We moved to using the ninaE promoter. Before that, we had used ey GMR
ninaE vs eyGMR
Here is a statement Prof Eissenberg made in answer to a referee comment
while reworking the Traffic ms Spring, 2010: "The rationale is to test
the role of GGA in all the cells of the eye, during
both differentiation and aging. If we just used "Rh1-Gal4," this
affect R1-R6, and only beginning at the end of pupal development. In our
design, we are able to test whether knockdown of GGA has a role in the
signaling necessary to specify the ommatidial array and the individual
retinula cells (it doesn't) and whether GGA is required for pigment cell
deposition (no role is evident from our experiments). This could not be
tested with the "Rh1-Gal4" driver."
We needed white eyes rather than pigmented eyes because (1) we could look
at fluorescence; and (2) we could make quantitative rhodopsin measurements.
In crosses that Prof Eissenberg set up and delivered, the ninaE promoter
was used to drive Rh1-GFP as well as the GGA kd into R1-6.
(1) Chinchore Y, Mitra A, Dolph PJ (2009) Accumulation of rhodopsin in late
endosomes triggers photoreceptor cell degeneration. PLoS Genet 5(2):
(2) Zinkl G, Maier L, Studer K, Sapp R, Chen DM, Stark WS (1990) Microphotometric,
ultrastructural and electrophysiological analyses of light dependent processes
on visual receptors in white-eyed wild-type and norpA (no receptor
potential) mutant Drosophila. Vis Neurosc 5: 429-439
and several lines of work this semester (for instance this)
indicate that rhodopsin is higher if flies are kept in the dark; also, rhodopsin
that has been cleared from the rhabdomere into the retinula cell is returned
to the rhabdomere quickly in the dark,
vitamin A deprivation and replacememt
Please refer to this
document. To determine whether the GGA kd altered rhodopsin import to the
rhabdomere, we deprived then replaced.
Importantly, crosses were set up on vitamin A deprivational medium. Because
of the late timing of action of the ninaE promoter, flies that emerged were
aged for twl days before being placed on the carrot juice in the dark.
At first we were struck with how normal the ninaE GGA kd looked (here
is 4 days of replacement). The difference from the ey GMR driven GGA kd
was profound (here
is day zero). I worried that the ninaE driver studies all utilized flies
kept in the dark while the ey GMR driver studies used flies kept in the
light; however day zero dark ey GMR flies also had abnormal rhabdomeres
as shown here.
(Again, the important thing is that the ninaE promoter Rh1-GFP white eyes
permitted confocal microscopy and that white eyes permitted quantitative
measurement of rhodopsin using microscope photometry).
In George Denny's poster (for the Biology Keath research award and the SLU
senior legacy symposium), he documents his evidence that GGA interferes
with the import of rhodopsin to the rhabdomere (middle left of poster).
This point is substantiated by a quantitative comparison of rhodopsin level
in 5 day vitamin A replaced flies (data
of Asmir Selimovic).
In contrast, GGA did not seem to alter clearance of rhodopsin from rhabdomeres