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 would only
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.

Dark rearing

Several references:
(1) Chinchore Y, Mitra A, Dolph PJ (2009) Accumulation of rhodopsin in late endosomes triggers photoreceptor cell degeneration. PLoS Genet 5(2): e1000377
and
(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.

Initial findings

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.

Major findings

(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 (George's poster, bottom left).