Microscopy of Drosophila's six rhodopsins
(seminar, Fall, 2002)
(emphasis on undergrad research assistants)

Background (Drosophila have at least 3 spectral receptor types, Harris, Stark and Walker, 1976)

A diagram showing R1-6, R7 and R8 photoreceptors, with their microvillar rhabdomeres, in each ommatidium of the compound eye

With "genetic dissection" (mutants lacking photoreceptors, like sevenless which we introduced) in my ERG (electroretinogram) setup, we demonstrated that there are 3 receptor spectra, R1-6 is a UV-blue receptor, R7 is a UV receptor, and R8 is a green receptor.

Methods (visual receptors can be imaged in live flies)

live white-eyed Drosophila could be fixed to a microscope slide to observe the pseudopupil.

A diagram explaining how the deep pseudopupil is a magnified virtual image of about 25 pooled rhabdomere tips. (The number depends on the numerical aperture of the microscope objective.).

I know I am getting ahead of myself by showing the following animation from the confocal microscope, but it so clearly dramatizes the above.

Movie - focus series from corneal surface to deep pseudopupil (292 K)

Here is my Leitz Dialux fluorescence microscope where electronic camera or microscope photometer can be interchanged. I built in shutters and locations for interference and neutral density filters so that it can be used to provide actinic stimulations for photopigment conversionaration.

Computer technology is credited to Bob Marietta (shown here with his family in 2002), SLU undergrad 1992-1996 (Bio Research award winner) and SLU Med School.

Here is Charles Thomas, LOCI (Laboratory for Optical and Computational Instrumentation), at the BioRad 600 confocal with a Drosophila pseudopupil imaged on the screen. (For his rock music, go to this site.)

Results 1 - Fluorescence and confocal microscope studies

A fluorescence micrograph showing vitamin A dependent fluorescence of R1-6 in the deep pseudopupil (deprived fly eye is on the right) like that from Stark et al. 1979

In the confocal microscope, R1-6 & R7 fluorescence is visualized in the deep pseudopupil using fluorescein optics.

Neither R1-6 nor R7 emitted in vitamin A deprived flies.

Only R7 fluoresced in R1-6 opsin mutants ninaE-ora and ninaE-oI17. (Also this mutant blocked Rh1 fluorescence to allow expression of the other 5 rhodopsins into R1-6 in transgenics provided by Charles Zuker and Steve Britt.)

In Musca, R7s fluoresce yellow (R7y) or pale (R7p); blue rhodopsin with accessory pigments vs. UV rhodopsin explained the respective classes (Hardie, TiNS 9 1986, 419-423). In the confocal microscope, optically neutralized white-eyed Musca (house fly) also shows bright and dim R7s.

We replicated this in Drosophila. With cornea optically neutralized by oil immersion, rhabdomeres were clear in a narrow band of focus; most R7s fluoresced brightly, while others were dim.

Going back to the diagram, what Franceschini and Hardie concluced in the 1980's was that there are 2 R7 opsins (later cloned in Drosophila as Rh3 and Rh4) and 2 R8 opsins (Rh5 and Rh6 only recently isolated).

This picture, from my Gateway middle school tutorial shows the 3 simple eyes (ocelli) where Rh2 dwells.

Here are the R1-6 fluorescences for Rh1-Rh6: Rh1 = Rh5 > Rh6 > Rh2 = Rh4 > Rh3 to be discussed -- I had hoped this approach would explain bright vs dim R7's (Rh3/Rh5 tandem pair has the brightest Rh5), but I do not believe it.

Charles Deutch won the Biology Undergraduate Research Award for work on this a few years ago.

A year ago, Carla Engelke helped bring this project to near completion; she won the college outstanding senior award for Biology.

Before I get back to a better approach, using GFP (green fluorescent protein), I want to show visual pigment conversions examined using these stocks.

Results 2 - visual pigment conversions.

Some of you may remember Dave Hunnius, now a resident, who worked on this project while he was an undergraduate here (1992-1996)

Using the deep pseudopupil, I could photograph the conversion of the blue-absorbing rhodopsin (R480) to yellow absorbing metarhodopsin in the deep pseudopupil of a live white-eyed Drosophila (Stark & Johnson,1980). Before I could get this good picture, a lesser picture seemed important enough that Doekele Stavenga used my earlier version in his chapter in the Handbook of Sensory Physiology (Chapter 7, Vol VII/6A, H. Autrum, ed. Berlin, Springer, 1979). At the time, I was really putting my neck on the chopping block since I disagreed with an explanation published by a very influential researcher (Lo and Pak, Nature, 273, 772-774, 1978).

Using my electronic color camera, I replicate the above experiment where Rh1 shows bright R1-6 with 579 nm light transmitted through the deep pseudopupil (left) but dim R1-6 when 450 nm light converts 480 nm rhodopsin to 570 nm metarhodopsin (right).

Andrew Ohar, who took my second semester intro course last semester, has been around this summer and is helping to put the finishing touches on this project.

We made a quicktime movie (92 K) of the conversion of R-480 to M-570 in R1-6 by 460 nm light; the deep pseudopupil of white-eyed (otherwise wild-type) flies was viewed using 579 nm light near M570's maximum.

Rh[1+2] flies (from Charles Zuker) show bright R1-6 with 520 nm light transmitted through the deep pseudopupil (left) but dim R1-6 when 436 nm light converts 420 nm rhodopsin to 520 nm metarhodopsin.

Rh[1+3] flies (from Charles Zuker) show bright R1-6 with blue light transmitted through the deep pseudopupil (left) but dim R1-6 when 350 nm light converts 345 nm rhodopsin to 460-465 nm metarhodopsin (right), my work with Dave Hunnius following Feiler et al.'s work (J. Neurosci. 12, 3862-3868, 1992).

Rh[1+4] flies (from Charles Zuker) show bright R1-6 with blue light transmitted through the deep pseudopupil (left) but dim R1-6 when 376 nm light converts 375 nm rhodopsin to 460-465 nm metarhodopsin (right), my work with Dave Hunnius following Feiler et al.'s work (J. Neurosci. 12, 3862-3868, 1992).

Rh[1+5] flies (from Steve Britt) show bright R1-6 with 505 nm transmitted through the deep pseudopupil (left) but dim R1-6 when 405 nm light converts 437 nm rhodopsin to 494 nm metarhodopsin (right), my recent work with Sanjay Agarwal following Salcedo et al.'s work (J. Neurosci. 19, 10716-10726, 1999).

The story for Rh6 is very interesting! (1) It is not closely related to the five other Drosophila rhodopsins; (2) It is the only one of six with a shorter wavelength metarhodopsin (R-508 nm, M-468 nm); and (3) The apparent metarhodopsin is less thermally stable (Salcedo et al., 1999. J. Neurosci. 19, 10716-10726). Also sev (sevenless) flies have Rh6 but not Rh5 (Chou et al., Development 126, 607-616, 1999), suggesting that all conclusions about R8 from my work (Harris et al., 1976, J. Physiol., 256, 415-439) applies to Rh6, not Rh5. At that time (Harris, 1976), we noted that R8 was "non-abdapting," i.e. we could not isolate rhodopsin vs metarhodopsin by chromatic adaptation. This is confirmed in Rh[1+6] flies here: Although R1-6 are bright (and slightly pink when compared with R7) when viewed with white light (left), R1-6 remain continuously dark when transilluminated with 514 nm light (no additional adaptation).

Results 3 - rhodopsin promoter - GFP (green fluorescent protein) reporter analyses

Quite a bit of this GFP work was done with Jeremy Beatty. I took him to ARVO (Association for Research in Vision and Ophthalmology in 1999; he won SLU Biology's Research award and graduated in 2000; he's in UMSL's optometry program; here he is in front of his ARVO-2002 poster.

Joe Polizzi, SLU Biology 1992-1996 then SLU Med School, now a cardio resident, is shown here (2001). He came back to my lab for a Med School research rotation and got this project started.

Charles Thomas (at LOCI) and I imaged GFP driven by Rh1's promoter using stocks made by Dr. Franck Pichaud and Prof. Claude Desplan: the "UAS-GAL4 system," crossing a stock homozygous for Rh1's promoter driving GAL4 on the third chromosome with a stock homozygous for UAS driving GFP on the second and third chromosomes. Because of the carotenoids in our fly food, GFP is expressed in R1-6 in the deep pseudopupil as well as in individual R1-6 rhabdomeres viewed with optical neutralization of the cornea. At first, I was disappointed with those images compared with our autofluorescence of deep pseudopupil and individual rhabdomeres. But then I concluded that GFP expression is not only in the rhabdomeres but also in the R1-6 cell bodies as judged by the smear of the image. Yeast-glucose flies (reared on "Tsubota food") have fluorescence since that food turns on the opsin gene even though they lack visual pigment (because of the lack of chromophore precursors in the food). These images relate to work on retinoid control of opsin gene transcription.

Opsin-promoter -- GFP-reporter analyses

For R1-6 (Rh1)

Most recently, using stocks made by Dr. Franck Pichaud in Prof. Claude Desplanís laboratory, my former undergraduate research assistant Jeremy Beatty used the "UAS-GAL4 system" to facilitate promoter-reporter analyses looking at GFP (green fluorescent protein). A stock homozygous for Rh1's promoter driving GAL4 on the third chromosome was crossed with a stock homozygous for UAS driving GFP on the second and third chromosomes. Vitamin A replete flies showed fluorescence of R1-6 in the deep pseudopupil (left); Transmission of 579 nm light, near metarhodopsin's maximum (middle), is decreased as 480 nm light converts rhodopsin-480 to metarhodopsin-570. This comparison shows that GFP expression does not interfere with visual pigment. Also, since the GFP image is larger, GFP expression is not only in the rhabdomere but also in the R1-6 cell bodies. Deprived flies lack this fluorescence and they lack visual pigment. Flies reared on yeast-glucose food have fluorescence even though they lack visual pigment because of the lack of chromophore precursors in the food.

For R7 (Rh3 and Rh4)

Kris Pineda is helping me to put the finishing touches on this work

More recently, Dr. Pichaud provided flies with the R7 opsin promoters driving GFP directly. Rh3's promoter (-345/+18) drives fluorescence in R7's rhabdomere. This fluorescence is decreased but not eliminated by vitamin A deprivation. Similarly, Rh4's promoter (-373/+83) drives fluorescence in R7, in this case in the rhabdomere and the cell body. Again, vitamin A deprivation decreases but does not eliminate this fluorescence. Earlier, we had shown that a yeast-glucose food that lacks chromophore precursors, and hence rhodopsin, does have substances that activate transcription. This is dramatized in Rh4-promoter - GFP reporter flies by the high fluorescence of R7. It is interesting to note that the difference in appearance of the deep pseudopupil for Rh3 vs Rh4 (rhabdomere only vs rhabdomere plus cell body) was also observed in the confocal microscope.

Confocal images

Charles Thomas (at LOCI) and I used confocal microscopy to examine Drosophila stocks with Rh3 vs. Rh4 promoters driving GFP (green fluorescent reporter), gifts of Claude Desplan and Franck Pichaud of New York University, help to solve the mediation of bright vs. dim R7 autofluorescence by specific rhodopsins. Here is a typical deep pseudopupil of Rh4GFP flies. Here (A and B) is a selection of images showing Rh4GFP marking some of the R7 rhabdomeres. GFP labels rhabdomeres and cell bodies. Analysis of multiple serial sections (by SLU undergraduate Carla Engelke, analysis facilitated by Charles Thomas's program "4D Turnaround") gives counts of 426 positives, 782 negatives, 256 unknowns, and 183 questionable positives. Previous studies suggest that there are 2:1::Rh4:Rh3 containing rhabdomeres. We put the ratio at a lower number, and we are fairly confident with this analysis. We obtained this typical deep pseudopupil of Rh3GFP. Here (A and B) are several views of rhabdomere tips in Rh3GFP. Analysis of Rh3GFP is equivocal: most R7's fluoresce, but only about half were strikingly positive. We conclude that, while GFP labels Rh3, Rh4 are seen (more dimly in the Rh3GFP stock) because of their autofluorescence. This would identify Rh4-containing rhabdomeres as the brightly fluorescing R7 subtype.

CONCLUSIONS
(1) The pathway to discovery is not always how you plan it at first.
(2) But it sure is fun, and creates enduring friendships with students.
(3) And you even stumble on some really neat observations.


Return to Stark home page

This page last revised on August 30, 2002