Drosophila Rhodopsin 7 can partially replace the structural role of Rhodopsin 1, but not its physiological function
- First Online:
Rhodopsin 7 (Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1–6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1–6, but does not exclude that it works in the inner photoreceptor cells.
KeywordsPhototransduction Electroretinograms Rhodopsins Compound eyes Drosophila melanogaster
Vision begins with the absorption of photons by visual pigment molecules. Rhodopsins are membrane-bound G-protein-coupled receptors that absorb photons, undergo conformational changes, and activate a G-protein to initiate visual signal transduction. Animal genomes typically contain multiple Rhodopsin genes coding for Rhodopsins with different spectral properties providing the basis for color vision. Each photoreceptor cell usually expresses a single rhodopsin, but exceptions are known in both vertebrates and invertebrates (Applebury et al. 2000; Hu et al. 2011, 2014; Mazzoni et al. 2004; Stavenga and Arikawa 2008). The fruit fly Drosophila melanogaster possesses six different well-characterized Rhodopsin molecules, Rh1 to Rh6. With the exception of Rh2, all Rhodopsins are found in the receptor cells of the compound eyes: Rh1 is expressed in the six outer receptor cells (R1–6) of each eye unit and Rh3 to Rh6 are expressed in the two inner receptor cells (R7, R8) (reviewed in Rister et al. 2013; Behnia and Desplan 2015). A seventh Rhodopsin, Rh7, of still unknown location and function was predicted from the genome in 2000 (Adams et al. 2000; Terakita 2005). qPCR studies showed that Rh7 is expressed at low levels in the compound eyes, suggesting that it may be co-expressed with one or several of the other Rhodopsins (Posnien et al. 2012; Senthilan and Helfrich-Förster 2016).
The aim of the present study was to investigate a potential function of Rh7 in the compound eyes. A suited method to reveal the properties of an unknown Rhodopsin is to express it in R1–6 instead of Rh1 (Feiler et al. 1988; 1992; Townson et al. 1998; Salcedo et al. 1999; Knox et al. 2003; Hu et al. 2014). Rh1 is required for proper rhabdomere morphogenesis and maintenance, in addition to its role as photopigment (O’Tousa et al. 1985; Kumar and Ready 1995; Kumar et al. 1997; Zuker et al. 1985). Thus, loss of Rh1 (in ninaE17 mutants) leads to the collapse of rhabdomeric microvilli inside the photoreceptor cytoplasm (Ahmad et al. 2007; Bentrop 1998; Kurada and O’Tousa 1995; Leonard et al. 1992). This can be prevented by expressing other functional Rhodopsins in R1–6 of ninaE17 mutants (Kumar et al. 1997). Here, we expressed Rh7 instead of Rh1 under the Rh1 promotor (Rh1–Rh7;ninaE17 flies) and investigated whether Rh7 can (1) rescue the retinal degradation provoked by the ninaE17 mutation and (2) lead to normal electroretinogram (ERG) responses. We also expressed Rh7 in addition to Rh1 (Rh1–Rh7 flies) to see whether this increases the ERG responses.
Materials and methods
Wild-type CantonS (WTCS) as well as flies with yellow body color and white eyes (yellow−white1118 = y−w1118) served as control for semi-thin sections and immunocytochemistry. For qPCR, deep pseudopupil, and ERG measurements, only flies in the y−w1118 background were used. In addition, ninaE17 and sevLY3 mutants were in the y−w1118 background. ninaE (=neither inactivation nor afterpotential E) codes for Rh1 and y− w1118; ninaE17 mutants are Rh1 null mutants (O’Tousa et al. 1985). sev (sevenless) codes for a tyrosin kinase that is critical for the development of the photoreceptor cell R7 (Basler and Hafen 1988) and y− w1118sevLY3 mutants lack the inner photoreceptor cell R7 (Harris et al. 1976). In the following, we will omit “y− w1118” and simply use ninaE17 and sevLY3.
Generation of Rh1–Rh7 transgenic flies
To generate flies expressing the Rh7 coding region under control of the Rh1 promotor, the full-length Rh7 CDS was amplified by PCR from a commercially available cDNA clone, GH14208 (Berkley Drosophila Genome Project), using a primer pair creating restriction enzyme sites for EcoRI or NotI, respectively. The digested PCR product was first ligated into vector pBRh1UTR providing an Rh1 minimal promoter and the Rh1–3′UTR (kind gift of A. Huber, Universität Hohenheim). From this vector, a NotI/XhoI-fragment containing Rh1 promotor + Rh7 coding sequence + Rh1 3′-UTR was excised and ligated into the P-element vector Yellow-C4 (P. Geyer, University of Iowa). To create transgenic fly lines, the construct was then microinjected into y− w1118 embryos resulting in several insertion lines. The insertion lines were tested for Rh7 expression by qPCR. One line with the transgene insertion on the second chromosome (y− w1118;Rh1–Rh7;+) yielded the highest Rh7 expression and was subsequently used for all experiments. This line was crossed into the ninaE17 mutant background (y− w1118;Rh1–Rh7;ninaE17) to get flies that express Rh7 instead of Rh1. Furthermore, the y− w1118;Rh1–Rh7;+ line was backcrossed into the y− w1118 background for several generations (=backcross) to get control flies with the same genetic background as the transgenics. In the following, we will simply call the lines Rh1–Rh7, Rh1–Rh7;ninaE17, and backcross. We checked the presence of all Rhodopsins by qPCR and did not find any differences to wild-type flies.
Rh1: TCTGTATTTCGAGACCTGGGTGCTC; GACATGAACCAGATGTAGGCAATCTTGC
cry: CGGAGTTGATGAATGTCC; GCATGTTTCGCTTTACGG.
The relative mRNA levels of Rh1 and Rh7 in different fly strains were quantified via qPCR in 1- and 10-day-old flies as described in Senthilan and Helfrich-Förster (2016). Total RNA was extracted from the retinas of five flies per strain and age using the Quick-RNA™ MicroPrep Kit from Zymo Research and reversely transcribed using the Qiagen QuantiTect Reverse Transcription Kit. qPCRs were then carried out with the Bioline SensiFAST SYBR No-ROX Kit in combination with the Qiagen Rotor-Gene Q machine and 0.1 µM PCR primers. For each strain and tissue, three biological replicates were examined, and for each replicate, two PCRs were run. The relative mRNA levels were calculated using the ΔCT equation and alpha-tubulin was used as the reference gene. To exclude gDNA contamination, we designed our Rhodopsin primers in an intron-spanning way, so that the PCR products obtained from the cDNA and the gDNA will differ by size and by melting temperature.
Rh1: GGAGTAGAAGATCAGGTATGAGCGTG; TGCCTACATCTGGTTCATGTCGAGC
Rh7: CATCTGCGACTTTCTGATGCTCATC; GGATGCACCACCACATTGTACCGATC
Alpha-tub: TCTGCGATTCGATGGTGCCCTTAAC; GGATCGCACTTGACCATCTGGTTGGC.
Rh7 antibody generation
Two different peptide antibodies against Rh7 were generated: one against a 21-mer peptide in the intracellular domain of Rh7 (aa411-431: TRSSYMTRSRSSFTHRLRTST) and one against an 18-mer peptide in the extracellular domain of Rh7 (aa54-71: TESSAVNVGKDHDKHVND). For the first antibody, the cDNA fragment encoding the intracellular peptide was cloned into the bacterial expression vector pQE40 (Qiagen). The recombinant protein (intracellular peptide coupled to dihydrofolate reductase) was expressed in Escherichia coli and used to immunize rabbits. These experiments were registered and conducted according to the legal animal care regulations (RP Karlsruhe, AZ. 35-9185.82/977/99). The second antibody was also generated in rabbits, but by Pineda Antikörper-Service (Berlin, Germany). It was affinity purified using the original peptide bound to Sepharose 6B columns prior to use. Before immunization, serum samples were taken to obtain preimmune serum as negative control for unspecific immunoreactivity. Dot blot analysis was used to confirm the selective binding of Rh7 antibody to the purified peptide.
Histology and immunocytochemistry
For evaluating the fine-structure of the retina, fly heads were embedded in Epon and 0.2 µm-thick semi-thin sections of the eyes (from distal to proximal) and were cut with a microtome (Leica Ultracut). After mounting on glass slides, the sections were stained with methylene-blue (Romeis 2015). Ten flies of WT controls, ninaE17 mutants, and ninaE17 mutants with Rh7 expression in R1–6 (Rh1–Rh7;ninaE17), respectively, were stained and analyzed at the age of 1, 6, 10 days, and 5 weeks. The rhabdomere size of R1–6 was determined in 10-day-old flies in ImageJ as stated below.
For anti-Rh7, anti-Rh1, and anti-RDHB (visualizing the pigment cells surrounding the photoreceptor cells; Wang et al. 2012) immunocytochemistry, retinas of 4–6-day-old flies were dissected and fixed as described in Hsiao et al. (2012). Primary antibodies were applied for 2 days at room temperature (rabbit anti-Rh7 at 1:100; mouse anti-Rh1 from Developmental Studies Hybridoma Bank at 1:100 and rabbit anti-RDHB, kindly donated by Craig Montell, at 1:100). Fluorescent secondary antibodies (Alexa Fluor 488, 555, 635, Invitrogen) were applied overnight (at 1:1000). Immunostainings were visualized by confocal microscopy (Leica TCS SPE).
Evaluation of rhabdomere size
Rhabdomere size of 1-day-old flies was determined by measuring the areas of single rhabdomeres in five ommatidia of three different retinas with ImageJ (function “measure” under “analyze”). This was done for R1–6 and R8 of ninaE17 mutants, Rh1–Rh7;ninaE17 and Rh1–Rh7 flies, respectively. In all cases, cross sections at the level of R8 were taken for the measurements. The sizes of R1–6 were averaged for each ommatidium. A mean size for a typical R1–6 rhabdomere was then determined by averaging the values for all 15 ommatidia.
The deep pseudopupil was determined in vivo in 6-day-old white-eyed flies under white orthodromic illumination as described in Franceschini and Kirschfeld (1971). Flies were anaesthetized and glued to a glass slide with the left eye oriented to the top and placed under a stereo microscope. The eye was illuminated with a cold light source (KL 1500 LED plus, Leica). The objective (Planapo 1.6×, n:A. 0.24) was focused onto a region below the crystal cones at which R7 was seen best.
Low expression levels of Rh7 in the retina can be enhanced by ectopic expression in R1–6
ninaE17 mutants carry a 1.6 kb deletion in the 5′ region of the Rh1 gene with no detectable Rh1 transcript on an agarose gel (O’Tousa et al. 1985). As expected, the genomic DNA PCR yielded no visible Rh1 gene product on the gel (Fig. 2c), while using qPCR, which is more sensitive, we still detected marginal mRNA signals in the ninaE17 mutants (Fig. 2a). This residual expression is probably a result of the incomplete deletion in the Rh1 gene. Residual Rh1 expression was elevated after expressing Rh7 in R1–6 of ninaE17 mutants (Fig. 2a) most probably because Rh7 expression prevented the degeneration of R1–6 (see below). Nevertheless, the Rh1 protein was not detectable at all in ninaE17 mutants nor in Rh1–Rh7;ninaE17 mutants (Fig. 2d). This coincides with previous studies showing that ninaE17 mutants lack a functional Rh1 protein and they are, therefore, considered as Rh1 null mutants (Bentrop et al. 1997).
Most interestingly, the degeneration of R1–6 in 10-day-old ninaE17 mutants did not reduce Rh7 mRNA levels (Fig. 2a), suggesting that Rh7 is normally not expressed in R1–6. Furthermore, Rh7 is still present in sevenlessLY3 (sevLY3) mutants that lack photoreceptor cell R7 (Fig. 2a) indicating that Rh7 is also not expressed in R7 and pinpointing to an expression of Rh7 in R8. Indeed, a reporter line carrying the gene for the green fluorescent protein (GFP) in the first intron of the Rh7 gene showed weak GFP expression in R8 (Kistenpfennig et al. 2017, in preparation).
Rh7 can partially replace Rh1 in maintaining rhabdomere structure of R1–6
Mean area of a rhabdomere determined from semi-thin cross sections of three retinas (5 ommatidia, each) at the level of R8 of 10-day-old flies
0.91 ± 0.03
0.47 ± 0.03
0.92 ± 0.02
0.47 ± 0.02
0.47 ± 0.02
0.47 ± 0.02
0.42 ± 0.02
In 5-week-old flies, the differences between ninaE17 mutants and Rh1–Rh7;ninaE17 flies became most evident. Whereas ninaE17 mutants of that age showed a severe degeneration of the entire retina and lacked all rhabdomeres, Rh1–Rh7;ninaE17 flies still retained small rhabdomeres in R1–6 and rather normal rhabdomeres of R7 and R8 (Fig. 3c). Holes between the rhabdomeres were now visible even in control flies, but occurred still more frequently in Rh1–Rh7;ninaE17 flies (asterisks in Fig. 3c). In ninaE17 mutants, even the target neuropils of the photoreceptor cells degenerated. This was most evident in the lamina, in which the axons of R1–6 terminate, and to a lesser degree in the medulla, in which the axons of R7 and R8 terminate (Fig. 3d). In contrast, Rh1–Rh7;ninaE17 flies showed no such signs of degeneration in the lamina and medulla (Fig. 3d).
We conclude that Rh7 can partially replace Rh1 in its rhabdomere and photoreceptor maintenance role. R1–6 photoreceptor cells and their rhabdomeres were present in Rh1–Rh7;ninaE17 flies, but they were significantly smaller than in control flies and obviously not arranged in a sufficient regular manner to enable a deep pseudopupil. This irregular arrangement is hard to see on our semi-thin sections even not at a bigger field of the retinal section (Fig. S1). Nevertheless, one must confess that a precise superposition depends on a formidable regularity in the architecture of the compound eye, such as a constancy of the center-to-center distance between the seven rhabdomere distal endings (Franceschini 1972). Already, small deviations may result in a failure to detect a deep pseudopupil.
Rh7 cannot rescue the electroretinogram (ERG) of ninaE17 mutants
Many animals express more than one Rhodopsin molecule in their eyes to enhance light sensitivity and to enable color vision. Mosquitoes express even several rhodopsins in one photoreceptor cell (R7) making them responsive to a broad spectrum of visible and UV light and enabling them to be active at very dim light (Hu et al. 2011, 2014). The fruit fly D. melanogaster possesses six well-characterized Rhodopsins and a seventh Rhodopsin (Rh7) with unknown function that might be co-expressed with the others. Rh7 seems to be an ancient type of Rhodopsin that is conserved among different arthropods, including species as Limulus polyphemus and Daphnia pulex (Senthilan and Helfrich-Förster 2016). Rh7 possesses nearly all important features for a visual functional protein, but it lacks the highly conserved QAKK motif, which is, together with the DRY motif, important for G-protein binding and consequently for the activation to the G-protein-coupled cascade. Therefore, it is unknown, whether it is a functional Rhodopsin. Here, we tested whether Rh7 can replace the function of Rh1 by expressing it instead of Rh1 in R1–6.
We found that Rh7 can partially overtake the functions of Rh1. It can fairly replace Rh1 in its rhabdomere maintenance function, but it cannot activate the phototransduction cascade in R1–6, at least not under our experimental conditions. We cannot exclude that Rh7 is a UV-sensitive Rhodopsin with a sensitivity maximum below 320 nm. Our halogen lamp emits almost no light at wavelengths below 400 nm (Fig. 1) and the UV-LED with λmax at 370 nm cannot excite Rhodopsins with sensitivity maxima below 320 nm given that the half width of a typical Rhodopsin absorption spectrum is ~100 nm (Salcedo et al. 1999). It is possible that Rh7 is a UV-sensitive Rhodopsin, because it retains primary protein sequence motifs that are characteristic of invertebrate UV pigments, such as the lysine at position 168 (position 110 in Rh1) and the DRY motif (Salcedo et al. 2003; Hu et al. 2014), but it remains questionable that Rh7 can have λmax below 320 nm. Drosophila UV Rhodopsins, Rh3 and Rh4, have sensitivity maxima at 345 and 375 nm, respectively (Feiler et al. 1992) and other invertebrate UV Rhodopsins are sensitive in the same wavelength range (Popp et al. 1996; Smith et al. 1997; Chase et al. 1997; Salcedo et al. 1999). On the other hand, there are also data supporting the notion that Rh7 is sensitive to visible light, because it contains a tyrosine at position 191 that is also present in all other visible-light-detecting Rhodopsins, such as Rh1, Rh2, Rh5, and Rh6, but changed into a phenylalanine in the UV-sensitive Rh3 and Rh4 (Chou et al. 1996). In sum, it is unlikely that we have been unable to excite Rh7 with our lighting system.
Furthermore, we cannot completely exclude that Rh7 shows extremely weak and slow ERG responses as found for the mosquito Rh7 ortholog Op10 when expressed in R1–6 of D. melanogaster (Hu et al. 2014). Hu et al. (2014) used a modified norpA;ninaE17 mutant background, in which norpA is activated only in R1–6. The norpA gene codes for the phospholipase C-β, which is an important component in the phototransduction cascade of all Drosophila photoreceptor cells (Bloomquist et al. 1988). Eliminating norpA in all photoreceptor cells except for R1–6 allowed measuring the activity of the ectopically expressed Op10 without interference from the R7 and R8 cells. In our experiments, R7 and R8 were still active and are responsible for the small receptor potential in ninaE17 and Rh1–Rh7;ninaE17 mutants (Fig. 4). These responses might have masked putative weak responses of Rh7. Nevertheless, we do not think that this is likely for the following reasons: (1) we compared the ERG responses between ninaE17 and Rh1–Rh7;ninaE17 mutants at different light intensities. Thus, at high intensities, we should have seen even small differences provoked by Rh7. (2) The activity of R1–6 provokes on- and off transients in the lamina that were clearly visible in the ERGs of WT flies, but completely absent in ninaE17 mutants (Fig. 4). In none of the measured ERGs of Rh1–Rh7;ninaE17 mutants, on- and off transients were present. This clearly indicates that Rh7 cannot activate the phototransduction cascade in R1–6.
The inability of Rh7 to activate the phototransduction cascade in R1–6 does not mean that Rh7 is not a functional photopigment. It may signal via a different phototransduction cascade that is absent in R1–6. Our data indicate that Rh7 is normally not expressed in R1–6. From sequence alignment, Rh7 is closely related to Drosophila Rh3, Rh4, and Rh5 genes (Senthilan and Helfrich-Förster 2016), suggesting that it is rather expressed in the inner instead the outer receptor cells, and we have first indications that Rh7 is expressed in the inner photoreceptor cell R8 (Kistenpfennig et al. in preparation). In mosquitos, the ortholog of Drosophila Rh7, Op10, is expressed in the inner receptor cell R7 together with Op8 (Hu et al. 2014). Although Op10 was able to activate the phototransduction cascade in R1–6 of D. melanogaster, it could do so only very weakly, suggesting that it usually operates with different signaling molecules that are not present in R1–6 (Hu et al. 2014). That the inner photoreceptor cells of D. melanogaster may use a different phototransduction cascade that works independently from the phospholipase C-ß which has been suggested by previous studies concerning circadian photoreception (Veleri et al. 2007; Szular et al. 2012).
It is also possible that Rh7 signals via a second Rhodopsin present in the inner photoreceptor cells. G-protein-coupled receptors (GPCRs), including Rhodopsin-like GPCRs, can form dimers (Gurevich and Gurevich 2008a, b; Hiller et al. 2013). The assumption that Rh7 may interact with another Rhodopsin to exert its function is supported by the fact that Rh7 and its homologues lack the highly conserved QAKK motif, which is, together with the DRY motif, important for G-protein binding and consequently for the activation of the G-protein-coupled cascade (see above). Thus, Rh7 might signal via a second Rhodopsin and not by itself. Most interestingly, Rh7 has unusually long C- and N-terminal tails that are well suited for the interaction with other proteins (Senthilan and Helfrich-Förster 2016).
At present, we cannot answer the question, in which photoreceptor cell Rh7 is working and what is the way of its action. Its presence in most groups of arthropods, excluding those living in aphotic environments, suggests that it has a conserved function in light signaling either as visual or as non-visual Rhodopsin (Senthilan and Helfrich-Förster 2016). Further studies are needed to answer this interesting question.
This study was funded by the German research foundation (DFG), GK 640 “Natural and artificial photoreceptors” and Collaborative research center SFB 1047 “Insect timing”, project A2. The work was started in 2003 at the University of Regensburg and continued from 2009 onward at the University of Würzburg. We thank Erich Buchner for help with setting up the ERG recordings, Susanne Clemens-Richter for excellent technical support with semi-thin sections, Janina Kempf and Heike Wecklein for performing qPCRs, and Susanne C. Schreiber and Andreas Heinold for help with Rh7 constructs. We are grateful to Susanne Fischer, Cornelia Bleyl, Wolfgang Bachleitner, and Steffen Just for their work on Rh7 that is not included in the present manuscript to Andreas Huber for providing constructs and to Craig Montell for donating the antibody against RDGB. R.G. was sponsored by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
Ethical approval is not required for the study.
Formal consent is not required for the study.
- Kistenpfennig C, Grebler R, Ogueta M, Hermann-Luibl C, Schlichting M, Stanewsky R, Senthilan PR, Helfrich-Förster C (2017) A new Rhodopsin influences light-dependent daily activity patterns of fruit flies. J Biol Rhythms. (in preparation)Google Scholar
- Kretschmar D, Hasan G, Sharma S, Heisenberg M, Benzer S (1997) The Swiss Cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila melanogaster. J Neurosci 17(19):7425–7432Google Scholar
- Posnien N, Hopfen C, Hibrant M, Ramos-Womack M, Murat S, Schönauer A, Herbert SL, Nunes MDS, Arif S, Breuker CJ, Schlötterer C, Mitteroecker P, McGregor AP (2012) Evolution of eye morphology and Rhodopsin expression in the Drosophila melanogaster species subgroup. PLoS One 7(5):e37346CrossRefPubMedPubMedCentralGoogle Scholar
- Romeis B (2015) Mikroskopische Technik. M Mulisch, U Welsch. 19 Auflage. Springer Spektrum, Heidelberg, ISBN 978-3-642-55189-5Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.