Genetic Network Regulating Visual Acuity Makes Limited Contribution

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.04.442661; this version posted May 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Genetic network regulating visual acuity makes limited contribution

2 to visually guided eye emmetropization

5 Tatiana V. Tkatchenko1 and Andrei V. Tkatchenko1,2*

8 1Department of Ophthalmology, Columbia University, New York, New York, USA;

9 3Department of Pathology and Cell Biology, Columbia University, New York, New York, USA

11 Tatiana V. Tkatchenko, E-mail: [email protected]

12 Andrei V. Tkatchenko, E-mail: [email protected]

18 RNA-seq data from this article have been deposited in the Gene Expression Omnibus database

19 under accession No. GSE158732

21 *Correspondence to: Andrei V. Tkatchenko, Edward S. Harkness Eye Institute, Research

22 Annex Room 415, 635 W. 165th Street, New York, NY 10032, USA. Phone: 212-342-5135;

23 Fax: 212-342-0094; E-mail: [email protected]

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24 Abstract

25 During postnatal development, the eye undergoes a refinement process whereby optical defocus

26 guides eye growth towards sharp vision in a process of emmetropization. Optical defocus

27 activates a signaling cascade originating in the retina and propagating across the back of the eye

28 to the sclera. Several observations suggest that visual acuity might be important for optical

29 defocus detection and processing in the retina; however, direct experimental evidence supporting

30 or refuting the role of visual acuity in refractive eye development is lacking. Here, we used

31 genome-wide transcriptomics to determine the relative contribution of the retinal genetic

32 network regulating visual acuity to the signaling cascade underlying visually guided eye

33 emmetropization.

34 Our results provide evidence that visual acuity is regulated at the level of molecular signaling in

35 the retina by an extensive genetic network. The genetic network regulating visual acuity makes

36 relatively small contribution to the signaling cascade underlying refractive eye development.

37 This genetic network primarily affects baseline refractive eye development and this influence is

38 primarily facilitated by the biological processes related to melatonin signaling, nitric oxide

39 signaling, phototransduction, synaptic transmission, and dopamine signaling. We also observed

40 that the visual-acuity-related genes associated with the development of human myopia are

41 chiefly involved in light perception and phototransduction. Our results suggest that the visual-

42 acuity-related genetic network primarily contributes to the signaling underlying baseline

43 refractive eye development, whereas its impact on visually guided eye emmetropization is

44 modest.

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46 Keywords: Refractive eye development, Emmetropization, Optical defocus, Visual acuity,

47 Signaling pathways, Gene expression profiling, Gene expression networks, RNA-seq.

49 Abbreviations

50 AMPK: AMP-activated protein kinase; ANOVA: analysis of variance; ARVO: Association for

51 Research in Vision and Ophthalmology; CACNA2D1: calcium voltage-gated channel auxiliary

52 subunit alpha2 delta 1; CAMK2G: calcium/calmodulin dependent protein kinase II gamma;

53 CB2R: cannabinoid receptor type 2; CFH: complement factor H; CNGB3: cyclic nucleotide

54 gated channel subunit beta 3; cpd: cycles per degree; DARPP32: protein phosphatase 1

55 regulatory inhibitor subunit 1B; DAVID: database for annotation, visualization and integrated

56 discovery; GP6: glycoprotein VI platelet; GRK1: G protein-coupled receptor kinase 1; GWAS:

57 genome-wide association study; IMPG2: interphotoreceptor matrix proteoglycan 2; IPA:

58 Ingenuity Pathway Analysis; JAK2: Janus kinase 2; LED: light-emitting diode; LRIT1: leucine

59 rich repeat, Ig-like and transmembrane domains 1; mTOR: mammalian target of rapamycin;

60 nNOS: neuronal nitric oxide synthase; OMIM: Online Mendelian Inheritance in Man database;

61 OR: odds ratio; PDE6A: phosphodiesterase 6A; PDE6C: phosphodiesterase 6C; PPARα:

62 Peroxisome proliferator-activated receptor alpha; PTEN: phosphatase and tensin homolog; RAR:

63 retinoic acid receptor; RCB: randomized complete block; RDH12: retinol dehydrogenase 12;

64 RhoGDI: Rho GDP-dissociation inhibitor; RIN: RNA integrity number; RNA-seq: massive

65 parallel RNA sequencing; RXRα: retinoid X receptor alpha; SNP: single-nucleotide

66 polymorphism; QTL: quantitative trait locus; VEP: visually evoked potential;

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68 1. Introduction

69 The general layout of the eye is established during embryonic development; however, the eye

70 undergoes a refinement process during postnatal period, whereby the optical geometry of the eye

71 and its refractive state are guided towards sharp vision in a process called emmetropization [1-8].

72 Various environmental factors influence refractive eye development during emmetropization [8-

73 11]; however, the leading environmental factor guiding refractive eye development is optical

74 defocus [8, 12]. The eye is very sensitive to the sign of optical defocus and can accurately

75 compensate for imposed defocus by modulating the growth of the posterior segment of the eye

76 [12-18]. Emmetropization uses optical defocus to match the eye’s axial length to its focal plane

77 and normally produces emmetropia (sharp vision) [8]. Prolonged exposure to positive optical

78 defocus leads to the development of hyperopia (farsightedness), whereas excessive exposure to

79 negative optical defocus leads to the development of myopia (nearsightedness) [13, 14, 16-27].

80 The role of optical defocus in eye emmetropization is well established [8]. It was also found that

81 molecular signaling underlying retinal response to positive and negative defocus propagate along

82 two largely distinct signaling cascades [12]. Genetic networks underlying baseline refractive eye

83 development and visually guided eye emmetropization are also beginning to come to light [8, 28,

84 29]. However, the molecular cascade responsible for the detection and processing of optical

85 defocus by the eye is still poorly understood. We have recently found that the gene expression

86 network subserving perception of contrast plays an important role in optical defocus detection

87 and visually guided eye emmetropization [30]; however, the role of visual acuity in optical

88 defocus detection and emmetropization remains a subject of debate.

89 Visual acuity is primarily determined by the density of photoreceptors in the central

90 retina and declines sharply with increasing retinal eccentricity [31, 32]. Several lines of evidence

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91 suggest that the absolute level of visual acuity does not play a significant role in optical defocus

92 detection and emmetropization because species with different levels of visual acuity ranging

93 from 1.3 cycles per degree (cpd) in fish [33], 0.6-1.4 cpd in mice [34-36], 2.4 cpd in tree shrews

94 [37, 38], 2.7 cpd in guinea pigs [39, 40], 5 cpd in cats [41, 42], 5 cpd chickens [43, 44] and 44

95 cpd in rhesus monkeys and humans [45-47] undergo emmetropization and can compensate for

96 imposed optical defocus [13-17, 48-52]. Interestingly, visual acuity in newborn human infants

97 during first months of life, i.e., when they undergo most active eye emmetropization, remains

98 low, 2.4 cpd in 1-month-old infants gradually increasing to 10-15 cpd in 8-month-olds [47, 53,

99 54]. In agreement with these observations, Mutti et al. found that emmetropization in infants

100 does not appear to be dependent on visual acuity [55]. Studies in a mutant chicken model with

101 diminished visual acuity also revealed that reduced visual acuity had limited impact on

102 susceptibility to either form-deprivation or lens-induced myopia, hence emmetropization [56]. It

103 was also found that accommodation and emmetropization are primarily dependent on low spatial

104 frequencies even in species with high visual acuity [57, 58]; and that the peripheral retina, which

105 has much lower visual acuity than the central retina [32, 53, 59-62], plays an important role in

106 defocus detection and emmetropization [8, 63-65]. Moreover, it was reported that inhibitory

107 inputs to the ganglion cells from the amacrine cells, which are critical for optical defocus

108 detection, are more predominant in the low-resolution peripheral retina, while the high-resolution

109 foveal retinal circuits primarily rely on excitatory inputs [66]. Nevertheless, there is evidence

110 that visual acuity might influence visually guided eye emmetropization. For example, certain

111 mutations affecting visual acuity in humans were also found to be linked to the development of

112 myopia [67]. It was also found that maturation of visually evoked potentials (VEPs), which

113 correlate with sensitivity to optical defocus, shows strong correlation with visual acuity [68].

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114 Similar to refractive eye development, visual acuity is controlled by both environmental

115 and genetic factors. Prusky et al. discovered that environmental enrichment during early

116 postnatal period, such as exposure to stationary and moving visual patterns, enhances visual

117 acuity, whereas visual deprivation reduces it [69, 70]. Genetic background also has a significant

118 impact on visual acuity. For example, different inbred strains of mice raised under the same

119 environmental conditions exhibit large differences in visual acuity and ability to discriminate

120 patterns [71]. Genetic polymorphism in the gene encoding complement factor H (CFH) was

121 found to affect visual acuity in humans [72]. It was also found that mutations in genes encoding

122 retinal synaptic proteins, such as the blu gene encoding a vesicular glutamate transporter and

123 Lrit1 encoding a leucine-rich transmembrane protein, affect visual acuity without changing the

124 density of photoreceptors [73, 74]. Glucose metabolism in the retina has been implicated in the

125 regulation of visual acuity [75, 76]. Finally, cannabinoids were shown to influence visual acuity

126 in mice via the cannabinoid receptor type 2 (CB2R) [77]. All this evidence suggests that visual

127 acuity is controlled by a genetic network, which integrates visual experience with the

128 downstream molecular signaling in the retina. Considering that some data suggest that visual

129 acuity might be linked to refractive eye development, it is critical to characterize the genetic

130 network and signaling pathways subserving visual acuity and determine their contribution to

131 baseline refractive eye development and visually guided eye emmetropization.

132 Here, we analyzed gene expression networks and signaling pathways regulating visual

133 acuity in a panel of genetically diverse mice, which have different visual acuity, different

134 baseline refractive errors, and different susceptibility to form-deprivation myopia, and

135 determined the contribution of the genetic network subserving visual acuity to baseline refractive

136 eye development and visually guided eye emmetropization.

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137

138 2. Materials and methods

139

140 2.1. Ethics statement

141

142 Inbred mouse strains (129S1/svlmj, A/J, C57BL/6J, CAST/EiJ, NOD/ShiLtJ, NZO/HlLtJ,

143 PWK/PhJ, and WSB/EiJ) were obtained from the Jackson Laboratory (Bar Harbor, ME) and

144 were maintained as an in-house breeding colony. Food and water were provided ad libitum. All

145 procedures adhered to the Association for Research in Vision and Ophthalmology (ARVO)

146 statement on the use of animals in ophthalmic and vision research and were approved by the

147 Columbia University Institutional Animal Care and Use Committee. Animals were anesthetized

148 via intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg) and were

149 euthanized using CO2 followed by cervical dislocation. The study was carried out in compliance

150 with the ARRIVE guidelines.

151

152 2.2. Analysis of visual acuity

153

154 Visual acuity was measured at P40 using a virtual optomotor system (Mouse OptoMotry System,

155 Cerebral Mechanics) (Supplementary Table S1), as previously described [78]. Briefly, the animal

156 to be tested was placed on a platform surrounded by four computer screens displaying a virtual

157 cylinder comprising a vertical sine wave grating in 3D coordinate space. The OptoMotry

158 software controlled the speed of rotation, direction of rotation, the frequency of the grating and

159 its contrast. To measure visual acuity, the initial spatial frequency of the grating was set at 0.1

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160 cycles/degree (cpd) and the contrast was set at maximum. The frequency was then systematically

161 increased using staircase procedure until the maximum spatial frequency capable of eliciting a

162 response (visual acuity) was determined. The staircase procedure was such that 3 correct answers

163 in a row advanced it to a higher spatial frequency, while 1 wrong answer returned it to a lower

164 frequency.

165

166 2.3. Analysis of baseline refractive error

167

168 We measured baseline refractive errors in both left and right eyes on alert animals at P40 using

169 an automated eccentric infrared photorefractor as previously described (Supplementary Table

170 S2) [79, 80]. The animal to be refracted was immobilized using a restraining platform, and each

171 eye was refracted along the optical axis in dim room light (< 1 lux), 20-30 minutes after the

172 instillation of 1% tropicamide ophthalmic solution (Alcon Laboratories) to ensure mydriasis and

173 cycloplegia. Refractive error measurements were automatically acquired by the photorefractor

174 every 16 msec. Each successful measurement series (i.e., Purkinje image in the center of the

175 pupil and stable refractive error for at least 5 sec.) was marked by a green LED flash, which was

176 registered by the photorefractor software. Five independent measurement series were taken for

177 each eye. Sixty individual measurements from each series, immediately preceding the green LED

178 flash, were combined, and a total of 300 measurements (60 measurements x 5 series = 300

179 measurements) were collected for each eye. Data for the left and right eyes were combined (600

180 measurements total) to calculate the mean refractive error and standard deviation for each

181 animal.

182

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183 2.4. Analysis of susceptibility to form-deprivation myopia

184

185 We measured the extent of myopia induced by diffuser-imposed retinal image degradation

186 (visual form deprivation) (Supplementary Table S3). The eye is the most sensitive to optical

187 defocus during a critical period in postnatal development, which continues in mice from the eye

188 opening (P12-P14) to approximately P60 [49, 79]. Therefore, visual form deprivation was

189 induced in P24 mice by applying plastic diffusers to one of the eyes and refractive development

190 of the treated eye was compared to that of the contralateral eye (contralateral eye was not treated

191 with a diffuser) as previously described [49, 81]. Diffusers represented low-pass optical filters,

192 which degraded the image projected onto the retina by removing high spatial frequency details.

193 Hemispherical plastic diffusers were made from zero power rigid contact lenses manufactured

194 from OP3 plastic (diameter = 7.0 mm, base curve = 7.0 mm; Lens.com) and Bangerter occlusion

195 foils (Precision Vision). Diffusers were inserted into 3D-printed plastic frames (Proto Labs). On

196 the first day of the experiment (P24), animals were anesthetized via intraperitoneal injection of

197 ketamine and xylazine, and frames with diffusers were attached to the skin surrounding the eye

198 with six stitches using size 5-0 ETHILON™ microsurgical sutures (Ethicon) and reinforced with

199 Vetbond™ glue (3M Animal Care Products) (the contralateral eye served as a control). Toenails

200 were covered with adhesive tape to prevent mice from removing the diffusers. Animals

201 recovered on a warming pad and were then housed under low-intensity constant light in

202 transparent plastic cages for the duration of the experiment as previously described [49, 81].

203 Following 21 days of visual form deprivation (from P24 through P45), diffusers were removed

204 and the refractive state of both treated and control eyes was assessed using an automated infrared

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205 photorefractor as previously described [79, 80]. The interocular difference in refractive error

206 between the treated and contralateral control eye was used as a measure of induced myopia.

207

208 2.5. RNA extraction and RNA-seq

209

210 Animals were euthanized following an IACUC-approved protocol. Eyes were enucleated, the

211 retinae were dissected from the enucleated eyes. The retinae were washed in RNAlater (Thermo

212 Fisher Scientific) for 5 min., frozen in liquid nitrogen, and stored at -80°C. To isolate RNA,

213 tissue samples were homogenized at 4°C in a lysis buffer using Bead Ruptor 24 tissue

214 homogenizer (Omni). Total RNA was extracted from each tissue sample using miRNAeasy mini

215 kit (QIAGEN) following the manufacturer’s protocol. The integrity of RNA was confirmed by

216 analyzing 260/280 nm ratios (Ratio260/280 = 2.11-2.13) on a Nanodrop (Thermo Scientific) and

217 the RNA Integrity Number (RIN = 9.0-10.0) using Agilent Bioanalyzer. Illumina sequencing

218 libraries were constructed from 1 μg of total RNA using the TruSeq Stranded Total RNA LT kit

219 with the Ribo-Zero Gold ribosomal RNA depletion module (Illumina). Each library contained a

220 specific index (barcode) and were pooled at equal concentrations using the randomized complete

221 block (RCB) experimental design before sequencing on Illumina HiSeq 2500 sequencing system.

222 The number of libraries per multiplexed sample was adjusted to ensure sequencing depth of ~70

223 million reads per library (paired-end, 2 x 100 bp). The actual sequencing depth was 76,773,554 ±

224 7,832,271 with read quality score 34.5 ± 0.4.

225

226 2.6. Post-sequencing RNA-seq data validation and analysis

227

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228 The FASTQ raw data files generated by the Illumina sequencing system were imported into

229 Partek Flow software package (Partek), libraries were separated based on their barcodes,

230 adapters were trimmed and remaining sequences were subjected to pre-alignment quality control

231 using the Partek Flow pre-alignment QA/QC module. After the assessment of various quality

232 metrics, bases with the quality score < 34 were removed (≤ 5 bases) from each end. Sequencing

233 reads were then mapped to the mouse reference genome Genome Reference Consortium Mouse

234 Build 38 (GRCm38/mm10, NCBI) using the STAR aligner resulting in 95.0 ± 0.4% mapped

235 reads per library, which covered 35.4 ± 1.0% of the genome. Aligned reads were quantified to

236 transcriptome using the Partek E/M annotation model and the NCBI’s RefSeq annotation file to

237 determine read counts per gene/genomic region. The generated read counts were normalized by

238 the total read count and subjected to the analysis of variance (ANOVA) to detect genes whose

239 expression correlates with either visual acuity, refractive error or susceptibility to form-

240 deprivation myopia. Differentially expressed transcripts were identified using a P-value

241 threshold of 0.05 adjusted for genome-wide statistical significance using Storey’s q-value

242 algorithm [82]. To identify sets of genes with coordinate expression, differentially expressed

243 transcripts were clustered using the Partek Flow hierarchical clustering module, using average

244 linkage for the cluster distance metric and Euclidean distance metric to determine the distance

245 between data points. Each RNA-seq sample was analyzed as a biological replicate, thus,

246 resulting in three (3) biological replicates per strain.

247

248 2.7. Gene ontology analysis and identification of canonical signaling pathways

249

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250 To identify biological functions (gene ontology categories), which were significantly associated

251 with the genes whose expression correlated with visual acuity, baseline refractive errors or

252 susceptibility to form-deprivation myopia, we used the database for annotation, visualization and

253 integrated discovery (DAVID) version 6.8 [83] and GOplot R package version 1.0.2 [84].

254 DAVID uses a powerful gene-enrichment algorithm and Gene Concept database to identify

255 biological functions (gene ontology categories) affected by differential genes. GOplot integrates

256 gene ontology information with gene expression information and predicts the effects of gene

257 expression changes on biological processes. DAVID uses a modified Fisher's exact test (EASE

258 score) with a P-value threshold of 0.05 to estimate the statistical significance of enrichment for

259 specific gene ontology categories. The IPA Pathways Activity Analysis module (Ingenuity

260 Pathway Analysis, QIAGEN) was used to identify canonical pathways encoded by the genes

261 involved in baseline refractive eye development, susceptibility to myopia or contrast perception,

262 and to predict the effects of gene expression differences in different mouse strains on the

263 pathways. The activation z-score was employed in the Pathways Activity Analysis module to

264 predict activation or suppression of the canonical pathways. The z-score algorithm is designed to

265 reduce the chance that random data will generate significant predictions. The z-score provides an

266 estimate of statistical quantity of change for each pathway found to be statistically significantly

267 affected by the changes in gene expression. The significance values for the canonical pathways

268 were calculated by the right-tailed Fisher's exact test. The significance indicates the probability

269 of association of molecules from a dataset with the canonical pathway by random chance alone.

270 The Pathways Activity Analysis module determines if canonical pathways, including functional

271 end-points, are activated or suppressed based on the gene expression data in a dataset. Once

272 statistically significant canonical pathways were identified, we subjected the datasets to the Core

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273 Functional Analysis in IPA to compare the pathways and identify key similarities and differences

274 in the canonical pathways underlying visual acuity, baseline refractive development,

275 susceptibility to form-deprivation myopia.

276

277 2.8. Identification of genes linked to human myopia and other eye-related human genetic

278 disorders

279

280 All mouse genes, which were found to be associated with visual acuity and baseline refractive

281 errors or susceptibility to form-deprivation myopia were analyzed for their association with eye-

282 related human genetic disorders. To identify genes linked to human myopia, we compared the

283 genes that we found in mice with a list of genes located in the human myopia quantitative trait

284 loci (QTLs). We first compiled a list of all single-nucleotide polymorphisms (SNPs) or markers

285 exhibiting a statistically significant association with myopia in the human linkage or genome-

286 wide association studies (GWAS) using the Online Mendelian Inheritance in Man (OMIM)

287 (McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University) and NHGRI-EBI

288 GWAS Catalog [85] databases. The LDlink’s LDmatrix tool (National Cancer Institute) was

289 used to identify SNPs in linkage disequilibrium and identify overlapping chromosomal loci. We

290 then used the UCSC Table Browser to extract all genes located within critical chromosomal

291 regions identified by the human linkage studies or within 200 kb (±200 kb) of the SNPs found by

292 GWAS. The list of genes located within human QTLs was compared with the list of genes that

293 we found in mice using Partek Genomics Suite (Partek). To identify genes associated with eye-

294 related human genetic disorders unrelated to myopia, we screened mouse genes that we found in

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295 this study against the Online Mendelian Inheritance in Man (OMIM) (McKusick-Nathans

296 Institute of Genetic Medicine, Johns Hopkins University) database.

297

298 2.9. Statistical data analysis and data graphing

299

300 Statistical analyses of the RNA-seq data were performed using statistical modules integrated into

301 Partek Flow (Partek), DAVID [83] and IPA (QIAGEN) software packages and described in the

302 corresponding sections. Other statistical analyses were performed using the STATISTICA

303 software package (StatSoft). The statistical significance of the overlaps between gene datasets

304 was estimated using probabilities associated with the hypergeometric distribution as

305 implemented in the Bioconductor R software package GeneOverlap and associated functions.

306 Data graphing and visualization was performed using Partek Flow (Partek) and IPA (QIAGEN)

307 graphing and visualization modules, as well as Prism 8 for Windows (GraphPad) and GOplot R

308 package [84].

309

310 3. Results

311

312 3.1. Visual acuity exhibits weak but significant association with baseline refractive error and

313 visually guided eye emmetropization in mice

314

315 In order to determine the role of visual acuity in baseline refractive eye development and visually

316 guided eye emmetropization, we analyzed the relationship between visual acuity, baseline

317 refractive error, and susceptibility to form-deprivation myopia (as a proxy for visually guided

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318 eye emmetropization) in eight genetically different inbred strains of mice characterized by

319 different levels of visual acuity, baseline refractive errors, and susceptibility to form-deprivation

320 myopia (Supplementary Tables S1-S3). Visual acuity (VA), baseline refractive error (RE), and

321 induced myopia (myopia) were measured in 129S1/svlmj, A/J, C57BL/6J, CAST/EiJ,

322 NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, and WSB/EiJ mice at P40-P45 (see Methods).

323 We found that all three attributes (visual acuity, refractive error, and susceptibility to

324 myopia) were inherited as quantitative traits (Figure 1A-C; Supplementary Tables S1-S3). We

325 also found that all three attributes were strongly influenced by genetic background (ANOVAVA,

326 F(7, 57)=1807.3, P < 0.00001; ANOVARE, F(7, 145) = 429.8, P < 0.00001; ANOVAmyopia, F(7,

327 48) = 9.8, P < 0.00001). In agreement with our recent report [30], correlation between baseline

328 refractive error and susceptibility to myopia was weak (r = 0.2686, P = 0.0305; Supplementary

329 Figure S1). Linear regression analyses revealed weak but statistically significant positive

330 correlation between visual acuity and baseline refractive error (r = 0.371, P = 0.0023) (Figure

331 1D), as well as between visual acuity and susceptibility to myopia (r = 0.3367, P = 0.0061)

332 (Figure 1E). Correlation coefficients for both regressions were similar suggesting essentially

333 equal, yet relatively modest, contribution of visual acuity to both baseline refractive eye

334 development and visually guided eye emmetropization.

335

336 3.2. Visual acuity is controlled by an extensive genetic network

337

338 Our data suggested that visual acuity is weakly associated with both baseline refractive eye

339 development and visually guided eye emmetropization. Therefore, we then set out to investigate

340 whether the signaling cascade underlying visual acuity is involved in the regulation of baseline

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341 refractive eye development and eye emmetropization. To gain insight into the signaling cascade

342 subserving visual acuity and examine its involvement in the regulation of baseline refractive eye

343 development and eye emmetropization, we performed a genome-wide gene expression profiling

344 in the retina of the eight mouse strains listed above and analyzed the transcriptomes underlying

345 visual acuity. The retinal acuity-related transcriptome was analyzed at P28 (age when refractive

346 eye development in mice is progressing towards its stable plateau, but visual input is still

347 influencing eye growth [49, 79]) using RNA-seq.

348 We found that visual acuity in mice correlates with expression of 1,073 retinal genes (Q-

349 value < 0.02) (Figure 2A, Supplementary Table S4). Expression of 394 of these genes was

350 positively correlated with visual acuity (i.e., expression was increased in animals with high

351 visual acuity and reduced in animals with low visual acuity), whereas expression of 679 of these

352 genes was negatively correlated with visual acuity (i.e., expression was decreased in animals

353 with high visual acuity and increased in animals with low visual acuity). Furthermore, visual

354 acuity was associated with biological processes related to protein phosphorylation, homophilic

355 cell adhesion, metabolic process, intracellular signal transduction, transcriptional regulation of

356 gene expression, axonogenesis, response to ischemia, and photoreceptor cell maintenance,

357 among other processes (Figure 2B, Supplementary Table S7). We also found that visual acuity

358 was linked to the activation or suppression of several canonical signaling pathways, including

359 protein kinase A signaling, RhoGDI signaling, oleate biosynthesis, cAMP-mediated signaling,

360 melatonin signaling, senescence pathway, Wnt/Ca+ pathway, opioid signaling pathway, prolactin

361 signaling, PPARα/RXRα activation pathway, PTEN signaling, nNOS signaling, and β-adrenergic

362 signaling (Figure 2C, Supplementary Table S10).

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363 These data suggest that visual acuity is regulated by an extensive genetic network

364 encoding a diverse set of biological processes and signaling pathways.

365

366 3.3. Comparison of the transcriptome for visual acuity with the transcriptome for baseline

367 refractive eye development reveals significant contribution of the genetic network subserving

368 visual acuity to baseline refractive development

369

370 To examine the contribution of the genetic network regulating visual acuity to baseline refractive

371 eye development, we analyzed the overlap between gene expression networks underlying visual

372 acuity and baseline refractive development. As reported previously [30], we identified 2,050

373 genes (Q-value < 0.01) whose expression was associated with baseline refractive eye

374 development (Supplementary Figure S2A, Supplementary Table S5). Gene ontology analysis

375 revealed 88 biological processes, which were involved in baseline refractive eye development

376 (Supplementary Figure S2B, Supplementary Table S8). The gene ontology data highlighted an

377 important role of visual perception, synaptic transmission, cell-cell communication, retina

378 development, and DNA methylation in baseline refractive eye development. Moreover, baseline

379 refractive development was associated with several canonical signaling pathways, including β-

380 adrenergic signaling, protein kinase A signaling, dopamine receptor signaling, HIPPO signaling,

381 mTOR signaling, phototransduction pathway, axonal guidance signaling, synaptic long-term

382 potentiation, tight junction signaling, and DNA methylation signaling (Supplementary Figure

383 S2C, Supplementary Table S11). We found that approximately 25% of genes involved in the

384 regulation of visual acuity (268 out of 1,073 genes) were also associated with baseline refractive

385 eye development (OR = 3.5, P = 3.391 × 10-78) (Figure 3A, Supplementary Table S13). These

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386 genes were involved in at least 47 biological processes and 31 molecular functions. The top 30

387 biological processes shown in Figure 3B (Supplementary Table S15) included such processes as

388 homophilic cell adhesion, metabolic process, lipid metabolism, protein autophosphorylation,

389 response to ischemia, visual perception, and neuron projection morphogenesis, i.e., many of the

390 key processes underlying visual acuity (Figure 2B). The majority of these processes were

391 suppressed in animals with high visual acuity and activated in animals with low visual acuity

392 (Figure 3B). In agreement with these observations, approximately 55% of the canonical signaling

393 pathways involved in the regulation of visual acuity were also involved in baseline refractive eye

394 development (Figure 3C). Most notable pathways included protein kinase A signaling, RhoGDI

395 signaling, melatonin signaling, senescence pathway, Wnt/Ca+ pathway, nNOS signaling, β-

396 adrenergic signaling, synaptic long-term potentiation, dopamine-DARPP32 feedback signaling,

397 α-adrenergic signaling, and mTOR Signaling.

398 Thus, although linear regression analysis demonstrated relatively modest influence of

399 visual acuity on baseline refractive eye development, we found a substantial overlap between

400 gene expression networks and canonical signaling pathways underlying these two processes.

401

402 3.4. Comparison of the transcriptome for visual acuity with the transcriptome for form-

403 deprivation myopia reveals modest contribution of the signaling cascade subserving visual

404 acuity to visually guided eye emmetropization

405

406 To examine the contribution of the genetic network regulating visual acuity to visually guided

407 eye emmetropization, we analyzed the overlap between gene expression networks underlying

408 visual acuity and susceptibility to form-deprivation myopia. As we reported previously [30],

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409 expression of 1,347 genes (Q-value < 0.01) was associated with susceptibility to form-

410 deprivation myopia (Supplementary Figure S3A, Supplementary Table S6). Gene ontology

411 analysis revealed involvement of several biological processes, including axonogenesis, transport,

412 fatty acid oxidation, aging, insulin secretion, cell proliferation, cell-cell adhesion, and cellular

413 response to hypoxia (Supplementary Figure S3B, Supplementary Table S9). Furthermore,

414 susceptibility to form-deprivation myopia was linked to several canonical signaling pathways,

415 including G2/M DNA damage checkpoint regulation, iron homeostasis signaling pathway, tight

416 junction signaling, sirtuin signaling pathway, β-adrenergic and α-adrenergic signaling, HIPPO

417 signaling, mTOR signaling, amyotrophic lateral sclerosis signaling, axonal guidance signaling,

418 and growth hormone signaling (Supplementary Figure S3C, Supplementary Table S12). We

419 discovered that approximately 19% of genes (205 out of 1,073 genes) involved in the regulation

420 of visual acuity were also involved in the regulation of susceptibility to form-deprivation myopia

421 (OR = 4.1, P = 5.219 × 10-70) (Figure 4A, Supplementary Table S14). These genes were involved

422 in approximately 31 biological process and 19 molecular functions. As shown in Figure 4B

423 (Supplementary Table S16), the top 30 biological processes included brain development,

424 response to hydrogen peroxide, spermine and spermidine biosynthesis, metabolic process,

425 response to ischemia, homophilic cell adhesion, regulation of autophagy, chloride

426 transmembrane transport, and synaptic vesicle recycling. The absolute majority of these

427 processes were suppressed in animals with high visual acuity and activated in animals with low

428 visual acuity. Only 28% of canonical signaling pathways involved in the regulation of visual

429 acuity were also involved in the regulation of susceptibility to form-deprivation myopia (Figure

430 4C). In addition to such pathways as tRNA charging, growth hormone signaling, methylmalonyl

431 pathway, RAR activation pathway, role of JAK2 in hormone-like cytokine signaling, 2-

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432 oxobutanoate degradation, and iron homeostasis signaling pathway, these pathways included

433 many of the same pathways involved in baseline refractive eye development, i.e., axonal

434 guidance signaling, β-adrenergic signaling, GP6 signaling pathway, superpathway of D-myo-

435 inositol (1,4,5)-trisphosphate metabolism, α-adrenergic signaling, 3-phosphoinositide

436 degradation, superpathway of inositol phosphate compounds, D-myo-inositol (1,4,5)-

437 trisphosphate degradation, mTOR signaling, and AMPK signaling.

438 In summary, these data suggest that the gene expression network regulating visual acuity

439 has relatively limited contribution to visually guided eye emmetropization. Moreover, many of

440 the canonical signaling pathways regulating both visual acuity and eye emmetropization are also

441 involved in baseline refractive eye development.

442

443 3.5. Genes regulating visual acuity are involved in human myopia development and other eye-

444 related genetic disorders

445

446 Our data suggested that genes involved the regulation of visual acuity contributed to both

447 baseline refractive eye development and visually guided eye emmetropization. Therefore, we

448 than evaluated the association of these genes with human myopia revealed by GWAS and gene-

449 based GWAS studies and other human eye-related genetic disorders listed in the Online

450 Mendelian Inheritance in Man (OMIM) database. We found that 61 out of 381 genes involved in

451 the regulation of visual acuity and refractive eye development was associated with known eye-

452 related human disorders (Figure 5, Supplementary Table S17). Approximately 51% of these

453 genes (31 gene) were associated with baseline refractive eye development, 33% (20 genes) were

454 involved in visually guided emmetropization, and 16% (10 genes) were associated with both

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455 baseline refractive eye development and emmetropization. The largest number of genes

456 implicated in human disorders was linked to myopia (57%), whereas other genes were associated

457 with retinal degeneration and retinal atrophy, neurodevelopmental disorders, and connective

458 tissue disorders. The most prevalent biological processes affected by these genes were visual

459 perception, calcium ion transport, metabolic processes, and neurogenesis (Supplementary Table

460 S18).

461 Cumulatively, these data suggest that the genetic network regulating visual acuity makes

462 a substantial contribution to the development of human myopia by modulating a number of

463 biological processes involved in refractive eye development, including phototransduction-related

464 signaling, signal transduction, and signaling pathways underlying various metabolic processes.

465

466 4. Discussion

467

468 Our recent data suggest that baseline refractive eye development and visually guided eye

469 emmetropization are controlled by overlapping but largely distinct genetic networks [28]. We

470 also found that the genetic network subserving contrast perception has a significant contribution

471 to optical defocus detection and emmetropization, while having a very little contribution to

472 baseline refractive eye development. Growing evidence indicates that detection of optical

473 defocus by the retina is mediated by luminance contrast and longitudinal chromatic aberrations

474 [8, 86]; however, some findings suggest that signaling cascades regulating visual acuity may also

475 be involved in eye emmetropization [67]. Our current data suggest that the retinal gene

476 expression network subserving visual acuity has a limited but significant involvement in both

477 baseline refractive eye development and visually guided eye emmetropization. However,

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478 contribution of the genetic network underlying visual acuity to baseline refractive eye

479 development appears to be both different and more significant compared to its contribution to

480 eye emmetropization.

481 We found that visual acuity is regulated by a large number of genes organized into a

482 rather elaborate genetic network contributing to a variety of biological processes, molecular

483 functions and signaling pathways. As expected, our data clearly suggest that visual acuity

484 depends on the density of photoreceptors because biological processes related to nervous system

485 development and positive regulation of cell proliferation were significantly overrepresented.

486 However, visual acuity seems to be also associated with transcriptional regulation of gene

487 expression, protein phosphorylation, and protein turnover, suggesting that visual acuity is also

488 actively modulated via dynamic changes in gene expression and protein signaling networks,

489 beyond a sheer number of photoreceptors per unit of retinal surface. Overrepresentation of

490 biological processes related to cell adhesion and axonogenesis points to a prominent role of

491 synaptic transmission in the regulation of visual acuity. We found that high visual acuity was

492 associated with activation of the RhoGDI signaling and PPARα/RXRα activation pathways and

493 strong suppression of the protein kinase A signaling pathway, oleate biosynthesis, opioid

494 signaling pathway, reelin signaling in neurons, Tec kinase signaling, and β-adrenergic signaling.

495 Although the majority of these biological processes and signaling pathways were implicated in

496 the regulation of visual acuity for the first time, the opioid signaling pathway was previously

497 shown to modulate visual acuity [28]. For example, cannabinoids were shown to influence visual

498 acuity through the cannabinoid receptor type 2 (CB2R), which closely interacts with the opioid

499 signaling pathway [77, 87-90]. Noteworthy, expression of CB2R was shown to be regulated by

500 nitric oxide, which has long been implicated in refractive eye development [12, 91-95].

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501 Although our linear regression analyses suggested that visual acuity has essentially equal

502 contribution to both baseline refractive eye development and visually guided eye

503 emmetropization, analysis of gene expression networks revealed a more complex relationship

504 between visual acuity, baseline refractive development, and emmetropization. Although 92 out

505 of 268 genes involved in the regulation of both visual acuity and baseline refractive development

506 were also implicated in emmetropization (OR = 48.2; P = 1.416 × 10-135), we found that the key

507 biological processes and signaling pathways impacted by the visual-acuity-related genetic

508 network in baseline refractive development and visually guided eye emmetropization were

509 substantially different. While visual-acuity-related biological processes involved in baseline

510 refractive development were primarily related to cell-cell communication, metabolic processes,

511 GTPase-mediated signaling, visual perception, and neuron projection morphogenesis, visual-

512 acuity-related biological processes involved in emmetropization were primarily related to

513 neurogenesis, ion transport, metabolic processes, cell-cell communication, response to ischemia,

514 and response to hydrogen peroxide. The visual-acuity-related genetic network contributes to

515 several canonical pathways common to both baseline refractive development and

516 emmetropization, including pathways involved in inositol metabolism, protein kinase A

517 signaling, α-adrenergic and β-adrenergic signaling, axon guidance, GP6 signaling, mTOR

518 signaling, and AMPK signaling. However, we also uncovered substantial differences. First, the

519 total number of canonical pathways underlying baseline refractive development and encoded by

520 the visual-acuity-related genetic network is much greater compared to the number of pathways

521 underlying eye emmetropization and encoded by the visual-acuity-related genetic network (35

522 versus 18). Second, despite the existence of shared pathways, the visual-acuity-related genetic

523 network contributes to largely different canonical pathways when it comes to baseline refractive

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524 development and visually guided eye emmetropization. Most notable pathways unique to

525 baseline refractive development include melatonin signaling, senescence pathway, Wnt/Ca+

526 pathway, nNOS signaling, phototransduction pathway, synaptic long-term potentiation, synaptic

527 long-term depression, and dopamine-DARPP32 feedback signaling. Conversely, pathways

528 unique to visually guided eye emmetropization are limited to tRNA charging, growth hormone

529 signaling, methylmalonyl pathway, RAR activation, JAK2 signaling, 2-oxobutanoate

530 degradation, and iron homeostasis signaling pathway. Taken together, these data suggest that the

531 visual-acuity-related gene network makes largely unique contributions to the retinal signaling

532 cascades underlying baseline refractive eye development and visually guided eye

533 emmetropization.

534 Several biological processes and signaling pathways regulating visual acuity, baseline

535 refractive eye development and eye emmetropization were previously implicated in refractive

536 eye development. For example, cell-cell communication and synaptic transmission were shown

537 to influence refractive eye development and response to optical defocus by several studies [12,

538 28, 29, 96-99]. Energy production and various metabolic processes were also shown to play an

539 important role in refractive eye development and eye’s response to optical defocus [12, 28, 29,

540 98-100]. The protein kinase A signaling, α-adrenergic signaling, β-adrenergic signaling, and

541 mTOR signaling pathways have been repeatedly found to be linked to retinal signaling

542 underlying refractive eye development [12, 28, 98, 100].

543 The biological processes and signaling pathways uniquely underlying either baseline

544 refractive development or visually guided eye emmetropization and regulated by the visual-

545 acuity-related genetic network provide several interesting insights into the biology of refractive

546 eye development. The finding that the genetic network regulating visual acuity and baseline

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547 refractive development contributes to melatonin signaling, senescence pathway,

548 phototransduction pathway, and synaptic long-term depression reveals a complex interplay

549 between genetic networks underlying visual acuity, perception of contrast, and baseline

550 refractive development because it was found that genes controlling both contrast perception and

551 baseline refractive development encode components of the same pathways. Interestingly, the

552 tRNA charging and JAK2 signaling pathways involved in both visual acuity regulation and

553 visually guided eye emmetropization are also involved in contrast perception, thus suggesting an

554 overlap between genetic networks underlying visual acuity regulation, contrast perception, and

555 visually guided eye emmetropization.

556 We discovered that approximately 16% of the genes, which we found to be involved in

557 visual acuity regulation and refractive eye development, were also implicated in human myopia

558 and other eye-related genetic disorders. Several genes were involved in visual perception. One of

559 the genes, GRK1, causing Oguchi disease is involved in light-dependent deactivation of

560 rhodopsin [101]. The myopia-associated gene CNGB3 encodes one of the subunits that form

561 cyclic nucleotide-gated ion channels required for sensory transduction in rod photoreceptors [28,

562 102]. The IMPG2 gene linked to retinitis pigmentosa plays a role in organization of the

563 interphotoreceptor matrix and promotes growth and maintenance of the photoreceptor outer

564 segment [103, 104]. The two genes, PDE6C known to cause cone dystrophy and PDE6A linked

565 to retinitis pigmentosa, encode subunits of cone and rod phosphodiesterases which play

566 important roles in phototransduction [105-109]. The RDH12 gene involved in the development

567 of Leber congenital amaurosis encodes retinol dehydrogenase which metabolizes both all-trans-

568 and cis-retinols, which play a key role in the phototransduction pathway [110, 111]. The two

569 myopia-associated genes CACNA2D1 and CAMK2G encode several subunits of the voltage-

Page 25 of 40

570 gated calcium channel and a calcium-dependent protein kinase, which play important roles in the

571 regulation of synaptic transmission in the retina downstream of photoreceptors [28, 112-115].

572 Interestingly, calcium signaling in the retina was shown to be modulated by dopamine and nitric

573 oxide, both of which have been implicated in refractive eye development [92-95, 116-122].

574 Taken together, these data suggest that visual acuity depends, to a significant degree, on the

575 signaling cascade underlying light perception and phototransduction. While deleterious

576 mutations in these genes often cause severe retinal damage associated with photoreceptor

577 degeneration, genetic variations modulating gene expression appear to primarily influence visual

578 acuity and refractive eye development.

579

580 5. Conclusions

581

582 In this study, we explored the genetic network regulating visual acuity and its contribution to

583 baseline refractive eye development and visually guided emmetropization. Our results provide

584 evidence that visual acuity is determined not only by the density of photoreceptors per unit of

585 retinal surface, but it is also regulated at the level of molecular signaling in the retina by an

586 extensive genetic network. This genetic network controls visual acuity via a diverse set of

587 biological processes primarily related to transcriptional regulation of gene expression, protein

588 phosphorylation, protein turnover, homophilic cell adhesion, metabolic processes, axonogenesis,

589 response to ischemia, photoreceptor signaling, and synaptic transmission. Our data suggest that

590 the genetic network regulating visual acuity makes modest but significant contributions to both

591 baseline refractive eye development and visually guided eye emmetropization; however, the

592 contribution to baseline refractive eye development is much more substantial. We found that

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593 visual-acuity-related pathways uniquely involved in baseline refractive eye development include

594 melatonin signaling, senescence pathway, Wnt/Ca+ pathway, nNOS signaling, phototransduction

595 pathway, synaptic long-term potentiation, synaptic long-term depression, and dopamine-

596 DARPP32 feedback signaling. Visual-acuity-related pathways uniquely underlying visually

597 guided eye emmetropization are primarily involved in tRNA charging, growth hormone

598 signaling, methylmalonyl pathway, RAR activation, JAK2 signaling, 2-oxobutanoate

599 degradation, and iron homeostasis signaling. A small number of genes involved in the regulation

600 of visual acuity are also involved in the development of human myopia. These genes are

601 overwhelmingly involved in light perception and phototransduction. Our results also suggest a

602 complex interplay between genetic networks that control visual acuity, contrast perception, and

603 refractive eye development.

604

605 Availability of data and materials

606 All data generated or analyzed during this study are included in this article and its supplementary

607 information files. The RNA-seq data have been deposited in the Gene Expression Omnibus

608 database [GSE158732] (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE158732).

609

610 Declaration of competing interests

611 TVT and AVT are named inventors on six US patent applications related to the development of a

612 pharmacogenomics pipeline for anti-myopia drug development.

613

614 Funding

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615 This work was supported by the National Institutes of Health grants R01EY023839 (AVT),

616 P30EY019007 (Core Support for Vision Research received by the Department of

617 Ophthalmology, Columbia University), and Research to Prevent Blindness (Unrestricted funds

618 received by the Department of Ophthalmology, Columbia University). The funders had no role in

619 study design, data collection and analysis, decision to publish, or preparation of the manuscript.

620

621 Author contributions

622 TVT and AVT conceptualized the study, analyzed refractive eye development, susceptibility to

623 form-deprivation myopia and visual acuity in mice, performed RNA-seq, and analyzed the data.

624 AVT supervised the entire study, analyzed and validated data, and wrote the original draft of the

625 manuscript. All authors read, edited, and approved the final version of the manuscript.

626

627 References

628 1. Tedja MS, Haarman AEG, Meester-Smoor MA, Kaprio J, Mackey DA, Guggenheim JA, et 629 al. IMI - Myopia Genetics Report. Invest Ophthalmol Vis Sci. 2019;60(3):M89-M105. 630 2. Fan Q, Verhoeven VJ, Wojciechowski R, Barathi VA, Hysi PG, Guggenheim JA, et al. 631 Meta-analysis of gene-environment-wide association scans accounting for education level 632 identifies additional loci for refractive error. Nature communications. 2016;7:11008. 633 3. Fan Q, Guo X, Tideman JW, Williams KM, Yazar S, Hosseini SM, et al. Childhood gene- 634 environment interactions and age-dependent effects of genetic variants associated with 635 refractive error and myopia: The CREAM Consortium. Sci Rep. 2016;6:25853. 636 4. Verhoeven VJ, Buitendijk GH, Consortium for Refractive E, Myopia, Rivadeneira F, 637 Uitterlinden AG, et al. Education influences the role of genetics in myopia. European 638 journal of epidemiology. 2013;28(12):973-80. 639 5. Chen YP, Hocking PM, Wang L, Povazay B, Prashar A, To CH, et al. Selective breeding 640 for susceptibility to myopia reveals a gene-environment interaction. Invest Ophthalmol Vis 641 Sci. 2011;52(7):4003-11. 642 6. Lyhne N, Sjolie AK, Kyvik KO, Green A. The importance of genes and environment for 643 ocular refraction and its determiners: a population based study among 20-45 year old twins. 644 Br J Ophthalmol. 2001;85(12):1470-6. 645 7. Tkatchenko AV, Tkatchenko TV, Guggenheim JA, Verhoeven VJ, Hysi PG, 646 Wojciechowski R, et al. APLP2 Regulates Refractive Error and Myopia Development in 647 Mice and Humans. PLoS Genet. 2015;11(8):e1005432.

Page 28 of 40

648 8. Troilo D, Smith EL, 3rd, Nickla DL, Ashby R, Tkatchenko AV, Ostrin LA, et al. IMI - 649 Report on Experimental Models of Emmetropization and Myopia. Invest Ophthalmol Vis 650 Sci. 2019;60(3):M31-M88. 651 9. French AN, Ashby RS, Morgan IG, Rose KA. Time outdoors and the prevention of myopia. 652 Exp Eye Res. 2013;114:58-68. 653 10. Rose KA, Morgan IG, Ip J, Kifley A, Huynh S, Smith W, et al. Outdoor activity reduces the 654 prevalence of myopia in children. Ophthalmology. 2008;115(8):1279-85. 655 11. Nickla DL. Ocular diurnal rhythms and eye growth regulation: where we are 50 years after 656 Lauber. Exp Eye Res. 2013;114:25-34. 657 12. Tkatchenko TV, Troilo D, Benavente-Perez A, Tkatchenko AV. Gene expression in 658 response to optical defocus of opposite signs reveals bidirectional mechanism of visually 659 guided eye growth. PLoS biology. 2018;16(10):e2006021. 660 13. Hung LF, Crawford ML, Smith EL. Spectacle lenses alter eye growth and the refractive 661 status of young monkeys. Nat Med. 1995;1(8):761-5. 662 14. Cottriall CL, McBrien NA. The M1 muscarinic antagonist pirenzepine reduces myopia and 663 eye enlargement in the tree shrew. Invest Ophthalmol Vis Sci. 1996;37(7):1368-79. 664 15. Metlapally S, McBrien NA. The effect of positive lens defocus on ocular growth and 665 emmetropization in the tree shrew. J Vis. 2008;8(3):1 -12. 666 16. Howlett MH, McFadden SA. Spectacle lens compensation in the pigmented guinea pig. 667 Vision Res. 2009;49(2):219-27. 668 17. Schaeffel F, Glasser A, Howland HC. Accommodation, refractive error and eye growth in 669 chickens. Vision Res. 1988;28(5):639-57. 670 18. Wiesel TN, Raviola E. Myopia and eye enlargement after neonatal lid fusion in monkeys. 671 Nature. 1977;266(5597):66-8. 672 19. Gwiazda J, Bauer J, Thorn F, Held R. A dynamic relationship between myopia and blur- 673 driven accommodation in school-aged children. Vision Res. 1995;35(9):1299-304. 674 20. Gwiazda JE, Hyman L, Norton TT, Hussein ME, Marsh-Tootle W, Manny R, et al. 675 Accommodation and related risk factors associated with myopia progression and their 676 interaction with treatment in COMET children. Invest Ophthalmol Vis Sci. 677 2004;45(7):2143-51. 678 21. Gwiazda J, Hyman L, Hussein M, Everett D, Norton TT, Kurtz D, et al. A randomized 679 clinical trial of progressive addition lenses versus single vision lenses on the progression of 680 myopia in children. Invest Ophthalmol Vis Sci. 2003;44(4):1492-500. 681 22. Sun YY, Li SM, Li SY, Kang MT, Liu LR, Meng B, et al. Effect of uncorrection versus full 682 correction on myopia progression in 12-year-old children. Graefes Arch Clin Exp 683 Ophthalmol. 2017;255(1):189-95. 684 23. Li SY, Li SM, Zhou YH, Liu LR, Li H, Kang MT, et al. Effect of undercorrection on 685 myopia progression in 12-year-old children. Graefes Arch Clin Exp Ophthalmol. 686 2015;253(8):1363-8. 687 24. Pararajasegaram R. VISION 2020-the right to sight: from strategies to action. Am J 688 Ophthalmol. 1999;128(3):359-60. 689 25. Holden BA, Fricke TR, Wilson DA, Jong M, Naidoo KS, Sankaridurg P, et al. Global 690 Prevalence of Myopia and High Myopia and Temporal Trends from 2000 through 2050. 691 Ophthalmology. 2016;123(5):1036-42. 692 26. Saw SM, Chua WH, Hong CY, Wu HM, Chan WY, Chia KS, et al. Nearwork in early- 693 onset myopia. Invest Ophthalmol Vis Sci. 2002;43(2):332-9.

Page 29 of 40

694 27. Huang HM, Chang DS, Wu PC. The Association between Near Work Activities and 695 Myopia in Children-A Systematic Review and Meta-Analysis. PLoS One. 696 2015;10(10):e0140419. 697 28. Tkatchenko TV, Shah RL, Nagasaki T, Tkatchenko AV. Analysis of genetic networks 698 regulating refractive eye development in collaborative cross progenitor strain mice reveals 699 new genes and pathways underlying human myopia. BMC Med Genomics. 2019;12(1):113. 700 29. Tkatchenko TV, Tkatchenko AV. Pharmacogenomic approach to antimyopia drug 701 development: pathways lead the way. Trends Pharmacol Sci. 2019;40(11):834-53. 702 30. Tkatchenko TV, Tkatchenko AV. Genome-wide analysis of retinal transcriptome reveals 703 common genetic network underlying perception of contrast and optical defocus detection. 704 BioRxiv. 2020:[Preprint]. 705 31. Levenson JH, Kozarsky A. Visual Acuity. In: rd, Walker HK, Hall WD, Hurst JW, editors. 706 Clinical Methods: The History, Physical, and Laboratory Examinations. Boston1990. 707 32. Green DG. Regional variations in the visual acuity for interference fringes on the retina. 708 The Journal of physiology. 1970;207(2):351-6. 709 33. Neumeyer C. Wavelength dependence of visual acuity in goldfish. J Comp Physiol A 710 Neuroethol Sens Neural Behav Physiol. 2003;189(11):811-21. 711 34. Pettigrew JD, Dreher B, Hopkins CS, McCall MJ, Brown M. Peak density and distribution 712 of ganglion cells in the retinae of microchiropteran bats: implications for visual acuity. 713 Brain, behavior and evolution. 1988;32(1):39-56. 714 35. Porciatti V, Pizzorusso T, Maffei L. The visual physiology of the wild type mouse 715 determined with pattern VEPs. Vision Res. 1999;39(18):3071-81. 716 36. Gianfranceschi L, Fiorentini A, Maffei L. Behavioural visual acuity of wild type and bcl2 717 transgenic mouse. Vision Res. 1999;39(3):569-74. 718 37. Petry HM, Fox R, Casagrande VA. Spatial contrast sensitivity of the tree shrew. Vision 719 Res. 1984;24(9):1037-42. 720 38. Norton TT, McBrien NA. Normal development of refractive state and ocular component 721 dimensions in the tree shrew (Tupaia belangeri). Vision Res. 1992;32(5):833-42. 722 39. Buttery RG, Hinrichsen CF, Weller WL, Haight JR. How thick should a retina be? A 723 comparative study of mammalian species with and without intraretinal vasculature. Vision 724 Res. 1991;31(2):169-87. 725 40. Howlett MH, McFadden SA. Emmetropization and schematic eye models in developing 726 pigmented guinea pigs. Vision Res. 2007;47(9):1178-90. 727 41. Berkley MA, Watkins DW. Grating resolution and refraction in the cat estimated from 728 evoked cerebral potentials. Vision Res. 1973;13(2):403-15. 729 42. Blake R, Cool SJ, Crawford ML. Visual resolution in the cat. Vision Res. 730 1974;14(11):1211-7. 731 43. Demello LR, Foster TM, Temple W. Discriminative performance of the domestic hen in a 732 visual acuity task. J Exp Anal Behav. 1992;58(1):147-57. 733 44. Diedrich E, Schaeffel F. Spatial resolution, contrast sensitivity, and sensitivity to defocus of 734 chicken retinal ganglion cells in vitro. Vis Neurosci. 2009;26(5-6):467-76. 735 45. Weinstein B, Grether WF. A comparison of visual acuity in the rhesus monkey and man. J 736 Comp Physiol. 1940;30:187-95. 737 46. Qiao-Grider Y, Hung LF, Kee CS, Ramamirtham R, Smith EL, 3rd. Normal ocular 738 development in young rhesus monkeys (Macaca mulatta). Vision Res. 2007;47(11):1424- 739 44.

Page 30 of 40

740 47. Hamilton R, Bach M, Heinrich SP, Hoffmann MB, Odom JV, McCulloch DL, et al. VEP 741 estimation of visual acuity: a systematic review. Doc Ophthalmol. 2020. 742 48. Shen W, Sivak JG. Eyes of a lower vertebrate are susceptible to the visual environment. 743 Invest Ophthalmol Vis Sci. 2007;48(10):4829-37. 744 49. Tkatchenko TV, Shen Y, Tkatchenko AV. Mouse experimental myopia has features of 745 primate myopia. Invest Ophthalmol Vis Sci. 2010;51(3):1297-303. 746 50. Jiang X, Kurihara T, Kunimi H, Miyauchi M, Ikeda SI, Mori K, et al. A highly efficient 747 murine model of experimental myopia. Sci Rep. 2018;8(1):2026. 748 51. Ni J, Smith EL, 3rd. Effects of chronic optical defocus on the kitten's refractive status. 749 Vision Res. 1989;29(8):929-38. 750 52. Mutti DO, Mitchell GL, Jones LA, Friedman NE, Frane SL, Lin WK, et al. Axial growth 751 and changes in lenticular and corneal power during emmetropization in infants. Invest 752 Ophthalmol Vis Sci. 2005;46(9):3074-80. 753 53. Courage ML, Adams RJ. Infant peripheral vision: the development of monocular visual 754 acuity in the first 3 months of postnatal life. Vision Res. 1996;36(8):1207-15. 755 54. Banks MS, Salapatek P. Acuity and contrast sensitivity in 1-, 2-, and 3-month-old human 756 infants. Invest Ophthalmol Vis Sci. 1978;17(4):361-5. 757 55. Mutti DO, Mitchell GL, Jones LA, Friedman NE, Frane SL, Lin WK, et al. 758 Accommodation, acuity, and their relationship to emmetropization in infants. Optom Vis 759 Sci. 2009;86(6):666-76. 760 56. Ritchey ER, Zelinka C, Tang J, Liu J, Code KA, Petersen-Jones S, et al. Vision-guided 761 ocular growth in a mutant chicken model with diminished visual acuity. Exp Eye Res. 762 2012;102:59-69. 763 57. Kruger PB, Mathews S, Aggarwala KR, Yager D, Kruger ES. Accommodation responds to 764 changing contrast of long, middle and short spectral-waveband components of the retinal 765 image. Vision Res. 1995;35(17):2415-29. 766 58. Schmid KL, Wildsoet CF. Contrast and spatial-frequency requirements for 767 emmetropization in chicks. Vision Res. 1997;37(15):2011-21. 768 59. Banks MS. The development of spatial and temporal contrast sensitivity. Curr Eye Res. 769 1982;2(3):191-8. 770 60. Kiorpes L, Kiper DC. Development of contrast sensitivity across the visual field in 771 macaque monkeys (Macaca nemestrina). Vision Res. 1996;36(2):239-47. 772 61. Geer I, Robertson KM. Measurement of central and peripheral dynamic visual acuity 773 thresholds during ocular pursuit of a moving target. Optom Vis Sci. 1993;70(7):552-60. 774 62. Sireteanu R, Fronius M, Constantinescu DH. The development of visual acuity in the 775 peripheral visual field of human infants: binocular and monocular measurements. Vision 776 Res. 1994;34(12):1659-71. 777 63. Diether S, Schaeffel F. Local changes in eye growth induced by imposed local refractive 778 error despite active accommodation. Vision Res. 1997;37(6):659-68. 779 64. Smith EL, 3rd, Hung LF, Huang J, Arumugam B. Effects of local myopic defocus on 780 refractive development in monkeys. Optom Vis Sci. 2013;90(11):1176-86. 781 65. Benavente-Perez A, Nour A, Troilo D. Axial eye growth and refractive error development 782 can be modified by exposing the peripheral retina to relative myopic or hyperopic defocus. 783 Invest Ophthalmol Vis Sci. 2014;55(10):6765-73.

Page 31 of 40

784 66. Sinha R, Hoon M, Baudin J, Okawa H, Wong ROL, Rieke F. Cellular and Circuit 785 Mechanisms Shaping the Perceptual Properties of the Primate Fovea. Cell. 786 2017;168(3):413-26 e12. 787 67. Preising MN, Friedburg C, Bowl W, Lorenz B. Unexpected Genetic Cause in Two Female 788 Siblings with High Myopia and Reduced Visual Acuity. BioMed research international. 789 2018;2018:1048317. 790 68. Lenassi E, Likar K, Stirn-Kranjc B, Brecelj J. VEP maturation and visual acuity in infants 791 and preschool children. Doc Ophthalmol. 2008;117(2):111-20. 792 69. Prusky GT, Douglas RM. Developmental plasticity of mouse visual acuity. Eur J Neurosci. 793 2003;17(1):167-73. 794 70. Prusky GT, Reidel C, Douglas RM. Environmental enrichment from birth enhances visual 795 acuity but not place learning in mice. Behav Brain Res. 2000;114(1-2):11-5. 796 71. Wong AA, Brown RE. Visual detection, pattern discrimination and visual acuity in 14 797 strains of mice. Genes Brain Behav. 2006;5(5):389-403. 798 72. Mooijaart SP, Koeijvoets KM, Sijbrands EJ, Daha MR, Westendorp RG. Complement 799 Factor H polymorphism Y402H associates with inflammation, visual acuity, and 800 cardiovascular mortality in the elderly population at large. Experimental gerontology. 801 2007;42(11):1116-22. 802 73. Smear MC, Tao HW, Staub W, Orger MB, Gosse NJ, Liu Y, et al. Vesicular glutamate 803 transport at a central synapse limits the acuity of visual perception in zebrafish. Neuron. 804 2007;53(1):65-77. 805 74. Ueno A, Omori Y, Sugita Y, Watanabe S, Chaya T, Kozuka T, et al. Lrit1, a Retinal 806 Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor 807 Cells and Visual Acuity. Cell reports. 2018;22(13):3548-61. 808 75. Mondal LK, Baidya KP, Bhattacharya B, Giri A, Bhaduri G. Relation between increased 809 anaerobic glycolysis and visual acuity in long-standing type 2 diabetes mellitus without 810 retinopathy. Indian J Ophthalmol. 2006;54(1):43-4. 811 76. Thangthaeng N, Rutledge M, Wong JM, Vann PH, Forster MJ, Sumien N. Metformin 812 Impairs Spatial Memory and Visual Acuity in Old Male Mice. Aging Dis. 2017;8(1):17-30. 813 77. Cecyre B, Bachand I, Papineau F, Brochu C, Casanova C, Bouchard JF. Cannabinoids 814 affect the mouse visual acuity via the cannabinoid receptor type 2. Sci Rep. 815 2020;10(1):15819. 816 78. Prusky GT, Alam NM, Beekman S, Douglas RM. Rapid quantification of adult and 817 developing mouse spatial vision using a virtual optomotor system. Invest Ophthalmol Vis 818 Sci. 2004;45(12):4611-6. 819 79. Tkatchenko TV, Shen Y, Tkatchenko AV. Analysis of postnatal eye development in the 820 mouse with high-resolution small animal magnetic resonance imaging. Invest Ophthalmol 821 Vis Sci. 2010;51(1):21-7. 822 80. Tkatchenko TV, Tkatchenko AV. Ketamine-xylazine anesthesia causes hyperopic refractive 823 shift in mice. J Neurosci Methods. 2010;193(1):67-71. 824 81. Tkatchenko TV, Shen Y, Braun RD, Bawa G, Kumar P, Avrutsky I, et al. Photopic visual 825 input is necessary for emmetropization in mice. Exp Eye Res. 2013;115C:87-95. 826 82. Storey JD, Tibshirani R. Statistical significance for genomewide studies. Proc Natl Acad 827 Sci U S A. 2003;100(16):9440-5. 828 83. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene 829 lists using DAVID bioinformatics resources. Nat Protoc. 2009;4(1):44-57.

Page 32 of 40

830 84. Walter W, Sanchez-Cabo F, Ricote M. GOplot: an R package for visually combining 831 expression data with functional analysis. Bioinformatics. 2015;31(17):2912-4. 832 85. Buniello A, MacArthur JAL, Cerezo M, Harris LW, Hayhurst J, Malangone C, et al. The 833 NHGRI-EBI GWAS Catalog of published genome-wide association studies, targeted arrays 834 and summary statistics 2019. Nucleic Acids Res. 2019;47(D1):D1005-D12. 835 86. Rucker FJ. The role of luminance and chromatic cues in emmetropisation. Ophthalmic 836 Physiol Opt. 2013;33(3):196-214. 837 87. Vigano D, Rubino T, Parolaro D. Molecular and cellular basis of cannabinoid and opioid 838 interactions. Pharmacol Biochem Behav. 2005;81(2):360-8. 839 88. Rios C, Gomes I, Devi LA. mu opioid and CB1 cannabinoid receptor interactions: 840 reciprocal inhibition of receptor signaling and neuritogenesis. Br J Pharmacol. 841 2006;148(4):387-95. 842 89. Korzh A, Keren O, Gafni M, Bar-Josef H, Sarne Y. Modulation of extracellular signal- 843 regulated kinase (ERK) by opioid and cannabinoid receptors that are expressed in the same 844 cell. Brain Res. 2008;1189:23-32. 845 90. Nadal X, La Porta C, Andreea Bura S, Maldonado R. Involvement of the opioid and 846 cannabinoid systems in pain control: new insights from knockout studies. European journal 847 of pharmacology. 2013;716(1-3):142-57. 848 91. Hervera A, Negrete R, Leanez S, Martin-Campos J, Pol O. The role of nitric oxide in the 849 local antiallodynic and antihyperalgesic effects and expression of delta-opioid and 850 cannabinoid-2 receptors during neuropathic pain in mice. J Pharmacol Exp Ther. 851 2010;334(3):887-96. 852 92. Nickla DL, Wildsoet CF. The effect of the nonspecific nitric oxide synthase inhibitor NG- 853 nitro-L-arginine methyl ester on the choroidal compensatory response to myopic defocus in 854 chickens. Optom Vis Sci. 2004;81(2):111-8. 855 93. Nickla DL, Damyanova P, Lytle G. Inhibiting the neuronal isoform of nitric oxide synthase 856 has similar effects on the compensatory choroidal and axial responses to myopic defocus in 857 chicks as does the non-specific inhibitor L-NAME. Exp Eye Res. 2009;88(6):1092-9. 858 94. Nickla DL, Lee L, Totonelly K. Nitric oxide synthase inhibitors prevent the growth- 859 inhibiting effects of quinpirole. Optom Vis Sci. 2013;90(11):1167-75. 860 95. Carr BJ, Stell WK. Nitric Oxide (NO) Mediates the Inhibition of Form-Deprivation Myopia 861 by Atropine in Chicks. Sci Rep. 2016;6(1):9. 862 96. Pardue MT, Faulkner AE, Fernandes A, Yin H, Schaeffel F, Williams RW, et al. High 863 susceptibility to experimental myopia in a mouse model with a retinal on pathway defect. 864 Invest Ophthalmol Vis Sci. 2008;49(2):706-12. 865 97. Tedja MS, Wojciechowski R, Hysi PG, Eriksson N, Furlotte NA, Verhoeven VJM, et al. 866 Genome-wide association meta-analysis highlights light-induced signaling as a driver for 867 refractive error. Nat Genet. 2018;50(6):834-48. 868 98. Stone RA, McGlinn AM, Baldwin DA, Tobias JW, Iuvone PM, Khurana TS. Image 869 defocus and altered retinal gene expression in chick: clues to the pathogenesis of ametropia. 870 Invest Ophthalmol Vis Sci. 2011;52(8):5765-77. 871 99. Riddell N, Giummarra L, Hall NE, Crewther SG. Bidirectional Expression of Metabolic, 872 Structural, and Immune Pathways in Early Myopia and Hyperopia. Front Neurosci. 873 2016;10:390. 874 100. Wu H, Chen W, Zhao F, Zhou Q, Reinach PS, Deng L, et al. Scleral hypoxia is a target for 875 myopia control. Proc Natl Acad Sci U S A. 2018;115(30):E7091-E100.

Page 33 of 40

876 101. Yamamoto S, Sippel KC, Berson EL, Dryja TP. Defects in the rhodopsin kinase gene in the 877 Oguchi form of stationary night blindness. Nat Genet. 1997;15(2):175-8. 878 102. Zhong H, Molday LL, Molday RS, Yau KW. The heteromeric cyclic nucleotide-gated 879 channel adopts a 3A:1B stoichiometry. Nature. 2002;420(6912):193-8. 880 103. Acharya S, Foletta VC, Lee JW, Rayborn ME, Rodriguez IR, Young WS, 3rd, et al. 881 SPACRCAN, a novel human interphotoreceptor matrix hyaluronan-binding proteoglycan 882 synthesized by photoreceptors and pinealocytes. J Biol Chem. 2000;275(10):6945-55. 883 104. Bandah-Rozenfeld D, Collin RW, Banin E, van den Born LI, Coene KL, Siemiatkowska 884 AM, et al. Mutations in IMPG2, encoding interphotoreceptor matrix proteoglycan 2, cause 885 autosomal-recessive retinitis pigmentosa. Am J Hum Genet. 2010;87(2):199-208. 886 105. Li TS, Volpp K, Applebury ML. Bovine cone photoreceptor cGMP phosphodiesterase 887 structure deduced from a cDNA clone. Proc Natl Acad Sci U S A. 1990;87(1):293-7. 888 106. Pittler SJ, Baehr W, Wasmuth JJ, McConnell DG, Champagne MS, vanTuinen P, et al. 889 Molecular characterization of human and bovine rod photoreceptor cGMP 890 phosphodiesterase alpha-subunit and chromosomal localization of the human gene. 891 Genomics. 1990;6(2):272-83. 892 107. Thiadens AA, den Hollander AI, Roosing S, Nabuurs SB, Zekveld-Vroon RC, Collin RW, 893 et al. Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone 894 photoreceptor disorders. Am J Hum Genet. 2009;85(2):240-7. 895 108. Huang SH, Pittler SJ, Huang X, Oliveira L, Berson EL, Dryja TP. Autosomal recessive 896 retinitis pigmentosa caused by mutations in the alpha subunit of rod cGMP 897 phosphodiesterase. Nat Genet. 1995;11(4):468-71. 898 109. Yau KW, Hardie RC. Phototransduction motifs and variations. Cell. 2009;139(2):246-64. 899 110. Haeseleer F, Jang GF, Imanishi Y, Driessen CA, Matsumura M, Nelson PS, et al. Dual- 900 substrate specificity short chain retinol dehydrogenases from the vertebrate retina. J Biol 901 Chem. 2002;277(47):45537-46. 902 111. Janecke AR, Thompson DA, Utermann G, Becker C, Hubner CA, Schmid E, et al. 903 Mutations in RDH12 encoding a photoreceptor cell retinol dehydrogenase cause childhood- 904 onset severe retinal dystrophy. Nat Genet. 2004;36(8):850-4. 905 112. Yanovitch T, Li YJ, Metlapally R, Abbott D, Viet KN, Young TL. Hepatocyte growth 906 factor and myopia: genetic association analyses in a Caucasian population. Mol Vis. 907 2009;15:1028-35. 908 113. Williams ME, Feldman DH, McCue AF, Brenner R, Velicelebi G, Ellis SB, et al. Structure 909 and functional expression of alpha 1, alpha 2, and beta subunits of a novel human neuronal 910 calcium channel subtype. Neuron. 1992;8(1):71-84. 911 114. de Quervain DJ, Papassotiropoulos A. Identification of a genetic cluster influencing 912 memory performance and hippocampal activity in humans. Proc Natl Acad Sci U S A. 913 2006;103(11):4270-4. 914 115. Gloyn AL, Desai M, Clark A, Levy JC, Holman RR, Frayling TM, et al. Human 915 calcium/calmodulin-dependent protein kinase II gamma gene (CAMK2G): cloning, 916 genomic structure and detection of variants in subjects with type II diabetes. Diabetologia. 917 2002;45(4):580-3. 918 116. Nickla DL, Totonelly K, Dhillon B. Dopaminergic agonists that result in ocular growth 919 inhibition also elicit transient increases in choroidal thickness in chicks. Exp Eye Res. 920 2010;91(5):715-20.

Page 34 of 40

921 117. Rohrer B, Spira AW, Stell WK. Apomorphine blocks form-deprivation myopia in chickens 922 by a dopamine D2-receptor mechanism acting in retina or pigmented epithelium. Vis 923 Neurosci. 1993;10(3):447-53. 924 118. Liu X, Grove JC, Hirano AA, Brecha NC, Barnes S. Dopamine D1 receptor modulation of 925 calcium channel currents in horizontal cells of mouse retina. Journal of neurophysiology. 926 2016;116(2):686-97. 927 119. Kurenny DE, Moroz LL, Turner RW, Sharkey KA, Barnes S. Modulation of ion channels 928 in rod photoreceptors by nitric oxide. Neuron. 1994;13(2):315-24. 929 120. Feldkaemper M, Schaeffel F. An updated view on the role of dopamine in myopia. Exp Eye 930 Res. 2013;114:106-19. 931 121. Iuvone PM, Tigges M, Stone RA, Lambert S, Laties AM. Effects of apomorphine, a 932 dopamine receptor agonist, on ocular refraction and axial elongation in a primate model of 933 myopia. Invest Ophthalmol Vis Sci. 1991;32(5):1674-7. 934 122. Stone RA, Lin T, Laties AM, Iuvone PM. Retinal dopamine and form-deprivation myopia. 935 Proc Natl Acad Sci U S A. 1989;86(2):704-6. 936

937 Supplementary materials

938 Supplementary Tables: Table S1. Visual acuity in different strains of mice (P40, cpd). Table

939 S2. Baseline refractive errors in different strains of mice (P40, diopters). Table S3. Form-

940 deprivation myopia in different strains of mice (deprived eye versus control eye, diopters). Table

941 S4. Genes whose expression correlates with visual acuity in mice (FC = fold change, high versus

942 low visual acuity). Table S5. Genes whose expression correlates with baseline refractive errors

943 in mice (FC = fold change, hyperopia versus myopia). Table S6. Genes whose expression

944 correlates with susceptibility to myopia in mice (FC = fold change, high versus low myopia).

945 Table S7. Gene ontology categories associated with genes whose expression correlates with

946 visual acuity in mice (BP, biological process; CC, cellular component; MF, molecular function).

947 Table S8. Gene ontology categories associated with genes whose expression correlates with

948 baseline refractive errors in mice (BP, biological process; CC, cellular component; MF,

949 molecular function). Table S9. Gene ontology categories associated with genes whose

950 expression correlates with susceptibility to myopia in mice (BP, biological process; CC, cellular

Page 35 of 40

951 component; MF, molecular function). Table S10. Canonical signaling pathways associated with

952 genes whose expression correlates with visual acuity in mice. Table S11. Canonical signaling

953 pathways associated with genes whose expression correlates with baseline refractive errors in

954 mice. Table S12. Canonical signaling pathways associated with genes whose expression

955 correlates with susceptibility to myopia in mice. Table S13. Genes whose expression correlates

956 with both visual acuity and baseline refractive errors in mice (FC = fold change, visual acuity =

957 high versus low visual acuity, RE = hyperopia versus myopia). Table S14. Genes whose

958 expression correlates with both visual acuity and susceptibility to myopia in mice (FC = fold

959 change, visual acuity = high versus low visual acuity, myopia = high versus low myopia). Table

960 S15. Gene ontology categories associated with genes whose expression correlates with both

961 visual acuity and baseline refractive errors in mice (BP, biological process; CC, cellular

962 component; MF, molecular function). Table S16. Gene ontology categories associated with

963 genes whose expression correlates with both visual acuity and susceptibility to myopia in mice

964 (BP, biological process; CC, cellular component; MF, molecular function). Table S17. Visual

965 acuity genes associated with refractive eye development in mice and linked to eye-related human

966 diseases (FC = fold change, visual acuity = high versus low visual acuity, RE = expression

967 correlates with RE, myopia = expression correlates with susceptibility to myopia). Table S18.

968 Gene ontology categories associated with genes whose expression correlates with visual acuity,

969 baseline refractive errors, and form-deprivation myopia and which are involved in human

970 diseases (see Table S17) (BP, biological process).

971

972 Supplementary Figure S1. Baseline refractive error correlates weakly with susceptibility to

973 form-deprivation myopia. Linear regression showing weak correlation between baseline

Page 36 of 40

974 refractive error and susceptibility to myopia. r, Pearson’s correlation coefficient; P, Pearson’s

975 correlation significance.

976

977 Supplementary Figure S2. Gene expression network underlying baseline refractive eye

978 development controls diverse biological functions and signaling pathways. (A) Expression of

979 2,050 genes (Q-value < 0.01) correlates with baseline refractive errors. Hierarchical clustering

980 shows that differential genes are organized in two clusters: one (top) cluster exhibits increased

981 expression in the myopic mice, and the second (bottom) cluster shows increased expression in

982 the hyperopic mice. (B) Top 30 biological processes affected by the genes involved in baseline

983 refractive eye development. Outer circle of the hierarchical clustering diagram shows

984 hierarchical clusters of biological processes (identified by different colors); inner circle shows

985 clusters of the associated genes up- or down-regulated in hyperopic mice versus myopic mice.

986 (C) Top 30 canonical pathways affected by the genes associated with baseline refractive eye

987 development. Horizontal yellow line indicates P = 0.05. Z-scores show activation or suppression

988 of the corresponding pathways in animals with hyperopia versus animals with myopia.

989

990 Supplementary Figure S3. Gene expression network subserving susceptibility to form-

991 deprivation myopia controls diverse biological functions and signaling pathways. (A) Expression

992 of 1,347 genes (Q-value < 0.01) correlates with susceptibility to form-deprivation myopia.

993 Hierarchical clustering shows that differential genes are organized in two clusters: one (top)

994 cluster exhibits increased expression in mice with low susceptibility to myopia, and the second

995 (bottom) cluster shows increased expression in mice with high susceptibility to myopia. (B) Top

996 30 biological processes affected by the genes involved in the development of form-deprivation

Page 37 of 40

997 myopia. Outer circle of the hierarchical clustering diagram shows hierarchical clusters of

998 biological processes (identified by different colors); inner circle shows clusters of the associated

999 genes up- or down-regulated in mice with high susceptibility to myopia versus mice with low

1000 susceptibility to myopia. (C) Top 30 canonical pathways affected by the genes associated with

1001 the development of form-deprivation myopia. Horizontal yellow line indicates P = 0.05. Z-scores

1002 show activation or suppression of the corresponding pathways in animals with high susceptibility

1003 to form-deprivation myopia versus animals with low susceptibility to form-deprivation myopia.

1004

1005

1006 Figure legends

1007 Fig. 1. Visual acuity exhibits weak correlation with baseline refractive error and form-

1008 deprivation myopia. (A) Visual acuity is influenced by genetic background. Horizontal red lines

1009 identify means of visual acuity for each strain, while each dot represents a mean visual acuity for

1010 individual animals. (B) Baseline refractive errors range from highly myopic to highly hyperopic

1011 in mice depending on the genetic background. Horizontal red lines show mean refractive errors

1012 for each strain, while each dot corresponds to mean refractive errors of individual animals. (C)

1013 Susceptibility to form-deprivation myopia in mice is determined by genetic background.

1014 Horizontal red lines identify means of induced myopia for each strain, while each dot represents

1015 a mean myopia for individual animals. (D) Visual acuity exhibits weak statistically significant

1016 correlation with baseline refractive error. Linear regression showing weak correlation between

1017 visual acuity and baseline refractive error. r, Pearson’s correlation coefficient; P, Pearson’s

1018 correlation significance. (E) Visual acuity exhibits weak statistically significant correlation with

Page 38 of 40

1019 form-deprivation myopia. Linear regression showing weak correlation between visual acuity and

1020 myopia. r, Pearson’s correlation coefficient; P, Pearson’s correlation significance.

1021

1022 Fig. 2. Gene expression network regulating visual acuity controls diverse biological functions

1023 and signaling pathways. (A) Expression of 1,073 genes (Q-value < 0.02) correlates with visual

1024 acuity. Hierarchical clustering shows that differential genes are organized in two clusters: one

1025 (top) cluster exhibits increased expression in mice with high visual acuity, and the second

1026 (bottom) cluster shows increased expression in mice with low visual acuity. (B) Top 30

1027 biological processes affected by the genes involved in the regulation of visual acuity. Outer

1028 circle of the hierarchical clustering diagram shows hierarchical clusters of biological processes

1029 (identified by different colors); inner circle shows clusters of the associated genes up- or down-

1030 regulated in mice with high visual acuity versus mice with low visual acuity. (C) Top 30

1031 canonical pathways affected by the genes involved in the regulation of visual acuity. Horizontal

1032 yellow line indicates P = 0.05. Z-scores show activation or suppression of the corresponding

1033 pathways in animals with high visual acuity versus animals with low visual acuity.

1034

1035 Fig. 3. Gene expression network regulating visual acuity makes significant contribution to

1036 baseline refractive eye development. (A) Venn diagram showing overlap between genes

1037 regulating visual acuity and genes underlying baseline refractive eye development. (B) Top 30

1038 biological processes affected by the genes involved in the regulation of visual acuity and baseline

1039 refractive development. Outer circle of the hierarchical clustering diagram shows hierarchical

1040 clusters of biological processes (identified by different colors); inner circle shows clusters of the

1041 associated genes up- or down-regulated in mice with high visual acuity versus mice with low

Page 39 of 40

1042 visual acuity. (C) Canonical signaling pathways involved in the regulation of visual acuity and

1043 baseline refractive eye development. Vertical red line and red dots indicate P = 0.05. Vertical

1044 grey line and grey dots indicate P = 0.1. Colors identify pathways associated with either visual

1045 acuity or baseline refractive development and correspond to the colors in the Venn diagram (A).

1046

1047 Fig. 4. Gene expression network regulating visual acuity makes modest contribution to visually

1048 guided eye emmetropization. (A) Venn diagram showing overlap between genes regulating

1049 visual acuity and genes underlying form-deprivation myopia. (B) Top 30 biological processes

1050 affected by the genes involved in the regulation of visual acuity and susceptibility to form-

1051 deprivation myopia. Outer circle of the hierarchical clustering diagram shows hierarchical

1052 clusters of biological processes (identified by different colors); inner circle shows clusters of the

1053 associated genes up- or down-regulated in mice with high visual acuity versus mice with low

1054 visual acuity. (C) Canonical signaling pathways involved in the regulation of visual acuity and

1055 susceptibility to form-deprivation myopia. Vertical red line and red dots indicate P = 0.05.

1056 Vertical grey line and grey dots indicate P = 0.1. Colors identify pathways associated with either

1057 visual acuity or susceptibility to form-deprivation myopia and correspond to the colors in the

1058 Venn diagram (A).

1059

1060 Fig. 5. Genes involved in the regulation of visual acuity are associated with the development of

1061 myopia and other human genetic disorders. Chord diagram showing genes (left semicircle) and

1062 human genetic disorders (right semicircle) associated with visual acuity regulation and refractive

1063 eye development in mice. Colored bars underneath gene names show up- or down-regulation of

1064 the corresponding genes in mice with high visual acuity versus mice with low visual acuity.

Page 40 of 40 bioRxiv preprint doi: https://doi.org/10.1101/2021.05.04.442661; this version posted May 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.04.442661; this version posted May 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.04.442661; this version posted May 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.04.442661; this version posted May 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.04.442661; this version posted May 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.