step. Through modeling and experimental analyses, the authors find that folding is likely mediated by a joint action of active cell shape changes in the mesoderm and apico-basal shrinking in the surrounding ectoderm and suggest that the mesoderm behave as a mechanically bistable tissue during gastrulation. https://doi.org/10.7554/eLife.69082.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Contractile forces generated by actin and myosin (actomyosin) networks are widely employed in embryogenesis to drive cell motility and cell shape change that pattern epithelial tissues (Martin and Goldstein, 2014; Munjal and Lecuit, 2014). During epithelial folding mediated by apical constriction, the actomyosin network in epithelial cells constricts cell apices, which leads to bending of the cell sheet (Sawyer et al., 2010). It remains unclear how ‘in-plane’ contractile forces generated at the apical surface drive ‘out-of-the-plane’ folding of the tissue. The invagination of the presumptive mesoderm during Drosophila gastrulation is a well-characterized epithelial folding process mediated by apical constriction (reviewed in Gilmour et al., 2017; Martin, 2020; Gheisari et al., 2020). During gastrulation, ventrally localized mesoderm precursor cells constrict their cell apices and become internalized into a ventral furrow. The folding process occurs in two steps (Leptin and Grunewald, 1990; Sweeton et al., 1991). During the first 10–12 min, the ventral cells undergo apical constriction and elongate in the apical-basal direction, while the cell apices remain near the surface of the embryo (the lengthening phase). In the next 6–8 min, the ventral cells rapidly internalize as they shorten back to a wedge-like morphology (the shortening phase) (Figure 1a). Ventral furrow formation is controlled by the dorsal-ventral (DV) patterning system. The maternally deposited morphogen Dorsal controls the expression of two transcription factors, Twist and Snail, in the presumptive mesoderm. Twist and Snail in turn cause myosin activation at the apical pole of ventral mesodermal cells through a sequential action of G-protein coupled receptor (GPCR) signaling (Leptin, 1991; Parks and Wieschaus, 1991; Costa et al., 1994; Kölsch et al., 2007; Manning et al., 2013; Kerridge et al., 2016) and RhoGEF2-Rho1-Rho associated kinase (Rok) pathway (Barrett et al., 1997; Häcker and Perrimon, 1998; Nikolaidou and Barrett, 2004; Dawes-Hoang et al., 2005; Martin et al., 2009; Mason et al., 2013). Activated myosin forms a supracellular actomyosin network across the apical surface of the prospective mesoderm and drives the constriction of cell apices (Martin et al., 2010; Martin et al., 2009; Martin and Goldstein, 2014). Figure 1 with 3 supplements see all Download asset Open asset Light-dependent membrane recruitment of CRY2-Rho1DN results in rapid myosin inactivation. (a) Drosophila mesoderm invagination occurs in distinct lengthening and shortening phases. Ventral side is up (same for all cross-section images in this work unless otherwise stated). A single ventral cell undergoing apical constriction is outlined in green. In this study, we sought to test whether myosin contractility is required throughout the folding process by acute, stage-specific inhibition of Rho1 (arrowheads). (b) Cartoon depicting the principle of the optogenetic tool used in this study. Upon blue light stimulation, CRY2-Rho1DN is translocated from the cytosol to the plasma membrane through the interaction between CRY2 and membrane anchored CIBN. (c) Confocal images showing the rapid membrane recruitment of CRY2-Rho1DN upon blue light illumination. (d) Relative abundance of membrane recruited CRY2-Rho1DN over time after blue light stimulation. Error bar: s.d., N=6 embryos. (e) A wild-type embryo expressing Sqh-GFP showing apical myosin accumulation during ventral furrow formation. N=4 embryos. (f) Activation of Opto-Rho1DN results in rapid dissociation of myosin from the ventral cell cortex (arrow) in a gastrulating embryo. N=8 embryos. (g) Confocal images showing the confined membrane recruitment of CRY2-Rho1DN within a region of interest (ROI, red box) that has been scanned by a focused beam of blue laser. N=6 embryos. All scale bars=20 μm. The essential role of apical constriction in ventral furrow formation has been well demonstrated. Genetic mutations or pharmacological treatments that inhibit myosin activity disrupt apical constriction and result in failure in ventral furrow formation (reviewed in Gheisari et al., 2020; Martin, 2020). Biophysical studies show that cell shape change occurred during the lengthening phase is a direct, viscous response of the tissue interior to apical constriction (Gelbart et al., 2012; He et al., 2014). However, accumulating evidence suggests that apical constriction does not directly drive invagination during the shortening phase. First, it has been observed that the maximal rate of apical constriction (or cell lengthening) and the maximal rate of tissue invagination occur at distinct times (Polyakov et al., 2014; Rauzi et al., 2015), and timed injection of Rok inhibitor indicates that the late stage of ventral furrow invagination is less sensitive to myosin inactivation (Krajcovic and Minden, 2012). Second, it has been previously proposed, and more recently experimentally demonstrated, that myosin accumulated at the lateral membranes of constricting cells (lateral myosin) facilitates furrow invagination by exerting tension along the apical-basal axis of the cell (Brodland et al., 2010; Conte et al., 2012; Gracia et al., 2019; John and Rauzi, 2021). Finally, a number of computational models predict that mesoderm invagination requires additional mechanical input from outside of the mesoderm, such as ‘pushing’ forces from the surrounding ectodermal tissue (Muñoz et al., 2007; Conte et al., 2009; Allena et al., 2010; Brodland et al., 2010). These models are in line with the finding that blocking the movement of the lateral ectoderm by laser cauterization inhibits mesoderm invagination (Rauzi et al., 2015). A similar disruption of ventral furrow formation can also be achieved by increasing actomyosin contractility in the lateral ectoderm (Perez-Mockus et al., 2017). While these pioneer studies highlight the importance of cross-tissue coordination during mesoderm invagination, the actual mechanical mechanism that drives the folding of the mesodermal epithelium and the potential role of the surrounding ectodermal tissue remain to be elucidated. In this study, we investigate the mechanics of ventral furrow formation by asking whether actomyosin contractility is required throughout the folding process. By developing an optogenetic tool to acutely inhibit actomyosin activity, we find that the dependence of furrow invagination on actomyosin contractility is strongly stage-dependent: Inhibition of actomyosin during apical constriction results in immediate relaxation of the constricted tissue, whereas similar treatment after a stereotyped transitional stage does not impede invagination, suggesting that the mesoderm epithelium has two stable mechanical status during gastrulation. The binary tissue response to actomyosin inhibition can be recapitulated by a 2D vertex model that combines apical constriction in the mesoderm and apicobasal shortening in the neighboring ectoderm, which generates in-plane compression due to volume conservation. Finally, we show evidence the lateral ectoderm undergoes apicobasal shortening around the transitional stage of ventral furrow formation. This ectodermal shortening process, as well as the associated ventrally directed movement of the ectoderm, could occur independently of mesoderm invagination. Taken together, we propose that Drosophila mesoderm epithelium is mechanically bistable during gastrulation. We further hypothesize that the mechanical bistability of the mesoderm is attributed to ectodermal compression and functions together with active cell shape change in the mesoderm to facilitate mesoderm invagination. Results Plasma membrane recruitment of Rho1DN results in rapid loss of apical myosin and F-actin network In order to acutely inhibit myosin activity in live embryos (Figure 1a), we generated an optogenetic tool, ‘Opto-Rho1DN,’ to inhibit Rho1 through light-dependent plasma membrane recruitment of a dominant negative form of Rho1 (Rho1DN). Like other Rho GTPases, Rho1 cycles between an active, GTP-bound state and an inactive, GDP-bound state (Etienne-Manneville and Hall, 2002). Rho1DN bears a T19N mutation, which abolishes the ability of the mutant protein to exchange GDP for GTP and thereby locks the protein in the inactive state (Barrett et al., 1997). A second mutation in Rho1DN, C189Y, abolishes its naive membrane targeting signal (Sebti and Der, 2003; Roberts et al., 2008). When Rho1DN is recruited to the plasma membrane, it binds to and sequesters Rho1 GEFs, thereby preventing activation of endogenous Rho1 as well as Rho1-Rok-mediated activation of myosin (Barrett et al., 1997; Feig and Cooper, 1988). Opto-Rho1DN is based on the CRY2-CIB light-sensitive dimerization system (Liu et al., 2008; Kennedy et al., 2010; Guglielmi et al., 2015; Figure 1b). The tool contains two components: a GFP-tagged N-terminal of CIB1 protein (CIBN) anchored at the cytoplasmic leaflet of the plasma membrane (CIBN-pmGFP) (Guglielmi et al., 2015), and a fusion protein with Rho1DN, a mCherry tag, and a blue light-reactive protein Cryptochrome 2 (CRY2-Rho1DN). CRY2-Rho1DN remained in the cytoplasm when the sample was kept in the dark or imaged with a yellow-green (561 nm) laser (Figure 1c, T=0). Upon blue (488 nm) laser stimulation, CRY2 undergoes a conformational change and binds to CIBN (Kennedy et al., 2010; Liu et al., 2008), resulting in rapid recruitment of CRY2-Rho1DN to the plasma membrane (Figure 1c and d; Figure 1—video 1). The average time for half-maximal membrane recruitment of CRY2-Rho1DN is 4 s (3.9±2.8 s, mean±s.d., n=5 embryos). Embryos expressing CIBN-pmGFP and CRY2-Rho1DN developed normally in a dark environment and could hatch (100%, n=63 embryos). In contrast, most embryos (>85%, n=70 embryos) kept in ambient room light failed to hatch, reflecting the essential role of Rho1 in Drosophila embryogenesis (Barrett et al., 1997; Magie et al., 1999; Johndrow et al., 2004). The effectiveness of Opto-Rho1DN on myosin inactivation was validated by stimulating embryos during apical constriction, which resulted in diminishing of apical myosin signal within 60 s (Figure 1e and f; Figure 1—video 2). Rapid disappearance of cortical myosin upon Rho1 inhibition is consistent with the notion that Rho1 and myosin cycle quickly through active and inactive statues due to the activity of GTPase activating proteins (GAPs) for Rho1 and myosin light chain phosphatases, respectively (Munjal et al., 2015; Coravos and Martin, 2016). Our data also provide direct evidence that sustained myosin activity during ventral furrow formation requires persistent activation of Rho1. In addition to myosin, we found that acute Rho1 inhibition also affects cortical F-actin networks during apical constriction, presumably through inactivation of the Rho1 effector formin Diaphanous (Johndrow et al., 2004; Homem and Peifer, 2008; Mason et al., 2013; Coravos and Martin, 2016). In unstimulated embryo, F-actin is enriched at the apical domain of the constricting cells (Figure 2a, red arrowheads and cyan arrows) and along the lateral membrane in both constricting and non-constricting cells (Figure 2a, yellow arrows and magenta arrows). Apical F-actin disappeared within 1.2 min after Opto-Rho1DN stimulation (Figure 2b). Lateral F-actin in the constricting cells was not immediately affected and only appeared to diminish 4 min after stimulation (Figure 2b). In contrast, lateral F-actin in the ectodermal cells was not significantly affected (Figure 2b). Taken together, our results indicate that Opto-Rho1DN is an effective tool for spatially- and temporally-confined inactivation of apical actomyosin contractility through simultaneous myosin inactivation and actin disassembly. Figure 2 Download asset Open asset Rapid disassembly of apical F-actin in the constricting cells after Opto-Rho1DN stimulation. (a) Cross-sections (left) and en face views (right) of a representative unstimulated embryo showing the localization of the F-actin marker UtrophinABD-Venus (UtrABD-Venus) during ventral furrow formation. At the onset of apical constriction, F-actin is enriched along the lateral membrane in both mesodermal and ectodermal cells (yellow and magenta arrows, respectively). During apical constriction, F-actin also accumulates at the apical domain of the constricting cells (cyan arrows). T=0 (min) is the onset of ventral furrow formation. +0 μm indicates the apex of the ventral most cells (red arrowheads) where the strongest accumulation of apical F-actin is observed. (b) Upon Opto-Rho1DN stimulation (immediately before 6.2 min), apical F-actin disappears within 1.2 min (cyan arrows). Lateral F-actin in the constricting cells was not immediately affected and only started to diminish 4 min after stimulation (yellow arrows). Lateral F-actin in the ectodermal cells was not significantly affected (magenta arrows). Note that the initial apical indentation (red arrowheads) quickly disappeared after stimulation upon relaxation of the cell apex. N=3 for unstimulated embryos and N=10 for stimulated embryos. Scale bars: cross-sections, 20 μm; en face views, 10 μm. Consistent with the actomyosin phenotype, Opto-Rho1DN stimulation before or during apical constriction resulted in prevention or reversal of apical constriction, respectively (Figure 1—figure supplement 1). In addition, Opto-Rho1DN stimulation resulted in an immediate loss of cortical tension at the ventral surface of the embryo during apical constriction (Figure 3), in accordance with the previous finding that the increase in tissue tension in the ventral mesodermal region during apical constriction is due to activation of apical myosin contractility and integration of contractile forces across the constriction domain (Martin et al., 2010). Finally, by stimulating the embryo within a specific region of interest (ROI), we showed that membrane recruitment of CRY2-Rho1DN and inhibition of apical constriction are restricted to cells within the stimulated region (Figure 1g, Figure 1—figure supplement 1c, d). Figure 3 Download asset Open asset Opto-Rho1DN stimulation during apical constriction results in an immediate loss of cortical tension at ventral surface of the embryo. (a) Cartoon depicting the experimental setup for laser ablation to detect cortical tension. Yellow shaded regions indicate the ablated regions. For stimulated embryos, light-activation of Opto-Rho1DN was performed 3 min before the laser ablation. Due to apical relaxation after stimulation, multiple z-planes were ablated (yellow shaded region) in order to ensure the ablation of the very apical surface of the ventral cells. (b) Width changes of the ablated region along the A-P axis during the first 20 s after laser ablation. A clear tissue recoil was observed after laser cutting in the unstimulated control embryos. In contrast, little to no tissue recoil was observed in the stimulated embryos, indicating lack of apical tension after Rho1 inhibition. p value was calculated using two-sided Wilcoxon rank-sum test. (c–d) Kymographs showing the comparison between unstimulated and stimulated embryos. No obvious tissue recoil was observed in the stimulated embryos (N=6 for unstimulated embryos and N=5 for stimulated embryos). Dotted line indicates ablation site. Acute inhibition of myosin contractility reveals mechanical bistability of the mesoderm during gastrulation Using Opto-Rho1DN, we determined the role of actomyosin contractility at different stages of ventral furrow formation. We first obtained non-stimulated control movies by imaging embryos bearing Opto-Rho1DN on a multiphoton microscope using a 1040-nm pulsed laser, which excites mCherry but does not stimulate CRY2 (Guglielmi et al., 2015). Without stimulation, the embryos underwent normal ventral furrow formation (Figure 4a). The transition from the lengthening phase to the shortening phase (TL-S trans) occurred around 10 min after the onset of gastrulation (9.3±1.6 min, mean±s.d., n=10 embryos), which was similar to the wild-type embryos (8.9±0.8 min, mean±s.d., n=6 embryos) (Figure 4—figure supplement 1a-c). This transition was characterized by a rapid inward movement of the apically constricted cells and could be readily appreciated by the time evolution of distance between the cell apices and the vitelline membrane of the eggshell (invagination depth ‘D,’ Figure 4—figure supplement 1d). Figure 4 with 4 supplements see all Download asset Open asset Acute inhibition of actomyosin contractility results in stage-dependent response during ventral furrow formation. (a) Still images from multiphoton movies showing different tissue responses to acute loss of myosin contractility during ventral furrow formation. Early Group, Mid Group, and Late Group embryos (N=8, 4, and 6 embryos, respectively) are defined based on their immediate response to myosin inhibition. For stimulated embryos, the first frame corresponds to the time point immediately after stimulation. The inset depicts the stimulation and imaging protocol. Arrowheads indicate the apex of the ventral most cells. (b) Time evolution of the invagination depth ‘D’ for the stimulated embryos and a representative unstimulated control embryo. For stimulated embryos, all movies were aligned in time to the representative control embryo based on furrow morphology at the time of stimulation. (c) Relationship between the transition phase for sensitivity to myosin inhibition (Ttrans) and lengthening-shortening transition (TL-S trans). (d) Scatter plot showing the relation between invagination depth at the time of stimulation (Ds) and the delay time (Tdelay) in furrow invagination compared to the representative control embryo. Tdelay is highly sensitive to Ds, with a switch-like change at Ds~6 μm (dashed line). Inset: Average Tdelay in Early (E, n=5 embryos that invaginated), Mid (M, n=4), and Late (L, n=6) Group embryos. Statistical comparisons were performed using two-sided Wilcoxon rank-sum test. (e) Cartoon depicting mechanical bistability of the mesoderm during gastrulation. Both the initial, pre-constriction state and the final, fully invaginated state are stable. During gastrulation, actomyosin contractility is critical for bringing the system from the initial state to an intermediate, transitional state, whereas the subsequent transition to the final state can occur independent of myosin contractility. Figure 4—source data 1 Cell length measurements for determining lengthening-shortening transition time. https://cdn.elifesciences.org/articles/69082/elife-69082-fig4-data1-v2.xlsx Download elife-69082-fig4-data1-v2.xlsx Next, we examined the effect of Opto-Rho1DN stimulation on furrow invagination (Figure 4b; Figure 4—figure supplement 1e; see Materials and methods). Measuring the rate of furrow invagination (dD/dt) immediately after stimulation revealed three types of tissue responses to stimulation (Figure 4a and b; Figure 4—figure supplement 1f, g; Figure 4—video 1). For most embryos that were stimulated before T=7 min (‘Early Group’ embryos), the constricting cells relaxed immediately. The initial apical indentation disappeared (dD/dt<0). The flanking, non-constricting cells, which were previously apically stretched by neighboring constricting cells, shrunk back, indicating that the apical cell cortex was elastic (Figure 4—figure supplement 2a, b). In contrast, for most embryos that were stimulated after T=7 min (‘Late Group’ embryos), furrow invagination proceeded at a normal rate and dD/dt remained positive. Finally, some embryos stimulated around T=7 min (6.5±1.4 min, mean±s.d., n=4 embryos; ‘Mid Group’ embryos) displayed an intermediate phenotype, where ventral furrow invagination paused for around 5 min (4.7±0.5 min; dD/dt≈0) before invagination resumed. We confirmed that there was no residual apical myosin in Late Group embryos upon stimulation (Figure 4—figure supplement 3a). Furthermore, lateral myosin, which has been shown to facilitate furrow invagination (Gracia et al., 2019; John and Rauzi, 2021), also quickly disappeared after stimulation (Figure 4—figure supplement 3b, c). Overall, furrow invagination in Late Group embryos is not due to myosin activities at the apical or lateral cortices of the mesodermal cells. Taken together, these results indicate that actomyosin contractility is required in the early but not the late phase of furrow formation. The time when Mid Group embryos were stimulated defined the for sensitivity to myosin inhibition which is immediately before (Figure While all Early Group embryos showed tissue relaxation after stimulation, of of were to after a delay (Figure 4b; Figure 4—figure supplement The invagination was associated with myosin at the cell apices that occurred 10 min after the initial stimulation (Figure 4—figure supplement It is unclear apical myosin in the of persistent membrane localization of the of furrow invagination when apical constriction is is consistent with previous studies and suggests that invagination is by additional mechanical input et al., 1994; Parks and Wieschaus, 1991). the response of ventral furrow formation to myosin inhibition to be we sought to of the furrow that with we examined the between immediately after stimulation (Ds) and the by which furrow invagination is affected by the delay time in furrow invagination, see Materials and methods). We found that for stimulated embryos, Tdelay was to Ds, with a transition at around 6 μm (Figure When was the Tdelay was to When was the Tdelay to 10 min min, mean±s.d., n=5 Early Group embryos) or to Early Group embryos). the response to acute myosin inhibition can be well by the tissue morphology associated with the stage of furrow The binary response of the embryo to acute myosin inhibition to propose that the mesoderm during gastrulation is mechanically In this the mesoderm epithelium has two stable the initial, and the final, fully invaginated (Figure input from apical constriction drives the transition from the initial state to an intermediate state which has the to transition into the final state further of actomyosin contractility. myosin is before the furrow back to the initial In contrast, myosin is after the furrow to (Figure of cell shape change after Opto-Rho1DN stimulation further into how cells to rapid myosin loss after Rho1 we performed cell using et al., For embryo, we examined a single of cells along the axis that the ventral mesodermal cells the lateral mesodermal cells and presumably some ectodermal cells and (Figure and Our analysis time with most cell shape changes after stimulation and min for Early Group embryos, and and min for Late Group embryos). time were used for Late embryos of the rapid furrow invagination the inhibition of actomyosin contractility. Figure 5 with 1 supplement see all Download asset Open asset reveals of cell response after Opto-Rho1DN stimulation. Early Group embryos and Late Group embryos. A of cells along the axis on side of the ventral was in box) over time after stimulation. constricting 10 and ectodermal cells. Early and Late and of three representative cells, cell 1 constricting cell 6 constricting and cell are shown in and cyan These cells are also on the cross-section views and the tissue with the invagination Cartoon depicting apical surface change and cell shape changes observed in Early Group (b) and Late Group (g) embryos. showing the apical surface change cell length change and cell change between and min or between and min from both Early and Late are shown in for show the measurements for immediately after stimulation Error bars: N=3 embryos for in Early Group embryos, there was a apical relaxation in the constricting cells and an apical in the cells stimulation Figure for a A number of were observed in Early Group embryos. First, there was no cell volume change Figure supplement similar to the for normal ventral furrow formation (Gelbart et al., 2012; et al., 2014). Second, the cells that were most apically stretched before stimulation underwent most apical which is cells Figure the of apical relaxation in the constriction domain was not In cells at the side of the constriction domain constricting cells relaxed more to the ventral constricting cells (Figure The of apical relaxation in the cells was with less of (Figure see Figure for a Finally, the in apical cell was associated with changes in cell along the apical-basal stimulation, the cells and the side constricting cells were all with their apical side bending the ventral cyan and cells in Figure stimulation, the cells up and their initial, cell This cell shape change is associated with a in the apical-basal cell length and lateral cell (Figure and Figure supplement 1). The ectodermal cells also showed some but their shape changes were in (Figure cells 10 and Interestingly, the tissue
Source: eLife
Authors: Hanqing Guo, Michael Swan, Bing He
Institutions: Dartmouth College, Princeton University
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4214893561