6. To mount wing discs for imaging, it is important to control the
degree to which the coverslip touches and compresses the wing
disc by suspending it at an appropriate height using supports
on either side. For cultured discs, which will have become more
three-dimensional following the morphogenetic movements of
eversion, we find one coverslip (held in place with small drops
of glycerol) on either side works well. This allows the coverslip
to lightly touch the discs, which helps prevent them from
moving around, without squashing them. To completely preserve the 3D shape something thicker is needed (e.g., we
sometimes use two coverslips on either side). For discs that
have not been cultured, one often wishes to flatten the disc to
more easily visualize the entire PE in one focal plane, and for
this something thinner than a coverslip is needed (e.g., we cut
square pieces of plastic from thin autoclave bags, ~0.05 mm
thick). Glycerol is then used to fill the space under the coverslip, and nail-polish used to hold everything in place (see Note
16).
7. Analyze the immunostained discs on a confocal microscope (see
Note 17). Key events of the pEMT that are expected to occur
include the degradation and retraction of the basement membrane (Fig. 4), the reduction, dismantling and fragmentation
of the adherens junctions (Fig. 4e), the activation of the JNK
pathway, and a reduction of Frazzled expression levels [21] (see
Note 16).
8. Alternatively, one can leave the discs overnight and score them
the next day. We find discs usually fall into three easily scored
categories. Successful eversion whereby the wing blade
becomes quite flattened and “wing-like” (Fig. 3b, e, h), unsuccessful eversion, where the wing disc remains relatively flat and
the peripodial epithelium is intact (Fig. 3c, f), and partially
successful eversion where the disc has clearly undergone some
morphogenetic movements and shows some signs of having
produced a wing blade, but it is still balled up and constrained
inside a partially retracted peripodial epithelium (Fig. 3d, g).
4 Notes
1. The degradation and downregulation of E-Cad during the
pEMT that one can see with immunostaining may not be as
apparent with this GFP fusion since it is under the control of a
strong, constitutive promoter.
2. Although there are direct UAS-Actin-GFP fusions available,
these may cause phenotypes. Reporters like UAS-Lifeact-GFP
and UAS-GFP-Moesin
ABD [38] (i.e., GFP fused to the actinbinding domain of Moesin) are preferable as they decorate the
filaments but do not incorporate into the filaments.
Using Drosophila Wing Eversion to Study EMT
129
degree to which the coverslip touches and compresses the wing
disc by suspending it at an appropriate height using supports
on either side. For cultured discs, which will have become more
three-dimensional following the morphogenetic movements of
eversion, we find one coverslip (held in place with small drops
of glycerol) on either side works well. This allows the coverslip
to lightly touch the discs, which helps prevent them from
moving around, without squashing them. To completely preserve the 3D shape something thicker is needed (e.g., we
sometimes use two coverslips on either side). For discs that
have not been cultured, one often wishes to flatten the disc to
more easily visualize the entire PE in one focal plane, and for
this something thinner than a coverslip is needed (e.g., we cut
square pieces of plastic from thin autoclave bags, ~0.05 mm
thick). Glycerol is then used to fill the space under the coverslip, and nail-polish used to hold everything in place (see Note
16).
7. Analyze the immunostained discs on a confocal microscope (see
Note 17). Key events of the pEMT that are expected to occur
include the degradation and retraction of the basement membrane (Fig. 4), the reduction, dismantling and fragmentation
of the adherens junctions (Fig. 4e), the activation of the JNK
pathway, and a reduction of Frazzled expression levels [21] (see
Note 16).
8. Alternatively, one can leave the discs overnight and score them
the next day. We find discs usually fall into three easily scored
categories. Successful eversion whereby the wing blade
becomes quite flattened and “wing-like” (Fig. 3b, e, h), unsuccessful eversion, where the wing disc remains relatively flat and
the peripodial epithelium is intact (Fig. 3c, f), and partially
successful eversion where the disc has clearly undergone some
morphogenetic movements and shows some signs of having
produced a wing blade, but it is still balled up and constrained
inside a partially retracted peripodial epithelium (Fig. 3d, g).
4 Notes
1. The degradation and downregulation of E-Cad during the
pEMT that one can see with immunostaining may not be as
apparent with this GFP fusion since it is under the control of a
strong, constitutive promoter.
2. Although there are direct UAS-Actin-GFP fusions available,
these may cause phenotypes. Reporters like UAS-Lifeact-GFP
and UAS-GFP-Moesin
ABD [38] (i.e., GFP fused to the actinbinding domain of Moesin) are preferable as they decorate the
filaments but do not incorporate into the filaments.
Using Drosophila Wing Eversion to Study EMT
129