Abstract
In the early stages of Drosophila melanogaster (Drosophila) metamorphosis, a partial epithelial-mesenchymal transition (pEMT) takes place in the peripodial epithelium of wing imaginal discs. Blocking this pEMT results in adults with internalized wings and missing thoracic tissue. Using peripodial GAL4 drivers, GAL80ts temporal control, and UAS RNAi transgenes, one can use these phenotypes to screen for genes involved in the pEMT. Dominant modifier tests can then be employed to identify genetic enhancers and suppressors. To analyze a gene’s role in the pEMT, one can then visualize peripodial cells in vivo at the time of eversion within the pupal case using live markers, and by dissecting, fixing, and immunostaining the prepupae. Alternatively, one can analyze the pEMT ex vivo by dissecting out wing discs and culturing them in the presence of ecdysone to induce eversion. This can provide a clearer view of the cellular processes involved and permit drug treatments to be easily applied.
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St Johnston D (2002) The art and design of genetic screens: Drosophila melanogaster. Nat Rev Genet 3:176–188. https://doi.org/10.1038/nrg751
Kaya-Copur A, Schnorrer F (2016) A guide to genome-wide in vivo RNAi applications in Drosophila. In: Drosophila: methods and protocols, Methods in molecular biology, vol 1478. Springer, New York, pp 117–143. https://doi.org/10.1007/978-1-4939-6371-3_6
Thiery JP, Acloque H, Huang RYJ, Nieto MA (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139:871–890. https://doi.org/10.1016/j.cell.2009.11.007
Leptin M, Grunewald B (1990) Cell shape changes during gastrulation in Drosophila. Development 110:73–84
Tepass U, Hartenstein V (1994) Epithelium formation in the Drosophila midgut depends on the interaction of endoderm and mesoderm. Development 120:579–590
Campbell K, Whissell G, Franch-Marro X et al (2011) Specific GATA factors act as conserved inducers of an endodermal-EMT. Dev Cell 21:1051–1061. https://doi.org/10.1016/j.devcel.2011.10.005
Gryzik T, Müller H-AJ (2004) FGF8-like1 and FGF8-like2 encode putative ligands of the FGF receptor Htl and are required for mesoderm migration in the Drosophila gastrula. Curr Biol 14:659–667. https://doi.org/10.1016/j.cub.2004.03.058
Crawford JM, Harden N, Leung T et al (1998) Cellularization in Drosophila melanogaster is disrupted by the inhibition of rho activity and the activation of Cdc42 function. Dev Biol 204:151–164. https://doi.org/10.1006/dbio.1998.9061
Barrett K, Leptin M, Settleman J (1997) The Rho GTPase and a putative RhoGEF mediate a signaling pathway for the cell shape changes in Drosophila gastrulation. Cell 91:905–915
Magie CR, Meyer MR, Gorsuch MS, Parkhurst SM (1999) Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development. Development 126:5353–5364
Ni J-Q, Zhou R, Czech B et al (2011) A genome-scale shRNA resource for transgenic RNAi in Drosophila. Nat Methods 8:405–407. https://doi.org/10.1038/nmeth.1592
Fristrom D, Fristrom JW (1993) The metamorphic development of the adult epidermis. In: Bate M, Alfonso Martinez Arias A (eds) The development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Plainview, pp 843–897
Pastor-Pareja JC, Grawe F, Martín-Blanco E, García-Bellido A (2004) Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade. Dev Cell 7:387–399. https://doi.org/10.1016/j.devcel.2004.07.022
Aldaz S, Escudero LM, Freeman M (2010) Live imaging of Drosophila imaginal disc development. Proc Natl Acad Sci U S A 107:14217–14222. https://doi.org/10.1073/pnas.1008623107
Aldaz S, Escudero LM, Freeman M (2013) Dual role of myosin II during Drosophila imaginal disc metamorphosis. Nat Commun 4:1761–1710. https://doi.org/10.1038/ncomms2763
Proag A, Monier B, Suzanne M (2019) Physical and functional cell-matrix uncoupling in a developing tissue under tension. Development 146:dev172577–9. https://doi.org/10.1242/dev.172577
Murray MJ (2015) Drosophila models of metastasis. AIMS Genet 2(1):25–53. https://doi.org/10.3934/genet.2015.1.25
Ishimaru S, Ueda R, Hinohara Y et al (2004) PVR plays a critical role via JNK activation in thorax closure during Drosophila metamorphosis. EMBO J 23:3984–3994. https://doi.org/10.1038/sj.emboj.7600417
Srivastava A, Pastor-Pareja JC, Igaki T et al (2007) Basement membrane remodeling is essential for Drosophila disc eversion and tumor invasion. Proc Natl Acad Sci U S A 104:2721–2726. https://doi.org/10.1073/pnas.0611666104
Martín-Blanco E, Pastor-Pareja JC, Garcia-Bellido A (2000) JNK and decapentaplegic signaling control adhesiveness and cytoskeleton dynamics during thorax closure in Drosophila. Proc Natl Acad Sci U S A 97:7888–7893
Manhire-Heath R, Golenkina S, Saint R, Murray MJ (2013) Netrin-dependent downregulation of Frazzled/DCC is required for the dissociation of the peripodial epithelium in Drosophila. Nat Commun 4:1–10. https://doi.org/10.1038/ncomms3790
Tripura C, Chandrika N-P, Susmitha V-N et al (2011) Regulation and activity of JNK signaling in the wing disc peripodial membrane during adult morphogenesis in Drosophila. Int J Dev Biol 55:583–590. https://doi.org/10.1387/ijdb.103275ct
Dietzl G, Chen D, Schnorrer F et al (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448:151–156. https://doi.org/10.1038/nature05954
Gibson MC, Lehman DA, Schubiger G (2002) Lumenal transmission of decapentaplegic in Drosophila imaginal discs. Dev Cell 3:451–460
Pallavi SK, Shashidhara LS (2003) Egfr/Ras pathway mediates interactions between peripodial and disc proper cells in Drosophila wing discs. Development 130:4931–4941. https://doi.org/10.1242/dev.00719
Larsen C, Franch-Marro X, Hartenstein V et al (2006) An efficient promoter trap for detection of patterned gene expression and subsequent functional analysis in Drosophila. Proc Natl Acad Sci U S A 103:17813–17817. https://doi.org/10.1073/pnas.0607652103
Calleja M, Moreno E, Pelaz S, Morata G (1996) Visualization of gene expression in living adult Drosophila. Science 274:252–255
McGuire SE, Mao Z, Davis RL (2004) Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila. Sci STKE 2004:pl6. https://doi.org/10.1126/stke.2202004pl6
Green EW, Fedele G, Giorgini F, Kyriacou CP (2014) A Drosophila RNAi collection is subject to dominant phenotypic effects. Nat Methods 11:222–223. https://doi.org/10.1038/nmeth.2856
Vissers JHA, Manning SA, Kulkarni A, Harvey KF (2016) A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth. Nat Commun 7:10368. https://doi.org/10.1038/ncomms10368
Ni J-Q, Liu L-P, Binari R et al (2009) A Drosophila resource of transgenic RNAi lines for neurogenetics. Genetics 182:1089–1100. https://doi.org/10.1534/genetics.109.103630
Ni J-Q, Markstein M, Binari R et al (2008) Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster. Nat Methods 5:49–51. https://doi.org/10.1038/nmeth1146
Port F, Strein C, Stricker M et al (2019) A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila. Elife 9:e53865. https://doi.org/10.7554/eLife.53865. PMC7062466. PMID: 32053108
Morin X, Daneman R, Zavortink M, Chia W (2001) A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila. Proc Natl Acad Sci U S A 98:15050–15055. https://doi.org/10.1073/pnas.261408198
Oda H, Tsukita S (1999) Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dalpha-catenin-GFP fusion protein. Dev Genes Evol 209:218–225
Oda H, Tsukita S (2001) Real-time imaging of cell-cell adherens junctions reveals that Drosophila mesoderm invagination begins with two phases of apical constriction of cells. J Cell Sci 114:493–501
Royou A, Field C, Sisson JC et al (2004) Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos. Mol Biol Cell 15:838–850. https://doi.org/10.1091/mbc.E03-06-0440
Hatan M, Shinder V, Israeli D et al (2011) The Drosophila blood brain barrier is maintained by GPCR-dependent dynamic actin structures. J Cell Biol 192:307–319. https://doi.org/10.1083/jcb.201007095
Ring JM, Martinez-Arias A (1993) Puckered, a gene involved in position-specific cell differentiation in the dorsal epidermis of the Drosophila larva. Dev Suppl:251–259
Mattila J, Omelyanchuk L, Kyttälä S et al (2005) Role of Jun N-terminal Kinase (JNK) signaling in the wound healing and regeneration of a Drosophila melanogaster wing imaginal disc. Int J Dev Biol 49:391–399. https://doi.org/10.1387/ijdb.052006jm
Su YC, Treisman JE, Skolnik EY (1998) The Drosophila Ste20-related kinase misshapen is required for embryonic dorsal closure and acts through a JNK MAPK module on an evolutionarily conserved signaling pathway. Genes Dev 12:2371–2380
Chatterjee N, Bohmann D (2012) A versatile ΦC31 based reporter system for measuring AP-1 and Nrf2 signaling in Drosophila and in tissue culture. PLoS One 7:e34063. https://doi.org/10.1371/journal.pone.0034063
Friedrich MV, Schneider M, Timpl R, Baumgartner S (2000) Perlecan domain V of Drosophila melanogaster. Sequence, recombinant analysis and tissue expression. Eur J Biochem 267:3149–3159. https://doi.org/10.1046/j.1432-1327.2000.01337.x
Wolfstetter G, Shirinian M, Stute C et al (2009) Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm. Mech Dev 126:721–736. https://doi.org/10.1016/j.mod.2009.05.001
Dai J, Estrada B, Jacobs S et al (2018) Dissection of Nidogen function in Drosophila reveals tissue-specific mechanisms of basement membrane assembly. PLoS Genet 14:e1007483–e1007431. https://doi.org/10.1371/journal.pgen.1007483
Kumagai T, Yokoyama H, Goto A et al (2000) Screening for Drosophila proteins with distinct expression patterns during development by use of monoclonal antibodies. Biosci Biotechnol Biochem 64:24–28. https://doi.org/10.1271/bbb.64.24
Milner MJ (1977) The eversion and differentiation of Drosophila melanogaster leg and wing imaginal discs cultured in vitro with an optimal concentration of beta-ecdysone. J Embryol Exp Morphol 37:105–117
Bainbridge SP, Bownes M (1981) Staging the metamorphosis of Drosophila melanogaster. J Embryol Exp Morphol 66:57–80
Acknowledgments
We thank members of the Murray lab for advice and support, Vishal Chaturvedi for critical reading of the manuscript, and Isabelle Lohrey for experimental tests. This work was supported by a National Collaborative Grant from the National Breast Cancer Foundation, and a University of Melbourne RGSS grant to M.J.M.
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Golenkina, S., Manhire-Heath, R., Murray, M.J. (2021). Exploiting Drosophila melanogaster Wing Imaginal Disc Eversion to Screen for New EMT Effectors. In: Campbell, K., Theveneau, E. (eds) The Epithelial-to Mesenchymal Transition. Methods in Molecular Biology, vol 2179. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0779-4_11
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DOI: https://doi.org/10.1007/978-1-0716-0779-4_11
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