Journal: bioRxiv

Article Title: Retinal Calcium Waves Coordinate Uniform Tissue Patterning of the Drosophila Eye

doi: 10.1101/2025.06.02.657513

Figure Lengend Snippet: S4A. Representative immunostaining images of Inx2 and Inx3 in 40hAPF retinas. These Z-stack images display the apical layer (A1–A6) , photoreceptor (PR) layer (A1’–A6’) , and basal layer (A1’’–A6’’) . Gap junction plaques containing Inx2 and Inx3 are observed at retinal support cell junctions, labeled with either Dlg1 (red) or E-cadherin (cyan). No Inx2 or Inx3 plaques were detected around PR cell bodies, indicated by empty space in the center of the ommatidium (A6’) , confirming that gap junctions are primarily in retinal non-neuronal cells. Scale bar: 10 μm (A1-6). S4B. Representative immunostaining images of Inx4–8 (Inx-X) in 40hAPF retinas, with innexins (white for B1’–5’) and adherens junctional marker E-cadherin (magenta for B1–4) or septate junctional marker Coracle (B5) . No significant Inx 4–8 signals were detected at ommatidial junctions at 40hAPF retina, with weak Inx5 signal from the early rhabdomere (B2) . Scale bar: 10 μm (B1-5). S4C. Immunostaining of Inx8 in retinas (C1) and photoreceptor axonal terminals (C2, C2’) at 48hAPF, using Inx8 ( green ) and neuronal membrane marker HRP ( blue ) , respectively. No meaningful signal was detected in the retina (C1) , suggesting the subcellular localization of Inx8 in PRs is confined to axonal terminals located in the optic lobe (C2) . Scale bar: 10 μm (C1,2). S4D. Representative images of retinal wave activity, displayed as color-coded 3-minute temporal calcium activity maps from control (D1) and inx8 mutant retinas (D2) during stage 2 waves. Scale bar: 10 μm (D1,2). See also Supplementary Movie S11 . S4E. Quantification of retinal calcium levels from S4D. n ≥ 6 retinas, ns: p ≥ 0.5. Error bars represent SD. S4F. Representative retinal images of transgenic inx1 mutants at 40hAPF, including control (F1, F1’), inx1-RNAi (F2, F2’), gRNA-inx1 (BDSC version) with longGMR-Cas9 (F3, F3’), gRNA-inx1 (custom version) with longGMR-Cas9 (referred as inx1 -sKO ) (F4, F4’), and gRNA-inx1 (BDSC version) with longGMR-G4 > UAS-Cas9 (F5, F5’) , using Inx1 (green) and junctional marker Dlg1 ( magenta ) , respectively. The images are Z-stacks of the retinal apical layer. Scale bar: 10 μm (F1-5). S4G–I. Quantification of Inx1 (G), Inx2 (H), and Inx3 (I) immunostaining signals in inx s-sKO mutant retinas from . n ≥ 14 retinas, ns: 0.5 ≥ p, *: p < 0.5, **: p < 0.01, ***: p < 0.001. Error bars represent SD. S4J. Quantification of vertical pathway frequency for each step indicated above in inx mutant retinas during late stage 1. All values were normalized to control. n ≥ 200 ommatidia from 5 retinas, ns: 0.5 ≥ p, *: p < 0.5, **: p < 0.01, ***: p < 0.001. Error bars represent SD.

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti-Dlg1 (1:200, 4F3, DSHB), rabbit anti-HA (1:200, Cell Signaling Technologies), rat anti-FLAG (1:200, Novus), mouse Anti-V5-tag:DyLight550 (1:200, BioRad), guinea pig anti-inx1 (1:1k, GenScript), rabbit anti-inx2 (1:2k, M. Hoch), guinea pig anti-inx3 (1:1k, GenScript), rabbit anti-inx4 (1:1k, A. Borst), rabbit anti-inx5 (1:1k, A. Borst), rabbit anti-inx6 (1:1k, A. Borst), rabbit anti-inx7 (1:1k, A. Borst), rabbit anti-inx8 (1:1k, A. Borst), chicken anti-GFP (1:1k, EMD), rat anti-Ecad (1:10, DSHB, DCAD2), AlexaFluor660 conjugated Phalloidin (Invitrogen, 1:200), and AlexaFluor405 conjugated Goat Anti-HRP (Jackson ImmunoResearch, 1:100).

Techniques: Immunostaining, Labeling, Marker, Membrane, Activity Assay, Control, Mutagenesis, Transgenic Assay