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. 2026 Feb 26;15(2):bio062463. doi: 10.1242/bio.062463

Gap junction-mediated signaling coordinates Rhodopsin coupling for Drosophila color vision

Xuanshuo Zhang 1, Ryoki Shinjo 1, Manabu Kitamata 2, Shinichi Otsune 1, Hideki Nakagoshi 1,✉
PMCID: PMC12969764  PMID: 41555831

ABSTRACT

The Drosophila compound eye is composed of approximately 800 ommatidia, and every ommatidium contains eight photoreceptor cells, six outer cells (R1-R6) and two inner cells (R7 and R8), and accessory cells (cone and pigment cells). The expression of rhodopsin genes in R7 and R8 is highly coordinated through an instructive signal from R7 to R8. The activity of the homeodomain protein Defective proventriculus in R1 is also required to transmit this instructive signal, suggesting that cell–cell communication between R7, R1, and R8 is important to generate the pattern of Rh expression in R7/R8 (Rhodopsin coupling). As cell junctions play crucial roles in maintaining the structural and functional integrity of tissues, we tested whether cell junction proteins are involved in the interactions between photoreceptor cells. Here, we demonstrate that gap junction proteins innexin 2 and innexin 7 in accessory cells are necessary for transmitting signals from R7 to R8. In addition, Notch-mediated accessory cell development and Rhodopsin coupling in R7/R8 are highly correlated. Our results provide evidence that functional coupling of two different neurons, R7 and R8, is established through gap junction-mediated signaling from adjacent accessory cells.

Keywords: Drosophila, Eye, Gap junction, Innexin, Opsin

Summary: Functional coupling of two different neurons in Drosophila photoreceptor cells is established through gap junction-mediated signaling from adjacent accessory cells.

INTRODUCTION

Cell-cell junctions regulate tissue homeostasis and are important for tissue barrier function, cell proliferation, and migration (Garcia et al., 2018; Guillot and Lecuit, 2013). Invertebrate epithelial cells have two main junctional domains: the apical adherens junction (AJ) and the lateral septate junction (SJ). The SJ serves as the occluding junction and is functionally similar to the vertebrate tight junction. In addition, SJ proteins have various non-occluding roles, for example, in wound healing, planar cell polarity, and apical-basal polarity (Rice et al., 2021). Drosophila Crumbs is a central regulator of epithelial apical-basal polarity, and it regulates AJ localization. This process is crucial for the morphogenesis of light-gathering organelles, rhabdomeres, in photoreceptor (PR) cells (Izaddoost et al., 2002; Pellikka et al., 2002; Pichaud, 2014). Thus, cell junction proteins not only mediate cell–cell adhesion but also can generate intracellular signals in various developmental contexts. Another important cell–cell junction is the gap junction that spans two plasma membranes, allowing cells to exchange ions and small molecules directly (Bauer et al., 2005; Guiza et al., 2018; Herve and Derangeon, 2013). These gap junction channels are formed by the alignment of two hexameric hemichannels of vertebrate connexins or invertebrate innexins. Mutations in human connexins can lead to diseases, supporting the physiological importance of gap junction channels. For example, mutations in connexin 26 (Cx26) and Cx30 are responsible for deafness (del Castillo et al., 2002; Denoyelle et al., 1998; Grifa et al., 1999; Kelsell et al., 1997), and mutations in Cx50 and Cx46 are responsible for inherited cataracts (Mackay et al., 1999; Shiels et al., 1998).

The Drosophila compound eye is composed of approximately 800 ommatidia, and every ommatidium contains a cluster of eight PR cells surrounded by accessory cells. Accessory cells consist of four cone cells, two primary pigment cells (PPCs), and a lattice of secondary (2°) and tertiary (3°) pigment cells (Fig. 1A). PR cells have a light-gathering structure (rhabdomere) and are classified into two groups based on the position of the rhabdomere. Six outer PR cells (R1-R6) have their rhabdomeres in the outer position and express Rhodopsin 1 (Rh1), which is involved in object (motion) detection. Two inner PR cells (R7 and R8) have their rhabdomeres in the inner position, vertically aligned on the same axis in an ommatidium, and express Rh3-Rh6, which are involved in color vision (Fig. 1B). During retinal development, specification of cone cell fate is induced by combinatorial epidermal growth factor receptor (EGFR) and Notch (N) pathways originating from PR cells (Nagaraj and Banerjee, 2007), and these cone cells have direct contact with PR cells during the four-cone cell stage and subsequent developmental stages (Fig. 1A). Thereafter, at 18 h after puparium formation (APF), the two interommatidial cells adjacent to the anterior–posterior cone cells receive high N signals, resulting in the expression of the immunoglobulin cell adhesion molecules Hibris (Hbs) and Sticks-and-Stones (Sns) to form PPCs (1°, light brown in Fig. 1A) (Bao, 2014). Interommatidial lattices of 2° and 3° pigment cells are formed with N-mediated signals to remove unneeded cells, while cone cells and PPCs oppose this signal through activation of EGFR signaling (Miller and Cagan, 1998). At 40 h APF, differentiation of all accessory cells is completed (Fig. 1A). Subsequently, rhabdomere morphogenesis and expression of rhodopsin genes are induced during the late pupal stage (Earl and Britt, 2006). R7 expresses stochastically either the UV-sensitive Rh3 or Rh4, whereas R8 expresses either blue-sensitive Rh5 or green-sensitive Rh6. Rh3 expression in R7 is coupled with Rh5 in R8, whereas Rh4 expression in R7 is coupled with Rh6 in R8. This Rhodopsin coupling is highly coordinated through instructive signals from R7 to R8 (Chou et al., 1999) and generates two major ommatidial subtypes, pale (Rh3/Rh5) and yellow (Rh4/Rh6) types. These two ommatidial subtypes are distributed randomly throughout the whole eye, and the ratio is about 30% pale type and 70% yellow type (Mollereau and Domingos, 2005; Wernet and Desplan, 2004) (Fig. 1B). Activity of the homeodomain protein defective proventriculus (Dve) is required to suppress rh3 in yellow-type R7, and that in R1 is required to transmit the instructive signal from R7 to R8 (Johnston et al., 2011; Kitamata et al., 2024) (Fig. 1B).

Fig. 1.

Fig. 1.

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Gap junctions in accessory cells are required for the instructive signal. (A) A schematic representation of accessory cell development. Photoreceptor cells R7, R8, and R1 (yellow circles) have direct contact with each other at the four-cone cell stage. Anterior–posterior (AP) and dorsal–ventral cone cells are shown in blue and light blue, respectively. Primary pigment cells (PPCs: light brown, 1°) are induced by AP cone cells, and secondary (2°) and tertiary (3°) pigment cells (dark brown) form a lattice of ommatidia. APF, after puparium formation. (B) Two major subtypes of ommatidia. Rectangles and circles represent rhabdomeres and nuclei of photoreceptor cells, respectively. Rhodopsin expression (Rh1, Rh3-Rh6) in rhabdomeres is shown in differently colored rectangles. Photoreceptor cells R1-R8 are shown in white characters. Dve expression in nuclei is shown with magenta circles. The instructive Rh5-inducing signal from R7 to R8 is shown by a curved light blue arrow. Horizontal sections of R7 and R8 layers show the position of rhabdomeres and cell bodies. (C) Rhodopsin expression in ommatidia of inx2 or inx7 knocked down in accessory cells using the spa-GAL4 driver. Ommatidia of yellow type (Rh4/Rh6) and pale type (Rh3/Rh5) are marked with yellow and blue circles, respectively. Ommatidia with mis-coupling (Rh3/Rh6) are marked with white squares. Expression of Rh3/Rh5 (blue) and Rh4-EGFP (green) is shown. In the R8 layer, Rh4-expressing R7 (green) is visible at the periphery. Rhabdomeres are labeled with phalloidin (red). Scale bars: 5 µm. (D) Rhodopsin coupling of inx KD ommatidia of the indicated genotypes. The ratio of yellow (Rh4/Rh6), pale (Rh3/Rh5), and mis-coupling ommatidia (Rh3/Rh6) is shown in green, blue, and orange, respectively. The number of scored ommatidia (n) and compound eyes (N) is shown. The ratio of mis-coupling in a compound eye is plotted below. Error bars show the standard error of the mean (s.e.m.).

In this study, we analyzed the requirements of cell junction-associated molecules for the instructive signal. We provided evidence showing that the gap junction molecules innexin 2 (Inx2) and innexin 7 (Inx7) in accessory cells are necessary for transmitting signals from R7 to R8.

RESULTS AND DISCUSSION

Gap junctions in accessory cells are required for the instructive signal

Rhodopsin expression in inner PR cells (R7/R8) is highly coordinated by the Rh5-inducing instructive signal from R7 to R8 (Fig. 1B). During pupal development, the cell body of R1 becomes located between those of R7 and R8. Furthermore, the Dve activity in R1 is required for transmission of this instructive signal. We tested whether cell junction molecules are involved in the transmission of the instructive signal. RNA interference (RNAi)-mediated knockdown (KD) of cell junction molecules for AJ (DE-cadherin), SJ (Discs large, Coracle), and gap junctions was carried out in whole eyes (Fig. S1). Ommatidia with KD of several gap junction genes, including Shaking-B (ShakB), innexin 2 (inx2), and innexin 7 (inx7), showed Rh3/Rh6 mis-coupling expression, which is thought to be a default state of Rhodopsin expression. Thus, these gap junction proteins seem to be required for transmission of the instructive signal. ShakB (also known as Inx8) has several isoforms, and ShakB (neural) is expressed in outer PR cells (R1-R6) (Stebbings et al., 2002). Inx2 is expressed in all eye disc cells during larval development, and its expression is restricted to the pigment cells during the pupal stage (Richard and Hoch, 2015). In addition, inx7 is also expressed in PPCs (Stebbings et al., 2002). Thus, we tested the requirements of Inx2 and Inx7 in accessory cells. The spa-GAL4 driver can induce upstream activating sequence (UAS)-mediated expression in cone cells and PPCs (Nagaraj and Banerjee, 2007) (Fig. S2). Ommatidia with inx2 or inx7 KD in accessory cells substantially induced mis-coupling (Rh3/Rh6) (Fig. 1C,D), suggesting that Inx2 and Inx7 are required in accessory cells to transmit the instructive signal from R7 to R8.

Cone cells form the corneal lens and cap the distal region of the rhabdomere, and they send inter-retinular fibers that contact the cell body of PR cells (Charlton-Perkins et al., 2021). The interaction between cone cells and PR cells also occurs in the four-cone cell stage during early pupal development (Tomlinson, 1985) (Fig. 1A). Cone cells express glia-associated genes, such as prospero and dPax2, and regulate rhabdomere morphogenesis and PR cell physiology (Charlton-Perkins et al., 2017). Pigment cells ensheathe the PR cell body and prevent light scattering between ommatidia, and they are also required for visual response (Wang and Montell, 2005). Because spa-Gal4-mediated KD for gap junction molecules affect both cone and pigment cells, it remains unclear which type of accessory cell is critical for Rhodopsin coupling.

Gap junction-mediated signaling is required during early pupal development

Gap junctions allow cells to exchange ions and small molecules directly. It remains unclear whether this transport activity is required between differentiated PR cells and accessory cells. Another possibility is that gap junction-mediated activity is required for functional development of both accessory cells and PR cells. To examine the roles of gap junction proteins, we carried out temporarily regulated inhibition of inx2 or inx7 in whole eyes using the temperature-sensitive GAL80 (GAL80ts) system (Fig. 2A). A temperature shift to 30°C relieves the blockade of GAL80-mediated inhibition of GAL4/UAS system and induces expression of double-stranded RNA for inx2 or inx7. A temperature shift from 0 h APF or 12 h APF to adult eclosion resulted in increased mis-coupling (Rh3/Rh6). However, a temperature shift from 24 h APF to adult eclosion did not affect the ratio of mis-coupling that is variable in different genetic backgrounds (Fig. 2B,C). Thus, the Inx2 or Inx7 activity after the mid-pupal stage is not required for establishment of the instructive signal. Considering the time lag of recovery from the GAL80-mediated blockade at 18°C and of RNAi induction after a temperature shift to 30°C, the required time window of Inx2 or Inx7 activity seems to be in the early pupal stages. These results suggest that gap junction proteins are required for functional accessory cell development to transmit the signal to the PR cells.

Fig. 2.

Fig. 2.

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Gap junction-mediated signaling is required during early pupal development. (A) Rhodopsin expression in ommatidia in which inx2 or inx7 is temporarily knocked down in whole eyes using the GMR-GAL4 driver with tub-GAL80ts. Test progenies were shifted to the restrictive temperature (30°C) from the indicated time points (0 h, 12 h, and 24 h APF) to adult eclosion. Control progenies (cont.) were reared at the permissive temperature (18°C). Ommatidia of yellow type (Rh4/Rh6) and pale type (Rh3/Rh5) are marked with yellow and blue circles, respectively. Ommatidia with mis-coupling (Rh3/Rh6) are marked with white squares. Expression of Rh3/Rh5 (blue) and Rh4-EGFP (green) is shown. Rhabdomeres are labeled with phalloidin (red). Scale bars: 5 µm. (B,C) The ratio of mis-coupling in a compound eye of the indicated genotypes is plotted. Error bars show s.e.m. The number of scored ommatidia (n) and compound eyes (N) is shown.

N-mediated accessory cell development correlates with the instructive signal

As N signaling plays crucial roles in accessory cell development, forced expression of a dominant-negative form of N (N[DN]) induces abnormal development of both cone and pigment cells. To examine the relationship between accessory cell development and Rhodopsin coupling, we carried out temporarily regulated inhibition of N in whole eyes (GMR>N[DN] GAL80ts, Fig. S3A,B) and in accessory cells (spa>N[DN] GAL80ts, Fig. S3A,C). A temperature shift from 0 h APF did not show morphological abnormality in accessory cell development, and Rhodopsin coupling was also normal (Fig. S3B,C). In contrast, a temperature shift from the late third larval instar (18 h before puparium formation, −18 h APF) showed abnormal accessory cell development associated with Rhodopsin mis-coupling (Fig. S3B,C). As described above, considering the time lag of recovery from the GAL80-mediated blockade at 18°C and of N[DN] induction after a temperature shift to 30°C, inhibition of N signaling in the early pupal stage appears to affect both accessory cell development and establishment of the instructive signal. These results suggest that N-mediated accessory cell development is required to establish the instructive signal between PR cells, R7, and R8. To determine the precise time window of N signal requirement for Rhodopsin coupling, a temperature-sensitive allele of N (Nts) was used to inhibit accessory cell development. Because accessory cell development is completed by 40 h APF, we tested four time windows of temperature shifts for N inhibition: 0-8 h, 10-16 h, 16-24 h, and 24-32 h APF (Fig. 3A). A temperature shift to 30°C during 10-16 h or 16-24 h APF caused abnormal accessory cell development, such as excess PPCs, disorganized 2° and 3° pigment cells, and duplicated bristles (Fig. 3B-E). In addition, ommatidia with a 30°C shift during 10-16 h APF resulted in incomplete rhabdomere development in R8. Thus, Rhodopsin coupling under this condition could not be tested. The other two conditions showed normal morphology of accessory cells with normal Rhodopsin coupling (Fig. 3F,G). Ommatidia with a 30°C shift during 16-24 h APF resulted in abnormal accessory cell development and showed mis-coupling of Rh3/Rh6 (Fig. 3F,G). As this time window corresponds to PPC induction, the disorganized 2° and 3° pigment cells and the duplicated bristles appear to be due to incomplete PPC development (Figs 1A and 3D,E). Taken together, these results strongly suggest that accessory cell development and Rhodopsin coupling in PR cells are highly coordinated through the gap junction-mediated signaling pathway.

Fig. 3.

Fig. 3.

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Notch (N)-mediated accessory cell development correlates with the instructive signal. (A) A schematic representation of eye development. The Inx activity during the early pupal stages (magenta) is required to establish the Rh5 instructive signal (blue). Accessory cell (AC) specification (green) is completed by 40 h APF, and this process requires N activities (black). L3, third-instar larva; PP, prepupa. (B-E) Eye morphology of Nts males at 40 h APF. Control progenies (B) were reared at the permissive temperature (18°C). (C-E) Test progenies shifted to the restrictive temperature (30°C) during 16-24 h APF have abnormal ACs (arrowheads), such as excess PPCs (C), disorganized 2° and 3° pigment cells (D), and duplicated bristles (E). The cell membrane is labeled with an anti-Arm antibody. (F) Rhodopsin expression in ommatidia of Nts males. Test progenies were shifted to the restrictive temperature (30°C) during the indicated periods (0-8 h, 16-24 h, and 24-32 h APF) to adult eclosion. Ommatidia of yellow type (Rh4/Rh6) and pale type (Rh3/Rh5) are marked with yellow and blue circles, respectively. Ommatidia with mis-coupling (Rh3/Rh6) are marked with white squares. Expression of Rh3/Rh5 (blue) and Rh4-EGFP (green) is shown. Rhabdomeres are labeled with phalloidin (red). Scale bars: 5 µm. (G) The ratio of mis-coupling in Nts males. A temperature shift during 10-16 h APF resulted in abnormal morphology of R8 rhabdomeres, and Rhodopsin coupling is not determined (ND). The ratio of mis-coupling in a compound eye of the indicated temperature shift conditions is plotted. Error bars show s.e.m. The number of scored ommatidia (n) and compound eyes (N) is shown.

Pigment cells might be more important, as Inx2 and Inx7 are highly expressed in pigment cells (Richard and Hoch, 2015; Stebbings et al., 2002). N signaling regulates accessory cell development with induction of Hbs in PPCs (Bao, 2014; Blackie et al., 2021), and Rh5 expression in R8 is disrupted in hbs mutants (Tan et al., 2020). These reports are consistent with our observation that N-mediated accessory cell development is closely associated with the Rh5-inducing instructive signal from R7 to R8. A hemichannel, one half of a gap junction channel (Guiza et al., 2018; Yasarbas et al., 2024), might transport a signaling molecule onto the PR cells just like glia–neuron intercommunication (Dossi et al., 2024; Visser et al., 2024). However, ShakB (Inx8) is highly expressed in PR cells (R1-R6) and is also required to establish the instructive signal. Thus, we favor the possibility that gap junction channels between accessory cells and PR cells are required to establish Rhodopsin coupling (Fig. 4B). Our results suggest that N-mediated accessory cell development and gap junction-mediated accessory cell–PR cell communication are required for PR cell maturation, such as rhabdomere morphogenesis and their Rhodopsin coupling.

Fig. 4.

Fig. 4.

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Calcium signaling is required for the instructive signal. (A) Rhodopsin coupling of ommatidia expressing a dominant-negative form of the IP3R (IP3R[DN]) or PKA (PKA[DN]) in whole eyes (GMR-GAL4) or in accessory cells (spa-GAL4). The ratio of yellow (Rh4/Rh6), pale (Rh3/Rh5), and mis-coupling ommatidia (Rh3/Rh6) is shown in green, blue, and orange, respectively. The ratio of mis-coupling in a compound eye of the indicated genotypes is plotted. Error bars show s.e.m. The number of scored ommatidia (n) and compound eyes (N) is shown. (B) A schematic model for gap junction-mediated instructive signal. In yellow-type ommatidia, Spineless (Ss) induces Rh4 and Dve expression in R7, and Dve represses Rh3 expression (Johnston et al., 2011). In pale-type ommatidia, Rh3-expressing R7 sends a signal to the adjacent R1. The activated form of Dve (Dve*) in R1 induces Rh5 expression in R8 through a relief of repression mechanism (Kitamata et al., 2024). In accessory cells, gap junction-mediated calcium waves (green circles) are induced (Choi et al., 2025). This intercommunication through gap junctions, including Inx2/Inx7 (yellow), might regulate accessory cell development and indirectly affect the Rh5-inducing instructive signal shown as a black arrow (1). Alternatively, homotypic junctions of ShakB (blue) between PR cells and heterotypic junctions of ShakB and Inx2/Inx7 might mediate transportation of some permeable molecules to regulate the instructive signal (2).

Calcium signaling is required for the instructive signal

Gap junctions can act as channels that exchange ions and small molecules directly, and calcium signaling or cAMP transportation through gap junctions regulates several types of cell differentiation. For example, gap junction-mediated calcium signaling regulates blood progenitor cell fate decisions in hematopoiesis (Ho et al., 2021) and specifies border cell fate during oogenesis (Sahu et al., 2017). Gap junction-mediated cAMP transportation from the niche controls stem cell progeny differentiation during oogenesis (Tu et al., 2023). To check the effect of calcium signaling and cAMP pathways on Rhodopsin coupling, a dominant-negative form of the IP3 receptor (IP3R) or protein kinase A (PKA) was induced. KD of IP3R, but not of PKA, apparently induced Rh3/Rh6 mis-coupling (Fig. 4A).

IP3 causes the release of Ca2+ into the cytoplasm and produces a calcium wave (Dupont et al., 2007). The specification of border cells requires Inx2 that mediates calcium flux between follicle cells. Moreover, among intestinal epithelial cells, high Ca2+ spreads through Inx2–Inx7 gap junctions during the healing process after injury (Petsakou et al., 2023). During eye development, calcium waves propagate through pigment cells, and they are required for endfeet stress fiber contraction (Ready and Chang, 2021). A recent report clearly shows that retinal calcium waves in accessory cells coordinate uniform tissue patterning through gap junctions (Choi et al., 2025). Thus, it is assumed that Inx2- and Inx7-mediated calcium signaling is involved in pigment cell development and subsequent specification of PR cells to establish Rhodopsin coupling. As retinal calcium waves are observed in accessory cells, but not in PR cells (Choi et al., 2025), there are two possible mechanisms for gap junction-mediated establishment of the Rh5-inducing instructive signal. (1) Inx2- and Inx7-mediated calcium signaling is required for accessory cell development, and completion of accessory cell development indirectly affects PR cell maturation independent of gap junction channel activities (Fig. 4B-1). (2) Homotypic junctions of ShakB between PR cells and heterotypic junctions of ShakB and Inx2/Inx7 might mediate transportation of some permeable molecules to PR cells and trigger the instructive signal (Fig. 4B-2).

Our results strongly suggest that the Rh5-inducing instructive signal from R7 to R8 is regulated by adjacent accessory cells through gap junction-mediated signaling. An intriguing possibility is that gap junction channel-associated molecules in accessory cells act as gatekeepers to control the precise timing of channel opening to PR cells and regulate subsequent Rhodopsin coupling. Further characterization of this signaling pathway will provide insights into the mechanism of functional specification of PR cells during retinal development.

MATERIALS AND METHODS

Drosophila strains

Flies were reared on a standard yeast and cornmeal-based diet under a 12 h light/dark cycle at 25°C. The following GAL4/UAS lines were used: GMR-GAL4 (Bloomington Drosophila Stock Center, BL#1104), spa-GAL4 (BL#26656), UAS-Stinger (BL#84277), UAS-IP3R[DN] (BL#602868), UAS-PKA[DN] (BL#35550), UAS-ogre (inx1)-IR JF02595 (BL#27283), UAS-ogre (inx1)-IR HMS02764, UAS-inx2-IR-2, UAS-inx4-IR-1, UAS-inx5-IR-1, UAS-inx6-IR-1, UAS-inx6-IR-3, UAS-Shaking-B (inx8)-IR-3, UAS-DE-Cad-IR-1 (NIG), UAS-inx3-IR v39094, UAS-inx7-IR v22949, UAS-dlg-IR v41134, UAS-cora-IR v9788 (VDRC), and UAS-d.n.N (N[DN]) (Go et al., 1998; Tanaka et al., 2007). For temporarily regulated inhibition of inx and N, tub-GAL80ts (BL#7017) and Nts (BL#2533) were used. GMR-wIR (a gift from Richard Carthew) was used to induce white RNAi in adult eyes (Lee and Carthew, 2003). Rh4 expression was monitored with Rh4-EGFP (BL#7456). Oregon-R (OR) or w;GMR-wIR Rh4-EGFP was used to obtain heterozygous control flies for GAL4 and UAS lines.

Immunohistochemistry

The compound eyes of adults and pupae were dissected in phosphate-buffered saline (PBS). Fixation of eyes was performed in a solution of 4% formaldehyde/PBS and 0.3% Triton X-100 for 15 min, and then they were washed three times with a solution of PBS and 0.3% Triton X-100. The following primary antibodies were used: mouse anti-Rh3 (2B1, a gift from Steven Britt, University of Texas, USA, 1:20), mouse anti-Rh5 (7F1, a gift from Steven Britt, 1:200), and mouse anti-Arm N2-7A1 (1:200, DSHB, RRID:AB_528089). The secondary antibody, goat anti-mouse IgG-Cy5 (#115-175-146, Jackson ImmunoResearch Laboratories, RRID:AB_2338713), was used for detection. Phalloidin-TRITC (Sigma-Aldrich, RRID:AB_2315148) was used to stain actin fibers of the rhabdomere. Confocal images of 0.2-1.22 µm sections were obtained with a confocal microscope (Olympus FV1200) and were processed using FluoView (Olympus) and Photoshop (Adobe).

Statistical analysis

Statistical analysis was performed as described previously (Kitamata et al., 2024). Statistical comparison of Rhodopsin coupling frequency distribution was calculated with a chi-square test from the total number of ommatidial subtypes. Deviation in the ratio of ommatidial subtypes in each compound eye is shown with the standard error of the mean (s.e.m.). Statistical comparison of mis-coupling was calculated from the total number of Rh6-expressing ommatidia in Rh3-expressing ommatidia with Fisher's exact test. Individual data points in the ratio of mis-coupling in each compound eye are also plotted with s.e.m. The significance of differences between the control and test progenies was analyzed using Prism 6 (GraphPad software). The levels of significance are indicated by asterisks: *P<0.05, **P<0.01, ***P<0.001, and ns (not significant).

Supplementary Material

biolopen-15-062463_review_history.pdf (413.6KB, pdf)
Supplementary information
biolopen-15-062463-s1.pdf (4.4MB, pdf)
DOI: 10.1242/biolopen.062463_sup1

Acknowledgements

We would like to express our gratitude to Steven Britt for providing the antibodies; Richard Carthew for providing the GMR-wIR; and to the Bloomington Drosophila Stock Center (BDSC), the Vienna Drosophila Resource Center (VDRC), the Drosophila Genomics and Genetic Resources (DGGR, Kyoto Stock Center), and the National Institute of Genetics (NIG) for supplying the fly strains; Core-Facility at Okayama University for the use of a confocal microscope (CFPOU DIA_717).

Footnotes

Author contributions

Conceptualization: H.N.; Data curation: X.Z., R.S., M.K., S.O.; Formal analysis: X.Z., S.O.; Funding acquisition: H.N.; Investigation: X.Z., R.S., M.K.; Methodology: M.K., S.O.; Project administration: X.Z., R.S.; Resources: H.N.; Supervision: H.N.; Visualization: X.Z., M.K., S.O.; Writing – original draft: X.Z.; Writing – review & editing: M.K., S.O., H.N.

Funding

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant No. JP15029244 awarded to H.N.). Open Access funding provided by Okayama University. Deposited in PMC for immediate release.

Data and resource availability

All relevant data and details of resources can be found within the article and its supplementary information.

Peer review history

The peer review history is available online at https://journals.biologists.com/bio/lookup/doi/10.1242/bio.062463.reviewer-comments.pdf

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DOI: 10.1242/biolopen.062463_sup1

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