List of HCR probes for cyp26 and rar genes

Primers used for qRT PCR

Regional differences in photoreceptor outer segment (OS) length are evident by 120 hours post-fertilization (hpf).

A, Schematic of the zebrafish head positioned laterally. The orange area in the eye indicates the acute zone (AZ). Dashed blue rectangles indicate the imaged regions. B, Timeline of cone development and visual behavior in zebrafish. C, Schematic of a photoreceptor and its cellular components: the OS, mitochondria (mito), and nucleus. A black line indicates the length of the OS, which was measured in (D). D, Confocal images of UV cones (Green) and UV opsin immunostaining (magenta) in the Tg(opn1sw1:GFP) transgenic line. Note weak UV opsin staining is seen outside of the OS (open arrowhead), beneath mitochondoria (arrow) at 120 hpf. E, A bar graph of mean UV cone OS length at 72, 96 and 120 hpf for the AZ, dorsal and nasal regions. n= 7 (AZ), 7 (dorsal), 8 (nasal) at 3 dpf, and n=8 for all other ages and regions. Open circles: data from individual fish. Error bars: Standard deviation. One-way ANOVA plus Tukey’s post-hoc test, *: p < 0.05.

cyp26a1 and c1 are expressed by Müller glia in the retinal areas with low Retinoic acid signalling activity which inversely correlates with regional differences in UV cone OS length

A, Schematic of retinoic acid signalling reporter construct in Tg(12xrare-ef1a:EGFP)sk71transgenic zebrafish. Retinoic acid receptor (RAR) and Retinoid X receptor (RXR) complex bind to Retinoic Acid Response Element (RARE) and transactivate downstream gene expression upon binding to Retinoic acid (RA). The reporter construct contains twelve repeats of RARE sequence followed by a minimum promoter ef1a and GFP coding sequence. B, A confocal image of a Tg(RARE:GFP) transgenic eye at 120 hpf. C, Line profiles of GFP intensity (green, arbitrary unit, a.u.) and UV cone OS length (magenta) across the eye, measured from the ventral region counter-clock direction as shown in the schematic. The data for UV cones OS length is from [3]. n=3 for RARE:GFP intensity. Line: mean, shades: standard deviations. D, RARE:GFP intensity as a function of UV cone OS length. Open circles: data from individual cone. Dashed line: linear trend line, r and p: Peason correlation coefficient and p-value. E, Confocal images of HCR in situ hybridisation for cyp26a1 (top) and cyp26c1 (bottom) at different time points during eye development. F, The cyp26a1 and cyp26c1 mRNAs (magenta) co-localise with Müller glia (MG) (open arrowheads), which are labeled (green) by Venus expression in Tg(tp1:Venus). The regions displayed are indicated as white rectangles in E. D, dorsal; N, nasal; T, temporal; V, ventral.

Cyp26 inhibition leads to shorter OS in the AZ.

A, Schematic illustrating Cyp26-mediated degradation of retinoic acid is blocked by Cyp26 inhibitor leading to increased retinoic acid levels. B, Experimental timeline of drug application from 72 to 120 hpf. C, Confocal images of UV opsin in the photoreceptor layer in the AZ, dorsal and nasal retinas labelled by immunostaining at 120 hpf. Zebrafish were treated with control vehicle or a Cyp26 inhibitor (inh.), R115866, from 72 to 120 hpf. D, UV cone OS length in the AZ, dorsal and nasal regions of the retina at 120 hpf. E, Comparisons of the ratios of the UV cone OS length of the AZ or nasal regions against the dorsal region with and without R115866. Bars and Lines: mean. Error bars: standard deviation. Open circles: data from individual fish. n=14 (Control) and 12 (Cyp26 inhibitor, inh.) One-way ANOVA plus Tukey’s post-hoc test, *: p < 0.05.

Cyp26 inhibition induces RA signalling predominantly in Müller glia and presumptive horizontal cells.

A, Experimental timeline of drug application. B, Confocal images of the eye of Tg(RARE:GFP) zebrafish at 120 hpf. C, Example high-magnification images of different regions of those eyes. D, dorsal; N, nasal; T, temporal; V, ventral. GCL, ganglion cell layer; HC, horizontal cell; INL, inner nuclear layer; ONL, outer nuclear later; RPE, retinal pigment epithelium. Bottom images are high-magnification images of the rectangle regions in the ONL (#) and INL (##), respectively, co-labeled with an antibody against Rlbp1 to identify the retinal pigment epithelium (RPE, arrowheads) and Müller glia (MG, arrowheads). D-F, Densities of GFP-positive (D, n=12 (control) and n=10 (Cyp26 inh.)), GFP and UV opsin double-positive (E, n=12 (control) and n=12 (Cyp26 inh.)), and GFP and Zpr-1 double positive (F, n=12 (control) and n=10 (Cyp26 inh.)) cells in different regions of the ONL measured as the number of cells per 100 μm in a section. G-J, Densities and fraction of GFP-positive MG (G, n=12 (control) and n=10 (Cyp26 inh.); H, n=12 (control) and n=10 (Cyp26 inh.)) and RPE (I, n=12 (control) and n=10 (Cyp26 inh.); J, n=12 (control) and n=10 (Cyp26 inh.)) in the indicated regions of the eye. In all experiments in this figure, zebrafish were exposed to either vehicle or Cyp26 inhibitor from 72 to 120 hpf, and either imaged or fixed at 120 hpf. Bars: Mean, error bars: standard deviation. One-way ANOVA plus Tukey’s post-hoc test, *: p < 0.05 comparing the same region with or without Cyp26 inhibitor.

RARα mediated Retinoic Acid signalling shortens OS in the AZ.

A, Experimental timeline of drug application and confocal images of the UV opsin immnostaining in the photoreceptor layer in the AZ, dorsal and nasal retinas labelled at 120 hpf. Zebrafish were treated with control vehicle or a RARα agonist, AM580, from 72 to 120 hpf. B, UV cone OS length in the AZ, dorsal and nasal regions of the retina at 120 hpf. C, Comparisons of the ratios of the UV cone OS length of the AZ or nasal regions against the dorsal region with and without AM580. Bars and lines: mean. Error bars: standard deviation. Open circles: data from individual fish. n=15 (Control) and 17 (RARα agonist). One-way ANOVA plus Tukey’s post-hoc test, *: p < 0.05.

RARα activation induces RA signalling in specific cell-types similar to Cyp26 inhibition.

A, Experimental timeline of drug applications and confocal images of the eye of Tg(RARE:GFP) zebrafish at 120 hpf after treatment with vehicle control, RARα and RARγ agonists. D, dorsal; N, nasal; T, temporal; V, ventral. B, High-magnification images of different regions of the RARα agonist-treated Tg(RARE:GFP) eyes. GCL, ganglion cell layer; HC, horizontal cell; INL, inner nuclear layer; ONL, outer nuclear later; RPE, retinal pigment epithelium. C-E, Densities of GFP-positive (C, n=8 (control) and n=7 (RARα agonist)), GFP and UV opsin double-positive (D, n=7 (control) and n=8 (RARα agonist)), and GFP and Zpr-1 double positive (E, n=7 (control) and n=8 (RARα agonist)) cells in different regions of the ONL measured as the number of cells per 100 μm in a section. F, Densities of GFP-positive RPE (n=8 (control) and n=8 (RARα agonist). G,H, Densities (G, n=7 (control) and n=8 (RARα agonist)) and fraction (H, n=7 (control) and n=7 (RARα agonist)) of GFP-positive MG in the indicated regions of the eye. Bars: Mean, error bars: standard deviation. One-way ANOVA plus Tukey’s post-hoc test, *: p < 0.05 comparing the same region with or without RARα agonist.

Light sensitivity is reduced in UV cones with shorter OSs after RARα agonist treatment.

A, Schematic and a 2-photon image of synaptically-targeted calcium sensor, SyGCaMP6f, in UV cones. B, Example single cone 2-photon calcium responses to varying duration of UV light flashes (1.5 × 104 photon/s/μm2). Light stim: UV light stimulation. C, Mean peak response amplitudes. Shadings indicate 95% confidence intervals. n= 5 and 4 fish for control and RARα agonist, respectively. n = 141, 111, 109, 63 UV cones for AZ (control), Dorsal (control), AZ (RARα agonist), Dorsal (RARα agonist), respectively.

Müller glia apical microvilli develop in a region-specific manner in a similar timeframe as UV cone outer segments.

A, Schematic of a cross-section of a larval zebrafish retina (left) displaying the apical microvilli of Müller glia (MG, green) in close contact with UV cone outer segments (light magenta). B, Confocal images of the photoreceptor OS layer in Tg(tp1:GFP-CAAX) zebrafish labelled with anti-UV cone opsin antibody in the AZ, dorsal and nasal retina at 72, 96 and 120 hpf. Arrowheads indicate apical microvilli of Müller glia.

Absence of Müller glia shortens UV cone OS in the entire retina.

A,B, Timeline of γ-secretase inhibitor (inh.) (from 45 hpf) and RARα agonist (from 72 hpf) exposures (A), and schematics of eyes displaying the presence and absence of Müller glia (MG, green) following eFont fixedxposure of zebrafish to control vehicle or the γ-secretase inhibitor, respectively, from 45 to 120 hpf (B). C, Confocal images of the UV cone OS stained with anti-UV opsin antibody in the AZ, dorsal and nasal retina at 120 hpf following pharmacological treatments as indicated in (A). D,E, UV cone OS length (D) and the ratios of the UV cone OS length (the AZ or nasal regions against dorsal region) (E) following pharmacological treatments as indicated in (A). n=10 (control); n=10 (γ-secretase inhibitor); n=13 (γ-secretase inhibitor and RARγ agonists). F,G, Timeline of γ-secretase inhibitor treatment (from 72 hpf after Müller glia genesis) and schematic depicting the resulting eye with Müller glia present (green). H,I, UV cone OS length (H) and the ratios of the UV cone OS length (the AZ or nasal regions against dorsal region) (I) following pharmacological treatments as indicated in (F). n=12 (control); n=12 (γ-secretase inhibitor). Bars and Lines: mean. Error bars: standard deviation. Open circles: data from individual fish. One-way ANOVA plus Tukey’s post-hoc test, *: p < 0.05.

Cyp26 inhibition induces differential expression of retinoic acid target/responsive genes.

Experimental timeline of drug application and confocal images of HCR in situ hybridisation against cyp26c1 (magenta) and cyp26b1 (orange) in Tg(RARE:GFP) (green) transgenic zebrafish at 120 hpf. Zebrafish were treated with or without Cyp26 inhibitor from 72 to 120 hpf.

RARγ activation does not change UV cone OS length.

A, Experimental timeline of drug application. B,C, UV cone OS length (B) and the ratios of the UV cone OS length (the AZ or nasal regions against dorsal region) (C) at 120 hpf following RARγ agonist treatment from 72 to 120 hpf. n=12 (control); n=12 (RARγ agonist). Bars and Lines: mean. Error bars: standard deviation. Open circles: data from individual fish.

RARα activation induces differential expression of retinoic acid target/responsive genes, similar to Cyp26 inhibition.

A, Experimental timeline of drug application and confocal images of HCR in situ hybridisation against cyp26c1 (magenta) and cyp26b1 (orange) in Tg(RARE:GFP) (green) transgenic zebrafish at 120 hpf. Zebrafish were treated with or without RARα agonist from 72 to 120 hpf. B, Relative mRNA expression changes for retinoic acid target/response genes cyp26b1, nr2f5 and sfrp1a between controls and RARα agonist-exposed samples. Mean ± S.E., n = 3, Student’s t-test, *p < 0.05.

Müller glia are absent in γ-secretase inhibitor treated retinas.

Experimental timeline of drug application and confocal images of the Tg(csl:mCherry) transgenic eyes at 96 hpf (top row) and high-magnification images in the inner nuclear layer in the AZ at 120 hpf immunolabelled with the Müller glial marker Rlbp1 (magenta, bottom row). Fish were exposed to γ-secretase inhibitor, DAPT or control vehicle from 45 hpf. At 72 hpf, the fish were additionally exposed either to the RARα agonist, AM580 or control vehicle. Note that some amacrine cells become RLBP1 positive in the γ-secretase inhibitor plus RARα agonist condition.

Inhibition of γ-secretase from 72 hpf does not change UV cone OS length.

Timeline of γ-secretase inhibitor treatment (from 72 to 120 hpf) and confocal images of the photoreceptor layer immunolabelled with anti-UV opsin antibody in the AZ, dorsal and nasal retina at 120 hpf.