A role for Myosin in triggering and executing amnioserosa cell delaminations during dorsal closure

A role for Myosin in triggering and executing amnioserosa cell delaminations during dorsal closure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A role for Myosin in triggering and executing amnioserosa cell delaminations during dorsal closure Nicole Gorfinkiel, Yanara Ferrer, Jon Recalde, Javier Gutiérrez, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6227955/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 9 You are reading this latest preprint version Abstract The remodeling of epithelial tissues is a critical process in morphogenesis, often involving the apoptotic removal of individual cells while preserving tissue integrity. In Drosophila , the amnioserosa—a highly dynamic extra-embryonic tissue—undergoes extensive remodeling, culminating in its complete elimination at the end of dorsal closure. While apoptotic cell delaminations in the amnioserosa have been proposed to contribute to dorsal closure, the cellular mechanisms underlying this process remain poorly understood. In this study, we have investigated actomyosin dynamics during cell delaminations and analyzed the consequences of perturbing non-muscle Myosin activity globally in the entire tissue as well as locally in groups of cells. We found that Myosin plays an essential role in both triggering and executing cell delaminations, with high Myosin contractility promoting cell extrusion even in the absence of caspase activity. Additionally, our results suggest that cell delaminations are governed by both cell-autonomous Myosin dynamics and mechanical cues from the tissue environment. Together, these findings provide new insights into the regulation of epithelial cell removal and the complex interplay between apoptotic and mechanical signals during tissue remodeling. Biological sciences/Cell biology Biological sciences/Developmental biology cell delamination actomyosin contractility Rho caspase activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION The remodeling of epithelial tissues is a fundamental process in morphogenesis, giving rise to a diverse array of structures, including sheets, tubes, and cysts, which are essential for organ formation and function. A key aspect of epithelial remodeling is the apoptotic removal of individual cells from the epithelium while maintaining overall tissue integrity. Apoptotic cell elimination has been observed in diverse contexts contributing both to tissue morphogenesis and to the maintenance of tissue homeostasis 1 – 3 . In some instances, entire epithelial tissues are removed and replaced, as occurs during embryogenesis in the extra-embryonic tissues of insects 4 and during metamorphosis 5 . The amnioserosa is the single extra-embryonic tissue present in Drosophila 6 . It is specified during early embryonic development by the dorso-ventral patterning system and plays essential functions during two vital morphogenetic movements, germband retraction and dorsal closure 7 . Amnioserosa cells undergo dramatic cell shape changes during embryogenesis. During germband elongation, these cells elongate and transition from a columnar to a squamous morphology through a process known as rotary cell elongation 8 . As development progresses into germband retraction, amnioserosa cells shorten, become isodiametric, and exert a pulling force that contributes to germband retraction 9 , 10 . Finally, during dorsal closure, amnioserosa cells exhibit dynamic fluctuations in apical surface area, driven by periodic contractions of the actomyosin cytoskeleton, and progressively reduce their apical surface 11 – 14 . Around 10% of the cells of the tissue basally delaminate before closure is completed 15 – 18 . These cellular processes, along with a decrease in volume, generate a morphogenetic force that drives the movement of the lateral epidermal sheets towards the dorsal midline. Ultimately, the entire amnioserosa tissue is internalized and degenerates by apoptosis 19 – 21 . Amnioserosa cell delaminations are apoptotic, and although they are not essential for closure, enhancing or supressing apoptosis increases or reduces cell delaminations respectively, leading to either an acceleration or a slowing down of closure 16 . This led to the suggestion that apoptotic cell delaminations in the amnioserosa provides an apoptotic force that contributes to closure. Additionally, inhibiting apoptosis in the amnioserosa also prevents cell volume decrease in the bulk of the tissue 22 , showing that the apoptotic pathway controls both individual delaminations and the remodeling of the entire tissue. The cellular mechanisms underlying the elimination of cells from an epithelium have been studied in several tissues, revealing the involvement of both cell autonomous and non-autonomous mechanisms. In the apoptotic cell, either an actomyosin ring 23 – 26 or medioapical actomyosin contractions 27 – 29 can contribute to autonomous-cell constriction, while neighbouring cells form a supracellular actomyosin cable that aids in the expulsion of the cell from the tissue 23 , 24 , 26 . In the amnioserosa, high levels of reactive oxygen species induce caspase activity and cell delaminations through the reorganization of non-muscle Myosin distribution 18 . During the early stages of dorsal closure, delaminating cells transition abruptly from a pulsatile behaviour to a rapidly, unpulsed, contracting behaviour 30 . This delamination transiently affects the oscillating behaviour of the neighbouring cells, and changes in Myosin dynamics have been observed in both the delaminating cell and its neighbours 30 . However, the precise cellular mechanisms underlying cell delamination remain unclear. Additionally, mechanical stresses have been proposed as potential triggers for both individual cell delamination of cells and the degeneration of the entire amnioserosa, but evidence for this has been lacking. In this work, we have investigated the cellular mechanisms driving cell delaminations in the amnioserosa and analyzed the consequences of perturbing non-muscle Myosin activity globally in the entire tissue as well as locally in groups of cells. Our results argue for an active role for Myosin in both triggering and executing cell delaminations and show that cells with high contractility can undergo delamination independently of caspase activity. RESULTS 1) Actomyosin dynamics during amnioserosa cell delaminations Cell delaminations in the amnioserosa can be observed from the end of germband retraction until the completion of dorsal closure. Although their number varies among embryos, they exhibit a consistent spatial pattern, occurring mostly in the anterior half of the amnioserosa and at the posterior canthi 15 , 17 , 18 (see below). In epithelial tissues, cell delaminations are usually mediated by T2 transitions, in which multiple vertices converge to form a single vertex, generating a rosette-like structure. In the amnioserosa, while some cells delaminate through T2 transitions (Fig. 1 A-A´´), we also observe delaminating cells that do not form rosettes. Instead, these cells leave a new junction once they are delaminated (Fig. 1 B-B´´). This type of delamination occurs in anisotropic cells where the longer junctions, usually dorso-ventrally oriented, approach and fuse, forming a new junction between new neighbours. We found that cells forming rosettes are more isotropic at the onset of delamination than cells leaving a junction (Fig. 1SA), suggesting that cell shape determines the geometry of the delamination process. Both types of cell delamination events are prevented upon expression of an apoptosis inhibitor such as p35 or Drosphila inhibitor of apoptosis 1, Diap1 15,16,18 . Cell delaminations are driven by either a cortical actomyosin ring or pulses of contractile medioapical actomyosin 24 , 26 – 29 . To investigate the mechanisms driving cell delaminations in the amnioserosa, we visualized non-muscle Myosin (MyoII) and actin distribution in delaminating cells from live embryos carrying the ECad:mTomato (ECad:mT) and the sqh:GFP knock-in alleles, or ECad:mT and the actin reporter utrophin fused to GFP (utr:GFP) (Fig. 1 C-I”). Since MyoII subcellular localization and dynamics changes as dorsal closure progresses, characterized by increasing junctional MyoII and stabilisation of the medioapical pool 12 , 13 , we examined cell delaminations at both early and late stages. During early stages, as the delaminating cell begins to apically constrict, we observed a stabilisation of medioapical MyoII, with no accumulation of junctional Myosin nor actin (Fig. 1 C, F). Early delaminating cells are pulsatile, and the onset of apical constriction coincides with the stabilisation of a medial MyoII pulse, giving rise to a non-pulsatile delaminating cell, as previously observed 30 . As the apical cell area reduces, cell junctions show an inward curvature (Fig. 1 C, C´, arrow), suggesting the existence of a pulling force from the medial part of the delaminating cell. Interestingly, we observed the presence of MyoII and actin puncta that start to form basally as the onset of the delamination process (Fig. 1 D´, D”, G, G´, arrows). As the apical cell area reduces, actin puncta can be seen to re-localize apically at the level of ECad (Fig. 1 F´, arrow) to form a continuous junctional belt. During the last steps of the process, an actomyosin junctional ring is observed subapically (Fig. 1 D”, E”, G”, H”), which persists basal to the plane of the epithelium for up to 20 minutes (not shown). During late dorsal closure, delaminating cells also show a stabilisation of medioapical MyoII that spans the whole apical cortex as the cell area reduces (Fig. 1 I). An actomyosin ring is also observed as the delamination process proceeds (Fig. 1 I´, I”). Such cytoskeletal dynamics has been observed in both isotropic and anisotropic cell delaminations (data not shown). In contrast to what has been observed in other epithelia, we did not observe the formation of a supracellular actomyosin ring in neighbouring cells. However, in some late delaminating cells, MyoII accumulated in neighbouring cells at the junctions with the delaminating cell (Fig. 1 I”, arrows) coincident with a decrease in ECad levels (Fig. 1SB). We propose that this could be a mechanism to maintain junction integrity during the delamination process 31 but would not be required to expulse the cell from the tissue. Altogether, these observations indicate that amnioserosa cells delaminate through a mechanism mediated by medioapical Myosin, with the formation of an actomyosin ring in the last steps of the delamination process. The Rho family of GTPases is a central regulator of F-actin, MyoII activity and contractility 32 . Both Rho-dependent and independent mechanisms for apoptotic cell elimination have been reported 33 . In the amnioserosa, using a Rho live sensor consisting of the Rho1 GTP-binding domain of anillin to GFP (aniRBD:GFP) 34 , we have observed an increase in medioapical Rho activity as soon as the apical cell area of a delaminating cell begins to decrease (Fig. 2 B). Thus, it is possible that apoptotic signals induce medioapical Rho activity, which in turn regulates the cytoskeletal changes described above that participate in the execution of the delamination process. Interestingly, during late stages of dorsal closure, all amnioserosa cells show medioapical accumulation of the Rho sensor (Fig. 1 A, A´). Since medioapical Rho activity is observed in apoptotic delaminating cells, and caspase activity has been detected throughout the amnioserosa tissue during late dorsal closure stages (Fig. 5 A-B”) 22 , 35 , 36 , we asked whether this late-stage medioapical Rho accumulation depends on caspase activity. The ectopic expression of Diap1 in the amnioserosa, while preventing cell delamination, does not prevent the medioapical accumulation of the Rho sensor in the bulk of the tissue during late dorsal closure (Fig. 2 C). This result suggests that Rho activity at the medioapical cortex of amnioserosa cells is regulated differently in delaminating cells versus the bulk of the tissue, where it appears to be independent of apoptotic signals. 2) Perturbing Myosin levels in the whole tissue does not alter the number of cell delaminations To investigate the requirement for Myosin in the triggering and the elimination of cells from the tissue, we aimed to perturb Myosin II activity in the whole tissue. We have previously shown that the ectopic expression of a constitutive active form of Mbs in the amnioserosa (Mbs is the Myosin binding subunit of the protein phosphatase PP1), decreases phosphorylated Myosin, decreases the rate of contraction of amnioserosa cells and slows dorsal closure 37 , 38 . Here, we analyzed whether cell delaminations are affected upon ectopic expression of MbsN300 (Fig. 3 B, D). Because dorsal closure takes longer in embryos ectopically expressing MbsN300, to compare the number of delaminations between the two genotypes, we considered the delaminations occurring during the same period of time, from − 30 min to 100 min of dorsal closure, and staging the embryos according to the distance between the posterior spiracles. Surprisingly, we found that the total number of cell delaminations in MbsN300 embryos is indistinguishable from that in control embryos. However, when comparing the timing of cell delaminations, we observed a delay: the number of cell delaminations between − 30 and 30 min of dorsal closure is lower in AS-GAL4, ECad:GFP/UAS-MbsN300 than in controls but is higher between 30 and 100 min of dorsal closure (Fig. 3 D). These results suggest an early requirement of MyoII for triggering cell delaminations, however, unexpectedly, during later stages, cell delaminations resume and become more frequent. To explore what could be driving late delaminations in these embryos, we analyzed MyoII localization in sqh:GFP; AS-GAL4, ECad:mT/UAS-MbsN300 embryos. While MyoII localizes in medioapical foci in early dorsal closure, in older embryos, MyoII shows an aberrant distribution, accumulating at bicellular junctions and cell vertices, without forming the dense medioapical mesh observed in control embryos (Fig. 3 H, compare with 3F and 3G, see also 31 ). Interestingly, the Rho sensor is also mislocalized, accumulating at cell junctions and cell vertices (Fig. 3 J). In these embryos, delaminating cells display junctional rather than medioapical Rho1 activity (Fig. 3 L-L”), along with strong junctional MyoII (Fig. 2SA). These observations suggest that, in these embryos, cell delamination does not involve medioapical MyoII activity but rather junctional Rho1 and MyoII. Reciprocally, we aimed to increase Myosin activity in the amnioserosa by ectopically expressing a constitutive active form of Myosin Light Chain Kinase (ctMLCK). We have previously shown that these cells are more contracted and accumulate both junctional and medioapical MyoII 12 , 39 . While in other tissues increasing Myosin activity perturbs cell delaminations 40 , in the amnioserosa neither the number nor the timing of cell delaminations are altered (Fig. 3 C, E). These cells exhibit an accumulation of Myosin at cell junctions and a persistent, dynamic medioapical network (Fig. 3 I), which further accumulates in extruding cells (Fig. 2SB). Interestingly, most of amnioserosa cells with ectopic ctMLCK show medioapical accumulation of the Rho sensor (Fig. 3 K), which becomes more pronounced in delaminating cells (Fig. 3 M-M”). These observations, together with the junctional localization of the Rho sensor upon ectopic expression of MbsN300, show that perturbing Myosin localization impacts Rho localization. These observations are in agreement with the existence of a positive feedback between the upstream regulator Rho and MyoII 34 (see Discussion). 3) In a tissue with tension heterogeneity, cells with higher levels of MyoII are more likely to delaminate We were intrigued by the observation that perturbing Myosin activity does not affect the overall rate of cell delaminations. While increased actomyosin contractility underlies cell ingression in several tissues 41 , 42 , a global increase of junctional MyoII has been found to inhibit both apoptotic basal cell delamination and apoptotic apical extrusion 40 , 43 . To further investigate this, we generated heterogeneities in MyoII levels across the tissue by selectively increasing or decreasing Myosin activity in groups of cells. For this, we ectopically expressed UAS-MbsN300 or UAS-ctMLCK using the prd-Gal4 driver, which is expressed in alternate segments in the epidermis and in the amnioserosa, along with a NLS:mCherry (NLS:mCh) reporter to identify MbsN300 or ctMLCK expressing cells. To assess whether changes in MyoII activity influence the likelihood of a cell delaminating, we calculated the ratio between the number of delaminating cells NLS-mCh positive and the number of delaminating cells NLS-mCh negative for each embryo. In ECad:GFP, prdGAL4/UAS-NLS:mCh, UAS-MbsN300 embryos, we observe that MbsN300 expressing cells show a larger apical cell area (Fig. 4 B, arrow, compare with 4A), confirming that these cells have lower actomyosin contractility. In these embryos, the ratio of NLS-mCh delaminating cells (and thus expressing MbsN300) to non NLS-mCh delaminating cells (with wild type MyoII levels) is lower than in control embryos (Fig. 4 D). These results confirm a requirement for medioapical MyoII in cell delamination. Conversely, in prdGAL4/UAS-NLS:mCh; UAS-ctMLCK embryos, we observe that ctMLCK positive cells show a smaller apical cell area (Fig. 4 C, arrow, compare with 4A). In this case, the ratio of NLS:mCh delaminating cells (with higher both junctional and medioapical MyoII) to non-NLS:mCh delaminating cells (with wild type MyoII levels) is greater than in control embryos (Fig. 4 D). Thus, although no increase in the number of delaminations is observed when Myosin activity is increased across the whole tissue, we find that cells with higher levels of Myo are more likely to delaminate when surrounded by cells with wild type MyoII activity. Importantly, delaminations of cells with high levels of MyoII occur throughout the entire process of dorsal closure, suggesting that high MyoII levels alone do not immediately induce delamination but increase the probability of a cell to delaminate. These results, together with the observation that increasing MyoII activity across the whole tissue does not result in an increase in the number of delaminations, suggest that high tissue stiffness could disrupt cell delamination. 4) Blocking endocytosis prevents cell delaminations In the pupal epidermis, a decrease in endocytic activity impairs or accelerates cell delaminations depending on the region of the segment where delaminations occur 5 , 29 . Therefore, we aimed to investigate the effect of blocking endocytic activity in the amnioserosa. We have previously shown that the ectopic expression of Rab5DN in the amnioserosa compromises apical constriction and apical membrane removal 44 . We observed a decrease in the number of cell delaminations with only few delaminations that occurring during late stages of dorsal closure (Fig. 5 A, B). The expression of Rab5DN in a mosaic manner using the prd-GAL4 driver shows that Rab5DN-expressing cells have a greater apical cell area than their neighbours (Fig. 5 C), confirming that blocking endocytosis affects apical contraction. In these embryos, cells expressing Rab5DN are less likely to delaminate than control cells (Fig. 5 D). We examined the subcellular localization of MyoII and found that although MyoII pulses are present, there is a decrease in junctional MyoII (Fig. 5 E, F). In fact, it has been shown that Rab5DN expressing amnioserosa cells show lower junctional tension, suggesting that endocytosis is important to maintain junctional tension 31 . These results show that endocytic activity is required for amnioserosa cell delaminations, which could be related, at least in part, to a decrease in tissue tension. 5) Interaction between contractility and caspase activity Our results show that cells with increased MyoII activity due to the ectopic expression of ctMLCK are more likely to delaminate when they are surrounded by cells with control levels of MyoII (Fig. 4 C, D). This observation raised the question of whether increased Myosin activity affects caspase activity. To investigate this possibility, we explored the relationship between Myosin and caspase activity. The apoptotic sensor Apoliner has been widely used in several systems to monitor capsase activity 45 . In this sensor, the fluorescent proteins mRFP and eGFP, are linked by a caspase-specific cleavage site from Diap1. Caspase activation leads to the cleavage of Apoliner and to the translocation of the eGFP from the cytoplasm to the nucleus. In the amnioserosa, during early stages of dorsal closure, the sensor is not active, except in the most posterior cells (Fig. 6 A). As dorsal closure progresses, the sensor is activated first in anterior cells and then in the whole tissue before closure is complete (Fig. 6 B, C and see also 22 , 35 ). The activation of the sensor is completely blocked in embryos ectopically expressing Apoliner and Diap1 even after the amnioserosa has been internalized underneath the epidermis (Fig. 3 S). Thus, Apoliner can detect low levels of caspase activity, that start to build up in the amnioserosa as dorsal closure progresses, but this activity does not inevitably lead to the cell delamination since most of the cells remain in the bulk of the tissue. We then turned to a different sensor, GC3Ai, which has been shown that can be detected in apoptotic cells throughout the whole process of programmed cell death 46 . In this sensor, the C and N termini of the GFP have been linked by a fragment containing a DEVD caspase cleavage site that must be cleaved for the GFP to fluoresce. In embryos ectopically expressing GC3Ai in the amnioserosa, sensor activity is only observed in delaminating cells (Fig. 6 C). The activity of the GC3Ai sensor in delaminating cells is detected as the cell starts to apically constrict until after it has ingressed and start to undergo fragmentation, a process that takes up to 40 minutes (Fig. 6 C´-C””). Thus, the GC3ai sensor detects apoptotic levels of caspase activity. We analyzed caspase activity with both sensors in embryos with perturbed Myosin activity. We wanted to determine whether, in AS-GAL4/UAS-MbsN300 embryos, the delay in the appearance of cell delaminations was due to an absence of caspase activity or to an impairment of the expulsion of the cell from the tissue. If this was the case, we expected to see an activation of the apoptotic sensors in the tissue with no delamination. In AS-GAL4/UASApoliner, UAS-MbsN300 embryos that complete dorsal closure, the activation of the sensor followed a similar temporal pattern than in control embryos (Fig. 6 D, compare with 6A), while the activity of the GC3ai sensor was only observed in delaminating cells (Fig. 6 F). These observations show that apoptotic levels of caspase activity do not arise independently of cell delamination and suggest that proper levels of Myosin activity are required to trigger apoptotic cell delaminations. Similarly, in embryos ectopically expressing ctMLCK, GC3Ai activity was only detected in cells that delaminate (Fig. 6 G). In contrast, in AS-GAL4/UASApoliner, UAS-ctMLCK embryos, we observed a consistent premature activation of Apoliner (Fig. 6 E, compare with 6A). These observations suggest that increasing MyoII activity can induce low levels of caspase activity. These observations lead us to analyze whether the preferential delamination of cells with high MyoII levels in prd-GAL4/UAS-NLS:mCh; UASctMLCK embryos was due to an increase of caspase activity induced by MyoII. For this, we asked whether delaminations of ctMLCK cells in these embryos could be prevented by the ectopic expression of Diap1. Surprisingly, in ECadGFP; prd-GAL4/UAS-NLS:mCh, UASDiap1; UASctMLCK, NLS:mCh embryos, we observe NLS:mCh positive cells that delaminate (Fig. 6 H). Although we cannot rule out the possibility that Diap1 levels in this experiment are insufficient to fully prevent caspase activity, this result suggests that cell delamination can occur in response to high levels of MyoII even in the absence of caspase activity. This finding reveals that in a mechanically heterogeneous tissue, cells with high contractility can be eliminated independently of caspase activity. DISCUSSION In this work, we have investigated the mechanisms underlying the triggering and execution of apototic cell delaminations in the amnioserosa during dorsal closure, before the tissue completely ingresses into the embryo and degenerates. Our results show that cell delaminations involve, firstly, the stabilisation of the medioapical actomyosin cortex, and secondly, the formation of an actomyosin ring, which completes the elimination of the apoptotic cell from the tissue. We have observed the appearance of actin puncta prior to the formation of the actomyosin ring, initially subapically and then co-localizing with ECadherin at the level of junctions. We propose that these puncta represent sites of actin nucleation for building the apoptotic actomyosin ring 47 . Although less prominent, Myosin puncta were also present, and we hypothesize they could be recruited by the nascent actin bundles. Our results also suggest that the small GTPase Rho regulates the reorganization of the actomyosin cytoskeleton to form the apoptotic actomyosin ring. Rho activity has mostly been shown to be required in neighbouring cells to the delaminating cells to form a supracellular actomyosin cable that squeezes extruding cells apically from the epithelium, but its requirement in the apoptotic cell itself is more controversial 23 , 33 . In the amnioserosa, all delaminating cells showed accumulation of a sensor of Rho activity in the medioapical cortex, strongly supporting the role of this small GTPase in regulating the cytoskeletal changes associated with cell delamination in the delaminating cell. Our observations do not provide evidence for the involvement of cell neighbours in the elimination of the delaminating cells. Although it has been shown that a delaminating cell induce a deformation in its neighbours and impacts their Myosin pulses 16 , 30 , we have not observed the formation of a supracellular actomyosin cable in neighbouring cells. An accumulation of MyoII close to cell junctions between the delaminating cell and its neighbours is only observed when ECad levels decrease significantly, suggesting that this might be a mechanism to preserve junction integrity. In fact, a mechanosensitive mechanism involving the actomyosin cytoskeleton to maintain junction integrity has been proposed 31 . Interestingly, we find that Rho subcellular localization changes upon perturbation of Myosin activity. In embryos ectopically expressing MbsN300, both MyoII and the Rho sensor accumulate at cell junctions, while in embryos ectopically expressing ctMLCK, Myo forms a dense apicomedial mesh, and the Rho sensor shows a more persistent apicomedial localization. These observations suggests that MyoII recruits Rho either to the junctional or to the medioapical region of cells, providing evidence for the existence of a positive feedback between Rho and MyoII. We have also observed oscillatory Rho activity in non-delaminating cells (unpublished observations) and as dorsal closure progresses, medioapical Rho activity is stabilised medioapically in the whole tissue. An advection-positive feedback mechanism between Rho and its downstream effectors Rock and MyoII has been shown to underlie actomyosin pulsatility in epidermal cells during germband elongation 34 . It is possible that such a mechanism is in place in the amnioserosa, contributing to the progressive stabilization of Rho and the medioapical acomyosin cortex at the level of the whole tissue. What triggers cell delaminations? The apoptotic removal of epithelial cells from a tissue can be regulated by both biochemical and mechanical signals 1 , 48 . Mechanical compression, local topological defects and the geometry of cell packing have all been shown to promote cell elimination from a tissue 49 – 51 . In the amnioserosa, it has been suggested that mechanical signals are involved in the triggering of cell delaminations 15 , 18 , 36 . Here, we have observed that decreasing MyoII activity in the entire tissue delays the appearance of cell delaminations. Since low MyoII activity is associated with low tissue tension 37 , our results argue for a role for mechanics in triggering of cell delaminations. However, increasing tissue tension through increasing Myo levels in the whole tissue does not increase cell delaminations. A global increase in junctional Myosin has also been shown to block cell delamination in the fly notum 40 , and to disrupt apoptotic extrusion in epithelial monolayers. In the latter, increasing Myosin in apoptotic cells or in the surrounding cells disrupted extrusion 43 . This contrasts with what we observe in the amnioserosa, where cells with higher MyoII than their neighbours are more likely to delaminate. Altogether, our results suggest that high tissue tension or a stiff mechanical environment may exert an inhibitory effect on the ability to delaminate. It is important to notice that cells with increased MyoII do not delaminate instantaneously but are more likely to do so over the entire process of dorsal closure. This suggests that other signals contribute to the decision of whether a cell will delaminate. In the fly notum, it has been found that the apical size of a cell, compared to other cells within the tissue and relative to the immediate neighbours, is a predictor of apoptotic cell delamination 52 . Interestingly, we have found here that cells with lower levels of Myosin and showing a greater apical cell area are less likely to delaminate, while cells with higher Myo levels and with a smaller apical cell area, are more likely to delaminate. Our results suggest that MyoII activity is a key determinant of apical cell size that could be mediating the relation between cell size and apoptotic fate. The fact that cells with higher MyoII levels than their neighbours can delaminate when caspase activity is prevented by the ectopic expression of Diap1 is intriguing. As mentioned above, we cannot rule out that the levels of Diap1 are not sufficient to fully prevent caspase activity due to a dilution of the GAL4 transcriptional activator. Nonetheless, it is worth pointing out that live basal cell delamination plays a crucial role in tumour cell invasion 53 . Interestingly, RasV12-transformed cells in epithelial monolayers are typically extruded apically through a mechanism called “epithelial defence against cancer” 54 . However, some cells evade this defence mechanism and instead delaminate basally, a process suggested to contribute to malignancy. The mechanisms underlying the shift in the direction of cell elimination from epithelia remain unclear, but one possible factor is Rho hyperactivation, which increases actomyosin contractility 53 . Our results provide further evidence for a role for actomyosin contractility in driving live basal cell delamination. The amnioserosa is a tissue in which the delamination of cells from the tissue occurs alongside the subsequent complete elimination of the tissue. This phenomenon also takes place in the larval epidermis, which is replaced by histoblasts during metamorphosis 55 , and is likely to occur in other insect extra-embryonic tissues 4 . Both in the amnioserosa and in the larval epidermis, all the cells show low, non-apoptotic, levels of caspase activity well before the tissue is eliminated. There is growing evidence that caspases have non-lethal functions and can influence a diverse array of cellular processes such as signalling, proliferation, differentiation and migration 56 . In the amnioserosa, a role for caspases in controlling cell volume decrease has been found 22 , and it is possible that low levels of caspases play a role in the remodelling of the cytoskeleton not directly linked to cell death. An important question, however, is how global tissue levels of caspases arise. We have found here that increasing Myo activity favours caspase activation. In the larval epidermis, it has also been proposed that MyoII potentiates caspase activity 5 . An interesting possibility is that actomyosin contractility influences caspase activity and thus the fate of amnioserosa cells. A relationship between mechanical cues and cell fate has been known for several years 57 . The Hippo pathway is involved in transforming mechanical stimuli into biochemical signalling pathways both in vertebrates and in Drosophila 58 – 60 . Notably, in wing imaginal disc cells, it has been found that medial actomyosin flows promote the medial accumulation of Kibra and the formation of a Hippo complex, which activates the Hippo pathway to repress the pro-growth transcriptional effector Yorkie 61 . The role of the Hippo pathway in the amnioserosa has not been explored yet but would be an interesting avenue for future research. MATERIALS AND METHODS Fly stocks and genetics : The following stocks were used in this work: ECad:GFP and ECad:mT knock-in alleles 62 ; sqh:GFP 2 ; aniRBD:GFP 34 ; AS-GAL4 (332.3 GAL4 line, RRID:BDSC_5398); prd-GAL4 (RRID:BDSC_1947); UAS-MbsN300 63 ; UAS-ctMLCK 64 ; UAS-Apoliner 45 ; UAS-GC3Ai 46 ; UAS-NLSmCherry (RRID:BDSC_38425); UAS-Diap1 (II); UAS-Rab5DN (RRID:BDSC_42704). Stocks built during this work: AS-GAL4, ECad:GFP and AS-GAL4, ECad:mT; sqh:GFP AS-GAL4, ECad:mT; aniRBD:GFP sqh:GFP; AS-GAL4, ECad:mT UAS-NLS:mCherry; UAS-MbsN300 UAS-NLS:mCherry; UAS-ctMLCK UAS-Apoliner; UAS-MbsN300 UAS-Apoliner; UAS-ctMLCK UAS-GC3Ai; UAS-MbsN300 UAS-GC3Ai; UAS-ctMLCK ECad:GFP; prdGAL4 UAS-Diap1; UAS-MbsN300 UAS-Diap1; UAS-ctMLCK UAS-Diap1, UAS-NLS:mCherry; UAS-ctMLCK Live-Imaging : Stage 12–13 Drosophila embryos were dechorionated, mounted in coverslips with the dorsal side glued to the glass and covered with Voltalef oil 10S (Attachem). The AS was imaged at 25–28ºC. using an inverted LSM 710 Meta laser scanning microscope or a Leica SP8 with a 40X or a 63X oil immersion Plan-Fluor (NA = 1.3) objective. GFP and mTomato cytoskeletal reporters were simultaneously imaged with an argon laser and a 651 diode laser. For whole AS imaging, 15–30 z sections 1-1.5µm apart were collected every 2 minutes. For cytoskeletal dynamics imaging, 10 z sections 1µm apart were collected every 30 seconds. Image analysis and statistics : Images were processed and assembled in Fiji (version 2.16.0) and Adobe Photoshop (version 23.5.0). Images were projected via the maximum intensity projection to cover the entire tissue or a single cell in case it was located in a curved region of the tissue. The identification of delaminating cells was done manually on the maximum intensity projections. Statistical analysis of the significance of differences in phenotype across genotypes was performed using the Wilcoxon-Mann-Whitney test using the R package. Declarations Author Contribution N.G. conceived the project, performed experiments, analysed data and wrote the paper, Y.F., J.G. and G.S. performed experiments, Y.F. and J.R. analysed data. ACKNOWLEDGEMENTS We are very grateful to Juan José Muñoz† for his help and support during the initial phase of this project. We thank Isabel Guerrero, Julia Duque and Guy Blanchard for critically reading the manuscript. We thank the Bloomington Stock Center, Bruno Monier and Jorg Großhans for Drosophila strains. Microscopy was performed at the Unidad de Citometría de Flujo y Microscopía de Fluorescencia of the Universidad Complutense de Madrid and at the Advanced Light Microscopy Facility at the Centro de Biología Molecular Severo Ochoa. This work was supported by grant PID2020-114533GB-C22 to NG from the Spanish Ministry of Economy and Competitiveness. Competing interests statement The authors declare no competing interests. Data Availability Data is provided within the manuscript or supplementary information files. All time-lapse movies are available on request to [email protected] References Valon, L. & Levayer, R. Dying under pressure: cellular characterisation and in vivo functions of cell death induced by compaction. Biol. Cell. 111 , 51–66 (2019). Ambrosini, A., Rayer, M., Monier, B. & Suzanne, M. Mechanical Function of the Nucleus in Force Generation during Epithelial Morphogenesis. Dev. Cell. 50 , 197–211e5 (2019). Tada, M. The morphogenetic changes that lead to cell extrusion in development and cell competition. Dev. Biol. 477 , (2021). Panfilio, K. A. & Roth, S. Epithelial reorganization events during late extraembryonic development in a hemimetabolous insect. Dev. Biol. 340 , (2010). Hoshika, S., Sun, X., Kuranaga, E. & Umetsu, D. Reduction of endocytic activity accelerates cell elimination during tissue remodeling of the Drosophila epidermal epithelium. Dev. (Cambridge) 147 , (2020). Schmidt-Ott, U. & Kwan, C. W. How two extraembryonic epithelia became one: serosa and amnion features and functions of Drosophila’s amnioserosa. Philosophical Trans. Royal Soc. B 377 , (2022). Lacy, M. E. & Hutson, M. S. Amnioserosa development and function in Drosophila embryogenesis: Critical mechanical roles for an extraembryonic tissue. Dev. Dyn. 245 , 558–568 (2016). Pope, K. L. & Harris, T. J. Control of cell flattening and junctional remodeling during squamous epithelial morphogenesis in Drosophila. Development 135 , 2227–2238 (2008). Lynch, H. E. et al. Cellular mechanics of germ band retraction in Drosophila. Dev. Biol. 384 , (2013). Schöck, F. & Perrimon, N. Cellular processes associated with germ band retraction in Drosophila. Dev. Biol. 248 , (2002). Solon, J., Kaya-Copur, A., Colombelli, J. & Brunner, D. Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure. Cell 137 , 1331–1342 (2009). Blanchard, G. B., Murugesu, S., Adams, R. J., Martinez-Arias, A. & Gorfinkiel, N. Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure. Development 137 , 2743–2752 (2010). David, D. J., Tishkina, A. & Harris, T. J. The PAR complex regulates pulsed actomyosin contractions during amnioserosa apical constriction in Drosophila. Development 137 , 1645–1655 (2010). Sokolow, A., Toyama, Y., Kiehart, D. P. & Edwards, G. S. Cell ingression and apical shape oscillations during dorsal closure in Drosophila. Biophys. J. 102 , 969–979 (2012). Gorfinkiel, N., Blanchard, G. B. & Adams, R. J. Martinez Arias, A. Mechanical control of global cell behaviour during dorsal closure in Drosophila. Development 136 , 1889–1898 (2009). Toyama, Y., Peralta, X. G., Wells, A. R., Kiehart, D. P. & Edwards, G. S. Apoptotic force and tissue dynamics during Drosophila embryogenesis. Sci. (1979) . 321 , 1683–1686 (2008). Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L. & Montague, R. A. Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila. J. Cell. Biol. 149 , 471–490 (2000). Muliyil, S., Krishnakumar, P. & Narasimha, M. Spatial, temporal and molecular hierarchies in the link between death, delamination and dorsal closure. Development 138 , 3043–3054 (2011). Gorfinkiel, N., Schamberg, S. & Blanchard, G. B. Integrative approaches to morphogenesis: lessons from dorsal closure. Genesis 49 , 522–533 (2011). Hayes, P. & Solon, J. Drosophila dorsal closure: An orchestra of forces to zip shut the embryo. Mech. Dev. 144 , 2–10 (2017). Kiehart, D. P., Crawford, J. M., Aristotelous, A., Venakides, S. & Edwards, G. S. Cell Sheet Morphogenesis: Dorsal Closure in Drosophila melanogaster as a Model System. Annual Rev. Cell. Dev. Biology Annu. Rev. Cell. Dev. Biol. 6 , 169–202 (2017). Saias, L. et al. Decrease in Cell Volume Generates Contractile Forces Driving Dorsal Closure. Dev. Cell. 33 , 611–621 (2015). Rosenblatt, J., Raff, M. C. & Cramer, L. P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr. Biol. 11 , 1847–1857 (2001). Kuipers, D. et al. Epithelial repair is a two-stage process driven first by dying cells and then by their neighbours. J. Cell. Sci. 127 , 1229–1241 (2014). Duszyc, K. et al. Mechanotransduction activates RhoA in the neighbors of apoptotic epithelial cells to engage apical extrusion. Curr. Biol. 31 , 1326–1336e5 (2021). Teng, X., Qin, L., Le Borgne, R. & Toyama, Y. Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium. Dev. (Cambridge) . 144 , 95–105 (2017). Atieh, Y., Wyatt, T., Zaske, A. M. & Eisenhoffer, G. T. Pulsatile contractions promote apoptotic cell extrusion in epithelial tissues. Curr. Biol. 31 , 1129–1140e4 (2021). An, Y. et al. Apical constriction is driven by a pulsatile apical myosin network in delaminating Drosophila neuroblasts. Dev. (Cambridge) . 144 , 2153–2164 (2017). Michel, M. & Dahmann, C. Tissue mechanical properties modulate cell extrusion in the Drosophila abdominal epidermis. Dev. (Cambridge) 147 , (2020). Saravanan, S., Meghana, C. & Narasimha, M. Local, cell-nonautonomous feedback regulation of myosin dynamics patterns transitions in cell behavior: a role for tension and geometry? Mol. Biol. Cell. 24 , 2350–2361 (2013). Sumi, A. et al. Adherens Junction Length during Tissue Contraction Is Controlled by the Mechanosensitive Activity of Actomyosin and Junctional Recycling. Dev. Cell. 47 , 453–463e3 (2018). Munjal, A. & Lecuit, T. Actomyosin networks and tissue morphogenesis. Development 141 , 1789–1793 (2014). Coleman, M. L. & Olson, M. F. Rho GTPase signalling pathways in the morphological changes associated with apoptosis. Cell Death & Differentiation 9, 493–504 (2002). (2002). Munjal, A., Philippe, J. M., Munro, E. & Lecuit, T. A self-organized biomechanical network drives shape changes during tissue morphogenesis. Nature 524 , 351–355 (2015). Shen, W. et al. Modulation of morphogenesis by Egfr during dorsal closure in Drosophila. PLoS One . 8 , e60180 (2013). Muliyil, S., Narasimha, M. & Mitochondrial, R. O. S. Regulates Cytoskeletal and Mitochondrial Remodeling to Tune Cell and Tissue Dynamics in a Model for Wound Healing. Dev. Cell. 28 , 239–252 (2014). Fischer, S. C. et al. Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics. PLoS One . 9 , e95695 (2014). Duque, J. & Gorfinkiel, N. Integration of actomyosin contractility with cell-cell adhesion during dorsal closure. Dev. (Cambridge) . 143 , 4676–4686 (2016). Machado, P. F. et al. Emergent material properties of developing epithelial tissues. BMC Biol. 13 , 1–15 (2015). Curran, S. et al. Myosin II Controls Junction Fluctuations to Guide Epithelial Tissue Ordering. Dev. Cell. 43 , 480–492e6 (2017). Priya, R. et al. Tension heterogeneity directs form and fate to pattern the myocardial wall. Nature 588 , 130–134 (2020). Simões, S., Oh, Y., Wang, M. F. Z., Fernandez-Gonzalez, R. & Tepass, U. Myosin II promotes the anisotropic loss of the apical domain during Drosophila neuroblast ingression. J. Cell Biol. 216 , 1387–1404 (2017). Mann, Z. et al. Preexisting tissue mechanical hypertension at adherens junctions disrupts apoptotic extrusion in epithelia. Mol. Biol. Cell. 35 , br3 (2024). Mateus, A. M., Gorfinkiel, N. & Schamberg, S. Martinez Arias, A. Endocytic and recycling endosomes modulate cell shape changes and tissue behaviour during morphogenesis in Drosophila. PLoS One . 6 , e18729 (2011). Bardet, P. L. et al. A fluorescent reporter of caspase activity for live imaging. Proc. Natl. Acad. Sci. U S A . 105 , 13901–13905 (2008). Schott, S. et al. A fluorescent toolkit for spatiotemporal tracking of apoptotic cells in living Drosophila tissues. Development 144 , 3840–3846 (2017). Michael, M. et al. Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions. Dev. Cell. 37 , 58–71 (2016). Fadul, J. & Rosenblatt, J. The forces and fates of extruding cells. Curr. Opin. Cell. Biol. 54 , 66–71 (2018). Levayer, R., Dupont, C. & Moreno, E. Tissue Crowding Induces Caspase-Dependent Competition for Space. Curr. Biol. 10.1016/j.cub.2015.12.072 (2016). Marinari, E. et al. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding. Nature 2012 484:7395 484, 542–545 (2012). Eisenhoffer, G. T. et al. Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature 2012 484:7395 484, 546–549 (2012). Cachoux, V. M. L. et al. Epithelial apoptotic pattern emerges from global and local regulation by cell apical area. Curr. Biol. 33 , 4807–4826e6 (2023). Slattum, G. M. & Rosenblatt, J. Tumour cell invasion: an emerging role for basal epithelial cell extrusion. Nature Reviews Cancer 2014 14:7 14, 495–501 (2014). Kon, S. & Fujita, Y. Cell competition-induced apical elimination of transformed cells, EDAC, orchestrates the cellular homeostasis. Dev. Biol. 476 , 112–116 (2021). Ninov, N., Chiarelli, D. A. & Martín-Blanco, E. Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis. Development 134 , 367–379 (2007). Baena-Lopez, L. A. All about the caspase-dependent functions without cell death. Semin Cell. Dev. Biol. 82 , 77–78 (2018). Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126 , 677–689 (2006). Halder, G., Dupont, S. & Piccolo, S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nature Reviews Molecular Cell Biology 13, 591–600 (2012). (2012). Kabigting, J. E. T. & Toyama, Y. Interplay between caspase, Yes-associated protein, and mechanics: A possible switch between life and death? Curr. Opin. Cell. Biol. 67 , 141–146 (2020). Davis, J. R. & Tapon, N. Hippo signalling during development. Development 146 , (2019). Tokamov, S. A. et al. Apical polarity and actomyosin dynamics control Kibra subcellular localization and function in Drosophila Hippo signaling. Dev. Cell. 58 , 1864–1879e4 (2023). Huang, J., Zhou, W., Dong, W. & Hong, Y. Targeted engineering of the Drosophila genome. Fly. (Austin) . 3 , 274–277 (2009). Lee, A. & Treisman, J. E. Excessive Myosin activity in mbs mutants causes photoreceptor movement out of the Drosophila eye disc epithelium. Mol. Biol. Cell. 15 , 3285–3295 (2004). Kim, Y. S., Fritz, J. L., Seneviratne, A. K. & VanBerkum, M. F. Constitutively active myosin light chain kinase alters axon guidance decisions in Drosophila embryos. Dev. Biol. 249 , 367–381 (2002). Supplementary Data Supplementary items Movie 16 and Movie 17 are not available with this version. Additional Declarations No competing interests reported. Supplementary Files Movie1.avi Movie2.avi Movie3.avi Movie4.avi Movie5.avi Movie6.avi Movie7.avi Movie8.avi Movie9.avi Movie10.avi Movie11.avi Movie12.avi Movie13.avi Movie14.avi Movie15.avi Movie18.avi SupplementaryInformationGorfinkieletal.pdf Cite Share Download PDF Status: Published Journal Publication published 01 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 16 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviews received at journal 13 Apr, 2025 Reviewers agreed at journal 23 Mar, 2025 Reviewers agreed at journal 22 Mar, 2025 Reviewers invited by journal 21 Mar, 2025 Editor assigned by journal 21 Mar, 2025 Submission checks completed at journal 20 Mar, 2025 First submitted to journal 20 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6227955","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":432735905,"identity":"30279910-e471-4372-b3c7-6b29bdb6b690","order_by":0,"name":"Nicole Gorfinkiel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYLCCCgMGA37StJwBapFsIE0LA4OBwQFiVZvzLz724UDBHWPjG8lPNzBU1BHWYjnjWfKMAwbPzMxupJndYDhzmLAWgxtnjJk/GBy2MbuRw3aDsY0I54G0MBwAajGeAdLyjwiHGZzvAWsxM5AAaWlgJsYWtmSQFmOJM8/MbiQcI8Yv5w8fZjjw57Bhf3vysxsfaohwGINEAhInAYciVMB/gChlo2AUjIJRMJIBALwrP2GeFkfjAAAAAElFTkSuQmCC","orcid":"","institution":"Complutense University of Madrid","correspondingAuthor":true,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Gorfinkiel","suffix":""},{"id":432735906,"identity":"52023673-9ba4-4e72-878d-da9cdf37af24","order_by":1,"name":"Yanara Ferrer","email":"","orcid":"","institution":"Complutense University of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Yanara","middleName":"","lastName":"Ferrer","suffix":""},{"id":432735907,"identity":"8ad1956e-c88f-4530-bb72-e690075c4173","order_by":2,"name":"Jon Recalde","email":"","orcid":"","institution":"Complutense University of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Jon","middleName":"","lastName":"Recalde","suffix":""},{"id":432735908,"identity":"1e1af76a-36d3-46b6-aac0-8827f4131d23","order_by":3,"name":"Javier Gutiérrez","email":"","orcid":"","institution":"Complutense University of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"","lastName":"Gutiérrez","suffix":""},{"id":432735909,"identity":"e12c03aa-5f19-422d-b6a9-053051d14431","order_by":4,"name":"Guillermo Sáez","email":"","orcid":"","institution":"Complutense University of Madrid","correspondingAuthor":false,"prefix":"","firstName":"Guillermo","middleName":"","lastName":"Sáez","suffix":""}],"badges":[],"createdAt":"2025-03-14 16:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6227955/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6227955/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-16032-2","type":"published","date":"2025-09-01T15:57:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79317313,"identity":"e82669fe-7001-4a3a-94f6-7ca694d13447","added_by":"auto","created_at":"2025-03-27 03:40:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1136514,"visible":true,"origin":"","legend":"\u003cp\u003eActomyosin dynamics during amnioserosa cell delamination. (A,B) Still images (maximum intensity projections) from a time-lapse movie (Movie 1) of a dorsal closure Drosophila embryo carrying the knock-in allele ECad:GFP at different time points prior to the delamination of the cell (asterisk) from the plane of the epithelium, showing the formation of a rosette-like structure (A) or the formation of a new junction (B, arrow). (C-E) Still images (individual Z planes) from a time-lapse movie of an early dorsal closure embryo carrying the knock-in alleles ECad:mT and sqh:GFP (C-E, Movie 2) or the knock-in allele ECad:mT and the reporter utrophin:GFP (F-H, Movie 3) showing an apical (C, F) and subapical (D, G) planes, as well as an orthogonal view (E, H)) at indicated times prior to the delamination. Notice the stabilisation of medioapical actomyosin during the apical-contraction phase (C, C´,F, arrows), the appearance of actin puncta (F´,G´, arrows) and the formation of a subapical actomysoin ring during the final phase (D”, E”, arrows). (I-H) Still images (maximum intensity projections) from a time-lapse movie of a late dorsal closure embryo carrying the knock-in alleles ECad:mT and sqh:GFP. Notice the accumulation of Myosin in neighbouring cells (I”, arrows), which coincides with a decrease of ECad:mT at cell junctions.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6227955/v1/95b435c7dec1581c206ee889.png"},{"id":79317305,"identity":"87401d74-5dde-4592-96dc-3a5a031537d6","added_by":"auto","created_at":"2025-03-27 03:40:36","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":439708,"visible":true,"origin":"","legend":"\u003cp\u003eRho activity in the amnioserosa. (A, A´) Still images (maximum intensity projections) from a time-lapse movie of a dorsal closure Drosophila embryo carrying the knock-in allele ECad:mT and the Rho sensor (aniRBD:GFP). During most of dorsal closure, the activity of the Rho sensor is only detected in delaminating cells (A). By the end of the process, most of the cells show medioapical accumulation of the Rho sensor (A´). (B) Magnification of the same time-lapse than in A (Movie 4), showing a delaminating cell (arrows) and the medioapical accumulation of the Rho sensor prior to cell delamination at the times indicated.\u003c/p\u003e","description":"","filename":"2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6227955/v1/345b094dff6b92974c346f1b.jpeg"},{"id":79317814,"identity":"310e07ee-09ff-4b1f-8003-1ac4bd3e0549","added_by":"auto","created_at":"2025-03-27 03:56:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1548328,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of perturbing Myosin activity in the amnioserosa on cell delaminations and actomyosin contractility. (A-C) Still image of a control ECad:GFP embryo (A, Movie 1), an AS-GAL4, ECad:GFP/UAS-MbsN300 (B, Movie 5) and an AS-GAL4, ECad:GFP/UAS-ctMLCK embryo (C, Movie 6) at -30 min of dorsal closure (staging relative to the distance of posterior spiracles). The cells labelled in red are cells that delaminate during dorsal closure. (D) Number of delaminating cells in AS-GAL4, ECadGFP (n=11) and AS-GAL4, ECadGFP/UAS-MbsN300 (n=5) embryos, between -30 and 30 min (left panel, p = 0.039) and between 30 and 90 min (right panel, p = 0.029). Although the total number of delaminating cells is similar between the two genotypes, AS-GAL4, ECadGFP/UAS-MbsN300 embryos show a delay in the appearance of cell delaminations, with most of them occurring from 30 min of dorsal closure. (E) Number of delaminating cells in AS-GAL4, ECadGFP (n=11) and AS-GAL4, ECadGFP/UAS-ctMLCK (n=5) embryos, between -30 and 30 min (left panel) and between 30 and 90 min (right panel). No differences in the total number of delaminating cells nor a change in the timing of appearance of cell delaminations is observed in these two conditions. (F, G) Still images from time-lapse movies of embryos carrying the knock-in alleles ECad:mT and sqh:GFP at early (F) and mid dorsal closure (G). (H, I) Still images from time-lapse movies of embryos carrying the knock-in alleles ECad:mT and sqh:GFP and ectopically expressing MbsN300 (H) or ctMLCK (I). (J, K) Still images from time-lapse movies of embryos carrying the knock-in allele ECad:mT and the Rho sensor and ectopically expressing MbsN300 (J, Movie 7) or ctMLCK (K, Movie 8). Notice the accumulation of Sqh:GFP and the Rho sensor at bicellular and tricellular junctions in H, J (arrow and arrowheads respectively) and their medioapical accumulation in I, K (arrows). (L, M) Still images of a delaminating cells from an embryo ectopically expressing MbsN300 (I) or ctMLCK (J) at the times indicated prior to cell delamination.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6227955/v1/d7fda42821acb5687c6c284e.png"},{"id":79317304,"identity":"1fb11b3b-a6a0-4745-a1d7-b4a1668a466d","added_by":"auto","created_at":"2025-03-27 03:40:36","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":540616,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of perturbing Myosin activity in groups of amnioserosa cells on cell delaminations. (A-C) Still images of from time-lapse movies of ECad:GFP; prd-GAL4/UAS-NLS:mCh (A, Movie 9), ECad:GFP; prd-GAL4/UAS-NLS:mCh; UAS-MbsN300 (B, Movie 10) and ECad:GFP; prd-GAL4/UAS-NLS:mCh; UAS-ctMLCK (C, Movie 11) embryos at 0 min of dorsal closure. (D) Ratio of NLS:mCherry to non-NLS:mCherry delaminating cells in ECad:GFP;prd-GAL4/UAS-NLS:mCh (n=11), ECad:GFP;prd-GAL4/UAS-NLS:mCh; UAS-MbsN300 (n=6) embryos and ECad:GFP;prd-GAL4/UAS-NLS:mCh; UAS-ctMLCK (n=5) embryos. Cells with lower levels of Myosin activity than their neighbours delaminate less than their neighbours (p = 0.049) while cells with higher levels of Myosin activity than their neighbours delaminate more (p = 0.009).\u003c/p\u003e","description":"","filename":"4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6227955/v1/7de2bf856bf234928c17cf44.jpeg"},{"id":79317317,"identity":"0e3c9963-78c1-4bd4-b46a-89bc5cd6df3e","added_by":"auto","created_at":"2025-03-27 03:40:37","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":512935,"visible":true,"origin":"","legend":"\u003cp\u003eBlocking endocytosis prevents cell delaminations. (A) Still image from a time-lapse movie of an AS-GAL4, ECad:GFP/UAS-Rab5DN embryo at -30 min of dorsal closure (Movie 12). (B) Number of delaminating cells between -30 min and 100 min in control (n=7) vs AS-GAL4, ECad:GFP/UAS-Rab5DN (n=3) embryos (p \u0026lt; 0.001). (C) Still image from a time-lapse movie (MIP) of an ECad:GFP; prd-GAL4/UAS-Rab5DN embryo at 0 min of dorsal closure (Movie 13). (D) Ratio of NLS:mCherry to non-NLS:mCherry delaminating cells in ECad:GFP;prd-GAL4/UAS-NLSmCh (n=11) and ECad:GFP;prd-GAL4/UAS-NLSmCh; UAS-Rab5DN (n=4) embryos (p = 0.03). (E, F) Still images from a time lapse movie from sqh:GFP; AS-GAL4, ECad:mT (E) and sqh:GFP; AS-GAL4, ECad:mT/UAS-Rab5DN (F) embryos.\u003c/p\u003e","description":"","filename":"5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6227955/v1/c780847d8ec55eea61c2135f.jpeg"},{"id":79317308,"identity":"53388cfd-0a18-4ea6-979c-07ced8be7301","added_by":"auto","created_at":"2025-03-27 03:40:36","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":862847,"visible":true,"origin":"","legend":"\u003cp\u003eCaspase activity in the amnioserosa. (A-B”) Still images (MIP) from a time-lapse movie of a dorsal closure embryo ectopically expressing the caspase sensor Apoliner in the amnioserosa (Movie 14). Early during dorsal closure, only posterior cells show caspase activity (A,B). As dorsal closure progresses, most amnioserosa cells located in the anterior part of the tissue start showing caspase activity while the majority of central cells still show low levels of nuclear GFP (A´,B´). During late dorsal closure, all amnioserosa cells have exhibit caspase activity (A”,B”). (C) Still images (MIP) from a time-lapse movie of an embryo ectopically expressing the apoptotic sensor GC3Ai (Movie 15). (C´-C””) Time course of GC3Ai activation during the delamination of a cell. (D) Still image of an AS-GAL4/UAS-GC3Ai; UAS-MbsN300 (Movie 16), with cells showing almost normal levels of caspase activity. (E) Still image of an AS-GAL4/UAS-Apoliner; UAS-ctMLCK (Movie 17), with cells showing higher levels of caspase activity. (F, G) Still images from a time-lapse movie of an AS-GAL4/UAS-GC3Ai; UAS-MbsN300 (F) and an AS-GAL4/UAS-GC3Ai; UAS-ctMLCK (G) embryo. Only cells that delaminate have GC3Ai signal. (H) Still image of an ECad:GFP; prd-GAL4/UAS-Diap1, UAS-NLS:mCh; UAS-ctMLCK (Movie 18). Delaminating cells are indicated with yellow asterisks.\u003c/p\u003e","description":"","filename":"6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6227955/v1/ef38ed301c202184fcc5f5ac.jpeg"},{"id":90827962,"identity":"ac4d008f-6da5-4882-9478-778fe53e17f5","added_by":"auto","created_at":"2025-09-08 16:04:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5420161,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6227955/v1/e6f2bf03-b785-4122-9164-0d0918774595.pdf"},{"id":79317815,"identity":"4c0cb8e0-f73b-441b-851e-5a48168f60dc","added_by":"auto","created_at":"2025-03-27 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03:40:38","extension":"pdf","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":324860,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationGorfinkieletal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6227955/v1/8854b2635e730b7dc922a98f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A role for Myosin in triggering and executing amnioserosa cell delaminations during dorsal closure","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe remodeling of epithelial tissues is a fundamental process in morphogenesis, giving rise to a diverse array of structures, including sheets, tubes, and cysts, which are essential for organ formation and function. A key aspect of epithelial remodeling is the apoptotic removal of individual cells from the epithelium while maintaining overall tissue integrity. Apoptotic cell elimination has been observed in diverse contexts contributing both to tissue morphogenesis and to the maintenance of tissue homeostasis\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In some instances, entire epithelial tissues are removed and replaced, as occurs during embryogenesis in the extra-embryonic tissues of insects\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and during metamorphosis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe amnioserosa is the single extra-embryonic tissue present in Drosophila\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. It is specified during early embryonic development by the dorso-ventral patterning system and plays essential functions during two vital morphogenetic movements, germband retraction and dorsal closure\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Amnioserosa cells undergo dramatic cell shape changes during embryogenesis. During germband elongation, these cells elongate and transition from a columnar to a squamous morphology through a process known as rotary cell elongation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. As development progresses into germband retraction, amnioserosa cells shorten, become isodiametric, and exert a pulling force that contributes to germband retraction\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Finally, during dorsal closure, amnioserosa cells exhibit dynamic fluctuations in apical surface area, driven by periodic contractions of the actomyosin cytoskeleton, and progressively reduce their apical surface\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Around 10% of the cells of the tissue basally delaminate before closure is completed\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These cellular processes, along with a decrease in volume, generate a morphogenetic force that drives the movement of the lateral epidermal sheets towards the dorsal midline. Ultimately, the entire amnioserosa tissue is internalized and degenerates by apoptosis\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmnioserosa cell delaminations are apoptotic, and although they are not essential for closure, enhancing or supressing apoptosis increases or reduces cell delaminations respectively, leading to either an acceleration or a slowing down of closure\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This led to the suggestion that apoptotic cell delaminations in the amnioserosa provides an apoptotic force that contributes to closure. Additionally, inhibiting apoptosis in the amnioserosa also prevents cell volume decrease in the bulk of the tissue\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, showing that the apoptotic pathway controls both individual delaminations and the remodeling of the entire tissue.\u003c/p\u003e \u003cp\u003eThe cellular mechanisms underlying the elimination of cells from an epithelium have been studied in several tissues, revealing the involvement of both cell autonomous and non-autonomous mechanisms. In the apoptotic cell, either an actomyosin ring\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e or medioapical actomyosin contractions\u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e can contribute to autonomous-cell constriction, while neighbouring cells form a supracellular actomyosin cable that aids in the expulsion of the cell from the tissue\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In the amnioserosa, high levels of reactive oxygen species induce caspase activity and cell delaminations through the reorganization of non-muscle Myosin distribution\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. During the early stages of dorsal closure, delaminating cells transition abruptly from a pulsatile behaviour to a rapidly, unpulsed, contracting behaviour\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. This delamination transiently affects the oscillating behaviour of the neighbouring cells, and changes in Myosin dynamics have been observed in both the delaminating cell and its neighbours\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, the precise cellular mechanisms underlying cell delamination remain unclear. Additionally, mechanical stresses have been proposed as potential triggers for both individual cell delamination of cells and the degeneration of the entire amnioserosa, but evidence for this has been lacking. In this work, we have investigated the cellular mechanisms driving cell delaminations in the amnioserosa and analyzed the consequences of perturbing non-muscle Myosin activity globally in the entire tissue as well as locally in groups of cells. Our results argue for an active role for Myosin in both triggering and executing cell delaminations and show that cells with high contractility can undergo delamination independently of caspase activity.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e1)\u0026nbsp; Actomyosin dynamics during amnioserosa cell delaminations\u003c/p\u003e\n\u003cp\u003eCell delaminations in the amnioserosa can be observed from the end of germband retraction until the completion of dorsal closure. Although their number varies among embryos, they exhibit a consistent spatial pattern, occurring mostly in the anterior half of the amnioserosa and at the posterior canthi\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e (see below).\u003c/p\u003e\n\u003cp\u003eIn epithelial tissues, cell delaminations are usually mediated by T2 transitions, in which multiple vertices converge to form a single vertex, generating a rosette-like structure. In the amnioserosa, while some cells delaminate through T2 transitions (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-A\u0026acute;\u0026acute;), we also observe delaminating cells that do not form rosettes. Instead, these cells leave a new junction once they are delaminated (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB-B\u0026acute;\u0026acute;). This type of delamination occurs in anisotropic cells where the longer junctions, usually dorso-ventrally oriented, approach and fuse, forming a new junction between new neighbours. We found that cells forming rosettes are more isotropic at the onset of delamination than cells leaving a junction (Fig.\u0026nbsp;1SA), suggesting that cell shape determines the geometry of the delamination process. Both types of cell delamination events are prevented upon expression of an apoptosis inhibitor such as p35 or Drosphila inhibitor of apoptosis 1, Diap1\u003csup\u003e15,16,18\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCell delaminations are driven by either a cortical actomyosin ring or pulses of contractile medioapical actomyosin\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. To investigate the mechanisms driving cell delaminations in the amnioserosa, we visualized non-muscle Myosin (MyoII) and actin distribution in delaminating cells from live embryos carrying the ECad:mTomato (ECad:mT) and the sqh:GFP knock-in alleles, or ECad:mT and the actin reporter utrophin fused to GFP (utr:GFP) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC-I\u0026rdquo;). Since MyoII subcellular localization and dynamics changes as dorsal closure progresses, characterized by increasing junctional MyoII and stabilisation of the medioapical pool\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, we examined cell delaminations at both early and late stages. During early stages, as the delaminating cell begins to apically constrict, we observed a stabilisation of medioapical MyoII, with no accumulation of junctional Myosin nor actin (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC, F). Early delaminating cells are pulsatile, and the onset of apical constriction coincides with the stabilisation of a medial MyoII pulse, giving rise to a non-pulsatile delaminating cell, as previously observed\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. As the apical cell area reduces, cell junctions show an inward curvature (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC, C\u0026acute;, arrow), suggesting the existence of a pulling force from the medial part of the delaminating cell. Interestingly, we observed the presence of MyoII and actin puncta that start to form basally as the onset of the delamination process (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026acute;, D\u0026rdquo;, G, G\u0026acute;, arrows). As the apical cell area reduces, actin puncta can be seen to re-localize apically at the level of ECad (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026acute;, arrow) to form a continuous junctional belt. During the last steps of the process, an actomyosin junctional ring is observed subapically (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD\u0026rdquo;, E\u0026rdquo;, G\u0026rdquo;, H\u0026rdquo;), which persists basal to the plane of the epithelium for up to 20 minutes (not shown). During late dorsal closure, delaminating cells also show a stabilisation of medioapical MyoII that spans the whole apical cortex as the cell area reduces (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI). An actomyosin ring is also observed as the delamination process proceeds (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI\u0026acute;, I\u0026rdquo;). Such cytoskeletal dynamics has been observed in both isotropic and anisotropic cell delaminations (data not shown).\u003c/p\u003e\n\u003cp\u003eIn contrast to what has been observed in other epithelia, we did not observe the formation of a supracellular actomyosin ring in neighbouring cells. However, in some late delaminating cells, MyoII accumulated in neighbouring cells at the junctions with the delaminating cell (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI\u0026rdquo;, arrows) coincident with a decrease in ECad levels (Fig.\u0026nbsp;1SB). We propose that this could be a mechanism to maintain junction integrity during the delamination process\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e but would not be required to expulse the cell from the tissue. Altogether, these observations indicate that amnioserosa cells delaminate through a mechanism mediated by medioapical Myosin, with the formation of an actomyosin ring in the last steps of the delamination process.\u003c/p\u003e\n\u003cp\u003eThe Rho family of GTPases is a central regulator of F-actin, MyoII activity and contractility\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Both Rho-dependent and independent mechanisms for apoptotic cell elimination have been reported\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In the amnioserosa, using a Rho live sensor consisting of the Rho1 GTP-binding domain of anillin to GFP (aniRBD:GFP)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, we have observed an increase in medioapical Rho activity as soon as the apical cell area of a delaminating cell begins to decrease (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Thus, it is possible that apoptotic signals induce medioapical Rho activity, which in turn regulates the cytoskeletal changes described above that participate in the execution of the delamination process. Interestingly, during late stages of dorsal closure, all amnioserosa cells show medioapical accumulation of the Rho sensor (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, A\u0026acute;). Since medioapical Rho activity is observed in apoptotic delaminating cells, and caspase activity has been detected throughout the amnioserosa tissue during late dorsal closure stages (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-B\u0026rdquo;)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, we asked whether this late-stage medioapical Rho accumulation depends on caspase activity. The ectopic expression of Diap1 in the amnioserosa, while preventing cell delamination, does not prevent the medioapical accumulation of the Rho sensor in the bulk of the tissue during late dorsal closure (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). This result suggests that Rho activity at the medioapical cortex of amnioserosa cells is regulated differently in delaminating cells versus the bulk of the tissue, where it appears to be independent of apoptotic signals.\u003c/p\u003e\n\u003cp\u003e2) Perturbing Myosin levels in the whole tissue does not alter the number of cell delaminations\u003c/p\u003e\n\u003cp\u003eTo investigate the requirement for Myosin in the triggering and the elimination of cells from the tissue, we aimed to perturb Myosin II activity in the whole tissue. We have previously shown that the ectopic expression of a constitutive active form of Mbs in the amnioserosa (Mbs is the Myosin binding subunit of the protein phosphatase PP1), decreases phosphorylated Myosin, decreases the rate of contraction of amnioserosa cells and slows dorsal closure\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Here, we analyzed whether cell delaminations are affected upon ectopic expression of MbsN300 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, D). Because dorsal closure takes longer in embryos ectopically expressing MbsN300, to compare the number of delaminations between the two genotypes, we considered the delaminations occurring during the same period of time, from \u0026minus;\u0026thinsp;30 min to 100 min of dorsal closure, and staging the embryos according to the distance between the posterior spiracles. Surprisingly, we found that the total number of cell delaminations in MbsN300 embryos is indistinguishable from that in control embryos. However, when comparing the timing of cell delaminations, we observed a delay: the number of cell delaminations between \u0026minus;\u0026thinsp;30 and 30 min of dorsal closure is lower in AS-GAL4, ECad:GFP/UAS-MbsN300 than in controls but is higher between 30 and 100 min of dorsal closure (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results suggest an early requirement of MyoII for triggering cell delaminations, however, unexpectedly, during later stages, cell delaminations resume and become more frequent.\u003c/p\u003e\n\u003cp\u003eTo explore what could be driving late delaminations in these embryos, we analyzed MyoII localization in sqh:GFP; AS-GAL4, ECad:mT/UAS-MbsN300 embryos. While MyoII localizes in medioapical foci in early dorsal closure, in older embryos, MyoII shows an aberrant distribution, accumulating at bicellular junctions and cell vertices, without forming the dense medioapical mesh observed in control embryos (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH, compare with 3F and 3G, see also\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e). Interestingly, the Rho sensor is also mislocalized, accumulating at cell junctions and cell vertices (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eJ). In these embryos, delaminating cells display junctional rather than medioapical Rho1 activity (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eL-L\u0026rdquo;), along with strong junctional MyoII (Fig.\u0026nbsp;2SA). These observations suggest that, in these embryos, cell delamination does not involve medioapical MyoII activity but rather junctional Rho1 and MyoII.\u003c/p\u003e\n\u003cp\u003eReciprocally, we aimed to increase Myosin activity in the amnioserosa by ectopically expressing a constitutive active form of Myosin Light Chain Kinase (ctMLCK). We have previously shown that these cells are more contracted and accumulate both junctional and medioapical MyoII\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. While in other tissues increasing Myosin activity perturbs cell delaminations\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, in the amnioserosa neither the number nor the timing of cell delaminations are altered (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, E). These cells exhibit an accumulation of Myosin at cell junctions and a persistent, dynamic medioapical network (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eI), which further accumulates in extruding cells (Fig. 2SB). Interestingly, most of amnioserosa cells with ectopic ctMLCK show medioapical accumulation of the Rho sensor (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eK), which becomes more pronounced in delaminating cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eM-M\u0026rdquo;). These observations, together with the junctional localization of the Rho sensor upon ectopic expression of MbsN300, show that perturbing Myosin localization impacts Rho localization. These observations are in agreement with the existence of a positive feedback between the upstream regulator Rho and MyoII\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (see Discussion).\u003c/p\u003e\n\u003cp\u003e3) In a tissue with tension heterogeneity, cells with higher levels of MyoII are more likely to delaminate\u003c/p\u003e\n\u003cp\u003eWe were intrigued by the observation that perturbing Myosin activity does not affect the overall rate of cell delaminations. While increased actomyosin contractility underlies cell ingression in several tissues\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, a global increase of junctional MyoII has been found to inhibit both apoptotic basal cell delamination and apoptotic apical extrusion\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. To further investigate this, we generated heterogeneities in MyoII levels across the tissue by selectively increasing or decreasing Myosin activity in groups of cells. For this, we ectopically expressed UAS-MbsN300 or UAS-ctMLCK using the prd-Gal4 driver, which is expressed in alternate segments in the epidermis and in the amnioserosa, along with a NLS:mCherry (NLS:mCh) reporter to identify MbsN300 or ctMLCK expressing cells. To assess whether changes in MyoII activity influence the likelihood of a cell delaminating, we calculated the ratio between the number of delaminating cells NLS-mCh positive and the number of delaminating cells NLS-mCh negative for each embryo.\u003c/p\u003e\n\u003cp\u003eIn ECad:GFP, prdGAL4/UAS-NLS:mCh, UAS-MbsN300 embryos, we observe that MbsN300 expressing cells show a larger apical cell area (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, arrow, compare with 4A), confirming that these cells have lower actomyosin contractility. In these embryos, the ratio of NLS-mCh delaminating cells (and thus expressing MbsN300) to non NLS-mCh delaminating cells (with wild type MyoII levels) is lower than in control embryos (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results confirm a requirement for medioapical MyoII in cell delamination. Conversely, in prdGAL4/UAS-NLS:mCh; UAS-ctMLCK embryos, we observe that ctMLCK positive cells show a smaller apical cell area (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, arrow, compare with 4A). In this case, the ratio of NLS:mCh delaminating cells (with higher both junctional and medioapical MyoII) to non-NLS:mCh delaminating cells (with wild type MyoII levels) is greater than in control embryos (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). Thus, although no increase in the number of delaminations is observed when Myosin activity is increased across the whole tissue, we find that cells with higher levels of Myo are more likely to delaminate when surrounded by cells with wild type MyoII activity. Importantly, delaminations of cells with high levels of MyoII occur throughout the entire process of dorsal closure, suggesting that high MyoII levels alone do not immediately induce delamination but increase the probability of a cell to delaminate. These results, together with the observation that increasing MyoII activity across the whole tissue does not result in an increase in the number of delaminations, suggest that high tissue stiffness could disrupt cell delamination.\u003c/p\u003e\n\u003cp\u003e4) Blocking endocytosis prevents cell delaminations\u003c/p\u003e\n\u003cp\u003eIn the pupal epidermis, a decrease in endocytic activity impairs or accelerates cell delaminations depending on the region of the segment where delaminations occur\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Therefore, we aimed to investigate the effect of blocking endocytic activity in the amnioserosa. We have previously shown that the ectopic expression of Rab5DN in the amnioserosa compromises apical constriction and apical membrane removal\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. We observed a decrease in the number of cell delaminations with only few delaminations that occurring during late stages of dorsal closure (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). The expression of Rab5DN in a mosaic manner using the prd-GAL4 driver shows that Rab5DN-expressing cells have a greater apical cell area than their neighbours (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), confirming that blocking endocytosis affects apical contraction. In these embryos, cells expressing Rab5DN are less likely to delaminate than control cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). We examined the subcellular localization of MyoII and found that although MyoII pulses are present, there is a decrease in junctional MyoII (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE, F). In fact, it has been shown that Rab5DN expressing amnioserosa cells show lower junctional tension, suggesting that endocytosis is important to maintain junctional tension\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. These results show that endocytic activity is required for amnioserosa cell delaminations, which could be related, at least in part, to a decrease in tissue tension.\u003c/p\u003e\n\u003cp\u003e5) Interaction between contractility and caspase activity\u003c/p\u003e\n\u003cp\u003eOur results show that cells with increased MyoII activity due to the ectopic expression of ctMLCK are more likely to delaminate when they are surrounded by cells with control levels of MyoII (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). This observation raised the question of whether increased Myosin activity affects caspase activity. To investigate this possibility, we explored the relationship between Myosin and caspase activity. The apoptotic sensor Apoliner has been widely used in several systems to monitor capsase activity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In this sensor, the fluorescent proteins mRFP and eGFP, are linked by a caspase-specific cleavage site from Diap1. Caspase activation leads to the cleavage of Apoliner and to the translocation of the eGFP from the cytoplasm to the nucleus. In the amnioserosa, during early stages of dorsal closure, the sensor is not active, except in the most posterior cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). As dorsal closure progresses, the sensor is activated first in anterior cells and then in the whole tissue before closure is complete (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, C and see also\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e). The activation of the sensor is completely blocked in embryos ectopically expressing Apoliner and Diap1 even after the amnioserosa has been internalized underneath the epidermis (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eS). Thus, Apoliner can detect low levels of caspase activity, that start to build up in the amnioserosa as dorsal closure progresses, but this activity does not inevitably lead to the cell delamination since most of the cells remain in the bulk of the tissue. We then turned to a different sensor, GC3Ai, which has been shown that can be detected in apoptotic cells throughout the whole process of programmed cell death\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In this sensor, the C and N termini of the GFP have been linked by a fragment containing a DEVD caspase cleavage site that must be cleaved for the GFP to fluoresce. In embryos ectopically expressing GC3Ai in the amnioserosa, sensor activity is only observed in delaminating cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). The activity of the GC3Ai sensor in delaminating cells is detected as the cell starts to apically constrict until after it has ingressed and start to undergo fragmentation, a process that takes up to 40 minutes (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC\u0026acute;-C\u0026rdquo;\u0026rdquo;). Thus, the GC3ai sensor detects apoptotic levels of caspase activity.\u003c/p\u003e\n\u003cp\u003eWe analyzed caspase activity with both sensors in embryos with perturbed Myosin activity. We wanted to determine whether, in AS-GAL4/UAS-MbsN300 embryos, the delay in the appearance of cell delaminations was due to an absence of caspase activity or to an impairment of the expulsion of the cell from the tissue. If this was the case, we expected to see an activation of the apoptotic sensors in the tissue with no delamination. In AS-GAL4/UASApoliner, UAS-MbsN300 embryos that complete dorsal closure, the activation of the sensor followed a similar temporal pattern than in control embryos (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD, compare with 6A), while the activity of the GC3ai sensor was only observed in delaminating cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). These observations show that apoptotic levels of caspase activity do not arise independently of cell delamination and suggest that proper levels of Myosin activity are required to trigger apoptotic cell delaminations.\u003c/p\u003e\n\u003cp\u003eSimilarly, in embryos ectopically expressing ctMLCK, GC3Ai activity was only detected in cells that delaminate (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG). In contrast, in AS-GAL4/UASApoliner, UAS-ctMLCK embryos, we observed a consistent premature activation of Apoliner (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE, compare with 6A). These observations suggest that increasing MyoII activity can induce low levels of caspase activity.\u003c/p\u003e\n\u003cp\u003eThese observations lead us to analyze whether the preferential delamination of cells with high MyoII levels in prd-GAL4/UAS-NLS:mCh; UASctMLCK embryos was due to an increase of caspase activity induced by MyoII. For this, we asked whether delaminations of ctMLCK cells in these embryos could be prevented by the ectopic expression of Diap1. Surprisingly, in ECadGFP; prd-GAL4/UAS-NLS:mCh, UASDiap1; UASctMLCK, NLS:mCh embryos, we observe NLS:mCh positive cells that delaminate (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH). Although we cannot rule out the possibility that Diap1 levels in this experiment are insufficient to fully prevent caspase activity, this result suggests that cell delamination can occur in response to high levels of MyoII even in the absence of caspase activity. This finding reveals that in a mechanically heterogeneous tissue, cells with high contractility can be eliminated independently of caspase activity.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this work, we have investigated the mechanisms underlying the triggering and execution of apototic cell delaminations in the amnioserosa during dorsal closure, before the tissue completely ingresses into the embryo and degenerates. Our results show that cell delaminations involve, firstly, the stabilisation of the medioapical actomyosin cortex, and secondly, the formation of an actomyosin ring, which completes the elimination of the apoptotic cell from the tissue. We have observed the appearance of actin puncta prior to the formation of the actomyosin ring, initially subapically and then co-localizing with ECadherin at the level of junctions. We propose that these puncta represent sites of actin nucleation for building the apoptotic actomyosin ring\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Although less prominent, Myosin puncta were also present, and we hypothesize they could be recruited by the nascent actin bundles. Our results also suggest that the small GTPase Rho regulates the reorganization of the actomyosin cytoskeleton to form the apoptotic actomyosin ring. Rho activity has mostly been shown to be required in neighbouring cells to the delaminating cells to form a supracellular actomyosin cable that squeezes extruding cells apically from the epithelium, but its requirement in the apoptotic cell itself is more controversial\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In the amnioserosa, all delaminating cells showed accumulation of a sensor of Rho activity in the medioapical cortex, strongly supporting the role of this small GTPase in regulating the cytoskeletal changes associated with cell delamination in the delaminating cell.\u003c/p\u003e \u003cp\u003eOur observations do not provide evidence for the involvement of cell neighbours in the elimination of the delaminating cells. Although it has been shown that a delaminating cell induce a deformation in its neighbours and impacts their Myosin pulses\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, we have not observed the formation of a supracellular actomyosin cable in neighbouring cells. An accumulation of MyoII close to cell junctions between the delaminating cell and its neighbours is only observed when ECad levels decrease significantly, suggesting that this might be a mechanism to preserve junction integrity. In fact, a mechanosensitive mechanism involving the actomyosin cytoskeleton to maintain junction integrity has been proposed\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly, we find that Rho subcellular localization changes upon perturbation of Myosin activity. In embryos ectopically expressing MbsN300, both MyoII and the Rho sensor accumulate at cell junctions, while in embryos ectopically expressing ctMLCK, Myo forms a dense apicomedial mesh, and the Rho sensor shows a more persistent apicomedial localization. These observations suggests that MyoII recruits Rho either to the junctional or to the medioapical region of cells, providing evidence for the existence of a positive feedback between Rho and MyoII. We have also observed oscillatory Rho activity in non-delaminating cells (unpublished observations) and as dorsal closure progresses, medioapical Rho activity is stabilised medioapically in the whole tissue. An advection-positive feedback mechanism between Rho and its downstream effectors Rock and MyoII has been shown to underlie actomyosin pulsatility in epidermal cells during germband elongation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. It is possible that such a mechanism is in place in the amnioserosa, contributing to the progressive stabilization of Rho and the medioapical acomyosin cortex at the level of the whole tissue.\u003c/p\u003e \u003cp\u003eWhat triggers cell delaminations? The apoptotic removal of epithelial cells from a tissue can be regulated by both biochemical and mechanical signals\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Mechanical compression, local topological defects and the geometry of cell packing have all been shown to promote cell elimination from a tissue\u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. In the amnioserosa, it has been suggested that mechanical signals are involved in the triggering of cell delaminations\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Here, we have observed that decreasing MyoII activity in the entire tissue delays the appearance of cell delaminations. Since low MyoII activity is associated with low tissue tension\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, our results argue for a role for mechanics in triggering of cell delaminations. However, increasing tissue tension through increasing Myo levels in the whole tissue does not increase cell delaminations. A global increase in junctional Myosin has also been shown to block cell delamination in the fly notum\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, and to disrupt apoptotic extrusion in epithelial monolayers. In the latter, increasing Myosin in apoptotic cells or in the surrounding cells disrupted extrusion\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This contrasts with what we observe in the amnioserosa, where cells with higher MyoII than their neighbours are more likely to delaminate. Altogether, our results suggest that high tissue tension or a stiff mechanical environment may exert an inhibitory effect on the ability to delaminate.\u003c/p\u003e \u003cp\u003eIt is important to notice that cells with increased MyoII do not delaminate instantaneously but are more likely to do so over the entire process of dorsal closure. This suggests that other signals contribute to the decision of whether a cell will delaminate. In the fly notum, it has been found that the apical size of a cell, compared to other cells within the tissue and relative to the immediate neighbours, is a predictor of apoptotic cell delamination\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Interestingly, we have found here that cells with lower levels of Myosin and showing a greater apical cell area are less likely to delaminate, while cells with higher Myo levels and with a smaller apical cell area, are more likely to delaminate. Our results suggest that MyoII activity is a key determinant of apical cell size that could be mediating the relation between cell size and apoptotic fate.\u003c/p\u003e \u003cp\u003eThe fact that cells with higher MyoII levels than their neighbours can delaminate when caspase activity is prevented by the ectopic expression of Diap1 is intriguing. As mentioned above, we cannot rule out that the levels of Diap1 are not sufficient to fully prevent caspase activity due to a dilution of the GAL4 transcriptional activator. Nonetheless, it is worth pointing out that live basal cell delamination plays a crucial role in tumour cell invasion\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Interestingly, RasV12-transformed cells in epithelial monolayers are typically extruded apically through a mechanism called \u0026ldquo;epithelial defence against cancer\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. However, some cells evade this defence mechanism and instead delaminate basally, a process suggested to contribute to malignancy. The mechanisms underlying the shift in the direction of cell elimination from epithelia remain unclear, but one possible factor is Rho hyperactivation, which increases actomyosin contractility\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Our results provide further evidence for a role for actomyosin contractility in driving live basal cell delamination.\u003c/p\u003e \u003cp\u003eThe amnioserosa is a tissue in which the delamination of cells from the tissue occurs alongside the subsequent complete elimination of the tissue. This phenomenon also takes place in the larval epidermis, which is replaced by histoblasts during metamorphosis\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, and is likely to occur in other insect extra-embryonic tissues\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Both in the amnioserosa and in the larval epidermis, all the cells show low, non-apoptotic, levels of caspase activity well before the tissue is eliminated. There is growing evidence that caspases have non-lethal functions and can influence a diverse array of cellular processes such as signalling, proliferation, differentiation and migration\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. In the amnioserosa, a role for caspases in controlling cell volume decrease has been found\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and it is possible that low levels of caspases play a role in the remodelling of the cytoskeleton not directly linked to cell death.\u003c/p\u003e \u003cp\u003eAn important question, however, is how global tissue levels of caspases arise. We have found here that increasing Myo activity favours caspase activation. In the larval epidermis, it has also been proposed that MyoII potentiates caspase activity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. An interesting possibility is that actomyosin contractility influences caspase activity and thus the fate of amnioserosa cells. A relationship between mechanical cues and cell fate has been known for several years\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The Hippo pathway is involved in transforming mechanical stimuli into biochemical signalling pathways both in vertebrates and in Drosophila\u003csup\u003e\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Notably, in wing imaginal disc cells, it has been found that medial actomyosin flows promote the medial accumulation of Kibra and the formation of a Hippo complex, which activates the Hippo pathway to repress the pro-growth transcriptional effector Yorkie\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. The role of the Hippo pathway in the amnioserosa has not been explored yet but would be an interesting avenue for future research.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFly stocks and genetics\u003c/span\u003e:\u003c/h2\u003e \u003cp\u003eThe following stocks were used in this work:\u003c/p\u003e \u003cp\u003eECad:GFP and ECad:mT knock-in alleles \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e; sqh:GFP \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e ; aniRBD:GFP \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e; AS-GAL4 (332.3 GAL4 line, RRID:BDSC_5398); prd-GAL4 (RRID:BDSC_1947); UAS-MbsN300 \u003csup\u003e63\u003c/sup\u003e; UAS-ctMLCK \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e; UAS-Apoliner \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e; UAS-GC3Ai \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e; UAS-NLSmCherry (RRID:BDSC_38425); UAS-Diap1 (II); UAS-Rab5DN (RRID:BDSC_42704).\u003c/p\u003e \u003cp\u003eStocks built during this work:\u003c/p\u003e \u003cp\u003eAS-GAL4, ECad:GFP and AS-GAL4, ECad:mT; sqh:GFP\u003c/p\u003e \u003cp\u003eAS-GAL4, ECad:mT; aniRBD:GFP\u003c/p\u003e \u003cp\u003esqh:GFP; AS-GAL4, ECad:mT\u003c/p\u003e \u003cp\u003eUAS-NLS:mCherry; UAS-MbsN300\u003c/p\u003e \u003cp\u003eUAS-NLS:mCherry; UAS-ctMLCK\u003c/p\u003e \u003cp\u003eUAS-Apoliner; UAS-MbsN300\u003c/p\u003e \u003cp\u003eUAS-Apoliner; UAS-ctMLCK\u003c/p\u003e \u003cp\u003eUAS-GC3Ai; UAS-MbsN300\u003c/p\u003e \u003cp\u003eUAS-GC3Ai; UAS-ctMLCK\u003c/p\u003e \u003cp\u003eECad:GFP; prdGAL4\u003c/p\u003e \u003cp\u003eUAS-Diap1; UAS-MbsN300\u003c/p\u003e \u003cp\u003eUAS-Diap1; UAS-ctMLCK\u003c/p\u003e \u003cp\u003eUAS-Diap1, UAS-NLS:mCherry; UAS-ctMLCK\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLive-Imaging\u003c/span\u003e: Stage 12\u0026ndash;13 Drosophila embryos were dechorionated, mounted in coverslips with the dorsal side glued to the glass and covered with Voltalef oil 10S (Attachem). The AS was imaged at 25\u0026ndash;28\u0026ordm;C. using an inverted LSM 710 Meta laser scanning microscope or a Leica SP8 with a 40X or a 63X oil immersion Plan-Fluor (NA\u0026thinsp;=\u0026thinsp;1.3) objective. GFP and mTomato cytoskeletal reporters were simultaneously imaged with an argon laser and a 651 diode laser. For whole AS imaging, 15\u0026ndash;30 z sections 1-1.5\u0026micro;m apart were collected every 2 minutes. For cytoskeletal dynamics imaging, 10 z sections 1\u0026micro;m apart were collected every 30 seconds.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImage analysis and statistics\u003c/span\u003e: Images were processed and assembled in Fiji (version 2.16.0) and Adobe Photoshop (version 23.5.0). Images were projected via the maximum intensity projection to cover the entire tissue or a single cell in case it was located in a curved region of the tissue. The identification of delaminating cells was done manually on the maximum intensity projections. Statistical analysis of the significance of differences in phenotype across genotypes was performed using the Wilcoxon-Mann-Whitney test using the R package.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eN.G. conceived the project, performed experiments, analysed data and wrote the paper, Y.F., J.G. and G.S. performed experiments, Y.F. and J.R. analysed data.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eWe are very grateful to Juan Jos\u0026eacute; Mu\u0026ntilde;oz\u0026dagger; for his help and support during the initial phase of this project. We thank Isabel Guerrero, Julia Duque and Guy Blanchard for critically reading the manuscript. We thank the Bloomington Stock Center, Bruno Monier and Jorg Gro\u0026szlig;hans for Drosophila strains. Microscopy was performed at the Unidad de Citometr\u0026iacute;a de Flujo y Microscop\u0026iacute;a de Fluorescencia of the Universidad Complutense de Madrid and at the Advanced Light Microscopy Facility at the Centro de Biolog\u0026iacute;a Molecular Severo Ochoa. This work was supported by grant PID2020-114533GB-C22 to NG from the Spanish Ministry of Economy and Competitiveness.\u003c/p\u003e \u003ch2\u003eCompeting interests statement\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files. All time-lapse movies are available on request to [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eValon, L. \u0026amp; Levayer, R. Dying under pressure: cellular characterisation and in vivo functions of cell death induced by compaction. \u003cem\u003eBiol. Cell.\u003c/em\u003e \u003cb\u003e111\u003c/b\u003e, 51\u0026ndash;66 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmbrosini, A., Rayer, M., Monier, B. \u0026amp; Suzanne, M. Mechanical Function of the Nucleus in Force Generation during Epithelial Morphogenesis. \u003cem\u003eDev. Cell.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e, 197\u0026ndash;211e5 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTada, M. The morphogenetic changes that lead to cell extrusion in development and cell competition. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cb\u003e477\u003c/b\u003e, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanfilio, K. A. \u0026amp; Roth, S. Epithelial reorganization events during late extraembryonic development in a hemimetabolous insect. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cb\u003e340\u003c/b\u003e, (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoshika, S., Sun, X., Kuranaga, E. \u0026amp; Umetsu, D. Reduction of endocytic activity accelerates cell elimination during tissue remodeling of the Drosophila epidermal epithelium. \u003cem\u003eDev. (Cambridge)\u003c/em\u003e \u003cb\u003e147\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmidt-Ott, U. \u0026amp; Kwan, C. W. How two extraembryonic epithelia became one: serosa and amnion features and functions of Drosophila\u0026rsquo;s amnioserosa. \u003cem\u003ePhilosophical Trans. Royal Soc. B\u003c/em\u003e \u003cb\u003e377\u003c/b\u003e, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLacy, M. E. \u0026amp; Hutson, M. S. Amnioserosa development and function in Drosophila embryogenesis: Critical mechanical roles for an extraembryonic tissue. \u003cem\u003eDev. Dyn.\u003c/em\u003e \u003cb\u003e245\u003c/b\u003e, 558\u0026ndash;568 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePope, K. L. \u0026amp; Harris, T. J. Control of cell flattening and junctional remodeling during squamous epithelial morphogenesis in Drosophila. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e135\u003c/b\u003e, 2227\u0026ndash;2238 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLynch, H. E. et al. Cellular mechanics of germ band retraction in Drosophila. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cb\u003e384\u003c/b\u003e, (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSch\u0026ouml;ck, F. \u0026amp; Perrimon, N. Cellular processes associated with germ band retraction in Drosophila. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cb\u003e248\u003c/b\u003e, (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSolon, J., Kaya-Copur, A., Colombelli, J. \u0026amp; Brunner, D. Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e137\u003c/b\u003e, 1331\u0026ndash;1342 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlanchard, G. B., Murugesu, S., Adams, R. J., Martinez-Arias, A. \u0026amp; Gorfinkiel, N. Cytoskeletal dynamics and supracellular organisation of cell shape fluctuations during dorsal closure. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e137\u003c/b\u003e, 2743\u0026ndash;2752 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavid, D. J., Tishkina, A. \u0026amp; Harris, T. J. The PAR complex regulates pulsed actomyosin contractions during amnioserosa apical constriction in Drosophila. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e137\u003c/b\u003e, 1645\u0026ndash;1655 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSokolow, A., Toyama, Y., Kiehart, D. P. \u0026amp; Edwards, G. S. Cell ingression and apical shape oscillations during dorsal closure in Drosophila. \u003cem\u003eBiophys. J.\u003c/em\u003e \u003cb\u003e102\u003c/b\u003e, 969\u0026ndash;979 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorfinkiel, N., Blanchard, G. B. \u0026amp; Adams, R. J. Martinez Arias, A. Mechanical control of global cell behaviour during dorsal closure in Drosophila. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e136\u003c/b\u003e, 1889\u0026ndash;1898 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToyama, Y., Peralta, X. G., Wells, A. R., Kiehart, D. P. \u0026amp; Edwards, G. S. Apoptotic force and tissue dynamics during Drosophila embryogenesis. \u003cem\u003eSci. (1979)\u003c/em\u003e. \u003cb\u003e321\u003c/b\u003e, 1683\u0026ndash;1686 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L. \u0026amp; Montague, R. A. Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila. \u003cem\u003eJ. Cell. Biol.\u003c/em\u003e \u003cb\u003e149\u003c/b\u003e, 471\u0026ndash;490 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuliyil, S., Krishnakumar, P. \u0026amp; Narasimha, M. Spatial, temporal and molecular hierarchies in the link between death, delamination and dorsal closure. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e138\u003c/b\u003e, 3043\u0026ndash;3054 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorfinkiel, N., Schamberg, S. \u0026amp; Blanchard, G. B. Integrative approaches to morphogenesis: lessons from dorsal closure. \u003cem\u003eGenesis\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e, 522\u0026ndash;533 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayes, P. \u0026amp; Solon, J. Drosophila dorsal closure: An orchestra of forces to zip shut the embryo. \u003cem\u003eMech. Dev.\u003c/em\u003e \u003cb\u003e144\u003c/b\u003e, 2\u0026ndash;10 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiehart, D. P., Crawford, J. M., Aristotelous, A., Venakides, S. \u0026amp; Edwards, G. S. Cell Sheet Morphogenesis: Dorsal Closure in Drosophila melanogaster as a Model System. \u003cem\u003eAnnual Rev. Cell. Dev. Biology Annu. Rev. Cell. Dev. Biol.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 169\u0026ndash;202 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaias, L. et al. Decrease in Cell Volume Generates Contractile Forces Driving Dorsal Closure. \u003cem\u003eDev. Cell.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 611\u0026ndash;621 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenblatt, J., Raff, M. C. \u0026amp; Cramer, L. P. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 1847\u0026ndash;1857 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuipers, D. et al. Epithelial repair is a two-stage process driven first by dying cells and then by their neighbours. \u003cem\u003eJ. Cell. Sci.\u003c/em\u003e \u003cb\u003e127\u003c/b\u003e, 1229\u0026ndash;1241 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuszyc, K. et al. Mechanotransduction activates RhoA in the neighbors of apoptotic epithelial cells to engage apical extrusion. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 1326\u0026ndash;1336e5 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeng, X., Qin, L., Le Borgne, R. \u0026amp; Toyama, Y. Remodeling of adhesion and modulation of mechanical tensile forces during apoptosis in Drosophila epithelium. \u003cem\u003eDev. (Cambridge)\u003c/em\u003e. \u003cb\u003e144\u003c/b\u003e, 95\u0026ndash;105 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtieh, Y., Wyatt, T., Zaske, A. M. \u0026amp; Eisenhoffer, G. T. Pulsatile contractions promote apoptotic cell extrusion in epithelial tissues. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 1129\u0026ndash;1140e4 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAn, Y. et al. Apical constriction is driven by a pulsatile apical myosin network in delaminating Drosophila neuroblasts. \u003cem\u003eDev. (Cambridge)\u003c/em\u003e. \u003cb\u003e144\u003c/b\u003e, 2153\u0026ndash;2164 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichel, M. \u0026amp; Dahmann, C. Tissue mechanical properties modulate cell extrusion in the Drosophila abdominal epidermis. \u003cem\u003eDev. (Cambridge)\u003c/em\u003e \u003cb\u003e147\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaravanan, S., Meghana, C. \u0026amp; Narasimha, M. Local, cell-nonautonomous feedback regulation of myosin dynamics patterns transitions in cell behavior: a role for tension and geometry? \u003cem\u003eMol. Biol. Cell.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 2350\u0026ndash;2361 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSumi, A. et al. Adherens Junction Length during Tissue Contraction Is Controlled by the Mechanosensitive Activity of Actomyosin and Junctional Recycling. \u003cem\u003eDev. Cell.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 453\u0026ndash;463e3 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunjal, A. \u0026amp; Lecuit, T. Actomyosin networks and tissue morphogenesis. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e141\u003c/b\u003e, 1789\u0026ndash;1793 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColeman, M. L. \u0026amp; Olson, M. F. Rho GTPase signalling pathways in the morphological changes associated with apoptosis. \u003cem\u003eCell Death \u0026amp; Differentiation\u003c/em\u003e 9, 493\u0026ndash;504 (2002). (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunjal, A., Philippe, J. M., Munro, E. \u0026amp; Lecuit, T. A self-organized biomechanical network drives shape changes during tissue morphogenesis. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e524\u003c/b\u003e, 351\u0026ndash;355 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen, W. et al. Modulation of morphogenesis by Egfr during dorsal closure in Drosophila. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, e60180 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuliyil, S., Narasimha, M. \u0026amp; Mitochondrial, R. O. S. Regulates Cytoskeletal and Mitochondrial Remodeling to Tune Cell and Tissue Dynamics in a Model for Wound Healing. \u003cem\u003eDev. Cell.\u003c/em\u003e \u003cb\u003e28\u003c/b\u003e, 239\u0026ndash;252 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFischer, S. C. et al. Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, e95695 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuque, J. \u0026amp; Gorfinkiel, N. Integration of actomyosin contractility with cell-cell adhesion during dorsal closure. \u003cem\u003eDev. (Cambridge)\u003c/em\u003e. \u003cb\u003e143\u003c/b\u003e, 4676\u0026ndash;4686 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachado, P. F. et al. Emergent material properties of developing epithelial tissues. \u003cem\u003eBMC Biol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 1\u0026ndash;15 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurran, S. et al. Myosin II Controls Junction Fluctuations to Guide Epithelial Tissue Ordering. \u003cem\u003eDev. Cell.\u003c/em\u003e \u003cb\u003e43\u003c/b\u003e, 480\u0026ndash;492e6 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePriya, R. et al. Tension heterogeneity directs form and fate to pattern the myocardial wall. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e588\u003c/b\u003e, 130\u0026ndash;134 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSim\u0026otilde;es, S., Oh, Y., Wang, M. F. Z., Fernandez-Gonzalez, R. \u0026amp; Tepass, U. Myosin II promotes the anisotropic loss of the apical domain during Drosophila neuroblast ingression. \u003cem\u003eJ. Cell Biol.\u003c/em\u003e \u003cb\u003e216\u003c/b\u003e, 1387\u0026ndash;1404 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMann, Z. et al. Preexisting tissue mechanical hypertension at adherens junctions disrupts apoptotic extrusion in epithelia. \u003cem\u003eMol. Biol. Cell.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, br3 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMateus, A. M., Gorfinkiel, N. \u0026amp; Schamberg, S. Martinez Arias, A. Endocytic and recycling endosomes modulate cell shape changes and tissue behaviour during morphogenesis in Drosophila. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e6\u003c/b\u003e, e18729 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBardet, P. L. et al. A fluorescent reporter of caspase activity for live imaging. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e105\u003c/b\u003e, 13901\u0026ndash;13905 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchott, S. et al. A fluorescent toolkit for spatiotemporal tracking of apoptotic cells in living Drosophila tissues. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e144\u003c/b\u003e, 3840\u0026ndash;3846 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichael, M. et al. Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions. \u003cem\u003eDev. Cell.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 58\u0026ndash;71 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFadul, J. \u0026amp; Rosenblatt, J. The forces and fates of extruding cells. \u003cem\u003eCurr. Opin. Cell. Biol.\u003c/em\u003e \u003cb\u003e54\u003c/b\u003e, 66\u0026ndash;71 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevayer, R., Dupont, C. \u0026amp; Moreno, E. Tissue Crowding Induces Caspase-Dependent Competition for Space. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cub.2015.12.072\u003c/span\u003e\u003cspan address=\"10.1016/j.cub.2015.12.072\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarinari, E. et al. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding. \u003cem\u003eNature 2012 484:7395\u003c/em\u003e 484, 542\u0026ndash;545 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisenhoffer, G. T. et al. Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. \u003cem\u003eNature 2012 484:7395\u003c/em\u003e 484, 546\u0026ndash;549 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCachoux, V. M. L. et al. Epithelial apoptotic pattern emerges from global and local regulation by cell apical area. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cb\u003e33\u003c/b\u003e, 4807\u0026ndash;4826e6 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSlattum, G. M. \u0026amp; Rosenblatt, J. Tumour cell invasion: an emerging role for basal epithelial cell extrusion. \u003cem\u003eNature Reviews Cancer 2014 14:7\u003c/em\u003e 14, 495\u0026ndash;501 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKon, S. \u0026amp; Fujita, Y. Cell competition-induced apical elimination of transformed cells, EDAC, orchestrates the cellular homeostasis. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cb\u003e476\u003c/b\u003e, 112\u0026ndash;116 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNinov, N., Chiarelli, D. A. \u0026amp; Mart\u0026iacute;n-Blanco, E. Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e134\u003c/b\u003e, 367\u0026ndash;379 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaena-Lopez, L. A. All about the caspase-dependent functions without cell death. \u003cem\u003eSemin Cell. Dev. Biol.\u003c/em\u003e \u003cb\u003e82\u003c/b\u003e, 77\u0026ndash;78 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEngler, A. J., Sen, S., Sweeney, H. L. \u0026amp; Discher, D. E. Matrix elasticity directs stem cell lineage specification. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e126\u003c/b\u003e, 677\u0026ndash;689 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalder, G., Dupont, S. \u0026amp; Piccolo, S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ. \u003cem\u003eNature Reviews Molecular Cell Biology\u003c/em\u003e 13, 591\u0026ndash;600 (2012). (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKabigting, J. E. T. \u0026amp; Toyama, Y. Interplay between caspase, Yes-associated protein, and mechanics: A possible switch between life and death? \u003cem\u003eCurr. Opin. Cell. Biol.\u003c/em\u003e \u003cb\u003e67\u003c/b\u003e, 141\u0026ndash;146 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavis, J. R. \u0026amp; Tapon, N. Hippo signalling during development. \u003cem\u003eDevelopment\u003c/em\u003e \u003cb\u003e146\u003c/b\u003e, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTokamov, S. A. et al. Apical polarity and actomyosin dynamics control Kibra subcellular localization and function in Drosophila Hippo signaling. \u003cem\u003eDev. Cell.\u003c/em\u003e \u003cb\u003e58\u003c/b\u003e, 1864\u0026ndash;1879e4 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, J., Zhou, W., Dong, W. \u0026amp; Hong, Y. Targeted engineering of the Drosophila genome. \u003cem\u003eFly. (Austin)\u003c/em\u003e. \u003cb\u003e3\u003c/b\u003e, 274\u0026ndash;277 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, A. \u0026amp; Treisman, J. E. Excessive Myosin activity in mbs mutants causes photoreceptor movement out of the Drosophila eye disc epithelium. \u003cem\u003eMol. Biol. Cell.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 3285\u0026ndash;3295 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, Y. S., Fritz, J. L., Seneviratne, A. K. \u0026amp; VanBerkum, M. F. Constitutively active myosin light chain kinase alters axon guidance decisions in Drosophila embryos. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cb\u003e249\u003c/b\u003e, 367\u0026ndash;381 (2002).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Supplementary Data","content":"\u003cp\u003eSupplementary items Movie 16 and Movie 17 are not available with this version.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"cell delamination, actomyosin contractility, Rho, caspase activity","lastPublishedDoi":"10.21203/rs.3.rs-6227955/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6227955/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe remodeling of epithelial tissues is a critical process in morphogenesis, often involving the apoptotic removal of individual cells while preserving tissue integrity. In \u003cem\u003eDrosophila\u003c/em\u003e, the amnioserosa\u0026mdash;a highly dynamic extra-embryonic tissue\u0026mdash;undergoes extensive remodeling, culminating in its complete elimination at the end of dorsal closure. While apoptotic cell delaminations in the amnioserosa have been proposed to contribute to dorsal closure, the cellular mechanisms underlying this process remain poorly understood. In this study, we have investigated actomyosin dynamics during cell delaminations and analyzed the consequences of perturbing non-muscle Myosin activity globally in the entire tissue as well as locally in groups of cells. We found that Myosin plays an essential role in both triggering and executing cell delaminations, with high Myosin contractility promoting cell extrusion even in the absence of caspase activity. Additionally, our results suggest that cell delaminations are governed by both cell-autonomous Myosin dynamics and mechanical cues from the tissue environment. Together, these findings provide new insights into the regulation of epithelial cell removal and the complex interplay between apoptotic and mechanical signals during tissue remodeling.\u003c/p\u003e","manuscriptTitle":"A role for Myosin in triggering and executing amnioserosa cell delaminations during dorsal closure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-27 03:40:30","doi":"10.21203/rs.3.rs-6227955/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-16T13:13:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T15:43:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-13T13:59:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8064452975674210739443523217759321100","date":"2025-03-23T15:23:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"203683711199952880268847736656555449808","date":"2025-03-22T16:53:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-21T10:10:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-21T09:43:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-20T13:16:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-20T13:15:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e58b8893-ed0e-44e1-a3a9-2c54aaa827ed","owner":[],"postedDate":"March 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46084329,"name":"Biological sciences/Cell biology"},{"id":46084330,"name":"Biological sciences/Developmental biology"}],"tags":[],"updatedAt":"2025-09-08T16:00:54+00:00","versionOfRecord":{"articleIdentity":"rs-6227955","link":"https://doi.org/10.1038/s41598-025-16032-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-01 15:57:43","publishedOnDateReadable":"September 1st, 2025"},"versionCreatedAt":"2025-03-27 03:40:30","video":"","vorDoi":"10.1038/s41598-025-16032-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-16032-2","workflowStages":[]},"version":"v1","identity":"rs-6227955","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6227955","identity":"rs-6227955","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}