During mouse postnatal eye development, the embryonic hyaloid vascular network regresses from the vitreous as an adaption for high-acuity vision. This process occurs with precisely controlled timing. Here, we show that opsin 5 (OPN5; also known as neuropsin)-dependent retinal light responses regulate vascular development in the postnatal eye. In Opn5-null mice, hyaloid vessels regress precociously. We demonstrate that 380-nm light stimulation via OPN5 and VGAT (the vesicular GABA/glycine transporter) in retinal ganglion cells enhances the activity of inner retinal DAT (also known as SLC6A3; a dopamine reuptake transporter) and thus suppresses vitreal dopamine. In turn, dopamine acts directly on hyaloid vascular endothelial cells to suppress the activity of vascular endothelial growth factor receptor 2 (VEGFR2) and promote hyaloid vessel regression. With OPN5 loss of function, the vitreous dopamine level is elevated and results in premature hyaloid regression. These investigations identify violet light as a developmental timing cue that, via an OPN5–dopamine pathway, regulates optic axis clearance in preparation for visual function.
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Source data for all figures have been provided as Supplementary Table 2. Additional experimental repeats for key retinal labelling experiments have been deposited on Figshare (https://doi.org/10.6084/m9.figshare.7450961). All other data supporting the findings of this study are available from the corresponding author on reasonable request.
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We thank P. Speeg (Lang lab) for excellent mouse colony management, L. Sankaran and P. Lyuboslavsky (Iuvone lab) for technical assistance, and D. Bredl and D. Copenhagen (UCSF) for providing tissue samples from the Drd2-eGFP mice. We also thank Y. Chen and Y.-C. Hu of the CCHMC Transgenic Animal and Genome Editing Core Facility for generating genetically modified mouse lines. This work was supported by NIH R01 GM124246 to E.D.B., NIH R01EY026921 to R.N.V.G., NIH P30EY001730 to the University of Washington, the Mark J. Daily, MD Research Fund to the University of Washington, and unrestricted grants to the University of Washington and Emory University Department of Ophthalmology from Research to Prevent Blindness. This work was also supported by NIH grants R01 EY027077 (R.A.L. and S.R.), R01 EY027711 (P.M.I. and R.A.L.), R01 EY022917 (R.S.H.) and R01 EY004864 (P.M.I.), by funds from the Goldman Chair of the Abrahamson Pediatric Eye Institute at Cincinnati Children’s Hospital Medical Center and by grant BIOCEV-CZ.1.05/1.1.00/02.0109 (Z.K.). This work was supported by NIH grant 2T32GM063483, which supports the UCCOM/CCHMC Medical Scientist Training Program.
Integrated supplementary information
a-e, Schematics of the Opn5 alleles used in this study. a, The Opn5 allele as targeted in ES cells by the International Knockout Mouse Consortium. Exons are numbered yellow boxes. FRT, FLP recombinase site-specific recombination sites. En2 SA, Engrailed 2 splice acceptor. IRES, internal ribosome entry sequence. Lacz, β-galactosidase open reading frame. pA, polyadenylation signal. loxP, cre recombinase site specific recombination sequences. hbactP, human β-actin promoter. Right-facing arrow indicates the start point of transcription for hBactP. neo, the neomycin resistance gene. b, The Opn5lacz allele generated after germ-line recombination at the loxP sites. c, the Opn5fl allele generated after germ-line recombination at the FRT sites. d, The Opn5- allele generated after germ-line recombination of the Opn5fl allele at the loxP sites. e, The Opn5cre allele generated by CRISPR targeting of Opn5 exon 1. Further description is available in Materials and Methods section.
a-f, Cryosections of retina from P8 Opn5+/+ control (a, c, e) and Opn5-/- null (b, d, f) mice labelled for nuclei with Hoechst 33258 (a-f, blue) for RBPMS (a-d, pink), calretinin (a, b, red), TH (a, b, green), ChAT (c, d, green), or DAT/SLC6A3 (e, f, red). g, h, Labelling of two examples of P8 retina from Opn5cre; Ai14 (tdTomato from this reporter in red), for nuclei with Hoechst 33258 (blue), and for the pan-RGC marker RBPMS (green). We could detect no substantial change in the distribution of RBPMS, TH, Calretinin, Chat or DAT/SLC6A3. Opn5cre positive RGCs are RBPMS+ regardless of whether they are within the ganglion cell layer (GCL) or are displaced ganglion cells (dRGC). In both examples, the boxed region is magnified in the adjacent panels to show more clearly the tdTomato-RBPMS colabeling. Scale bars are 50 μm. Image panels are representative of at least three separate experiments.
a-c, tdTomato signal (red) in Opn5cre; Ai14 P28 mouse brain cryosections in the optic tracts (a), superior colliculus (b) and lateral geniculate nucleus (c). These projections are characteristic of retinal ganglion cells. d-g, Opn5cre; Ai6 P8 retinal cryosections imaged for nuclei with DAPI (d, grayscale), for ZsGreen1 from Ai6 (d, e, green), for RBPMS (d, f, red) and for Calretinin (d, g, blue). White outlines of green cells are displayed on the red RBPMS (f) and blue Calretinin (g) channels. in (d) a displaced RGC and some of the amacrine cells are indicated. RBPMS is exclusive to RGCs and Calretinin identifies both amacrine cells and a subset of RGCs. We determined the RBPMS and calretinin status of Opn5cre; Ai6 positive cells from n-3 mice at P8. White outlines of Opn5cre; Ai14 positive cell bodies were moved to images of the RBMPS and Calretinin labelling channels and counts performed. In these sections there were 88 Opn5cre; Ai6 positive cells. In these same sections, there were 1446 RBPMS positive cells and 910 Calretinin positive cells. All 88 Opn5cre; Ai6 positive cells were positive for RBPMS and 50/88 were also positive for Calretinin. Importantly, there were zero Opn5cre; Ai14 positive cells that expressed only calretinin; they invariably also expressed RBPMS. This analysis provides evidence that Opn5cre is expressed is RBPMS positive RGCs, but not in amacrine cells. Scale bars 500 μm (a) 200 μm (b, c) 50 μm (d). Image panels are representative of at least three separate experiments.
Supplementary Figure 4 Flt1 (VEGFR1) and dopamine pathway agonists and antagonists regulate hyaloid vessel regression.
a, b, Hyaloid vessel preparations from P8 control Chx10-cre; Flt1+/+ (a) and Chx10-cre; Flt1fl/fl (b) mice. Scale bars 200 μm. c, Quantification of hyaloid vessel numbers for mice from (a) but over a P3-P8 time-course. The melanopsin (OPN4)-dependent response pathway that regulates hyaloid vessel regression uses VEGFA as an intermediate. Furthermore, when FLT1 (aka VEGFR1), a naturally occurring inhibitor of VEGFA is conditionally deleted from the retina, hyaloid persistence is the result (a-c). Thus, it was possible that the precocious hyaloid regression of the Opn5 null could be explained by reduced levels of VEGFA or by elevated levels of FLT1. d, e, ELISA quantification of VEGFA and FLT1 in the P6 vitreous of Opn5+/+ control (d, e, grey bars), Opn5± heterozygote (d, e, light blue bars), and Opn5-/- homozygote (d, e, light blue bars). Levels of vitreal VEGFA and FLT1 were not significantly changed in the Opn5 null suggesting a mechanism of hyaloid regression distinct from that of the OPN4 pathway. f, Quantification of P8 hyaloid vessels in Opn5+/+, Opn5+/-, and Opn5-/- mice injected with the dopamine receptor agonist SKF38393 from P1-P8. Controls include uninjected and vehicle injected mice as indicated. g, Quantification of P8 hyaloid vessels in wild type mice injected with vehicle or with the dopamine receptor antagonists L741,626 or 2-CMDO as indicated. (f). Sample numbers (n) are shown at the base of each histogram bar and represent mice. p-values by (c, g), Student’s t-test, (d, e) One Way ANOVA, (f) Two Way ANOVA. Image panels are representative of at least three separate experiments.
Supplementary Figure 5 Superficial retinal vasculature in Pdgfb-icreERT2, Drd2fl/fl conditional null mice, and immunoblot densitometry for β-Tubulin, VEGFR2 and phospho-VEGFR2.
a, b, d, e, Flat-mount, isolectin-labeled, P8 retinae from control Drd2fl/fl (a, b, whole retinal disc) and Pdgfb-icreERT2; Drd2fl/fl (d, e, 20 x magnification) mice. c, d, Quantification of superficial vascular plexus migration (c) and branchpoint density (f). Sample size (n) is shown at the base of the histogram bar and represents mice. No significant differences in migration distance or vascular density were identified. Image panels are representative of at least three separate experiments. Scale bars 500 μm (a, b) 50 μm (d, e). g-i, quantification, in arbitrary units, of immunoblot band intensity for the β-tubulin (TUBB) loading control (g), as well as for VEGFR2 (h) and phospho-VEGFR2 (i) for hyaloid vessel lysates from Drd2fl/fl control (grey) and Drd2fl/fl; Pdgfb-icreERT2 experimental (blue) mice. The horizontal axis indicates the lysate volume that was loaded for each band quantification. Pearson correlation coefficients for each set of three points are indicated. These data represent one sample of the n = 3 mice used for quantification of phospho-Y1173-VEGFR2.
(4i) Unprocessed scans of immunoblot data from Fig 4i detecting phospho-T53-DAT (p-DAT), total DAT and TUBB from retina showing lower levels of phospho-T53-DAT in Opn5 control and null retina. (6k) Unprocessed scans of immunoblot data from Fig 6k detecting VEGFR2, pY1173-VEGFR2 and TUBB in hyaloid vessels from Drd2fl/fl and Drd2fl/fl;PDGFB-icreERT2 mice. A three step, two-fold loading dilution is indicated on the blot as 20, 10, 5 ul. (6m-n) Unprocessed scans of immunoblot data from Fig 6m, n detecting (m) VEGFR2, pY1173-VEGFR2 and (n) AKT, pS473-AKT in hyaloid vessels from Opn5 control and null animals. The genotype of sample loaded in each lane is indicated at the top of each panel, animal ID indicated on pDAT immunoblot.