Lineage tracing of <i>Prss56</i> expressing cells during ocular development.

<p><b>(A)</b><i>Prss56</i>^<i>Cre/+</i> mice were crossed to <i>R26</i>^{<i>tdTomato/+</i>} reporter mice that express tdTomato following CRE-mediated excision of a stop codon to label <i>Prss56</i> expressing cells and their derivatives. (<b>B</b>-<b>F)</b> Representative images showing lineage tracing of <i>Prss56</i> expressing cells (red) in <i>Prss56</i>^<i>Cre/+</i>; <i>R26</i>^{<i>tdTomato/+</i>} eyes throughout ocular development. (<b>B</b>) tdTomato expression is first detected in the retina at embryonic day (E) 16.5 in retinal progenitor cells (RPCs). (<b>C</b>, <b>D</b>) The number of tdTomato positive RPCs increases with age, shown are (<b>C</b>) E18.5 and (<b>D</b>) P2 retinas. (<b>E</b>) By P7, when retinal laminar organization is visible, tdTomato expression was predominantly observed in cells exhibiting characteristic features of Müller glia, with cell bodies located in the inner nuclear layer and apicobasal processes extending across the retina. tdTomato expression was also detected in the inner segment of rod photoreceptors. (<b>F</b>) tdTomato expression continues to be detected in Müller cells and rod photoreceptors following complete maturation of retinal cell types at P13. Interestingly, tdTomato-labeled cells were enriched in the peripheral region and relatively sparser in the central region of the retina. (<b>G</b>) Ki67 immunolabeling of P0 <i>Prss56</i>^<i>Cre/+</i>; <i>R26</i>^{<i>tdTomato/+</i>} eyes demonstrate Ki67 expression in tdTomato positive retinal cells. E, embryonic day; GCL, ganglionic cell layer; INL, inner nuclear layer; ONBL, outer neuroblastic layer; ONL, outer nuclear layer; P, postnatal day. Scale bars: 100μm (<b>B</b>-<b>F</b>) and 50μm (<b>G</b>).</p

PRSS56 activity is required during both the vision-independent and dependent stages of ocular growth.

<p>To determine the temporal window critical for the PRSS56-mediated effect on ocular axial growth, <i>Prss56</i> conditional mutant mice (<i>Prss56</i>^<i>F/F</i>) were crossed to mice expressing the ubiquitous inducible Ubc-Cre recombinase (<i>Ubc-Cre</i>^<i>ERT2</i>). (<b>A</b>) Schematic of tamoxifen treatment at distinct developmental stages preceding and following the opening of the eyes. Tamoxifen injection at two different time points, P6 and P8, was performed to ablate <i>Prss56</i> after the earliest detectable effect of mutant <i>Prss56</i> on ocular axial length. (<b>B</b>-<b>D</b>) OCT-based ocular biometry demonstrates that following tamoxifen injection, <i>Prss56</i>^<i>F/F</i><i>; Ubc-Cre</i>^<i>ERT2</i> mice display a significantly reduced ocular axial length (<b>B</b>) and increased retinal thickness (<b>C</b>) and a significant decrease in VCD (<b>D</b>) compared to the control <i>Prss56</i>^<i>F/+</i>; Ubc-Cre^<i>ERT2</i> mice (measured at P17). The ocular axial length, retinal thickness, and VCD of uninjected <i>Prss56</i>^<i>F/F</i><i>; Ubc-Cre</i>^<i>ERT2</i> <i>and Prss56</i>^<i>F/+</i><i>; Ubc-Cre</i>^<i>ERT2</i> mice were indistinguishable. Administration of tamoxifen at P6 caused a greater decrease in ocular axial length compared to administration at P8 suggesting a requirement for continuous PRSS56 activity during ocular development to sustain normal ocular growth, N = 6 to 8 per group for <b>A</b> and <b>B</b>. (<b>E</b>-<b>G</b>) OCT measurements demonstrate that following tamoxifen injection at P13 (a time point when the eyes are open), <i>Prss56</i>^<i>F/F</i><i>; Ubc-Cre</i>^<i>ERT2</i> mice display a slight but significant decrease in ocular axial length (<b>E</b>) and increase in retinal thickness (<b>F</b>) at P30 and P45. Reduced ocular axial length was associated with a significant decrease in VCD in <i>Prss56</i>^<i>F/F</i><i>; Ubc-Cre</i>^<i>ERT2</i> mice compared to <i>Prss56</i>^<i>F/+</i><i>; Ubc-Cre</i>^<i>ERT2</i> and uninjected controls at P30 and P45, N = 5 to 10 per group (<b>G</b>). <b>(H</b>-<b>J)</b> Ocular biometry following tamoxifen injection at the beginning of a critical emmetropization period (P18) shows that the ocular axial length is not significantly different between the <i>Prss56</i>^<i>F/F</i><i>; Ubc-Cre</i>^<i>ERT2</i> and control mice at any of the three ages examined (P30, P45, and P60). However, <i>Prss56</i>^<i>F/F</i><i>; Ubc-Cre</i>^<i>ERT2</i> mice display a slightly thicker retina and significantly reduced VCD compared to the control groups. (<b>K</b>) Following <i>Prss56</i> ablation at P13 and P18, the eyes display a decrease in the combined value of retinal thickness and VCD. (<b>L</b>) qPCR analysis revealed elevated <i>Prss56</i> mRNA levels in <i>Prss56</i>^<i>F/F</i><i>; Ubc-Cre</i>^<i>ERT2</i> retina compared to <i>Prss56</i>^<i>F/+</i><i>; Ubc-Cre</i>^<i>ERT2</i> retina following tamoxifen injection at P8, P13, or P18 (shown are data from mice harvested at P17 and P60, respectively). (<b>M</b>) Following <i>Prss56</i> ablation at P18, the eyes display a hyperopic shift in refraction compared to control eyes at 3 months (N = 6 per group). Values are presented as mean ± SD (or mean ± SEM in <b>L</b>); * p<0.05, ** p<0.01, *** p<0.001, t-test.</p

Conditional RAX-Cre-mediated ablation of <i>Prss56</i> from fully differentiated Müller glia leads to ocular size reduction.

<p><i>Prss56</i> was conditionally ablated from Müller cells in a time-specific manner by crossing <i>Prss56</i>^<i>F/F</i> to the inducible RAX-Cre mouse strain (<i>Rax-Cre</i>^<i>ERT2</i>). CRE expression was induced by tamoxifen injection at P8, a time point preceding complete Müller glia differentiation. (<b>A</b>) Representative images of slit lamp examination by broad-beam illumination following tamoxifen injection at P8. <i>Prss56</i>^<i>F/F</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes were indistinguishable from control <i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes at 2 months of age. (<b>B</b>, <b>C</b>) Following tamoxifen injection (TAM +; horizontal axis) at P8, <i>Prss56</i>^<i>F/F</i><i>Rax-Cre</i>^<i>ERT2</i> mice display a significant decrease in ocular axial length (<b>B</b>) and increase in retinal thickness (<b>C</b>) compared to control eyes (<i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> or <i>Prss56</i>^<i>F/F</i> mice without <i>Rax-Cre</i>^<i>ERT2</i>), N = 6 to 8 per group. (<b>D</b>) Following tamoxifen injection at P8, a significant reduction in VCD was detected in <i>Prss56</i>^<i>F/F</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes compared to control <i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes, N = 6 per group. Ocular axial length, retinal thickness, and VCD in uninjected <i>Prss56</i>^<i>F/F</i><i>; Rax-Cre</i>^<i>ERT2</i> and <i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> mice were indistinguishable. (<b>E</b>) Representative OCT images showing reduced axial length and VCD in <i>Prss56</i>^<i>F/F</i><i>; Rax-Cre</i>^<i>ERT2</i> eyes compared to the control <i>Prss56</i>^<i>F/+</i><i>; Rax-Cre</i>^<i>ERT2</i> or <i>Prss56</i>^<i>F/F</i> mice. (<b>F</b>) qPCR analysis following tamoxifen injection at P8 revealed that <i>Prss56</i> mRNA was significantly upregulated in <i>Prss56</i>^<i>F/F</i> <i>; Rax-Cre</i>^<i>ERT2</i> retina compared to their <i>Prss56</i>^<i>F/+</i> <i>; Rax-Cre</i>^<i>ERT2</i> counterparts or uninjected controls, N≥ 6 per group. Values are presented as mean ±SD; * p<0.05, ** p<0.01, *** p<0.001, t-test. (<b>G-I</b>) Müller glia endfeet organization of <i>Prss56</i>^<i>Cre</i>; <i>R26</i>^{<i>tdTomato</i>} reporter mice during retinal development. Representative images of retinal section (<b>G, I</b>) or whole mount (<b>H</b>) showing Müller glia endfeet from control and <i>Prss56</i> mutant mice at P6. (<b>I</b>) Magnified images of the retinal endfeet are shown. The ILM of <i>Prss56</i> mutant mice (<i>Prss56</i>^{<i>Cre/gclr4</i>}; <i>R26</i>^{<i>tdTomato</i>}) at P6 is marked by regions of increased endfeet complexity (arrow head) compared to the ILM of control mice (<i>Prss56</i>^<i>Cre/+</i>; <i>R26</i>^{<i>tdTomato</i>}). A substantial proportion of endfeet appear more spread out, occupying a larger area in the mutant compared to control retinal whole mounts (occupying smaller area). Red boxes highlight individual endfoot. N = 4 per genotype and scale bars = 17μm in <b>G</b> and <b>I</b>, and 50μm in <b>H</b>. ACD, anterior chamber depth; AL, axial length; VCD, vitreous chamber depth.</p

<i>Prss56</i> ablation rescues myopia in mice.

<p>(<b>A</b>) Representative OCT images demonstrating that <i>Prss56</i> ablation rescues myopia in <i>Egr1</i>^<i>-/-</i> mice (compare <i>Prss56</i>^<i>+/-</i><i>;Egr1</i>^<i>-/-</i> <i>to Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>-/-</i>, shown are P30 eyes). Reciprocally, <i>Egr1</i> deficiency rescues hyperopia in <i>Prss56</i>^<i>-/-</i> mice (compare <i>Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>+/-</i> <i>to Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>-/-</i><i>)</i>. The red and blue lines indicate ocular axial length (AL) and vitreous chamber depth (VCD), respectively. (<b>B</b>) <i>Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>+/-</i> eyes display a significant reduction in axial length, whereas <i>Prss56</i>^<i>+/-</i><i>;Egr1</i>^<i>-/-</i> exhibit significantly elongated axial length compared to the control eyes (<i>Prss56</i>^<i>+/-</i><i>;Egr1</i>^<i>+/-</i> <i>)</i>. The eyes of double mutants (<i>Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>-/-</i><i>)</i> attain a size that is not significantly different from control eyes (<i>Prss56</i>^<i>+/-</i><i>;Egr</i>^<i>-/-</i>), at all ages examined (P10 to P60). (<b>C</b>) Consistent with modulation of ocular axial length by <i>Prss56</i> and <i>Egr1</i> mutations, hyperopic refraction observed in <i>Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>+/-</i> eyes was rescued in the double mutants (<i>Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>-/-</i>). Conversely, <i>Prss56</i> ablation rescued myopic refraction observed in <i>Prss56</i>^<i>+/-</i><i>;Egr1</i>^<i>-/-</i> eyes (compare <i>Prss56</i>^<i>+/-</i><i>;Egr1</i>^<i>-/-</i> <i>to Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>-/-</i>, shown are data from 2-months old mice). (<b>D</b>) The VCD of double mutant eyes (<i>Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>-/-</i><i>)</i> was significantly reduced compared to <i>Egr1</i> single mutant eyes (<i>Prss56</i>^<i>+/-</i><i>;Egr1</i>^<i>-/-)</i> and increased compared to <i>Prss56</i> single mutant eyes (<i>Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>+/-</i>). The VCD of double mutant eyes was not significantly different from the control eyes. (<b>E)</b> The retina is thicker in double mutants (<i>Prss56</i>^<i>-/-</i><i>;Egr1</i>^<i>-/-</i><i>)</i> compared to control mice <i>(Prss56</i>^<i>+/-</i><i>;Egr1</i>^<i>+/-</i>) at P30 and P60, despite their ocular axial length being similar. Values are presented as mean ± SD. For comparison between single mutant and controls: * p<0.05; ** p<0.01;*** p<0.001, t-test. For comparison between single mutant and double mutants: ^# p<0.05; ^## p<0.01; ^###p<0.001, t-test. <b>B</b>-<b>D</b>: N ≥ 6 per group; <b>E</b>: N ≥ 8 per group.</p

<i>Prss56</i>^-/- eyes exhibit reduced ocular axial length and hyperopia.

<p>(<b>A</b>) Representative images of slit-lamp examination by broad-beam illumination to assess ocular structures including the iris, pupil, and lens at 1 and 3 months of age. <i>Prss56</i>^<i>-/-</i> eyes did not exhibit any obvious structural abnormalities and were indistinguishable from <i>Prss56</i>^<i>+/-</i> eyes. (<b>B</b>) Representative OCT images demonstrating a reduction in ocular size in <i>Prss56</i>^<i>-/-</i> compared to <i>Prss56</i>^<i>+/-</i> mice (shown are P30 eyes). The red, blue and yellow lines indicate ocular axial length (AL), vitreous chamber depth (VCD) and anterior chamber depth (ACD), respectively. (<b>C</b>-<b>D</b>) <i>Prss56</i>^<i>-/-</i> eyes exhibit a modest but highly significant reduction in axial length (<b>C</b>) and equatorial diameter (<b>D</b>) at P15 and P25. (<b>E</b>) A significant reduction in VCD and increase in ACD was detected in <i>Prss56</i>^<i>-/-</i> compared to <i>Prss56</i>^+/- eyes (shown are data from P30 eyes). (<b>F</b>) Consistent with reduced ocular size, <i>Prss56</i>^<i>-/-</i> mice display a hyperopic refraction compared to <i>Prss56</i>^<i>+/-</i> littermates (shown are data from 2-months old mice). (<b>G</b>) Retinal thickness was significantly increased in <i>Prss56</i>^<i>-/-</i> compared to <i>Prss56</i>^<i>+/-</i> eyes. Values are presented as mean ± SD, *** p<0.001, t-test. <b>C</b> and <b>D:</b> N > 10 per group; <b>E</b> and <b>F:</b> N ≥ 6 per group; and <b>G:</b> N≥ 4. (<b>H</b>-<b>I</b>) Representative B-scan images of eyes from an unaffected individual (<b>H</b>) and an individual with a <i>PRSS56</i> mutation (<b>I</b>). VCD is substantially reduced in the eye of the individual carrying a mutant <i>PRSS56</i> allele compared to a normal emmetropic eye.</p

Earliest <i>Prss56</i> expression occurs in late retinal progenitor cells.

<p>(<b>A</b>, <b>B</b>) Representative images of <i>Prss56</i>^<i>Cre/+</i>; <i>R26</i>^{<i>tdTomato/+</i>} retina immunolabeled for SOX2 (<b>A</b>) or PKCα (<b>B</b>). (<b>A</b>) tdTomato expression (red) is present in SOX2 immunopositive Müller cells (green). The peripheral and central regions of the retina are oriented left to right. (<b>B</b>) Representative images showing low tdTomato expression in a subset of PKCα immunolabeled bipolar cells in <i>Prss56</i>^<i>Cre/+</i>; <i>R26</i>^{<i>TdTomato/+</i>} retina (arrows). (<b>C</b>) Flow cytometry analysis of Glutamine Synthetase (GS) and Rhodopsin expression in <i>Prss56</i>^<i>Cre/+</i>; <i>R26</i>^{<i>tdTomato/+</i>} retinal cell suspensions. GS expression was predominantly detected in tdTomato negative (green) and high tdTomato expressing cells (purple). Rhodopsin expression was predominantly detected in tdTomato negative (green) and low tdTomato expressing (orange) cells. A minimum of 4 eyes per group was pooled for each retinal cell suspension and flow cytometry analyses were repeated 2–3 times on independent samples. Gating was established based on <i>Prss56</i>^<i>Cre/+</i>; <i>R26</i>^{<i>tdTomato/+</i>} retinal cell suspension incubated with AlexaFluor 488 conjugated secondary antibody only. Together, these data demonstrate that <i>Prss56</i> is expressed by late RPCs that give rise to bipolar cells, rod photoreceptors, and Müller cells. Scale bars = 100μm(<b>A</b>) and 50μm (<b>B</b>).</p

Early requirement for PRSS56 in ocular size determination.

<p>(<b>A</b>, <b>B</b>) Ocular biometric analysis by OCT revealed reduced ocular axial length (<b>A</b>) and increased retinal thickness (<b>B</b>) in <i>Prss56</i>^{<i>glcr4/glcr4</i>} eyes compared to <i>Prss56</i>^{<i>glcr4/+</i>} eyes at distinct developmental time points ranging from P6 to P17. Although both mutant and control mice exhibit an age-dependent increase in ocular size, ocular axial length is significantly reduced in <i>Prss56</i> mutant mice compared to control mice. The reduction in ocular axial length was detected as early as P6 (<b>A</b>). (<b>B</b>) OCT analysis revealed that <i>Prss56</i> mutant retina is significantly thicker than control retina (shown is P17). (<b>C</b>-<b>D</b>) Histological analysis revealed that the number of nuclear stacks in both the inner and outer retinal nuclear layers (INL and ONL, respectively) was consistently greater in the <i>Prss56</i> mutant retina (<b>D</b>) compared to control retina (<b>C</b>). For comparison between <i>Prss56</i> mutant and control mice: *p<0.05, ** p<0.01, *** p<0.001, t-test. Scale bars = 100μm. Values are presented as mean ± SD. N ≥ 12 per group for P6 measurements and N > 6 for P8 and P17 (<b>A</b>, <b>B</b>).</p

Association analysis between <i>ABCC5</i> rs1401999 and primary angle closure glaucoma in all chip-typed sample collections (top panel), de-novo genotyped sample collections (middle panel), and PACG cases and clinically certified controls with open angles (bottom panel).

<p>MAF case: Minor allele frequency in PACG cases.</p><p>MAF control: Minor allele frequency in controls.</p><p>OR: Odds ratio.</p><p><i>P</i>: <i>P</i>-value for association with PACG.</p><p>I²: I-squared index for between-collection heterogeneity.</p><p>* Results here are presented based on raw minor allele frequency counts without further adjustment.</p>†<p>PACG patients were recruited from the Beijing Tongren Hospital and controls were recruited from the Handan Eye Study (HES), a population-based study of eye disease in rural Chinese aged 30 years and over.</p

Association analysis between <i>ABCC5</i> rs1401999 and susceptibility to primary angle closure glaucoma (PACG).

<p>The PACG sample collections have been described elsewhere <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004089#pgen.1004089-Vithana1" target="_blank">[6]</a>. The vertical line represents a per-allele odds ratio of 1.00. The oblongs represent point estimates (referring to the per-allele odds ratio), with the height of the oblongs inversely proportional to the standard error of the point estimates. Horizontal lines indicate the 95% confidence interval for each point estimate. Meta-analyses of samples are reflected by blue diamonds. The width of the diamonds indicates their 95% confidence intervals. All point estimates in Stage 1 have been adjusted for the top axes of genetic stratification using logistic regression.</p

Quantitative trait analysis between <i>ABCC5</i> rs1401999 and anterior chamber depth in SIMES, SINDI, and BES.

<p>SIMES: Singapore Malay Eye Study (typed with Illumina 610K GWAS chip).</p><p>SINDI: Singapore Indian Eye Study (typed with Illumina 610K GWAS chip).</p><p>BES1: Beijing Eye Study typed with Illumina 610K GWAS chip.</p><p>BES2: Beijing Eye Study typed with direct sequencing.</p><p>β: Per-allele effect size of <i>ABCC5</i> rs1401999 on anterior chamber depth.</p><p>SE: Standard error for β.</p><p><i>P</i>gc: Genomic control corrected <i>P</i>-value.</p><p>MAF: Minor allele frequency.</p><p>*: I²-index for heterogeneity = 0%.</p