3 Methods
3.1 RNAi Screen
for Genes Involved
in Eversion
1. Establish a GAL4 driver stock that incorporates GAL80
ts (see
Note 3). One can also include UAS-Dicer-2 to enhance the
strength of the RNAi effect [23] (see Note 4).
2. In a vial, cross five female virgins of the driver stock, between
2 and 5 days old, to 5 males (2–5 days old) and leave at the
restrictive temperature (e.g., ~22–23
C) for 3 days (see
Note 5).
3. Turn adult parent flies into a new vial each day for 3 days, i.e.,
Day 1—vial#1; Day 2—vial#2; Day 3—vial#3. On Day
4, remove adults from vial#3. Shift vials to 29
C 2 days after
the adults have been removed (i.e., Day 4—move vial#1 to
29
C, Day 5—vial#2, Day 6—vial#3) (see Note 6).
4. From Day 11 onwards, phenotypically score adult flies. This
includes both eclosed adults and pharate adults that are still
within the pupal case (Fig. 2 and see Note 7). To score pharate
adults, one can remove prepupae from the vial by carefully
dislodging them by taking hold of the anterior spiracles with
forceps or pushing laterally with a paintbrush. Place prepupae
in 80% ethanol for scoring. If air bubbles impair optical clarity
(Fig. 2d), pierce the case to allow ethanol to displace the air.
Although there is a wide spectrum of phenotypes, we find it
useful to categorize them as follows (in increasing level of
severity):
l
Mild defects of one or two wings (e.g., smaller, crumpled,
mispositioned) but thorax normal (Fig. 2g).
l
Both wings everted but thorax disrupted, usually in the
form of a midline cleft (Fig. 2a, d, e).
l
Single eversion failure resulting in an internalized wing and
missing half of the thorax (Fig. 2b, f).
l
Double eversion failure, where both wings are internalized
and completely lacking a thorax.
l
Early pupal lethal—when a pupa has failed to develop to
adulthood (see Note 8).
5. When it is clear that no further pupae will develop to adulthood
(typically ~Day 15), one can fill the vial with 80% ethanol,
dislodge pupae from walls with a paintbrush, and tip out the
contents into a 90 mm petri dish to complete the scoring
(Fig. 2c and see Note 9).
3.2 Screening
for Enhancers/
Suppressors
Having identified a gene whose knockdown causes partially penetrant eversion failure, one can then use this genotype as a convenient vehicle for screening for suppressors and enhancers. We find
that genotypes whose eversion phenotypes are partially penetrant
Using Drosophila Wing Eversion to Study EMT
123
3.1 RNAi Screen
for Genes Involved
in Eversion
1. Establish a GAL4 driver stock that incorporates GAL80
ts (see
Note 3). One can also include UAS-Dicer-2 to enhance the
strength of the RNAi effect [23] (see Note 4).
2. In a vial, cross five female virgins of the driver stock, between
2 and 5 days old, to 5 males (2–5 days old) and leave at the
restrictive temperature (e.g., ~22–23
C) for 3 days (see
Note 5).
3. Turn adult parent flies into a new vial each day for 3 days, i.e.,
Day 1—vial#1; Day 2—vial#2; Day 3—vial#3. On Day
4, remove adults from vial#3. Shift vials to 29
C 2 days after
the adults have been removed (i.e., Day 4—move vial#1 to
29
C, Day 5—vial#2, Day 6—vial#3) (see Note 6).
4. From Day 11 onwards, phenotypically score adult flies. This
includes both eclosed adults and pharate adults that are still
within the pupal case (Fig. 2 and see Note 7). To score pharate
adults, one can remove prepupae from the vial by carefully
dislodging them by taking hold of the anterior spiracles with
forceps or pushing laterally with a paintbrush. Place prepupae
in 80% ethanol for scoring. If air bubbles impair optical clarity
(Fig. 2d), pierce the case to allow ethanol to displace the air.
Although there is a wide spectrum of phenotypes, we find it
useful to categorize them as follows (in increasing level of
severity):
l
Mild defects of one or two wings (e.g., smaller, crumpled,
mispositioned) but thorax normal (Fig. 2g).
l
Both wings everted but thorax disrupted, usually in the
form of a midline cleft (Fig. 2a, d, e).
l
Single eversion failure resulting in an internalized wing and
missing half of the thorax (Fig. 2b, f).
l
Double eversion failure, where both wings are internalized
and completely lacking a thorax.
l
Early pupal lethal—when a pupa has failed to develop to
adulthood (see Note 8).
5. When it is clear that no further pupae will develop to adulthood
(typically ~Day 15), one can fill the vial with 80% ethanol,
dislodge pupae from walls with a paintbrush, and tip out the
contents into a 90 mm petri dish to complete the scoring
(Fig. 2c and see Note 9).
3.2 Screening
for Enhancers/
Suppressors
Having identified a gene whose knockdown causes partially penetrant eversion failure, one can then use this genotype as a convenient vehicle for screening for suppressors and enhancers. We find
that genotypes whose eversion phenotypes are partially penetrant
Using Drosophila Wing Eversion to Study EMT
123