Chapter 11
Exploiting Drosophila melanogaster Wing Imaginal Disc
Eversion to Screen for New EMT Effectors
Sofia Golenkina, Rosemary Manhire-Heath, and Michael J. Murray
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,
GAL80
ts 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.
Key words Epithelial-mesenchymal transition, Wing, Imaginal disc, Eversion, RNAi, Culture
1 Introduction
Genetic screens in Drosophila melanogaster (Drosophila) have
provided enormous insight into a wide range of cellular and developmental processes, and molecular pathways. These include both
forward screens employing chemical mutagenesis and transposon
insertions [1] and reverse genetic screens using sequence-specific
gene disruption methods such as RNAi [2]. However, few screens
have specifically targeted epithelial-mesenchymal transition (EMT)
events, despite the enormous importance of EMTs in development,
cancer metastasis, and wound healing [3]. Part of the reason for
this is that the two best documented EMTs in Drosophila, the
formation of the mesoderm and endoderm [4–6], occur early in
embryogenesis, which creates challenges for loss-of-function
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_11,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
Sofia Golenkina and Rosemary Manhire-Heath contributed equally with all other contributors.
115
Exploiting Drosophila melanogaster Wing Imaginal Disc
Eversion to Screen for New EMT Effectors
Sofia Golenkina, Rosemary Manhire-Heath, and Michael J. Murray
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,
GAL80
ts 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.
Key words Epithelial-mesenchymal transition, Wing, Imaginal disc, Eversion, RNAi, Culture
1 Introduction
Genetic screens in Drosophila melanogaster (Drosophila) have
provided enormous insight into a wide range of cellular and developmental processes, and molecular pathways. These include both
forward screens employing chemical mutagenesis and transposon
insertions [1] and reverse genetic screens using sequence-specific
gene disruption methods such as RNAi [2]. However, few screens
have specifically targeted epithelial-mesenchymal transition (EMT)
events, despite the enormous importance of EMTs in development,
cancer metastasis, and wound healing [3]. Part of the reason for
this is that the two best documented EMTs in Drosophila, the
formation of the mesoderm and endoderm [4–6], occur early in
embryogenesis, which creates challenges for loss-of-function
Kyra Campbell and Eric Theveneau (eds.), The Epithelial-to Mesenchymal Transition: Methods and Protocols,
Methods in Molecular Biology, vol. 2179, https://doi.org/10.1007/978-1-0716-0779-4_11,
© Springer Science+Business Media, LLC, part of Springer Nature 2021
Sofia Golenkina and Rosemary Manhire-Heath contributed equally with all other contributors.
115