Characterization of the ancient photoreceptor RHODOPSIN 7 in Drosophila melanogaster

• Applicant: Dr. Pingkalai Senthilan
• Subject Area: Sensory and Behavioural Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 550267117

Drosophila melanogaster, widely studied for visual processes, contains 7 rhodopsins, of which the first six are well characterized. Despite being known for 24 years, the function of RHODOPSIN 7 (RH7) remains poorly explored and controversial. Our recent findings indicate that RH7 is an ancient rhodopsin that is not directly involved in image formation. Rather, it appears to be involved in basic visual tasks such as contrast vision and circadian clock synchronization, akin to the function of mammalian melanopsins. We observed that Rh7[0] mutants have reduced activity in the dark and have problems to shift their activity into darkness (e.g., during short days or high temperatures in light phases). Additionally, Rh7[0] mutants have a significantly diminished startle response (a brief activity spike) after sudden light-off. TAPIN-seq profiles indicate that Rh7 is not expressed the retina but present in cells that process visual information, including the lamina monopolar cells (LMCs). Our recent electroretinogram (ERG) measurements confirm Rh7 expression in the lamina since the ON transient potential stemming from the LMCs is affected in Rh7[0] mutants but not the receptor potential originating from the retina. In addition, we could rescue the light-off startle response of the Rh7[0] mutants by reintroducing Rh7 in into their LMCs. By protein interaction assays we could show that RH7 functions through a hitherto unknown signaling pathway independent of the Gαq protein, which fits to its distinct protein motifs and its expression in cells lacking the phototransduction machinery. In this project, we will test our hypothesis that RH7 works in LMCs. First, we will apply advanced in situ hybridization techniques and generate T2A-Gal4 lines, which visualize not only transcription but also translation into the RH7 protein. By using MultiColor FlpOut and split-Gal4 techniques we will identify those LMCs that express Rh7, and by targeted RNAi we will knock-down RH7 in these cells and test the effects on the ERG and the behavioral lights-off response. Rescue-experiments in the relevant LMCs will show whether RH7 can reinstall the wild-type ERG and behavioral responses in the Rh7[0] mutant background, and calcium imaging in control and Rh7[0] flies will evaluate the role of RH7 in contrast and motion vision. Additionally, we will examine the dark-fly, which has been kept for 1400 generations in complete darkness and likely possesses a RH7 that is consistently active. Finally, we will identify the molecules interacting with RH7 via protein-protein interaction assays and mass spectrometry, thus revealing its distinct signaling pathway. We believe that these studies will not only enhance our comprehension of RH7 but also broaden our understanding of rhodopsins and their evolutionary history. The evident similarity to mammalian melanopsins, which are expressed in ganglion cells that process visual information, underscores the significance of this research.

• DFG Programme: Research Grants