8. Although it is not necessarily the case that phenotypes such as
crumpled wings and thoracic clefts would be caused by failure
in the pEMT, we believe that this is often the case. RNAi lines
that we know block the pEMT and cause wing eversion failure
(e.g., UAS-netA.IR), also produce these milder phenotypes.
We speculate that partial failure of the pEMT may impede
subsequent epithelial migration resulting in clefts, or
subsequent development of the wing, resulting in weak wing
phenotypes.
9. If one is not ready to score the phenotypes, the ethanol-filled
vial can be sealed with saran wrap to prevent evaporation and
kept for later analysis.
10. Although the suction effect of the oil is often sufficient to hold
the case securely to the coverslip, one can also use physical
objects (e.g., small pieces of aluminum or glass) to support
the pupal case at a suitable angle.
11. An alternative approach has been developed by Aldaz et al. [14]
whereby one partially dissects the prepupa into “quarters” in
culture medium, orients the pupal fragment with the open side
towards the glass and images from the “inside out” to avoid
imaging through the pupal case. This method produces high
quality results but requires significant skill in dissection
techniques.
12. Capturing movements of a three-dimensional in vivo event
involves trade-offs between the conflicting demands of obtaining clear images at each time point (i.e., high x–y pixel resolution, small z-step size, strong laser, long-dwell times),
achieving good temporal resolution, and avoiding photodamage to the tissue. The choice of objective may be constrained by
the three-dimensional structure of the pupal case. For example,
when imaging thorax closure through the operculum, the
anterior spiracles stick out, preventing the surface of the operculum from coming closely in contact with the glass, necessitating a longer working distance objective, typically of lower
magnification.
13. Others have used overnight fixation in 1Â PBS + 0.3
Triton X [20].
14. Removing tissue is critical as it will absorb antibodies and
staining reagents and inhibit them from penetrating into the
region where the wing disc attaches to the body wall.
15. A common method used to dissect out wing discs is to hold the
middle of the larva with one pair of forceps and the mouthparts
with another pair, and then pull out the mouthparts, bringing
with it the CNS, associated imaginal discs, and wing discs.
Alternatively, one can hold the larva in the middle with both
forceps side-by-side and pull the larva apart into two halves.
Using Drosophila Wing Eversion to Study EMT
131
crumpled wings and thoracic clefts would be caused by failure
in the pEMT, we believe that this is often the case. RNAi lines
that we know block the pEMT and cause wing eversion failure
(e.g., UAS-netA.IR), also produce these milder phenotypes.
We speculate that partial failure of the pEMT may impede
subsequent epithelial migration resulting in clefts, or
subsequent development of the wing, resulting in weak wing
phenotypes.
9. If one is not ready to score the phenotypes, the ethanol-filled
vial can be sealed with saran wrap to prevent evaporation and
kept for later analysis.
10. Although the suction effect of the oil is often sufficient to hold
the case securely to the coverslip, one can also use physical
objects (e.g., small pieces of aluminum or glass) to support
the pupal case at a suitable angle.
11. An alternative approach has been developed by Aldaz et al. [14]
whereby one partially dissects the prepupa into “quarters” in
culture medium, orients the pupal fragment with the open side
towards the glass and images from the “inside out” to avoid
imaging through the pupal case. This method produces high
quality results but requires significant skill in dissection
techniques.
12. Capturing movements of a three-dimensional in vivo event
involves trade-offs between the conflicting demands of obtaining clear images at each time point (i.e., high x–y pixel resolution, small z-step size, strong laser, long-dwell times),
achieving good temporal resolution, and avoiding photodamage to the tissue. The choice of objective may be constrained by
the three-dimensional structure of the pupal case. For example,
when imaging thorax closure through the operculum, the
anterior spiracles stick out, preventing the surface of the operculum from coming closely in contact with the glass, necessitating a longer working distance objective, typically of lower
magnification.
13. Others have used overnight fixation in 1Â PBS + 0.3
Triton X [20].
14. Removing tissue is critical as it will absorb antibodies and
staining reagents and inhibit them from penetrating into the
region where the wing disc attaches to the body wall.
15. A common method used to dissect out wing discs is to hold the
middle of the larva with one pair of forceps and the mouthparts
with another pair, and then pull out the mouthparts, bringing
with it the CNS, associated imaginal discs, and wing discs.
Alternatively, one can hold the larva in the middle with both
forceps side-by-side and pull the larva apart into two halves.
Using Drosophila Wing Eversion to Study EMT
131