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• W3159084180 abstract "•Toll receptors generate planar-polarized patterns of Src and PI3K activity•Src and PI3K are required for localized myosin contractility and convergent extension•Src kinases promote Toll-2 phosphorylation and Toll-2/PI3K-reg complex formation•Toll-2 phosphorylation is necessary for planar polarity and convergent extension Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. We show that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity. Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. We show that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity. Cell-surface receptors convert extracellular signals into changes in cell behavior that are essential for the formation, remodeling, and repair of multicellular tissues. Toll-like receptors (TLRs) are a conserved family of receptors with roles in dorsal-ventral patterning (Morisato and Anderson, 1995Morisato D. Anderson K.V. Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo.Annu. Rev. Genet. 1995; 29: 371-399Crossref PubMed Google Scholar) and cell-pathogen recognition in the innate immune system (Anderson, 2000Anderson K.V. Toll signaling pathways in the innate immune response.Curr. Opin. Immunol. 2000; 12: 13-19Crossref PubMed Scopus (0) Google Scholar; Leulier and Lemaitre, 2008Leulier F. Lemaitre B. Toll-like receptors--taking an evolutionary approach.Nat. Rev. Genet. 2008; 9: 165-178Crossref PubMed Scopus (346) Google Scholar). More recently, members of the Toll-receptor family have also been shown to regulate cell behavior during the development of epithelial tissues and the nervous system (Anthoney et al., 2018Anthoney N. Foldi I. Hidalgo A. Toll and toll-like receptor signalling in development.Development. 2018; 145: dev156018Crossref PubMed Scopus (39) Google Scholar; Paré and Zallen, 2020Paré A.C. Zallen J.A. Cellular, molecular, and biophysical control of epithelial cell intercalation.Curr. Top. Dev. Biol. 2020; 136: 167-193Crossref PubMed Scopus (6) Google Scholar). Toll receptors organize cell movement (Paré et al., 2014Paré A.C. Vichas A. Fincher C.T. Mirman Z. Farrell D.L. Mainieri A. Zallen J.A. A positional Toll receptor code directs convergent extension in Drosophila.Nature. 2014; 515: 523-527Crossref PubMed Google Scholar; Benton et al., 2016Benton M.A. Pechmann M. Frey N. Stappert D. Conrads K.H. Chen Y.T. Stamataki E. Pavlopoulos A. Roth S. Toll genes have an ancestral role in axis elongation.Curr. Biol. 2016; 26: 1609-1615Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar; Sun et al., 2017Sun Z. Amourda C. Shagirov M. Hara Y. Saunders T.E. Toyama Y. Basolateral protrusion and apical contraction cooperatively drive Drosophila germ-band extension.Nat. Cell Biol. 2017; 19: 375-383Crossref PubMed Scopus (55) Google Scholar), direct nervous system wiring (McIlroy et al., 2013McIlroy G. Foldi I. Aurikko J. Wentzell J.S. Lim M.A. Fenton J.C. Gay N.J. Hidalgo A. Toll-6 and Toll-7 function as neurotrophin receptors in the Drosophila melanogaster CNS.Nat. Neurosci. 2013; 16: 1248-1256Crossref PubMed Scopus (55) Google Scholar; Ballard et al., 2014Ballard S.L. Miller D.L. Ganetzky B. Retrograde neurotrophin signaling through Tollo regulates synaptic growth in Drosophila.J. Cell Biol. 2014; 204: 1157-1172Crossref PubMed Scopus (30) Google Scholar; Ward et al., 2015Ward A. Hong W. Favaloro V. Luo L. Toll receptors instruct axon and dendrite targeting and participate in synaptic partner matching in a Drosophila olfactory circuit.Neuron. 2015; 85: 1013-1028Abstract Full Text Full Text PDF PubMed Google Scholar; Foldi et al., 2017Foldi I. Anthoney N. Harrison N. Gangloff M. Verstak B. Nallasivan M.P. AlAhmed S. Zhu B. Phizacklea M. Losada-Perez M. et al.Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila.J. Cell Biol. 2017; 216: 1421-1438Crossref PubMed Scopus (17) Google Scholar), repair wounds (Carvalho et al., 2014Carvalho L. Jacinto A. Matova N. The Toll/NF-κB signaling pathway is required for epidermal wound repair in Drosophila.Proc. Natl. Acad. Sci. USA. 2014; 111: E5373-E5382Crossref PubMed Scopus (30) Google Scholar; Capilla et al., 2017Capilla A. Karachentsev D. Patterson R.A. Hermann A. Juarez M.T. McGinnis W. Toll pathway is required for wound-induced expression of barrier repair genes in the Drosophila epidermis.Proc. Natl. Acad. Sci. USA. 2017; 114: E2682-E2688Crossref PubMed Scopus (6) Google Scholar), and eliminate less fit cells through cell competition (Meyer et al., 2014Meyer S.N. Amoyel M. Bergantiños C. de la Cova C. Schertel C. Basler K. Johnston L.A. An ancient defense system eliminates unfit cells from developing tissues during cell competition.Science. 2014; 346: 1258236Crossref PubMed Scopus (108) Google Scholar), defining new functions for this receptor family. In the immune system, Toll-like receptor (TLR) signaling activates the expression of immune-response genes by promoting the nuclear translocation of transcriptional regulators in the NF-κB, AP-1, and IRF families (Kawai and Akira, 2011Kawai T. Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.Immunity. 2011; 34: 637-650Abstract Full Text Full Text PDF PubMed Scopus (2203) Google Scholar; Fitzgerald and Kagan, 2020Fitzgerald K.A. Kagan J.C. Toll-like receptors and the control of immunity.Cell. 2020; 180: 1044-1066Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). However, how Toll receptors elicit fast-acting and localized responses in cells to generate spatially regulated changes in cell behavior is not understood. Tyrosine phosphorylation is a rapid and reversible mechanism that transduces extracellular information into molecular changes within cells. Cell-surface receptors such as growth-factor receptors initiate signaling cascades through their intrinsic tyrosine-kinase activity, which is activated by ligand binding and receptor dimerization (Lemmon and Schlessinger, 2010Lemmon M.A. Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2010; 141: 1117-1134Abstract Full Text Full Text PDF PubMed Scopus (2553) Google Scholar; Kovacs et al., 2015Kovacs E. Zorn J.A. Huang Y. Barros T. Kuriyan J. A structural perspective on the regulation of the epidermal growth factor receptor.Annu. Rev. Biochem. 2015; 84: 739-764Crossref PubMed Scopus (149) Google Scholar). In addition, receptors that lack kinase activity can also participate in tyrosine-kinase signaling by recruiting nonreceptor tyrosine kinases. Src-family nonreceptor tyrosine kinases are essential for signaling by growth-factor receptors, T and B cell receptors, integrins, and cadherins (Thomas and Brugge, 1997Thomas S.M. Brugge J.S. Cellular functions regulated by Src family kinases.Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Crossref PubMed Scopus (2087) Google Scholar), and have diverse roles in cell-cell adhesion (Takahashi et al., 2005Takahashi M. Takahashi F. Ui-Tei K. Kojima T. Saigo K. Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila.Development. 2005; 132: 2547-2559Crossref PubMed Scopus (61) Google Scholar; McLachlan et al., 2007McLachlan R.W. Kraemer A. Helwani F.M. Kovacs E.M. Yap A.S. E-cadherin adhesion activates c-Src signaling at cell-cell contacts.Mol. Biol. Cell. 2007; 18: 3214-3223Crossref PubMed Scopus (115) Google Scholar; Shindo et al., 2008Shindo M. Wada H. Kaido M. Tateno M. Aigaki T. Tsuda L. Hayashi S. Dual function of Src in the maintenance of adherens junctions during tracheal epithelial morphogenesis.Development. 2008; 135: 1355-1364Crossref PubMed Scopus (63) Google Scholar; Förster and Luschnig, 2012Förster D. Luschnig S. Src42A-dependent polarized cell shape changes mediate epithelial tube elongation in Drosophila.Nat. Cell Biol. 2012; 14: 526-534Crossref PubMed Scopus (68) Google Scholar; Hunter et al., 2018Hunter M.V. Willoughby P.M. Bruce A.E.E. Fernandez-Gonzalez R. Oxidative stress orchestrates cell polarity to promote embryonic wound healing.Dev. Cell. 2018; 47: 377-387.e4Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), cell-matrix adhesion (Huveneers and Danen, 2009Huveneers S. Danen E.H. Adhesion signaling–crosstalk between integrins, Src, and Rho.J. Cell Sci. 2009; 122: 1059-1069Crossref PubMed Scopus (0) Google Scholar), and cytoskeletal organization (Thomas and Wieschaus, 2004Thomas J.H. Wieschaus E. src64 and tec29 are required for microfilament contraction during Drosophila cellularization.Development. 2004; 131: 863-871Crossref PubMed Scopus (0) Google Scholar; Andreeva et al., 2014Andreeva A. Lee J. Lohia M. Wu X. Macara I.G. Lu X. PTK7-Src signaling at epithelial cell contacts mediates spatial organization of actomyosin and planar cell polarity.Dev. Cell. 2014; 29: 20-33Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar; Sun et al., 2017Sun Z. Amourda C. Shagirov M. Hara Y. Saunders T.E. Toyama Y. Basolateral protrusion and apical contraction cooperatively drive Drosophila germ-band extension.Nat. Cell Biol. 2017; 19: 375-383Crossref PubMed Scopus (55) Google Scholar; Hunter et al., 2018Hunter M.V. Willoughby P.M. Bruce A.E.E. Fernandez-Gonzalez R. Oxidative stress orchestrates cell polarity to promote embryonic wound healing.Dev. Cell. 2018; 47: 377-387.e4Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). A critical event in receptor-initiated tyrosine kinase signaling is often phosphorylation of the receptor itself, which generates binding sites that recruit downstream effector proteins (Pawson, 2004Pawson T. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems.Cell. 2004; 116: 191-203Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar; Lemmon and Schlessinger, 2010Lemmon M.A. Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2010; 141: 1117-1134Abstract Full Text Full Text PDF PubMed Scopus (2553) Google Scholar). Although canonical TLR signaling involves a serine/threonine kinase cascade triggered by the recruitment of TLR-associated adaptor proteins, tyrosine kinases in the Src family have also been shown to facilitate the response to TLR signaling in cultured cells (Abram and Lowell, 2008Abram C.L. Lowell C.A. The diverse functions of Src family kinases in macrophages.Front. Biosci. 2008; 13: 4426-4450Crossref PubMed Scopus (0) Google Scholar; Page et al., 2009Page T.H. Smolinska M. Gillespie J. Urbaniak A.M. Foxwell B.M.J. Tyrosine kinases and inflammatory signalling.Curr. Mol. Med. 2009; 9: 69-85Crossref PubMed Scopus (65) Google Scholar; Chattopadhyay and Sen, 2014Chattopadhyay S. Sen G.C. Tyrosine phosphorylation in toll-like receptor signaling.Cytokine Growth Factor Rev. 2014; 25: 533-541Crossref PubMed Google Scholar). However, the effects of Src-family kinases on Toll-receptor signaling in vivo, and whether tyrosine-kinase pathways mediate the rapid and localized effects of Toll receptors on cell behavior, are not known. Convergent extension is a conserved morphogenetic process that shapes developing organs and elongates the body axis of multicellular organisms (Huebner and Wallingford, 2018Huebner R.J. Wallingford J.B. Coming to consensus: a unifying model emerges for convergent extension.Dev. Cell. 2018; 46: 389-396Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In Drosophila, three Toll-family receptors expressed in distinct striped patterns provide critical spatial cues that orient cell rearrangements and elongate the embryo along the head-to-tail axis (Paré et al., 2014Paré A.C. Vichas A. Fincher C.T. Mirman Z. Farrell D.L. Mainieri A. Zallen J.A. A positional Toll receptor code directs convergent extension in Drosophila.Nature. 2014; 515: 523-527Crossref PubMed Google Scholar). Toll receptors guide cell movements by directing the planar-polarized localization of proteins involved in actomyosin contractility and cell adhesion (Paré et al., 2014Paré A.C. Vichas A. Fincher C.T. Mirman Z. Farrell D.L. Mainieri A. Zallen J.A. A positional Toll receptor code directs convergent extension in Drosophila.Nature. 2014; 515: 523-527Crossref PubMed Google Scholar; Paré and Zallen, 2020Paré A.C. Zallen J.A. Cellular, molecular, and biophysical control of epithelial cell intercalation.Curr. Top. Dev. Biol. 2020; 136: 167-193Crossref PubMed Scopus (6) Google Scholar). However, the signaling pathways that generate spatially regulated changes in the organization and activity of the force-generating machinery downstream of Toll receptors are unknown. Here, we show that a Src-mediated tyrosine-kinase pathway is essential for planar polarity and Toll-receptor signaling during convergent extension in Drosophila. Toll receptors generate a planar-polarized pattern of Src kinase activity, and Src in turn phosphorylates the Toll-2 C-terminal domain, promoting the association of Toll-2 with PI3-kinase (PI3K). Drosophila Src-family kinases, the regulatory and catalytic subunits of the PI3K complex, and tyrosine phosphorylation of the Toll-2 cytoplasmic domain are all necessary for planar polarity, cell intercalation, and convergent extension. These results identify a localized signaling mechanism by which Toll receptors induce rapid changes in cell polarity and behavior during development. To understand how Toll receptors direct cell rearrangements during convergent extension, we sought to identify proteins that are required for receptor activity. The immediate effectors of Toll-receptor signaling are predicted to physically associate with Toll receptors, act in a spatially localized fashion within cells, and regulate cell polarity and behavior during convergent extension. The three Toll-family receptors required for convergent extension, Toll-2, Toll-6, and Toll-8, do not possess intrinsic tyrosine-kinase activity, but we hypothesized that they could interact with nonreceptor tyrosine kinases to transmit polarity signals within cells. The two Drosophila Src-family kinases, Src42 and Src64, are both expressed and active during convergent extension (Takahashi et al., 2005Takahashi M. Takahashi F. Ui-Tei K. Kojima T. Saigo K. Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila.Development. 2005; 132: 2547-2559Crossref PubMed Scopus (61) Google Scholar; Sopko et al., 2014Sopko R. Foos M. Vinayagam A. Zhai B. Binari R. Hu Y. Randklev S. Perkins L.A. Gygi S.P. Perrimon N. Combining genetic perturbations and proteomics to examine kinase-phosphatase networks in Drosophila embryos.Dev. Cell. 2014; 31: 114-127Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Src42 and Src64 are uniformly distributed throughout the plasma membrane of embryonic epithelial cells (Figures 1A and S1A–S1C). By contrast, a phosphospecific antibody that binds to the active form of Src42 (Shindo et al., 2008Shindo M. Wada H. Kaido M. Tateno M. Aigaki T. Tsuda L. Hayashi S. Dual function of Src in the maintenance of adherens junctions during tracheal epithelial morphogenesis.Development. 2008; 135: 1355-1364Crossref PubMed Scopus (63) Google Scholar) detected a planar-polarized enrichment of active Src42 kinase at adherens junctions between anterior and posterior cells (Figures 1A and S1D; see Table S1 for full statistical information). To determine whether this pattern of Src42 activity requires Toll-receptor signaling, we analyzed the localization of active Src42 in embryos that lack all three Toll receptors. The planar-polarized pattern of active Src42 was severely disrupted in Toll-2,6,8 triple mutants (Figures 1B, 1C, and S1E), whereas total Src42 localization was unaffected (Figures S1J and S1K). Defects in active Src42 localization were detected in Toll-2 single mutants and Toll-6,8 double mutants, indicating that multiple Toll receptors contribute to this pattern (Figure S1F). Active Src42 localization was defective in a subset of cells in Toll-2 mutants (Figures S1G–S1I), consistent with the region-specific expression and function of Drosophila Toll receptors (Paré et al., 2014Paré A.C. Vichas A. Fincher C.T. Mirman Z. Farrell D.L. Mainieri A. Zallen J.A. A positional Toll receptor code directs convergent extension in Drosophila.Nature. 2014; 515: 523-527Crossref PubMed Google Scholar). These results demonstrate that Toll receptors generate a planar-polarized pattern of Src42 activity during convergent extension. An early event in receptor-activated tyrosine-kinase-signaling pathways is often phosphorylation of the receptor itself (Pawson, 2004Pawson T. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems.Cell. 2004; 116: 191-203Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar; Lemmon and Schlessinger, 2010Lemmon M.A. Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2010; 141: 1117-1134Abstract Full Text Full Text PDF PubMed Scopus (2553) Google Scholar). We therefore tested whether Src kinases promote Toll-receptor phosphorylation. We found that Src42 and Src64 significantly enhanced the tyrosine phosphorylation of Toll-2 and Toll-6, but not Toll-8, in Drosophila S2R+ cells (Figures 1D, 1F, S2A, and S2B). Toll-2 and Toll-6 also co-immunoprecipitated with both kinases in vitro. To identify the residues that are targeted by Src kinases, we mutated different sets of tyrosines in Toll-2. The Toll-2 cytoplasmic domain contains a conserved Toll/interleukin-1 receptor (TIR) domain and a unique C-terminal extension that is present in a subset of Drosophila Toll receptors (Figure 1E) (Leulier and Lemaitre, 2008Leulier F. Lemaitre B. Toll-like receptors--taking an evolutionary approach.Nat. Rev. Genet. 2008; 9: 165-178Crossref PubMed Scopus (346) Google Scholar). Mutating all 5 tyrosines in the Toll-2 TIR domain to unphosphorylatable phenylalanine residues (Toll-2TIR-YF) had no effect on Toll-2 tyrosine phosphorylation when coexpressed with Src42 or Src64 in S2R+ cells (Figures 1F and S2B). By contrast, mutating the 7 tyrosines in the Toll-2 C-terminal domain, which are present in two clusters, significantly reduced Toll-2 tyrosine phosphorylation and disrupted its interaction with both kinases (Figures 1E, 1F, S2B, and S2E). The more C-terminal tyrosine cluster (C2) was particularly important for the association between Toll-2 and Src42, whereas tyrosine cluster C1 was dispensable for this interaction (Figure 1F). These results demonstrate that Src-family kinases promote the phosphorylation of a subset of Drosophila Toll receptors in vitro. To examine whether Toll-2 is tyrosine phosphorylated in vivo, we used CRISPR approaches to generate embryos that express C-terminally tagged Toll-2 proteins from the Toll-2 locus (Zhang et al., 2014Zhang X. Koolhaas W.H. Schnorrer F. A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila.G3 (Bethesda). 2014; 4: 2409-2418Crossref PubMed Scopus (58) Google Scholar; Paré et al., 2019Paré A.C. Naik P. Shi J. Mirman Z. Palmquist K.H. Zallen J.A. An LRR receptor-teneurin system directs planar polarity at compartment boundaries.Dev. Cell. 2019; 51: 208-221.e6Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Toll-2–HA or Toll-2–V5 expressed from the endogenous locus were present in the expected striped pattern (Figures S2F and S2G). Both proteins were tyrosine phosphorylated in embryos during convergent extension, detected with a pan-phosphotyrosine antibody and with phosphospecific antibodies to specific tyrosines in the Toll-2 C-terminal domain (Figures 1G, S2C, and S2D). By contrast, Toll-2 tyrosine phosphorylation was significantly reduced in embryos that lack either C-terminal tyrosine cluster of Toll-2 or that express a catalytically inactive Src42 variant, Src42K276R, which is predicted to act as a dominant negative for both Src kinases (Src DN) (Figures 1G–1I). These results demonstrate that Toll-2 tyrosine phosphorylation requires Src-family kinase activity in vivo. The findings that Toll receptors generate planar-polarized Src activity, and that Src-family kinases promote Toll-receptor phosphorylation, raised the possibility that Toll receptors could direct planar polarity through a Src-dependent signaling pathway. To investigate this possibility, we analyzed whether Src kinases are required for cell polarity and behavior during convergent extension. As Src42 and Src64 both enhance Toll-2 and Toll-6 tyrosine phosphorylation in vitro, and often function redundantly in vivo (Takahashi et al., 2005Takahashi M. Takahashi F. Ui-Tei K. Kojima T. Saigo K. Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila.Development. 2005; 132: 2547-2559Crossref PubMed Scopus (61) Google Scholar; Shindo et al., 2008Shindo M. Wada H. Kaido M. Tateno M. Aigaki T. Tsuda L. Hayashi S. Dual function of Src in the maintenance of adherens junctions during tracheal epithelial morphogenesis.Development. 2008; 135: 1355-1364Crossref PubMed Scopus (63) Google Scholar), we wanted to examine the effects of disrupting both kinases simultaneously. However, disrupting the expression of both Src kinases in the embryo is technically challenging, as maternal Src42 expression cannot be eliminated using standard genetic methods, due to the proximity of Src42 to the centromere (Chou and Perrimon, 1996Chou T.B. Perrimon N. The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster.Genetics. 1996; 144: 1673-1679Crossref PubMed Google Scholar). To circumvent this limitation, we generated embryos that were defective for both proteins using CRISPR/Cas9 to target the Src42 locus in the female germline (Port et al., 2014Port F. Chen H.M. Lee T. Bullock S.L. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.Proc. Natl. Acad. Sci. USA. 2014; 111: E2967-E2976Crossref PubMed Scopus (435) Google Scholar) and a maternally expressed shRNA to deplete maternal Src64 transcripts (Ni et al., 2011Ni J.Q. Zhou R. Czech B. Liu L.P. Holderbaum L. Yang-Zhou D. Shim H.S. Tao R. Handler D. Karpowicz P. et al.A genome-scale shRNA resource for transgenic RNAi in Drosophila.Nat. Methods. 2011; 8: 405-407Crossref PubMed Scopus (476) Google Scholar). Embryos with a >85% reduction in Src42 and Src64 levels by western blot or in cortical Src42 localization by immunofluorescence (Figures S3A–S3D) were selected for further analysis and are referred to as Src KD embryos. Toll receptors direct convergent extension by activating myosin contractility at interfaces between anterior and posterior cells (vertical edges) and targeting the adherens junction regulator Par-3 to complementary interfaces between dorsal and ventral cells (horizontal edges) (Bertet et al., 2004Bertet C. Sulak L. Lecuit T. Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation.Nature. 2004; 429: 667-671Crossref PubMed Scopus (625) Google Scholar; Zallen and Wieschaus, 2004Zallen J.A. Wieschaus E. Patterned gene expression directs bipolar planar polarity in Drosophila.Dev. Cell. 2004; 6: 343-355Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar; Blankenship et al., 2006Blankenship J.T. Backovic S.T. Sanny J.S. Weitz O. Zallen J.A. Multicellular rosette formation links planar cell polarity to tissue morphogenesis.Dev. Cell. 2006; 11: 459-470Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar; Rauzi et al., 2008Rauzi M. Verant P. Lecuit T. Lenne P.F. Nature and anisotropy of cortical forces orienting Drosophila tissue morphogenesis.Nat. Cell Biol. 2008; 10: 1401-1410Crossref PubMed Scopus (391) Google Scholar; Fernandez-Gonzalez et al., 2009Fernandez-Gonzalez R. Simoes Sde M. Röper J.C. Eaton S. Zallen J.A. Myosin II dynamics are regulated by tension in intercalating cells.Dev. Cell. 2009; 17: 736-743Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar; Simões et al., 2010Simões S. de M. Blankenship J.T. Weitz O. Farrell D.L. Tamada M. Fernandez-Gonzalez R. Zallen J.A. Rho-kinase directs Bazooka/Par-3 planar polarity during Drosophila axis elongation.Dev. Cell. 2010; 19: 377-388Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Consistent with a role for Src kinases in Toll-receptor signaling, reducing the expression of both kinases severely disrupted planar polarity. In contrast with wild-type embryos, myosin II cortical localization was significantly reduced and Par-3 localization expanded to occupy interfaces at all orientations in Src KD embryos (Figures 2A and 2B ). Similar defects were observed in embryos that express dominant negative Src42 (Figures S3E–S3G). By contrast, loss of either Src42 or Src64 alone did not significantly affect planar polarity (Figures S3J–S3O). Expression of constitutively active Src42Y511F (Src42 CA) (Tateno et al., 2000Tateno M. Nishida Y. Adachi-Yamada T. Regulation of JNK by Src during Drosophila development.Science. 2000; 287: 324-327Crossref PubMed Scopus (110) Google Scholar) had the opposite effect, producing ectopic myosin II localization at the cortex and reducing the level of cortical Par-3, disrupting the planar-polarized distributions of both proteins (Figures 2C and 2D). Despite these defects, the localization of adherens junction proteins to the apical junctional domain occurred normally in embryos with increased or decreased Src activity, indicating that apical-basal polarity was not affected (Figures S3H and S3I). These results demonstrate that Src activity is necessary and sufficient for the localized assembly of myosin at the cell cortex, consistent with a model in which Src plays an instructive role in directing planar polarity. The critical outputs of Toll-receptor signaling in the Drosophila embryo are planar-polarized cell rearrangements that lead to body axis elongation (Irvine and Wieschaus, 1994Irvine K.D. Wieschaus E. Cell intercalation during Drosophila germband extension and its regulation by pair-rule segmentation genes.Development. 1994; 120: 827-841Crossref PubMed Google Scholar; Paré et al., 2014Paré A.C. Vichas A. Fincher C.T. Mirman Z. Farrell D.L. Mainieri A. Zallen J.A. A positional Toll receptor code directs convergent extension in Drosophila.Nature. 2014; 515: 523-527Crossref PubMed Google Scholar). To determine whether Src activity is necessary for these cell- and tissue-level behaviors, we generated time-lapse movies of Src KD embryos that express E-cadherin tagged with the green fluorescent protein (GFP) to track cell behavior. Convergent extension is driven by cell intercalation, in which vertical edges contract to produce 4-cell vertices (Bertet et al., 2004Bertet C. Sulak L. Lecuit T. Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation.Nature. 2004; 429: 667-671Crossref PubMed Scopus (625) Google Scholar) and rosettes where 5 or more cells meet (Blankenship et al., 2006Blankenship J.T. Backovic S.T. Sanny J.S. Weitz O. Zallen J.A. Multicellular rosette formation links planar cell polarity to tissue morphogenesis.Dev. Cell. 2006; 11: 459-470Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). These structures resolve in a perpendicular direction, driving tissue elongation (Figure 2E). Src KD embryos displayed a significant disruption of convergent extension, elongating to less than half the extent of wild type (Figures 2F and 2G; Videos S1 and S2). This defect required the loss of both kinases, as the double mutant was more severe than loss of either kinase alone (Figures S3P and S3Q). Src KD embryos also displayed defects in cell intercalation, including slowed edge contraction (Figure 2H) and an increase in the percentage of cells that failed to initiate rearrangement (no contraction), 4-cell vertices or rosettes that failed to resolve (no resolution), and new edges tha" @default.
• W3159084180 created "2021-05-10" @default.
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• W3159084180 date "2021-06-01" @default.
• W3159084180 modified "2023-10-10" @default.
• W3159084180 title "Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity" @default.
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