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• W3045069165 endingPage "101413" @default.
• W3045069165 startingPage "101413" @default.
• W3045069165 abstract "•NADPH oxidases are responsible for cell delamination in Drosophila pupal notum•Nox is upregulated in delaminating cells prior to caspase-3 activation•Nox promotes caspase-3 activation and cell delamination•H2O2 suppresses apoptotic nuclear fragmentation during delamination Thorax fusion occurs in the midline of the Drosophila pupal notum and involves epithelial cell delamination requiring apoptotic signaling. By genetic screening, we found that NADPH oxidases (Nox and Duox) associated with superoxide anion (O˙-2) are responsible for caspase-3 activation and delamination. We observed that Nox is upregulated in cells that undergo delamination and that delamination depends on caspase activation. However, the cell morphology and the almost complete lack of propidium iodide incorporation suggested little membrane disruption and signified apoptotic modulation. These results demonstrate that most delaminating cells undergo caspase activation, but this activation is not sufficient for apoptosis. We showed that the expression of Catalase, encoding an H2O2 scavenger in the cytosol, increases delamination and induces apoptotic nuclear fragmentation in caspase-3-activated cells. These findings suggest that the roles of O˙-2 and intracellular H2O2 for delamination differs before and after caspase-3 activation, which involves live cell delamination. Thorax fusion occurs in the midline of the Drosophila pupal notum and involves epithelial cell delamination requiring apoptotic signaling. By genetic screening, we found that NADPH oxidases (Nox and Duox) associated with superoxide anion (O˙-2) are responsible for caspase-3 activation and delamination. We observed that Nox is upregulated in cells that undergo delamination and that delamination depends on caspase activation. However, the cell morphology and the almost complete lack of propidium iodide incorporation suggested little membrane disruption and signified apoptotic modulation. These results demonstrate that most delaminating cells undergo caspase activation, but this activation is not sufficient for apoptosis. We showed that the expression of Catalase, encoding an H2O2 scavenger in the cytosol, increases delamination and induces apoptotic nuclear fragmentation in caspase-3-activated cells. These findings suggest that the roles of O˙-2 and intracellular H2O2 for delamination differs before and after caspase-3 activation, which involves live cell delamination. For organ morphogenesis, two distant tissues approach and combine to form one continuous structure. Various organs such as the palate, neural tube, heart, eyes, face, and body wall are developed by this process of “tissue fusion” (Ray and Niswander, 2012Ray H.J. Niswander L. Mechanisms of tissue fusion during development.Development. 2012; 139: 1701-1711Crossref PubMed Scopus (88) Google Scholar). A characteristic feature of tissue fusion is the appearance of many apoptotic cells near the fusion sites. However, the regulatory mechanisms and functions of apoptosis in this context remain to be elucidated (Cuervo and Covarrubias, 2004Cuervo R. Covarrubias L. Death is the major fate of medial edge epithelial cells and the cause of basal lamina degradation during palatogenesis.Development. 2004; 131: 15-24Crossref PubMed Scopus (142) Google Scholar; Cuervo et al., 2002Cuervo R. Valencia C. Chandraratna R.A.S. Covarrubias L. Programmed cell death is required for palate shelf fusion and is regulated by retinoic acid.Dev. Biol. 2002; 245: 145-156Crossref PubMed Scopus (113) Google Scholar; Farbman, 1968Farbman A.I. Electron microscope study of palate fusion in mouse embryos.Dev. Biol. 1968; 18: 93-116Crossref PubMed Scopus (97) Google Scholar; Hinrichsen, 1985Hinrichsen K. The early development of morphology and patterns of the face in the human-embryo.Adv. Anat. Embryol. Cell. 1985; 98: 1-76Crossref PubMed Google Scholar; Martinez-Alvarez et al., 2000Martinez-Alvarez C. Tudela C. Perez-Miguelsanz J. O'Kane S. Puerta J. Ferguson M.W.J. Medial edge epithelial cell fate during palatal fusion.Dev. Biol. 2000; 220: 343-357Crossref PubMed Scopus (157) Google Scholar; Mori et al., 1994Mori C. Nakamura N. Okamoto Y. Osawa M. Shiota K. Cytochemical identification of programmed cell-death in the fusing fetal mouse palate by specific labeling of DNA fragmentation.Anat. Embryol. 1994; 190: 21-28Crossref PubMed Scopus (117) Google Scholar; Yamaguchi et al., 2011Yamaguchi Y. Shinotsuka N. Nonomura K. Takemoto K. Kuida K. Yosida H. Miura M. Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure.J. Cell Biol. 2011; 195: 1047-1060Crossref PubMed Scopus (132) Google Scholar). Drosophila melanogaster thorax fusion is a remarkable model for epithelial tissue fusion (Martin-Blanco and Knust, 2001Martin-Blanco E. Knust E. Epithelial morphogenesis: filopodia at work.Curr. Biol. 2001; 11: R28-R31Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar; Martin-Blanco et al., 2000Martin-Blanco E. Pastor-Pareja J.C. Garcia-Bellido A. JNK and decapentaplegic signaling control adhesiveness and cytoskeleton dynamics during thorax closure in Drosophila.Proc. Natl. Acad. Sci. U S A. 2000; 97: 7888-7893Crossref PubMed Scopus (98) Google Scholar). We have previously shown that sub-lethal caspase activation regulates the speed of thorax closure (Fujisawa et al., 2019Fujisawa Y. Kosakamoto H. Chihara T. Miura M. Non-apoptotic function of Drosophila caspase activation in epithelial thorax closure and wound healing.Development. 2019; 146: dev160937Crossref Scopus (10) Google Scholar). Following thorax fusion in the midline, a monolayered epithelium called the pupal notum is formed. Epithelial cells along the fusion site of the midline frequently undergo basal extrusion (delamination, Koto et al., 2011Koto A. Kuranaga E. Miura M. Apoptosis ensures spacing pattern formation of Drosophila sensory organs.Curr. Biol. 2011; 21: 278-287Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar; Figure 1A). The delamination rate escalates with increasing cell size via P110 overexpression and diminishes with decreasing cell size via Tsc1/2 overexpression (Marinari et al., 2012Marinari E. Mehonic A. Curran S. Gale J. Duke T. Baum B. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding.Nature. 2012; 484: 542-545Crossref PubMed Scopus (261) Google Scholar). This suggests that delamination is correlated with the local crowding status of the epithelium. Furthermore, upon laser wounding, the calculation of cell movement and direction via particle image velocimetry has demonstrated that delamination is caused by mechanical compaction of the midline cells (Levayer et al., 2016Levayer R. Dupont C. Moreno E. Tissue crowding induces caspase-dependent competition for space.Curr. Biol. 2016; 26: 670-677Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This delamination is therefore designated “crowding-induced cell delamination.” Only approximately 30% of delamination is reported to be caspase dependent (Marinari et al., 2012Marinari E. Mehonic A. Curran S. Gale J. Duke T. Baum B. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding.Nature. 2012; 484: 542-545Crossref PubMed Scopus (261) Google Scholar). However, this caspase-dependent fraction has been found to be an underestimation (Levayer et al., 2016Levayer R. Dupont C. Moreno E. Tissue crowding induces caspase-dependent competition for space.Curr. Biol. 2016; 26: 670-677Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Levayer et al. showed that major stress-sensitive pathways including p53, JNK, or Hippo Yap/Taz signaling are not involved in the delamination process (Levayer et al., 2016Levayer R. Dupont C. Moreno E. Tissue crowding induces caspase-dependent competition for space.Curr. Biol. 2016; 26: 670-677Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The same group demonstrated that cell delamination is coupled with ERK downregulation and pro-apoptotic gene Hid upregulation (Moreno et al., 2019Moreno E. Valon L. Levillayer F. Levayer R. Competition for space induces cell elimination through compaction-driven ERK downregulation.Curr. Biol. 2019; 29: 23-34 e8Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Although ERK inactivation is limited to approximately 60% of delaminating cells with caspase-3 activation, other pathways involved in cell delamination have not yet been identified. NADPH oxidases are involved in the generation of reactive oxygen species (ROS). These can attack a large number of biomolecules and are, therefore, associated with apoptotic cell death (Redza-Dutordoir and Averill-Bates, 2016Redza-Dutordoir M. Averill-Bates D.A. Activation of apoptosis signalling pathways by reactive oxygen species.Biochim. Biophys. Acta. 2016; 1863: 2977-2992Crossref PubMed Scopus (1091) Google Scholar; Wang et al., 2018Wang Y. Branicky R. Noe A. Hekimi S. Superoxide dismutases: dual roles in controlling ROS damage and regulating ROS signaling.J. Cell Biol. 2018; 217: 1915-1928Crossref PubMed Scopus (335) Google Scholar). For example, spatiotemporal ROS production by NADPH oxidase is critical for tapetal programmed cell death in Arabidopsis (Xie et al., 2014Xie H.T. Wan Z.Y. Li S. Zhang Y. Spatiotemporal production of reactive oxygen species by NADPH Oxidase is critical for tapetal programmed cell death and pollen development in Arabidopsis.Plant Cell. 2014; 26: 2007-2023Crossref PubMed Scopus (125) Google Scholar) and is essential for follicle cell rupture during Drosophila ovulation (Li et al., 2018Li W. Young J.F. Sun J.J. NADPH oxidase-generated reactive oxygen species in mature follicles are essential for Drosophila ovulation.Proc. Natl. Acad. Sci. U S A. 2018; 115: 7765-7770Crossref PubMed Scopus (14) Google Scholar). Most studies exploring the involvement of NADPH oxidase in caspase activation and apoptosis are based on in vitro or ex vivo experimental models, and regulation of cellular functions by NADPH oxidases have not been specifically examined in vivo at the single cell level. The NADPH oxidase Nox is involved in superoxide anion (O˙-2) production. In this study, we found, by genetic screening, that Nox regulates caspase-3 activation and delamination, independent of ERK downregulation. Furthermore, we showed that intracellular hydrogen peroxide (H2O2) enables cells to undergo delamination without apoptotic features downstream of caspase-3 activation. ROS generated by Nox (O˙-2) and intracellular H2O2 can thus differentially regulate cell delamination both upstream and downstream of caspase-3 activation. There are differing opinions regarding the requirement of caspase activation for crowding-induced cell delamination (Levayer et al., 2016Levayer R. Dupont C. Moreno E. Tissue crowding induces caspase-dependent competition for space.Curr. Biol. 2016; 26: 670-677Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar; Marinari et al., 2012Marinari E. Mehonic A. Curran S. Gale J. Duke T. Baum B. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding.Nature. 2012; 484: 542-545Crossref PubMed Scopus (261) Google Scholar). We therefore tested whether apoptotic signaling is required for delamination (Figure 1B). Overexpressing Diap1 was sufficient to suppress the delamination rate in the midline region, which has been previously defined (M region; Figures 1C and 1D) (Levayer et al., 2016Levayer R. Dupont C. Moreno E. Tissue crowding induces caspase-dependent competition for space.Curr. Biol. 2016; 26: 670-677Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In addition, the delamination rate was significantly reduced when Reaper, Hid, and Grim (RHG, three antagonists for an endogenous caspase inhibitor Diap1)-RNAi, Dark-RNAi, Dronc-RNAi, or p35 was expressed (Figure 1D). This confirmed that apoptotic signaling is necessary for delamination. Flies that overexpressed Diap1 showed a “broaden (Br) phenotype” on the adult notum (Figure 1E). This is consistent with a previous report, wherein the absence of caspase activation-induced delamination increased the final size of the M region (Levayer et al., 2016Levayer R. Dupont C. Moreno E. Tissue crowding induces caspase-dependent competition for space.Curr. Biol. 2016; 26: 670-677Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Using this phenotype as an indicator for the lack of cell delamination, we sought to identify the molecules involved in cell delamination. To this end, we conducted a genetic screen using Pnr-Gal4 as a control (Figure S1A). We crossed overexpression or inhibition lines based on genes selected from cellular functions such as cell death, mechano-sensing, cytoskeleton formation, and oxidative stress. As shown by “Br” in the first screening (Figures S1A and S1B), we found that some manipulations led to a Br phenotype. Among the first candidates, we found that the manipulation of apoptotic genes, ROS-related genes, Foxo-RNAi, and Atf3 led to a Br phenotype. None of the mechano-sensing genes were picked up in the first screening, suggesting differing mechanisms of cell delamination between Drosophila pupal notum and other models of live-cell delamination reported in vertebrates (Eisenhoffer et al., 2012Eisenhoffer G.T. Loftus P.D. Yoshigi M. Otsuna H. Chien C.B. Morcos P.A. Rosenblatt J. Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia.Nature. 2012; 484: 546-549Crossref PubMed Scopus (424) Google Scholar). To test whether these phenotypes could be attributed to the suppression of delamination, we performed live imaging on the pupal notum. We identified that overexpression of Atf3, Sod1, or Sod2 or knockdown of Foxo, Nox, or Duox suppressed delamination. In contrast, Catalase or Keap1 overexpression and Hayan-RNAi did not suppress delamination. Although further analyses are required for each gene manipulation, given that Br phenotype is an integrated phenotype of both cell delamination and cell proliferation, these manipulations could enhance cell proliferation (which was not assessed in the screening). It is also possible that enhancement of cell death in the M region affects the crowding status and balance of cell proliferation. Cell elimination could therefore lead to a shift to the Br phenotype. Catalase overexpression is examined in detail below. Among the selected genes, we focused on the suppressive effect of Nox or Duox knockdown. Both genes belong to the NADPH oxidase family and are the only two members present in Drosophila. NADPH oxidases have an NADPH-binding domain close to the C terminal, transfer an electron from NADPH to O2, and produce O˙-2. We confirmed the involvement of NADPH oxidases in delamination using four independent Nox-RNAi lines or three Duox-RNAi lines (Figures 2A, 2B, S1C). To investigate the contribution of NADPH oxidases to caspase-3 activation, we monitored the ECFP/Venus ratio of the FRET-based caspase activity indicator SCAT3 (Takemoto et al., 2003Takemoto K. Nagai T. Miyawaki A. Miura M. Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects.J. Cell Biol. 2003; 160: 235-243Crossref PubMed Scopus (247) Google Scholar) at 16 h after puparium formation (APF), continuing until 36 h APF (Figure 1A). This is the point at which cells begin to undergo delamination in the M region. We found that Nox-RNAi or Duox-RNAi inhibited caspase-3 activation, although these inhibitory effects were smaller than those of Dark-RNAi (Figures 2C and 2D ). These data suggest the involvement of NADPH oxidases in caspase-3 activation, which is required for delamination in the M region, in addition to the involvement of Hid modulation via ERK downregulation (Moreno et al., 2019Moreno E. Valon L. Levillayer F. Levayer R. Competition for space induces cell elimination through compaction-driven ERK downregulation.Curr. Biol. 2019; 29: 23-34 e8Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To validate the involvement of an NADPH oxidase in cell delamination, we first monitored Nox expression. This was done using a gene-trap Gal4 {CRIMIC}Nox-Gal4, in which the splicing acceptor, T2A, and the Gal4 sequence are inserted in-frame into the first intron of nox (Lee et al., 2018Lee P.T. Zirin J. Kanca O. Lin W.W. Schulze K.L. Li-Kroeger D. Tao R. Devereaux C. Hu Y.H. Chung V. et al.A gene-specific T2A-GAL4 library for Drosophila.Elife. 2018; 7: e35574Crossref PubMed Scopus (59) Google Scholar). As Gal4 is separated from the truncated (first exon) Nox protein after translation, this expression pattern is expected to reflect the endogenous transcription and translation patterns of the gene. Combining this construction with G-TRACE (Evans et al., 2009Evans C.J. Olson J.M. Ngo K.T. Kim E. Lee N.E. Kuoy E. Patananan A.N. Sitz D. Tran P. Do M.T. et al.G-TRACE: rapid Gal4-based cell lineage analysis in Drosophila.Nat. Methods. 2009; 6: 603-605Crossref PubMed Scopus (193) Google Scholar; Figure 3A), we monitored the spatiotemporal pattern of Nox expression from 13 h APF. This stage occurs approximately 3 h before the beginning of cell delamination (Figure 3B). Despite the time lag of approximately a few hours between Nox expression and Gal4/UAS-dependent labeling via fluorescence protein expression, cells marked with magenta (DsRed::nls) nuclei reflected “present” expression, whereas those with green (nuc::GFP) nuclei indicated cells that previously (potentially during larval stage) showed Nox expression (described as “past”; Figures 3A and 3B). Prior to the delamination stage (13–16 h APF; Figure 1A), GFP-expressing cells are rarely observed in the thorax fusion site of the M region. DsRed expression instead gradually became stronger in a subset of cells at specific locations, including the M region. Such DsRed+ cells with “present” Nox expression frequently underwent delamination in the M region (83.0%; Figure 3C, Video S1). These observations suggest that, in this region, Nox functions in a cell-autonomous manner during delamination. At the delamination stage (after 16 h APF), Nox expression in cells of the M region was confirmed in the pupal notum of flies carrying Nox::V5::TurboID, in which the C terminus of Nox is fused with a promiscuous biotin ligase (TurboID, Branon et al., 2018Branon T.C. Bosch J.A. Sanchez A.D. Udeshi N.D. Svinkina T. Carr S.A. Feldman J.L. Perrimon N. Ting A.Y. Efficient proximity labeling in living cells and organisms with TurboID.Nat. Biotechnol. 2018; 36: 880-887Crossref PubMed Scopus (280) Google Scholar; Figure S2A) that biotinylates neighboring proteins and can sensitively label TurboID-expressing cells (Figures S2B–S2C′). To examine whether Nox-Gal4 “present” cells undergo delamination through caspase activation, we injected the pan-caspase inhibitor Z-VAD-fmk into the pupal notum and monitored the delamination process (Figures S3A and S3B). Consequently, Z-VAD-fmk treatment did not block the appearance of Nox-Gal4 “present” cells but prevented delamination and increased the number of these cells in the M region at 28 h APF (Figure S3B). This further supports the idea that Nox functions upstream of caspase-3 activation to promote delamination. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwNTY2NTk1ZWEzYTcyNGRkNmJiNmFhMjYzNWU0ODZlYiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM5MzgzMzc4fQ.lVpkCISK5vOWgMRbjTZnmNuH0MlGjNNFxIajN5t4WBIPsggbR5x0xzkI5CmqcqLDHGhtpkI8qIpL78lBd6HxhHEuYjRTB3VuxsN-Ijr4d4jF77qSPz6jis8IFXf7mHGhlwppv9ygBnr0RX_z4ayMICy53d9rZG2nOlnh8eVeqpKrwb-DHuBBkix9RlyB0x4ip0Lr0ZRAdyiyTSUhfFTAngGGD3EMbmC2UZFSDADe_fKXry66-fF_Le2okvPt6n6B58I5NTjyKXfv8Zujx7UDmpEC7oXbbUJl8cHTr4psDuRIdbaFSL3nKlbwRDGXdefj9pyggC0G2kaceHS4AUiwPw Download .mp4 (0.61 MB) Help with .mp4 files Video S1. Cell Delamination of Nox Expressing Cells, Related to Figure 3DsRed::nls was monitored from 18 to 35.5 h APF after puparium formation (APF) using an EMCCD camera (ImagEM X2, Hamamatsu) with an Olympus MVX10 macroscope. A previous study showed that ERK downregulation occurs in delaminating cells (Moreno et al., 2019Moreno E. Valon L. Levillayer F. Levayer R. Competition for space induces cell elimination through compaction-driven ERK downregulation.Curr. Biol. 2019; 29: 23-34 e8Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). This is based on the fact that the mCherry signal of the nls::C1C2Cic::mCherry (miniCic) reporter, which is exported from the nucleus upon phosphorylation by ERK signaling, is observed in the nucleus during delamination. This reporter reveals that, among delaminating cells, ERK downregulation occurs in approximately 60% of the caspase-3-activated cells (Moreno et al., 2019Moreno E. Valon L. Levillayer F. Levayer R. Competition for space induces cell elimination through compaction-driven ERK downregulation.Curr. Biol. 2019; 29: 23-34 e8Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We therefore used this reporter to determine whether Nox regulates ERK downregulation. We combined heat shock-dependent flippase expression (Hs-flp) and Ay-Gal4 systems to conduct a mosaic analysis. We first expressed a constitutively active form of EGFR (EGFR-CA) with GFP. The mCherry signal was observed in a cytoplasmic or uniform manner in GFP+ cell populations with EGFR-CA expression but in a nuclear or uniform manner in those without gene manipulation (control; Figures 4A and 4B ). This confirmed the translocation of the miniCic reporter upon ERK upregulation. We detected almost no modulation of mCherry localization in GFP+ cells with Nox knockdown compared with that in the control (Figures 4A and 4B). Consistent with this result, we found that Nox-RNAi via Pnr-Gal4 did not induce mCherry cytoplasmic signals in cells around the midline (Figures 4C and 4D). These findings indicate that Nox is not responsible for ERK downregulation and that these parallel pathways regulate cell delamination in the M region of the pupal notum. NADPH oxidases involve ROS production. To examine the ROS levels in this tissue, we used the in vivo sensor for oxidative stress, GstD-GFP (Sykiotis and Bohmann, 2008Sykiotis G.P. Bohmann D. Keap1/Nrf2 signaling regulates oxidative stress tolerance and lifespan in Drosophila.Dev. Cell. 2008; 14: 76-85Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). The GFP signal pattern indicated a high level of ROS around the midline (Figure 5A). We thus tested the correlation between ROS generation and cell delamination. Although only approximately 40% of the cells positive for GstD-GFP underwent delamination (Figure 5B), almost all delaminating cells were labeled with GstD-GFP (Figure 5C). As in mammals, there are three superoxide dismutases (Sods) present in Drosophila. These include Sod1, Sod2, and Sod3, which are involved in the conversion of intracellular, mitochondrial, and extracellular O˙-2 to hydrogen peroxide (H2O2), respectively (Figure 5D). H2O2 oxidizes the sensor cysteine residues of Keap1, an E3-ligase of Nrf2. The oxidization of Keap1 inactivates E3-ligase activity and then Nrf2 is stabilized. Nrf2 translocates to nuclei, activates antioxidant response element, and induces the expression of genes such as GstD (Kansanen et al., 2013Kansanen E. Kuosmanen S.M. Leinonen H. Levonen A.L. The Keap1-Nrf2 pathway: mechanisms of activation and dysregulation in cancer.Redox Biol. 2013; 1: 45-49Crossref PubMed Scopus (704) Google Scholar; Suzuki et al., 2019Suzuki T. Muramatsu A. Saito R. Iso T. Shibata T. Kuwata K. Kawaguchi S.I. Iwawaki T. Adachi S. Suda H. et al.Molecular mechanism of cellular oxidative stress sensing by Keap1.Cell Rep. 2019; 28: 746-758 e744Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Thus, H2O2 is likely to be accumulated in GstD-GFP-positive delaminating cells. To examine O˙-2 production, we injected dihydroethidium (DHE) with Hoechst 33342 (Figure 5E). When normalized to that of Hoechst 33342, the DHE signal demonstrated a higher level of O˙-2 generation in cells around the midline (Figure 5F). Besides, given that overexpressing each Sod solely suppressed the delamination rate (Figures 5G and 5H) and caspase-3 activation (Figures 5I and 5J), O˙-2 reduction and an increase of H2O2 could suppress delamination. To further confirm the requirement of O˙-2 production on caspase activation, we normalized the SCAT3 ratio of the M region by that of the OM region for comparison. Similar to in Dark-RNAi, we found that the relative ratios of SCAT3 in Nox-RNAi, Sod1, or Sod3 were significantly increased in M region cells compared with that of the control (Figure 5K). These data suggest an involvement of O˙-2 in caspase-3 activation around the midline. H2O2 is known to act in redox signaling without executing cell death (Sies, 2017Sies H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress.Redox Biol. 2017; 11: 613-619Crossref PubMed Scopus (713) Google Scholar). Given that the expression of the O˙-2 scavenger Sod promotes H2O2 generation, we can assume that delamination consists of the positive involvement of O˙-2 and negative involvement of H2O2. Indeed, we found that the H2O2 scavenger Catalase increased delamination (Figures 6A and 6B ), suggesting the inhibitory impact of H2O2 on delamination. We therefore wished to determine the means by which H2O2 suppresses caspase-mediated cell delamination. SCAT3 monitoring revealed that Catalase expression did not affect caspase-3 activation (Figures S4A and S4B). Consistently, H2O2 and paraquat, which produce O˙-2 and H2O2, similarly triggered caspase-3 activation administered to cultured S2 Drosophila cells (Figures S5A and S5B). These data support the notion that both O˙-2 and H2O2 have the positive potential to drive caspase-3 activation. These results therefore suggest that O˙-2 reduction by Sods inhibits caspase-3 activation and H2O2 prevents delamination after caspase-3 activation. CaspaseTracker/CasExpress (thereafter, described as “CasT”) is a Gal4-based caspase-3 activation sensor (Ding et al., 2016Ding A.X. Sun G. Argaw Y.G. Wong J.O. Easwaran S. Montell D.J. CasExpress reveals widespread and diverse patterns of cell survival of caspase-3 activation during development in vivo.Elife. 2016; 5: e10936Crossref PubMed Scopus (52) Google Scholar; Tang et al., 2015Tang H.L. Tang H.M. Fung M.C. Hardwick J.M. In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity.Sci. Rep. 2015; 5: 9015Crossref PubMed Scopus (58) Google Scholar). Here, 56.4% of the CasT+ cells detected at 25 h APF did not undergo delamination, even after 6 h (Figures 6C–6E). We then investigated whether CasT-induced genetic manipulations alter the cell delamination rate by expressing p35, thus inhibiting the caspase activation required for delamination. As expected, p35 expression under the control of CasT effectively prevented delamination (Figure 6G). Notably, CasT-driven Catalase expression significantly increased the delamination rate (Figures 6F and 6G). These findings demonstrate the inhibitory effect of H2O2 on cell delamination after caspase-3 activation. Next, we found that less than 10% of the delaminating CasT+ cells showed nuclear fragmentation, a feature of typical apoptosis (Figures 6F and 6H, Video S2). In addition, the injection of a marker of late apoptosis, namely, propidium iodide (PI), into the pupa revealed that only 4.3% of the delaminating cells turned PI-positive (Figures 6I and 6J, Video S3). These results suggest that, although caspase-3 is activated in delaminating cells, this activation could be insufficient for executing apoptotic cell death. When Catalase was expressed in CasT+ cells, nuclear fragmentation was increased (Figures 6F and 6H), supporting the idea that H2O2 inhibits events occurring after initial caspase-3 activation (such as nuclear fragmentation). Therefore, cells undergo delamination without apparent apoptotic features. Taking this into consideration, the presence of both O˙-2 and intracellular H2O2 before and after caspase-3 activation leads to live cell delamination (Figure 7). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJkYjg3MzRhMTA4MTE1ZDFkYmU4YThmYjhjZTk4MDc4NCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM5MzgzMzc4fQ.sMucGdbyxipUfdqId78BVdgWQOXKfd0fikeknwyNMRYiZM1OBNi73Lq54MXl8V0xEwc8t65mSLou9GpxmQQWY-Tg6q3sfO3kTJaSH7_GR-XJEGD3vcGTOfB3qxvhgFsKYcCxXcjbkq_X9JP09euUhMhQn3gED0yAz6ylPk6REMHfrz_DoLEhexSKHqdQrrdVPp5QjfOZzA5RBNigEshlJT5wp4PdP2xc0zFAJyHzBU2vpjJTja8SvafDIh_uJAm3QF8BJMAGphBw9AYa8WZ97j5slPwySRYaCgM_I807Ghu48OhF5B1E4oYylYtpUyC1v_LxqseVwjuDe2q5gQWnxw Download .mp4 (9.14 MB) Help with .mp4 files Video S2. Cell Delamination of caspase-3 Activated Cells, Related to Figure 6GFP and mCherry signal were monitored after puparium formation (APF) eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJjOTA1YjE1Y2M5OTMyMTdjZTg2ZTU4YzM3MTUyMzJkZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjM5MzgzMzc4fQ.rQmgCAGcZ9D4knLI7sXs4dFWasY8sj-1UboSkpw2p4w4TZ5HtMpEfYuieukNJ90zIk-JPSbuQbI6HjsSMaV8h1ORwQ4kDAH30r9sqwEMe3Jz-XBpC2tYYiNKkMvr0nokWp4iKnCQQVht68RPz2qbrvN8y6QZDdncNEpsKDdjQIzYqEyZnVCiOCJhdQQFVhgSr1GD7JgBqCW1pZOCIgYsDASqTJSoBSpX6z34KZ-GY7Obbyowk3NbzXJIQF3fpVGR6ETIDdaJUw-MLavOOev2KHP6ugYzO6v8Y2ITQ_LvcshIjMw8geLs7h6IOYXWp1IuR13225mF6UUOTNOXGF4t4A" @default.
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• W3045069165 date "2020-08-01" @default.
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• W3045069165 title "ROS Regulate Caspase-Dependent Cell Delamination without Apoptosis in the Drosophila Pupal Notum" @default.
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• W3045069165 doi "https://doi.org/10.1016/j.isci.2020.101413" @default.
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