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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 3 4 5 Tatiana V. Tkatchenko1 and Andrei V. Tkatchenko1,2* 6 7 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 10 11 Tatiana V. Tkatchenko, E-mail: [email protected] 12 Andrei V. Tkatchenko, E-mail: [email protected] 13 14 15 16 17 18 RNA-seq data from this article have been deposited in the Gene Expression Omnibus database 19 under accession No. GSE158732 20 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] Page 1 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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. 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. 45 Page 2 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. 46 Keywords: Refractive eye development, Emmetropization, Optical defocus, Visual acuity, 47 Signaling pathways, Gene expression profiling, Gene expression networks, RNA-seq. 48 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; 67 Page 3 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. 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 Page 4 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. 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]. Page 5 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.