Predicted RH7 structure and expression pattern in the Drosophila optic system.

(A) Schematic representation of the predicted 3D structure of RH7 (GH14208p) based on AlphaFold (Jumper et al., 2021). The structure is color-coded using a continuous spectral gradient ranging from blue (N-terminus) to red (C-terminus). The three intracellular loops are highlighted and labeled in numbered circles: (1) ICL1 with the LRTPXN motif, (2) ICL2 with the highly conserved DRY motif, and (3) ICL3, which lacks the typical QAKKMNV motif. (B) Left: Schematic representation of the Drosophila melanogaster optical system. The presumed Rh7-expressing cells, according to Davis et al, (2020) are shown (in color) in addition to retinal receptor cells (in black). Right: Heatmap of relative gene expression across Drosophila optic lobe cell types. TPM values were obtained by TAPIN sequencing (Davis et al., 2020) and normalized per gene (100% = maximum TPM per gene). Gray scale: from white (0%), to black (100%). Genes are shown on the x-axis, and cell types on the y-axis.

Fly lines

Primers

Antibodies used for immunohistochemistry

Evolutionary conservation, distribution, and signaling properties of RH7.

(A) Phylogenetic tree of 470 r-opsin protein sequences from 142 selected species, highlighting the clades containing the Drosophila rhodopsins: RH1, RH2, and RH6 (dark blue); RH3, RH4, and RH5 (purple); and RH7 (red). Early-diverging r-opsins are shown in brown, and vertebrate melanopsins in orange. Drosophila opsins are marked with stars in the corresponding colors. The four tardigrade RH7-like opsins are represented with a red triangle, and the human melanopsin is indicated by an orange square. (B) Distribution of RH7 and DCRY across 360 analyzed panarthropod species: 57% possess both genes (black), 12% possess only RH7 (red), 1% possess only DCRY (blue), and 32% lack both proteins (grey). (C) GsX assay showing that RH7 robustly activates the Gαz chimera in a light-dependent manner, with minimal differences between 9-cis-retinal and all-trans-retinal. No activation was detected with the Gαq chimera.

Altered locomotor activity and light-off responses in Rh70 mutant flies.

(A) Left, average locomotor activity profiles of wild-type (WT) and Rh70 mutant flies under standard conditions (12 h light (30 µW/cm²):12 h dark, 25 °C). Right, box plots showing daytime average activity, siesta onset, siesta duration, maximum activity after lights-off (LOFF), and LOFF response duration. (B) Average activity profiles of WT, Rh70, per0, and per0;;Rh70 flies during a 12 h light (30 µW/cm²):12 h dark cycle at 25 °C. During the light phase, the light was switched off for 30 min every 1.5 h to assess the light-off startle response. Activity was normalized to the mean activity of each fly across the entire 24 h recording period. Non-normalized activity profiles are shown in Figure 3–figure supplement 2A. (C) Light-off startle responses after 3.5 h of light exposure at 5, 30, or 120 µW/cm², followed by a 30 min dark period. (D) 4h assay with dimmed light. After 3.5 h of light exposure, illumination was reduced for 30 min to either 5 or 30 µW/cm² instead of being switched off completely (****p <0.0001, ***p <0.001, **p <0.01, *p <0.05, Mann-Whitney U-Test, BH correction, R-studio).

Expression pattern and cellular localization of Rh7 in the optic lobe and circadian neurons.

(A) Relative qPCR analysis revealed strong Rh7 expression in the lamina, followed by the brain, with minimal background expression in the retina. (B) Rh7 in situ HCR in wild-type (WT) controls revealed strong Rh7 signals in the lamina (LA) and medulla (ME), and weaker signals in the central brain (CB). (C) Rh70 mutants showed no detectable Rh7 in situ HCR signal, confirming the specificity of the probe and the loss of Rh7 expression in the mutant. (D) Rh7-Gal4-driven UAS-GFP expression revealed robust labeling in the optic system, particularly in the lamina and medulla, as well as in various brain cells. (E) Rh7-Gal4-driven MCFO labeling identified lamina neurons L5 and the medulla neuron Mi1. Right: schematic illustration of the detected neurons in the optic lobe. (F) Immunostaining of Rh7-Gal4>UAS-GFP flies with anti-GFP and anti-PER antibodies revealed co-labeling in clock neurons, suggesting that a subset of circadian neurons expresses Rh7. Open arrows mark DN1p and arrows mark DN2; double arrows mark DN1a. (G) & (H) in situ HCR in Rh7-Gal4>UAS-GFP flies revealed that all GFP-labeled cells in the lamina (G) and medulla (H) also showed Rh7 transcript signal, confirming Rh7 expression in these neurons. Scale bar in B, C, D represents 100 µm, in E, G, H 25 µm, in F, G’ 10 µm and in G’’ 1 µm.

Functional characterization of Rh7 rescue and Darkfly rescue lines.

(A) & (B) Normalized average daily activity of Darkfly and rescue fly lines recorded under standard conditions (12 h light (30 µW/cm²):12 h dark, 25 °C). During the light phase, lights were turned off for 30 min every 1.5 h. (A) Darkfly flies exhibit an enhanced startle response to light-off. (B) No-rescue (Rh7-Gal4; Rh70>UAS-empty; Rh7⁰) display a phenotype similar to Rh7 mutants, whereas the Rh7 rescue line (Rh7-Gal4; Rh70>UAS-Rh7; Rh7⁰) resembles WT controls. The Rh7Darkfly (Rh7-Gal4; Rh70>UAS-Rh7Darkfly; Rh7⁰) rescue shows an enhanced startle response similar to Darkfly, particularly during the second half of the day. (C) RH7 rescue alters daytime activity and siesta parameters under LD conditions. Both Rh7 rescue and Rh7Darkfly rescue flies showed reduced average daytime activity compared to no-rescue flies. Similar effects were observed for siesta length, including a significant difference between Rh7 rescue and Rh7Darkfly rescue flies. For siesta beginning (h), significant differences were observed between no-rescue fliesand Rh7Darkfly rescue flies, as well as between Rh7 rescue and Rh7Darkfly rescue flies. (D) To measure the startle response, the 4-hour assay was performed. The Rh7 rescue line restored the enhanced startle response that was absent in the no-rescue mutants, which behave like Rh7⁰ mutants (p < 0.0001). The Rh7Darkfly rescue showed an even stronger startle response, significantly higher than the Rh7 rescue line (p < 0.001). (E) Optomotor response assays show that no-rescue flies behave like Rh7⁰ mutants, with no significant difference between them. Rh7 rescue flies respond like WT controls. Both Rh7⁰ mutants and no-rescue flies differ significantly from WT and Rh7 rescue flies (p < 0.01). Darkfly and Rh7Darkfly rescue flies do not differ from each other but show significant differences compared to WT and Rh7 rescue flies (***p < 0.001, **p < 0.01, *p < 0.05, Mann-Whitney U-Test, BH correction, R-studio).

Structural features and hypothetical activation states of RH7.

(A) Protein lengths of Drosophila and human opsins are shown alongside randomly selected non-opsin GPCRs. General GPCRs are significantly longer, primarily due to extended N- and C-termini, whereas opsins are shorter and largely confined to the transmembrane core. RH7 and OPN4 (melanopsin) display intermediate protein lengths relative to both groups. The red bars represent the N- and C-termini of the proteins, and the black bars represent the core protein, spanning from the 1st to the 7th transmembrane domain. Additional information, including full protein names and UniProt IDs, is provided in Supplementary table 1. (B) Left: Predicted 3D structure of RH7 based on AlphaFold, representing a closed conformation. Center: A putative open conformation that may result from light-induced conformational changes and subsequent C-terminal phosphorylation (magenta), potentially promoting G protein binding, as suggested by Maggio et al. (2023). Right: Hypothetical structure of RH7 in the Darkfly variant, possibly stabilized in an open, constitutively active state, independent of light.