3. Since adding an extra UAS line could potentially suppress
phenotypes by titrating GAL4 away from the target UAS
RNAi construct, it is worthwhile including some negative controls such as UAS-GFP, in your tests.
3.3 Analyzing
the pEMT
Having identified genes required for eversion, one can then investigate how they affect the pEMT. To analyze the cellular events at
the time of the pEMT, there are several approaches. Most straightforward is to allow a prepupa to develop until the time of pEMT
(~2–3 h APF) and then either image it live, through the pupal case
(see Subheading 3.4), or dissect and fix it and apply immunostaining
(see Subheading 3.5). An alternative is to induce the pEMT ex vivo
by dissecting larvae/prepupae and culturing them in the presence
of ecdysone (see Subheading 3.6). This method was established by
Milner over 40 years ago [47] and has since been substantially
updated and improved by Aldaz et al. [14]. This method can be
applied to wing discs from wandering third-instar larvae, or prepupae that have been allowed to develop until just before
the pEMT.
3.4 Live Visualization
of the Peripodial pEMT
In Vivo
To visualize peripodial cells in vivo as they undergo the pEMT and
break through the epidermis, one must carefully stage prepupae
from the time of pupariation, i.e., the white prepupa stage when
larvae have ceased movement and everted their spiracles, but not
yet begun to tan [48]. Analysis can be carried out either on living
prepupae, using fluorescent transgene reporters, or by dissecting
and fixing prepupae at the time of eversion. Both approaches are
detailed below. The simplest approach is to leave the prepupa intact
and image through the pupal case with a laser confocal. This
approach has been used to visualize the moment when the pEMT
occurs and at the later stages during the epithelial migration of
thorax closure [13, 21]. Although this is the least intrusive
approach, and technically the simplest, image quality is significantly
impacted by the pupal case.
1. Remove prepupae from the vial by dislodging them gently with
a paintbrush or using forceps to take hold of the anterior
spiracles. Take care not to puncture or deform them. Clean
any food off with water and a paintbrush.
2. Adhere the prepupa to the base of the iBidi dish with a small
drop of liquid paraffin (or other oil such as voltalef 10S) orienting the prepupa such that the events one is examining are
closest to the glass base. For example, to visualize the pEMT
events, one must have the lateral anterior region downwards.
To visualize the epithelial migration during thorax closure, the
operculum should be downwards (see Notes 10 and 11).
Using Drosophila Wing Eversion to Study EMT
125
phenotypes by titrating GAL4 away from the target UAS
RNAi construct, it is worthwhile including some negative controls such as UAS-GFP, in your tests.
3.3 Analyzing
the pEMT
Having identified genes required for eversion, one can then investigate how they affect the pEMT. To analyze the cellular events at
the time of the pEMT, there are several approaches. Most straightforward is to allow a prepupa to develop until the time of pEMT
(~2–3 h APF) and then either image it live, through the pupal case
(see Subheading 3.4), or dissect and fix it and apply immunostaining
(see Subheading 3.5). An alternative is to induce the pEMT ex vivo
by dissecting larvae/prepupae and culturing them in the presence
of ecdysone (see Subheading 3.6). This method was established by
Milner over 40 years ago [47] and has since been substantially
updated and improved by Aldaz et al. [14]. This method can be
applied to wing discs from wandering third-instar larvae, or prepupae that have been allowed to develop until just before
the pEMT.
3.4 Live Visualization
of the Peripodial pEMT
In Vivo
To visualize peripodial cells in vivo as they undergo the pEMT and
break through the epidermis, one must carefully stage prepupae
from the time of pupariation, i.e., the white prepupa stage when
larvae have ceased movement and everted their spiracles, but not
yet begun to tan [48]. Analysis can be carried out either on living
prepupae, using fluorescent transgene reporters, or by dissecting
and fixing prepupae at the time of eversion. Both approaches are
detailed below. The simplest approach is to leave the prepupa intact
and image through the pupal case with a laser confocal. This
approach has been used to visualize the moment when the pEMT
occurs and at the later stages during the epithelial migration of
thorax closure [13, 21]. Although this is the least intrusive
approach, and technically the simplest, image quality is significantly
impacted by the pupal case.
1. Remove prepupae from the vial by dislodging them gently with
a paintbrush or using forceps to take hold of the anterior
spiracles. Take care not to puncture or deform them. Clean
any food off with water and a paintbrush.
2. Adhere the prepupa to the base of the iBidi dish with a small
drop of liquid paraffin (or other oil such as voltalef 10S) orienting the prepupa such that the events one is examining are
closest to the glass base. For example, to visualize the pEMT
events, one must have the lateral anterior region downwards.
To visualize the epithelial migration during thorax closure, the
operculum should be downwards (see Notes 10 and 11).
Using Drosophila Wing Eversion to Study EMT
125