One can then grab the mouthparts on-end with one pair of
forceps, stabilize the body wall with the other pair, and push
the larva inside out. We find this method is less likely to damage
the delicate peripodial epithelium. Isolated wing discs can be
conveniently transferred in a P1000 tip. If the disc is still
attached to the body wall, we transfer using a P1000 tip
which has had some of the tip removed with scissors to create
a larger bore. If discs are sticking to the inside of the tip, it is
worth trying other brands as some use rougher plastic than
others.
16. It is imperative that partly everted wing discs are handled very
gently to avoid damaging the very delicate PE. Since one is
screening for the formation of holes, it is important not to
introduce artifactual holes. Normally, one can distinguish these
from real holes as they will have occurred after fixation and have
sharply demarcated edges.
17. Since the PE can become highly convoluted and threedimensional by the time of eversion analysis of the PE breakdown requires a z-stack with high resolution in z (typically
0.5–1 μm z-step) so that one can have continuity in the zdirection to detect changes to the AJs (i.e., diminution, fragmentation, and delocalization of DE-Cadherin) even when the
tissue is oriented orthogonally to the image plane.
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.
References
1. 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
2. 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/10.1007/978-1-4939-63713_6
3. 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
4. Leptin M, Grunewald B (1990) Cell shape
changes during gastrulation in Drosophila.
Development 110:73–84
5. Tepass U, Hartenstein V (1994) Epithelium
formation in the Drosophila midgut depends
on the interaction of endoderm and mesoderm. Development 120:579–590
6. 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
7. Gryzik T, Mu ¨ller H-AJ (2004) FGF8-like1 and
FGF8-like2 encode putative ligands of the FGF
receptor Htl and are required for mesoderm
132
Sofia Golenkina et al.
forceps, stabilize the body wall with the other pair, and push
the larva inside out. We find this method is less likely to damage
the delicate peripodial epithelium. Isolated wing discs can be
conveniently transferred in a P1000 tip. If the disc is still
attached to the body wall, we transfer using a P1000 tip
which has had some of the tip removed with scissors to create
a larger bore. If discs are sticking to the inside of the tip, it is
worth trying other brands as some use rougher plastic than
others.
16. It is imperative that partly everted wing discs are handled very
gently to avoid damaging the very delicate PE. Since one is
screening for the formation of holes, it is important not to
introduce artifactual holes. Normally, one can distinguish these
from real holes as they will have occurred after fixation and have
sharply demarcated edges.
17. Since the PE can become highly convoluted and threedimensional by the time of eversion analysis of the PE breakdown requires a z-stack with high resolution in z (typically
0.5–1 μm z-step) so that one can have continuity in the zdirection to detect changes to the AJs (i.e., diminution, fragmentation, and delocalization of DE-Cadherin) even when the
tissue is oriented orthogonally to the image plane.
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.
References
1. 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
2. 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/10.1007/978-1-4939-63713_6
3. 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
4. Leptin M, Grunewald B (1990) Cell shape
changes during gastrulation in Drosophila.
Development 110:73–84
5. Tepass U, Hartenstein V (1994) Epithelium
formation in the Drosophila midgut depends
on the interaction of endoderm and mesoderm. Development 120:579–590
6. 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
7. Gryzik T, Mu ¨ller H-AJ (2004) FGF8-like1 and
FGF8-like2 encode putative ligands of the FGF
receptor Htl and are required for mesoderm
132
Sofia Golenkina et al.