Christina Zeitz

J´erome E Roger

Isabelle Audo

Christelle Michiels

Nuria Sánchez-Farías

Juliette Varin

Helen Frederiksen

Baptiste Wilmet

Jacques Callebert

Marie-Laure Gimenez

Nassima Bouzidi

Frederic Blond

Xavier Guilllonneau

Stéphane Fouquet

Thierry Léveillard

Vasily Smirnov

Ajoy Vincent

Elise H2on

José-Alain Sahel

Barbara Kloeckener-Gruissem

Florian Sennlaub

Catherine W Morgans

Robert M Duvoisin

Andrei V Tkatchenko

Serge Picaud

Shedding light on myopia by studying complete congenital stationary night blindness

Keywords: congenital stationary night blindness, myopia, retina, transcriptome sequencing, meta-analysis

Myopia is the most common eye disorder, caused by heterogeneous genetic and environmental factors. Rare progressive and stationary inherited retinal disorders are often associated with high myopia. Genes implicated in myopia encode proteins involved in a variety of biological processes including eye morphogenesis, extracellular matrix organization, visual perception, circadian rhythms, and retinal signaling. Differentially expressed genes (DEGs) identified in animal models mimicking myopia are helpful in suggesting candidate genes implicated in human myopia. Complete congenital stationary night blindness (cCSNB) in humans and animal models represents an ON-bipolar cell signal transmission defect and is also associated with high myopia. Thus, it represents also an interesting model to identify myopia-related genes, as well as disease mechanisms. While the origin of night blindness is molecularly well established, further research is needed to elucidate the mechanisms of myopia development in subjects with cCSNB. Using whole transcriptome analysis on three different mouse models of cCSNB (in Gpr179 -/-, Lrit3 -/-and Grm6 -/-), we identified novel actors of the retinal signaling cascade, which are also novel candidate genes for myopia. Meta-analysis of our transcriptomic data with published transcriptomic databases and genome-wide association studies from myopia cases led us to propose new biological/cellular processes/mechanisms potentially at the origin of myopia in cCSNB subjects. The results provide a foundation to guide the development of pharmacological myopia therapies.

Introduction

Myopia (short-sightedness) is the most common human eye disorder worldwide, in which the image is focused in front of the retina, resulting usually from an excessive growth of the eye posterior segment (increased axial length) (reviewed by e.g. [START_REF] Goss | Retinal-image mediated ocular growth as a mechanism for juvenile onset myopia and for emmetropization. A literature review[END_REF][START_REF] Mcbrien | Role of the sclera in the development and pathological complications of myopia[END_REF], generally as a consequence of accelerated post-natal eye growth [START_REF] Mcbrien | Role of the sclera in the development and pathological complications of myopia[END_REF] but also set by the optical power of the cornea and the lens [START_REF] Iribarren | Crystalline lens and refractive development[END_REF]. It can be due to structural abnormalities of the cornea as found in keratoconus or due to changes primarily in the lens power found e.g. in older people developing cataracts (reviewed by [START_REF] Flitcroft | IMI -Defining and Classifying Myopia: A Proposed Set of Standards for Clinical and Epidemiologic Studies[END_REF][START_REF] Iribarren | Crystalline lens and refractive development[END_REF]). Recent animal studies, combined with clinical studies, have revealed that the mechanisms of optically guided eye growth are influenced by the retina [START_REF] Flitcroft | The complex interactions of retinal, optical and environmental factors in myopia aetiology[END_REF].

High axial myopia, defined as a refractive error of more than -6.0 diopters (D) or ocular axial length of more than 26-26.5 mm [START_REF] Flitcroft | IMI -Defining and Classifying Myopia: A Proposed Set of Standards for Clinical and Epidemiologic Studies[END_REF], can lead to blinding complication such as myopic macular degeneration (MMD), retinal tears and retinal detachment, and predispose to other ocular diseases such as glaucoma and early onset cataracts [START_REF] Hornbeak | Myopia genetics: a review of current research and emerging trends[END_REF]. A subset of subjects with high myopia also demonstrate diffuse retinal changes beyond the macula, so-called "diffuse chorioretinal atrophy", which is defined as pathologic myopia [START_REF] Ohno-Matsui | Updates of pathologic myopia[END_REF]; pathologic myopia can be associated with fundus changes, regional retinal and choroidal thinning [START_REF] Ohno-Matsui | Posterior staphyloma in pathologic myopia[END_REF][START_REF] Ye | Visual Acuity in Pathological Myopia Is Correlated With the Photoreceptor Myoid and Ellipsoid Zone Thickness and Affected by Choroid Thickness[END_REF], and increased retinal complications, such as retinal detachment [START_REF] Ohno-Matsui | Updates of pathologic myopia[END_REF]. Epidemiological studies estimated that half the world's population will be myopic by 2050, with 10% showing high myopia [START_REF] Holden | Global Prevalence of Myopia and High Myopia and Temporal Trends from 2000 through 2050[END_REF]. Indeed, it was shown that the incidence of myopia is increasing, affecting now 20%-40% of children at the age of 17-18 in developed western countries and up to 80% -90% in East and Southeast Asia (reviewed by [START_REF] Morgan | The epidemics of myopia: Aetiology and prevention[END_REF]) (e.g. [START_REF] Ang | Review: Myopia control strategies recommendations from the 2018 WHO/IAPB/BHVI Meeting on Myopia[END_REF][START_REF] Cumberland | Frequency and Distribution of Refractive Error in Adult Life: Methodology and Findings of the UK Biobank Study[END_REF][START_REF] Foster | Epidemiology of myopia[END_REF][START_REF] French | Prevalence and 5to 6-year incidence and progression of myopia and hyperopia in Australian schoolchildren[END_REF][START_REF] Holden | Myopia, an underrated global challenge to vision: where the current data takes us on myopia control[END_REF][START_REF] Holden | Global Prevalence of Myopia and High Myopia and Temporal Trends from 2000 through 2050[END_REF][START_REF] Hysi | Genome-wide association studies of refractive error and myopia, lessons learned, and implications for the future[END_REF][START_REF] Kempen | The prevalence of refractive errors among adults in the United States, Western Europe, and Australia[END_REF][START_REF] Lam | Prevalence of myopia in local and international schools in Hong Kong[END_REF][START_REF] Lin | Prevalence of myopia in Taiwanese schoolchildren: 1983 to 2000[END_REF][START_REF] Morgan | The epidemics of myopia: Aetiology and prevention[END_REF]Morgan et al., 2012;[START_REF] Pan | Worldwide prevalence and risk factors for myopia[END_REF][START_REF] Vitale | Increased prevalence of myopia in the United States between 1971-1972 and 1999-2004[END_REF]). However, in less developed countries, with less developed education systems, the prevalence of myopia in young adults is estimated to be only 5% -10% (reviewed by [START_REF] Morgan | The epidemics of myopia: Aetiology and prevention[END_REF]) (e.g. [START_REF] Anera | Prevalence of refractive errors in school-age children in Morocco[END_REF][START_REF] Casson | Exceptionally low prevalence of refractive error and visual impairment in schoolchildren from Lao People's Democratic Republic[END_REF]Dandona et al., 2002a;Dandona et al., 2002b;[START_REF] Gao | Refractive error in school children in an urban and rural setting in Cambodia[END_REF][START_REF] Jimenez | Prevalence of myopia in an adult population of two different ethnic groups in the Ecuadorian Amazon[END_REF][START_REF] Jimenez | Ametropias in school-age children in Fada N'Gourma (Burkina Faso, Africa)[END_REF][START_REF] Khandekar | Magnitude and determinants of refractive error in Omani school children[END_REF][START_REF] Lewallen | A population-based survey of the prevalence of refractive error in Malawi[END_REF][START_REF] Lindquist | Screening for uncorrected refractive error in secondary school-age students in Fiji[END_REF][START_REF] Lithander | Prevalence of myopia in school children in the Sultanate of Oman: a nation-wide study of 6292 randomly selected children[END_REF][START_REF] Pokharel | Refractive Error Study in Children: results from Mechi Zone, Nepal[END_REF].

High myopia is a complex disorder, which is mediated by genetic and more importantly by environmental factors [START_REF] Baird | The GEnes in Myopia (GEM) study in understanding the aetiology of refractive errors[END_REF][START_REF] Morgan | How genetic is school myopia?[END_REF][START_REF] Morgan | The biological basis of myopic refractive error[END_REF][START_REF] Morgan | The epidemics of myopia: Aetiology and prevention[END_REF][START_REF] Wojciechowski | Nature and nurture: the complex genetics of myopia and refractive error[END_REF][START_REF] Young | Molecular genetics of human myopia: an update[END_REF]. More than 300 genes were associated with refractive error development in humans [START_REF] Flitcroft | Novel Myopia Genes and Pathways Identified From Syndromic Forms of Myopia[END_REF][START_REF] Hysi | Meta-analysis of 542,934 subjects of European ancestry identifies new genes and mechanisms predisposing to refractive error and myopia[END_REF][START_REF] Kiefer | Genome-wide analysis points to roles for extracellular matrix remodeling, the visual cycle, and neuronal development in myopia[END_REF][START_REF] Tedja | IMI -Myopia Genetics Report[END_REF]Tedja et al., 2018;Verhoeven et al., 2013). They encode proteins of vastly different functions, including eye morphogenesis, extracellular matrix organization, visual perception, circadian rhythms, pigmentation and retinal signaling [START_REF] Flitcroft | Novel Myopia Genes and Pathways Identified From Syndromic Forms of Myopia[END_REF][START_REF] Hysi | Meta-analysis of 542,934 subjects of European ancestry identifies new genes and mechanisms predisposing to refractive error and myopia[END_REF]Tedja et al., 2018). The contribution of heritability to myopia is difficult to determine [START_REF] Dirani | Heritability of refractive error and ocular biometrics: the Genes in Myopia (GEM) twin study[END_REF][START_REF] Hammond | Genes and environment in refractive error: the twin eye study[END_REF][START_REF] Lopes | Estimating heritability and shared environmental effects for refractive error in twin and family studies[END_REF][START_REF] Lyhne | The importance of genes and environment for ocular refraction and its determiners: a population based study among 20-45 year old twins[END_REF][START_REF] Peet | Heritability and familial aggregation of refractive error in the Old Order Amish[END_REF][START_REF] Tedja | IMI -Myopia Genetics Report[END_REF]Tedja et al., 2018;[START_REF] Teikari | Heritability estimate for refractive errors--a population-based sample of adult twins[END_REF][START_REF] Wojciechowski | Nature and nurture: the complex genetics of myopia and refractive error[END_REF] and estimated to be rather low [START_REF] Morgan | How genetic is school myopia?[END_REF], since it was shown that environmental factors play a very important role in the occurrence of myopia, especially illumination conditions. This is underpinned by the increasing prevalence of myopia taking place in East and Southeast Asia, where children spend more time inside studying as compared to less developed countries [START_REF] Morgan | The epidemics of myopia: Aetiology and prevention[END_REF]. Indeed, the intensity of ambient light was found to influence the development of myopia.

Outdoor activities have a protective effect against the development and progression of myopia [START_REF] He | Effect of Time Spent Outdoors at School on the Development of Myopia Among Children in China: A Randomized Clinical Trial[END_REF][START_REF] Rose | Outdoor activity reduces the prevalence of myopia in children[END_REF][START_REF] Wu | Outdoor activity during class recess reduces myopia onset and progression in school children[END_REF][START_REF] Xiong | Time spent in outdoor activities in relation to myopia prevention and control: a metaanalysis and systematic review[END_REF]. Similarly for animal models raised under different lighting conditions, high illuminances have been found to slow the development of induced myopia [START_REF] Smith | Protective effects of high ambient lighting on the development of form-deprivation myopia in rhesus monkeys[END_REF]. Interestingly, intermittent exposure to bright light was more efficient in suppressing myopia progression than continuous light exposure [START_REF] Lan | Intermittent episodes of bright light suppress myopia in the chicken more than continuous bright light[END_REF].

The identification of differentially expressed genes (DEG) in animals with induced myopia has been helpful in suggesting candidate genes associated with the development of human myopia [START_REF] Brand | A microarray analysis of retinal transcripts that are controlled by image contrast in mice[END_REF][START_REF] Frost | Alterations in protein expression in tree shrew sclera during development of lens-induced myopia and recovery[END_REF][START_REF] Gao | Patterns of mRNA and protein expression during minus-lens compensation and recovery in tree shrew sclera[END_REF][START_REF] Mcglinn | Form-deprivation myopia in chick induces limited changes in retinal gene expression[END_REF][START_REF] Riddell | Bidirectional Expression of Metabolic, Structural, and Immune Pathways in Early Myopia and Hyperopia[END_REF][START_REF] Schippert | Microarray analysis of retinal gene expression in chicks during imposed myopic defocus[END_REF][START_REF] Shelton | Microarray analysis of choroid/RPE gene expression in marmoset eyes undergoing changes in ocular growth and refraction[END_REF][START_REF] Siegwart | The time course of changes in mRNA levels in tree shrew sclera during induced myopia and recovery[END_REF][START_REF] Tkatchenko | Large-Scale microRNA Expression Profiling Identifies Putative Retinal miRNA-mRNA Signaling Pathways Underlying Form-Deprivation Myopia in Mice[END_REF][START_REF] Tkatchenko | Form deprivation modulates retinal neurogenesis in primate experimental myopia[END_REF].

High myopia can also be syndromic. Pathogenic variants affecting proteins expressed in the retina, either in photoreceptors or in the downstream bipolar cells (BCs) can lead to progressive and non-progressive retinal monogenetic disorders, which can be also associated with other ocular disorders including myopia. One such disease is X-linked retinitis pigmentosa (RP) (MIM #300029), one of the most frequent and severe progressive form of RP, with symptoms of night blindness that occur during the first decade of life and progress to legal blindness by the third to fourth decade in most affected males [START_REF] Flaxel | Difference between RP2 and RP3 phenotypes in X linked retinitis pigmentosa[END_REF]. In this disease, high myopia is often observed from childhood in addition to retinal degeneration and electrophysiological abnormalities [START_REF] Flaxel | Difference between RP2 and RP3 phenotypes in X linked retinitis pigmentosa[END_REF][START_REF] Koenekoop | Novel RPGR mutations with distinct retinitis pigmentosa phenotypes in French-Canadian families[END_REF].

Mutations in RPGR (MIM #312610) account for the majority of X-linked RP, and these patients show high myopia in addition to RP [START_REF] Sanchez Tocino | Phenotypic high myopia in X-linked retinitis pigmentosa secondary to a novel mutation in the RPGR gene[END_REF]. Similarly, mutations in ARR3 (e.g. [START_REF] Van Mazijk | Early onset Xlinked female limited high myopia in three multigenerational families caused by novel mutations in the ARR3 gene[END_REF][START_REF] Xiao | X-linked heterozygous mutations in ARR3 cause female-limited early onset high myopia[END_REF] and OPN1LW, mainly expressed in cone-photoreceptors, were also associated with high myopia (reviewed by [START_REF] Neitz | Intermixing the OPN1LW and OPN1MW Genes Disrupts the Exonic Splicing Code Causing an Array of Vision Disorders[END_REF]. Other syndromic and non-syndromic eye disorders associated with high myopia include Marfan syndrome (MIM #154700) [START_REF] Pyeritz | The Marfan syndrome: diagnosis and management[END_REF], with mutations in FBN1 (MIM #134797), Knobloch (MIM #267750) (summarized by [START_REF] Aldahmesh | Identification of ADAMTS18 as a gene mutated in Knobloch syndrome[END_REF], with mutations in COL18A1 (MIM #120328), Wagner (MIM #143200), with mutations in VCAN (MIM #118661), Stickler syndrome (MIM #108300), with mutations in COL2A1 (MIM #120140), Ehlers-Danlos syndrome (MIM#130000), with mutations in COL5A1 (MIM# 120215) and Donnai-Barrow syndrome (MIM #222448) [START_REF] Ohlsson | Congenital Renal Disease, Deafness and Myopia in One Family[END_REF], with mutations in LRP2 (MIM #600073) [START_REF] Kantarci | Mutations in LRP2, which encodes the multiligand receptor megalin, cause Donnai-Barrow and facio-oculo-acoustico-renal syndromes[END_REF]. Based on these findings, it appears that disruption of the retinal signaling pathway from photoreceptors to the inner retina can lead to myopia. Interestingly, common genetic variants within or nearby genes that are associated with syndromic myopia are enriched for variants that cause non-syndromic, common myopia [START_REF] Flitcroft | Novel Myopia Genes and Pathways Identified From Syndromic Forms of Myopia[END_REF]. For most of the implicated genes, expression in the retina is documented. Reinforcing these findings, degeneration of photoreceptors and/or amacrine cells (ACs) in chicken retinas, has been associated with increased axial growth of the eye, leading to myopia [START_REF] Ehrlich | Effects of selective neurotoxins on eye growth in the young chick[END_REF][START_REF] Fischer | Colchicine causes excessive ocular growth and myopia in chicks[END_REF]. E.g. this was shown using colchicine to destroy amacrine cells, which normally suppress axial growth of the eye [START_REF] Fischer | Colchicine causes excessive ocular growth and myopia in chicks[END_REF].

Studies in chicken and mice showed that light dependent ON-bipolar cell (BC) dopamine signaling is a major player in the development and protection of myopia [START_REF] Boatright | Inhibition of endogenous dopamine release in amphibian retina by L-2-amino-4-phosphonobutyric acid (L-AP4) and trans-2aminocyclopentane-1,3-dicarboxylate (ACPD)[END_REF][START_REF] Boelen | Light-stimulated release of dopamine from the primate retina is blocked by 1-2-amino-4-phosphonobutyric acid (APB)[END_REF][START_REF] Dumitrescu | Ectopic retinal ON bipolar cell synapses in the OFF inner plexiform layer: contacts with dopaminergic amacrine cells and melanopsin ganglion cells[END_REF][START_REF] Iuvone | Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons[END_REF][START_REF] Schaeffel | Animal models in myopia research[END_REF][START_REF] Stone | Image defocus and altered retinal gene expression in chick: clues to the pathogenesis of ametropia[END_REF]. Retinal dopamine content is decreased in the absence of light and during myopia development [START_REF] Schaeffel | Animal models in myopia research[END_REF]. It was shown, that intravitreal or topical application of levodopa, which is widely used in the treatment of neurological disorders involving dysregulation of the dopaminergic system, inhibits the development of experimental myopia in chickens [START_REF] Thomson | Effectiveness and safety of topical levodopa in a chick model of myopia[END_REF]. In addition, it has been demonstrated that non-myopic children treated for attention deficit hyperactivity disorder (ADHD) with dopaminergic agents have slower developmental myopic refraction than untreated peers, providing a more direct suggestion that dopamine plays a role in human refractive development [START_REF] Gurlevik | Effect of methylphenidate as a dopaminergic agent on myopia: Pilot study[END_REF].

Patients with complete congenital stationary night blindness (cCSNB), which represents a complete ON-BC dysfunction, with unaltered OFF-BC function present as well with high myopia (median error of -7.4 D) [START_REF] Bijveld | Genotype and phenotype of 101 dutch patients with congenital stationary night blindness[END_REF][START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Multiple mutations in cCSNB-associated genes such as NYX, GRM6, GPR179, TRPM1 and LRIT3 lead to this condition in patients and mouse models [START_REF] Audo | Whole-Exome Sequencing Identifies Mutations in GPR179 Leading to Autosomal-Recessive Complete Congenital Stationary Night Blindness[END_REF][START_REF] Bech-Hansen | Mutations in NYX, encoding the leucinerich proteoglycan nyctalopin, cause X-linked complete congenital stationary night blindness[END_REF][START_REF] Dryja | Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6[END_REF][START_REF] Gregg | Identification of the gene and the mutation responsible for the mouse nob phenotype[END_REF][START_REF] Koike | TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade[END_REF][START_REF] Li | Recessive mutations of the gene TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness in humans[END_REF][START_REF] Maddox | Allelic variance between GRM6 mutants, Grm6nob3 and Grm6nob4 results in differences in retinal ganglion cell visual responses[END_REF][START_REF] Masu | Specific deficit of the ON response in visual transmission by targeted disruption of the mGluR6 gene[END_REF][START_REF] Morgans | TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells[END_REF][START_REF] Neuille | Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB)[END_REF][START_REF] Orhan | A New Mouse Model for Complete Congenital Stationary Night Blindness Due to Gpr179 Deficiency[END_REF][START_REF] Orlandi | GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes[END_REF][START_REF] Pardue | Mouse b-wave mutants[END_REF][START_REF] Peachey | GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Pusch | The complete form of X-linked congenital stationary night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein[END_REF][START_REF] Qian | Identification of a new mutant allele, Grm6(nob7), for complete congenital stationary night blindness[END_REF][START_REF] Van Genderen | Mutations in TRPM1 are a common cause of complete congenital stationary night blindness[END_REF][START_REF] Varin | Better understanding of the ON-bipolar cells signaling cascade and development of therapies for Congenital Stationary Night Blindness[END_REF][START_REF] Zeitz | Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF][START_REF] Zeitz | Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz flicker electroretinogram[END_REF]. cCSNB mouse models lacking Nyx (nob) and Grm6 with ON-BC dysfunction, showed increased susceptibility to myopia and decreased dopamine content [START_REF] Chakraborty | ON pathway mutations increase susceptibility to form-deprivation myopia[END_REF][START_REF] Pardue | High susceptibility to experimental myopia in a mouse model with a retinal on pathway defect[END_REF]. While human subjects with cCSNB and high myopia have no ON-BC transmission, their OFF-BC transmission is largely intact [START_REF] Miyake | Congenital stationary night blindness with negative electroretinogram. A new classification[END_REF]Zeitz et al., 2015). By contrast, subjects with incomplete CSNB (iCSNB) show variable degrees of myopia (median refractive error of -4.8 D), associated with both reduced ON-and OFFpathway dysfunction [START_REF] Bijveld | Genotype and phenotype of 101 dutch patients with congenital stationary night blindness[END_REF][START_REF] Miyake | Congenital stationary night blindness with negative electroretinogram. A new classification[END_REF][START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. We are not aware that high myopia has been reported with other forms of CSNB, leading to a rod dysfunction and a Riggs-ERG [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Aleman et al showed that stimulation of the ON-pathway may thicken the choroid and thus perhaps inhibit myopia in humans [START_REF] Aleman | Reading and Myopia: Contrast Polarity Matters[END_REF]. Together, these findings suggest that ON-pathway disruption contributes to the development of myopia and that studies on cCSNB can be used to better understand myopia.

In recent years, gene identification studies in CSNB patients led to the identification of key proteins mediating photoreceptor to ON-BC signal transmission. Because of the prevalence of myopia in CSNB patients, these ON-pathway genes are also candidate genes for myopia.

Seeking new myopia genes through this purely genetic approach has its limitations since CSNB is a rare retinal condition [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]; for example our data suggest that the prevalence of CSNB in France is ~1:400,000 [START_REF] Varin | Better understanding of the ON-bipolar cells signaling cascade and development of therapies for Congenital Stationary Night Blindness[END_REF], though it is likely to be underdiagnosed since correct diagnosis requires the application of a specific clinical examination (e.g. electroretinography) and/or molecular genetic testing. Given these limitations, we followed an alternative approach using whole transcriptome sequencing to seek novel CSNBassociated or CSNB-myopia related genes. This allowed us to add novel proteins to the retinal signaling pathway, identify new candidate genes for myopia, and strengthen the evidence for several previously proposed myopia-associated genes. These results provide a framework for the development of future therapeutic strategies to treat myopia.

Phenotype in human cases and animal models with complete congenital stationary night blindness (cCSNB) and associated myopia

Signal transmission from photoreceptors to ON-bipolar cells

The clinical characterization of subjects with CSNB, and more specifically cCSNB, was recently reviewed [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Electroretinography is crucial for a correct positive diagnosis of CSNB and distinguish its different forms, both in human subjects and animal models [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. A clear diagnostic electroretinogram (ERG) feature in cCSNB is the absence of the b-wave in dark adapted conditions (DA 0.01) (Figure 1). In light-adapted conditions (LA 3.0 30 Hz and LA 3.0), ERGs are altered differently (see for details (Zeitz et al., 2015)) (Figure 1). ERG findings under scotopic conditions highlight similarities between cCSNB animal models and the human phenotype. In contrast, under photopic conditions, cCSNB animal models show more variability between species (Figure 1). The photopic ERG phenotype of cCSNB mouse models is generally more severe, including the absence of the bwave, compared to that of human cases or larger animal models (Figure 1) (e.g. [START_REF] Neuille | Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB)[END_REF][START_REF] Orhan | A New Mouse Model for Complete Congenital Stationary Night Blindness Due to Gpr179 Deficiency[END_REF]). In contrast, cone-driven responses are comparable between CSNB dogs, horses and human cases, i.e. mildly reduced and delayed (reviewed in [START_REF] Kondo | A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness[END_REF]Varin et al., 2021b;[START_REF] Witzel | Congenital stationary night blindness: an animal model[END_REF][START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF] (Figure 1). These differences may be a result of species-specific physiological differences, but could also be due to the use of different ERG protocols (Varin et al., 2021b).

ERG features associated with myopia in human subjects and animal models are less well studied [START_REF] Koh | Correlation of structural and electrophysiological changes in the retina of young high myopes[END_REF][START_REF] Westall | Values of electroretinogram responses according to axial length[END_REF].. Some studies in myopic chicken suggested that the oscillatory potentials are altered [START_REF] Fujikado | Retinal function with lensinduced myopia compared with form-deprivation myopia in chicks[END_REF]. By performing multifocal ERG measurements Ho et al., showed that ERG responses originating from the human paracentral retina negative defocus are reduced compared to ERG responses originating from the central retina [START_REF] Ho | Signdependent changes in retinal electrical activity with positive and negative defocus in the human eye[END_REF]. This paracentral region of the retina is believed to predominately reflect the activity from ON-and OFF-BCs, with relatively small contributions from photoreceptors and other inner retina cells [START_REF] Chu | Porcine global flash multifocal electroretinogram: possible mechanisms for the glaucomatous changes in contrast response function[END_REF]. Because genetic data are often unavailable for myopia subjects, it remains uncertain if myopia cases that present specific ERG-alterations represent syndromic myopia cases, as found e.g. in cCSNB. For those genes that have been implicated in myopia, they may also have roles in ON-pathway signaling. For example, a mouse model of myopia lacking Aplp2, coding for the amyloid beta precursor like protein 2, which is highly expressed in bipolar cells, revealed a reduction in the amplitude of the ERG b-wave and oscillatory potentials [START_REF] Tkatchenko | APLP2 Regulates Refractive Error and Myopia Development in Mice and Humans[END_REF]. A genomewide-association study (GWAS) revealed an association between APLP2 variants and myopia in humans; thus, the ERG defects in the Aplp2 -/-mouse were thought to be linked to myopia [START_REF] Tkatchenko | APLP2 Regulates Refractive Error and Myopia Development in Mice and Humans[END_REF] (Figure 2). However, the electrophysiological properties of the retina of the Aplp2 -/-mouse model are also consistent with a role of APLP2 in synaptic transmission. It would be interesting to document ERGs of myopia cases linked to APLP2.

Visual acuity in cCSNB and myopia

Subjects with cCSNB have decreased visual acuity with a median of 0.30 log MAR (Minimum Angle of Resolution; about 20/40) [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Relatively preserved mean best corrected visual acuity of 0.02 log MAR (about 20/20; [START_REF] Ye | Visual Acuity in Pathological Myopia Is Correlated With the Photoreceptor Myoid and Ellipsoid Zone Thickness and Affected by Choroid Thickness[END_REF]) and 0.88 decimals (better than 20/25; [START_REF] Liu | Prevalence and progression of myopic retinopathy in Chinese adults: the Beijing Eye Study[END_REF]) is observed in myopic (including high myopic) subjects without underlying retinal changes. Pathologic myopia, however, is associated with a reduced mean best corrected visual acuity of 0.56 ± 0.35 decimals (about 20/35; [START_REF] Liu | Prevalence and progression of myopic retinopathy in Chinese adults: the Beijing Eye Study[END_REF]) and 0.19 ± 0.20 log MAR (about 20/30; [START_REF] Ye | Visual Acuity in Pathological Myopia Is Correlated With the Photoreceptor Myoid and Ellipsoid Zone Thickness and Affected by Choroid Thickness[END_REF]). About 30% of cases with myopic retinal changes have visual acuity worse than 20/60. Hence, although cCSNB subjects demonstrate high axial myopia [START_REF] Al-Hujaili | Long-term follow-up of retinal function and structure in TRPM1-associated complete congenital stationary night blindness[END_REF] their visual acuity loss is generally more severe, due to cone ON-bipolar cell dysfunction, than in subjects with isolated myopia.

Visual acuity in mice can be measured using an optomotor test [START_REF] Jellali | The optomotor response: a robust first-line visual screening method for mice[END_REF]. All tested mouse models for cCSNB reveal severely reduced visual acuity under scotopic conditions [START_REF] Neuille | Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB)[END_REF][START_REF] Orhan | A New Mouse Model for Complete Congenital Stationary Night Blindness Due to Gpr179 Deficiency[END_REF][START_REF] Pinto | Generation, identification and functional characterization of the nob4 mutation of Grm6 in the mouse[END_REF]. Visual acuity under photopic conditions may be also altered [START_REF] Neuille | Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB)[END_REF][START_REF] Pinto | Generation, identification and functional characterization of the nob4 mutation of Grm6 in the mouse[END_REF]Varin et al., 2021b). A few studies have measured visual acuity in myopia mouse models; For example, in Aplp2 knock-outs visual acuity was normal in form-deprivation myopia, but contrast vision was reduced and correlated with myopia [START_REF] Tkatchenko | APLP2 Regulates Refractive Error and Myopia Development in Mice and Humans[END_REF]. Similarly, patients with myopia exhibited reduced sensitivity to contrast in comparison to emmetropes and contrast sensitivity decreased with an increasing degree of myopia [START_REF] Stoimenova | The effect of myopia on contrast thresholds[END_REF].

Retinal structure in cCSNB and myopia

In contrast to progressive rod-cone dystrophy, the retinal structure is quite well preserved in human subjects with cCSNB. This is due to the stationary, functional, but not degenerative nature of disease of cCSNB. A few reports describe thinning of inner retinal layers (INLs) in cases with cCSNB as measured by spectral domain Optical Coherence Tomography (SD-OCT). However, this thinning is different from that observed in subjects with progressive rodcone dystrophy (Al [START_REF] Oreany | Congenital stationary night blindness with hypoplastic discs, negative electroretinogram and thinning of the inner nuclear layer[END_REF][START_REF] Godara | Assessing retinal structure in complete congenital stationary night blindness and Oguchi disease[END_REF][START_REF] Ivanova | Novel frameshift mutation in NYX gene in a Russian family with complete congenital stationary night blindness[END_REF]. SD-OCT measurements done in the Lrit3 mouse model of cCSNB also revealed thinning of the INL [START_REF] Neuille | Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB)[END_REF]. It is not clear if the INL thinning observed in human cCSNB subjects and in a few mouse models is related to the development of high myopia or if it is only caused by the mutated gene causing cCSNB. A systematic analysis of cCSNB cases compared to myopia control cohorts is needed to determine the exact cause of this thinning [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF].

In subjects with myopia without outer retinal changes, regional changes in central retinal thickness have be described by SD-OCT; these include thinning of the INL and outer nuclear layer, and thickening of OPL and photoreceptor outer segments [START_REF] Liu | Macular Thickness Profiles of Intraretinal Layers in Myopia Evaluated by Ultrahigh-Resolution Optical Coherence Tomography[END_REF][START_REF] Ye | Visual Acuity in Pathological Myopia Is Correlated With the Photoreceptor Myoid and Ellipsoid Zone Thickness and Affected by Choroid Thickness[END_REF]. In subjects with pathologic myopia, additional changes described under the term of myopic maculopathy are observed [START_REF] Ohno-Matsui | Updates of pathologic myopia[END_REF].

Studying myopia in mouse models

Animal models have been increasingly used to investigate mechanisms of myopia. Myopia can be induced using negative lenses or form deprivation in a variety of species as diverse as monkeys [START_REF] Qiao-Grider | Normal ocular development in young rhesus monkeys (Macaca mulatta)[END_REF][START_REF] Weinstein | A comparison of visual acuity in the rhesus monkey and man[END_REF], tree shrews [START_REF] Norton | Normal development of refractive state and ocular component dimensions in the tree shrew (Tupaia belangeri)[END_REF][START_REF] Petry | Spatial contrast sensitivity of the tree shrew[END_REF], cats [START_REF] Berkley | Grating resolution and refraction in the cat estimated from evoked cerebral potentials[END_REF][START_REF] Blake | Visual resolution in the cat[END_REF], guinea pigs [START_REF] Buttery | How thick should a retina be? A comparative study of mammalian species with and without intraretinal vasculature[END_REF][START_REF] Howlett | Emmetropization and schematic eye models in developing pigmented guinea pigs[END_REF], chickens [START_REF] Irving | Chick eye optics: zero to fourteen days[END_REF][START_REF] Jarvis | Measuring and modelling the spatial contrast sensitivity of the chicken (Gallus g. domesticus)[END_REF][START_REF] Schmid | Assessment of visual acuity and contrast sensitivity in the chick using an optokinetic nystagmus paradigm[END_REF], and fish [START_REF] Shen | Eyes of a lower vertebrate are susceptible to the visual environment[END_REF]. However, most these models are not well suited for genetic research.

The mouse, has become a popular animal model for the myopia research [START_REF] Barathi | Involvement of GABA transporters in atropine-treated myopic retina as revealed by iTRAQ quantitative proteomics[END_REF][START_REF] Barathi | Muscarinic cholinergic receptor (M2) plays a crucial role in the development of myopia in mice[END_REF][START_REF] Beleggia | CRIM1 haploinsufficiency causes defects in eye development in human and mouse[END_REF][START_REF] Cases | Foxg1-Cre Mediated Lrp2 Inactivation in the Developing Mouse Neural Retina, Ciliary and Retinal Pigment Epithelia Models Congenital High Myopia[END_REF][START_REF] Hudson | Post-translationally abnormal collagens of prolyl 3-hydroxylase-2 null mice offer a pathobiological mechanism for the high myopia linked to human LEPREL1 mutations[END_REF][START_REF] Ma | Wnt signaling in form deprivation myopia of the mice retina[END_REF][START_REF] Miyake | Identification of myopia-associated WNT7B polymorphisms provides insights into the mechanism underlying the development of myopia[END_REF][START_REF] Pardue | High susceptibility to experimental myopia in a mouse model with a retinal on pathway defect[END_REF][START_REF] Song | Enlargement of the Axial Length and Altered Ultrastructural Features of the Sclera in a Mutant Lumican Transgenic Mouse Model[END_REF][START_REF] Storm | Megalin-deficiency causes high myopia, retinal pigment epithelium-macromelanosomes and abnormal development of the ciliary body in mice[END_REF][START_REF] Tekin | SLITRK6 mutations cause myopia and deafness in humans and mice[END_REF][START_REF] Tkatchenko | Genetic background modulates refractive eye development and susceptibility to myopia in the mouse[END_REF][START_REF] Tkatchenko | APLP2 Regulates Refractive Error and Myopia Development in Mice and Humans[END_REF][START_REF] Tran-Viet | Mutations in SCO2 are associated with autosomal-dominant high-grade myopia[END_REF]Wu et al., 2015a;Wu et al., 2015b). The mouse offers a number of important advantages compared to other species, including well-established techniques allowing the alteration of its genome.

Moreover, both genes and environment can be manipulated in the same mouse [START_REF] Barathi | Two models of experimental myopia in the mouse[END_REF][START_REF] Schaeffel | Measurement of refractive state and deprivation myopia in two strains of mice[END_REF]Tejedor and de la Villa, 2003a;[START_REF] Tkatchenko | Mouse experimental myopia has features of primate myopia[END_REF].

Behavioral studies suggest that vision is critical for accurate spatial navigation in mice [START_REF] Buhot | Exploration, anxiety, and spatial memory in transgenic anophthalmic mice[END_REF][START_REF] Cook | Anxiety-related behaviors in the elevated zero-maze are affected by genetic factors and retinal degeneration[END_REF][START_REF] De La Cera | Optical aberrations in the mouse eye[END_REF][START_REF] Faulstich | Comparison of plasticity and development of mouse optokinetic and vestibulo-ocular reflexes suggests differential gain control mechanisms[END_REF]. Although mice are classified as nocturnal animals they are also active during the day and have diurnal retinal features [START_REF] Flores | Pattern recognition of sleep in rodents using piezoelectric signals generated by gross body movements[END_REF][START_REF] Jacobs | Losses of functional opsin genes, short-wavelength cone photopigments, and color vision-A significant trend in the evolution of mammalian vision[END_REF][START_REF] Yang | Genetic analysis of daily physical activity using a mouse chromosome substitution strain[END_REF]. Mice undergo emmetropization driven by photopic visual stimulation similar to humans [START_REF] Tkatchenko | Photopic visual input is necessary for emmetropization in mice[END_REF]. Basic spatial and temporal vision characteristics in mice are similar to those in humans [START_REF] Histed | Psychophysical measurement of contrast sensitivity in the behaving mouse[END_REF][START_REF] Umino | Speed, spatial, and temporal tuning of rod and cone vision in mouse[END_REF] but with lower visual function compared to humans [START_REF] Prusky | Characterization of mouse cortical spatial vision[END_REF]. Due to the above-mentioned differences, thresholds for rod-dominated (scotopic) and cone-dominated (photopic) vision in mice, as well as the structure of the retinal scotopic and photopic pathways, maybe different but still show high similarities to those in humans [START_REF] Leamey | Comparative survey of the mammalian visual system with reference to the mouse[END_REF][START_REF] Umino | Speed, spatial, and temporal tuning of rod and cone vision in mouse[END_REF]. Although mice lack the fovea found in primates, the mouse retina has an area of increased density of photoreceptors and retinal ganglion cells (RGC) just temporal to the optic disc, which represents an anatomical and functional analog of the area centralis found in all non-primate mammals [START_REF] Drager | Ganglion cell distribution in the retina of the mouse[END_REF][START_REF] Jeon | The major cell populations of the mouse retina[END_REF][START_REF] Leamey | Comparative survey of the mammalian visual system with reference to the mouse[END_REF]. Induced changes in genetic background and gene-targeted mutations affect refractive eye development and susceptibility to myopia in mice [START_REF] Schippert | Relative axial myopia in Egr-1 (ZENK) knockout mice[END_REF][START_REF] Tkatchenko | APLP2 Regulates Refractive Error and Myopia Development in Mice and Humans[END_REF]Tkatchenko et al., 2019;[START_REF] Troilo | IMI -Report on Experimental Models of Emmetropization and Myopia[END_REF][START_REF] Wisard | Exaggerated eye growth in IRBP-deficient mice in early development[END_REF][START_REF] Zhou | Modulation of retinal cell populations and eye size in retinoic acid receptor knockout mice[END_REF][START_REF] Zhou | Genetic deletion of the adenosine A2A receptor confers postnatal development of relative myopia in mice[END_REF]. Experimental myopia can be induced in mice using visual form deprivation (form deprivation myopia, FDM) or negative spectacle lenses (lens-induced myopia, LIM), similar to other animal models (Chakraborty et al., 2014a;[START_REF] Pardue | High susceptibility to experimental myopia in a mouse model with a retinal on pathway defect[END_REF][START_REF] Pardue | Investigating mechanisms of myopia in mice[END_REF][START_REF] Park | Retinal degeneration increases susceptibility to myopia in mice[END_REF][START_REF] Schaeffel | Measurement of refractive state and deprivation myopia in two strains of mice[END_REF]Tejedor and de la Villa, 2003b;[START_REF] Tkatchenko | APLP2 Regulates Refractive Error and Myopia Development in Mice and Humans[END_REF] (Figure 3). Susceptibility to FDM was increased in mouse models with a defect in the ON-pathway [START_REF] Chakraborty | ON pathway mutations increase susceptibility to form-deprivation myopia[END_REF][START_REF] Pardue | High susceptibility to experimental myopia in a mouse model with a retinal on pathway defect[END_REF], highlighting that ON-pathway disruption may be an important element in refractive error development. More specifically, cCSNBassociated myopia as shown by myopic shifts in eyes with FDM was observed in mice lacking Nyx (nob) [START_REF] Gregg | Identification of the gene and the mutation responsible for the mouse nob phenotype[END_REF] and Grm6 [START_REF] Masu | Specific deficit of the ON response in visual transmission by targeted disruption of the mGluR6 gene[END_REF] [START_REF] Chakraborty | ON pathway mutations increase susceptibility to form-deprivation myopia[END_REF][START_REF] Pardue | High susceptibility to experimental myopia in a mouse model with a retinal on pathway defect[END_REF]. We hypothesize that the cCSNB models used in the present study will similarly show a greater susceptibility to FDM-or LIM-induced myopia, although the genetic background of the mice must be considered, as this can strongly influence both the baseline and susceptibility to induced myopia. For example, wild-type mice in a "129 background" show high baseline myopia (~ -20D) compared to those in a C57BL/6J background (Tkatchenko et al., 2019). Our cCSNB models lacking Gpr179 and Lrit3 are in a C57BL/6J background; in contrast, our cCSNB mouse model lacking Grm6 is in a different genetic background (129S6.129S(Cg)) than the one used by [START_REF] Chakraborty | ON pathway mutations increase susceptibility to form-deprivation myopia[END_REF][START_REF] Chakraborty | ON pathway mutations increase susceptibility to form-deprivation myopia[END_REF]. Thus, we estimate that the difference of the refractive error following FDM or LIM may not be measurable in our mice lacking Grm6. Recently we were indeed able to show that our cCSNB model lacking Gpr179 is more susceptible to LIM (Wilmet et al., 2022 under review).

All cCSNB mouse models share the same gene defects as human subjects with cCSNB [START_REF] Audo | Whole-Exome Sequencing Identifies Mutations in GPR179 Leading to Autosomal-Recessive Complete Congenital Stationary Night Blindness[END_REF][START_REF] Bech-Hansen | Mutations in NYX, encoding the leucinerich proteoglycan nyctalopin, cause X-linked complete congenital stationary night blindness[END_REF][START_REF] Dryja | Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6[END_REF][START_REF] Gregg | Identification of the gene and the mutation responsible for the mouse nob phenotype[END_REF][START_REF] Pardue | A naturally occurring mouse model of X-linked congenital stationary night blindness[END_REF][START_REF] Peachey | GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Pusch | The complete form of X-linked congenital stationary night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein[END_REF][START_REF] Zeitz | Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF][START_REF] Zeitz | Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz flicker electroretinogram[END_REF]. All cCNSB genetic defects result in a defective ON-BC transmission. In contrast, disruption of mainly the OFF-pathway caused by a mutation in the Vsx1 gene did not significantly alter refractive eye development or the susceptibility to experimentally induced FDM (Chakraborty et al., 2014b). These findings lend further support to the selective contribution of the ON-pathway to myopia at least in animal models. Recently it was shown that Vsx1 is expressed in both cone ON-and OFF-BCs, but not in rod ON-BCs [START_REF] Shekhar | Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics[END_REF]. We are not aware of studies showing myopia in mice with iCSNB, which affects both ON-and OFF-BCs. However, in a recent study, patients with a variant near GJD2 had high myopia, supporting a potential role for altered signaling in cone-driven OFF pathways in myopia development. Therefore, it is possible that multiple retinal pathways contribute to myopia [START_REF] Jiang | Electrical responses from human retinal cone pathways associate with a common genetic polymorphism implicated in myopia[END_REF].

Numerous retinal signaling pathways have been shown to be involved in refractive eye development and myopia in animal models [START_REF] Summers | Functional integration of eye tissues and refractive eye development: Mechanisms and pathways[END_REF]Tkatchenko and Tkatchenko, 2019;[START_REF] Troilo | IMI -Report on Experimental Models of Emmetropization and Myopia[END_REF]. Dopamine signaling plays an important role in these processes and dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC) have been implicated in refractive error development in mice [START_REF] Schaeffel | Animal models in myopia research[END_REF]. Stimulation of DA biosynthesis in light is due to the activation of tyrosine hydroxylase (TH) [START_REF] Iuvone | Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons[END_REF]. Dopaminergic ACs receive excitatory glutamatergic input from ON-BCs [START_REF] Boatright | Inhibition of endogenous dopamine release in amphibian retina by L-2-amino-4-phosphonobutyric acid (L-AP4) and trans-2aminocyclopentane-1,3-dicarboxylate (ACPD)[END_REF][START_REF] Boelen | Light-stimulated release of dopamine from the primate retina is blocked by 1-2-amino-4-phosphonobutyric acid (APB)[END_REF][START_REF] Dumitrescu | Ectopic retinal ON bipolar cell synapses in the OFF inner plexiform layer: contacts with dopaminergic amacrine cells and melanopsin ganglion cells[END_REF][START_REF] Stone | Image defocus and altered retinal gene expression in chick: clues to the pathogenesis of ametropia[END_REF] and transmit the signal further to RGCs in the retina. Retinal DA and DOPAC content drop during the development of FDM and remain at reduced levels [START_REF] Schaeffel | Animal models in myopia research[END_REF]. Further support for the role of DA in myopia development comes from the cCSNB mouse models lacking Nyx (nob) and Grm6, as they are characterized by not only an increased susceptibility to FDM but also by lower levels of DA and DOPAC (nob mice) and DOPAC (Grm6 -/-) compared to wild-type animals [START_REF] Chakraborty | ON pathway mutations increase susceptibility to form-deprivation myopia[END_REF][START_REF] Pardue | High susceptibility to experimental myopia in a mouse model with a retinal on pathway defect[END_REF]. Compared to measuring the refractive error in animal models with FDM or LIM, measuring DA and DOPAC by ultra-performance liquid chromatography is less time consuming, since the induction of experimental myopia is not needed. Indeed, recent findings revealed that levels of DOPAC are reduced in genetic retinal disease mouse models and are not affected by myopia induction [START_REF] Chakraborty | ON pathway mutations increase susceptibility to form-deprivation myopia[END_REF][START_REF] Park | Retinal degeneration increases susceptibility to myopia in mice[END_REF]. As shown in Figure 4, we detected lower levels of DA and DOPAC after light adaptation in mice lacking Gpr179, Lrit3 and Grm6 compared to wild-type animals, further demonstrating that these cCSNB mouse models provide a valid basis to study myopia.

Differentially expressed genes in cCSNB

RNA-Seq studies in mouse models for cCSNB

Several studies have identified novel myopia-associated candidate genes by whole transcriptome sequencing in several animal models of myopia [START_REF] Riddell | Novel evidence for complement system activation in chick myopia and hyperopia models: a meta-analysis of transcriptome datasets[END_REF][START_REF] Riddell | Bidirectional Expression of Metabolic, Structural, and Immune Pathways in Early Myopia and Hyperopia[END_REF]Tkatchenko et al., 2019;Tkatchenko and Tkatchenko, 2021a, b;[START_REF] Tkatchenko | Gene expression in response to optical defocus of opposite signs reveals bidirectional mechanism of visually guided eye growth[END_REF][START_REF] Vocale | RNAseq and GSEA identifies suppression of ligand-gated chloride efflux channels as the major gene pathway contributing to form deprivation myopia[END_REF][START_REF] Wu | Scleral hypoxia is a target for myopia control[END_REF]. Here, we performed whole transcriptome sequencing (RNA-Seq) in three different adult mouse models of cCSNB (Gpr179 -/-, Lrit3 -/-and Grm6 -/-) and their age-matched littermates (each, n = 5) to investigate the molecular basis of myopia in cCSNB and to identify novel proteins involved in the retinal signaling cascade, including the ON-pathway. After identification and validation of differentially expressed genes (DEGs), a meta-analysis was performed by comparing our data with i) published transcriptomic data from purified retinal cells and single cell RNA-Seq data [START_REF] Macosko | Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets[END_REF][START_REF] Shekhar | Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics[END_REF][START_REF] Siegert | Transcriptional code and disease map for adult retinal cell types[END_REF][START_REF] Siegert | Genetic address book for retinal cell types[END_REF][START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF] and ii) pathway analyses and myopia databases and/or publications concerning the role of identified DEGs in normal vision. Figure 5 shows DEGs with fold changes (FC) of at least 1.2, P-values of ≤ 0.01 and an expression value of at least 5 transcripts per million reads (TPM) from RNA-Seq data of adult Gpr179 -/-, Lrit3 -/-and Grm6 -/-mice compared to data from age-matched wild-type littermates (Supplementary Table S1-S4). As expected, all three mouse models exhibited significantly lower expression of their respective "knocked out" genes (Gpr179, Lrit3, Grm6) compared to the corresponding wild-type strains (Supplementary Tables S1-S4). Overall, 155, 133, and 222 genes were differentially expressed in the Gpr179 -/-, Lrit3 -/-and Grm6 -/-mouse strains, respectively (Supplementary Tables S1-S4 and Figure 5a). Differences in numbers and types of DEGs between the strains may be due in part to differences in genetic background, which may also influence differential regulation of the gene network (Tkatchenko et al., 2019). While cCSNB mouse models lacking Gpr179 and Lrit3 are on a C57BL/6J background, mice lacking Grm6 are on a 129S6.129S(Cg) background. However, to minimize background related issues, for each knock-out strain, the respective littermate wild-type strain was used for comparison. DEGs, appearing in all three cCSNB mouse strains may be implicated in a common physiological role, while DEGs appearing in only one cCSNB mouse strain may have a unique role important for the specific gene missing in the model. Here, we focused mainly on DEGs shared by at least two of the three mutant strains.

Validation of RNA-Seq studies

To identify genes playing a role in the cCSNB phenotype, DEGs with at least 1.2 FCs and Pvalue ≤ 0.01 were investigated, including their expression in specific retinal cell-types and their association with the term "myopia". Overall, there were 52 DEGs, that appeared in at least two cCSNB mouse strains (Supplementary Table S5 andS6) and 7 in all three (Supplementary Table S6, bold, Figure 5A andB). The Gpr179 -/-and Lritr3 -/-mouse strains shared the majority of the DEGs (38), followed by Gpr179 -/-and Grm6 -/-( 16) and Grm6 -/-and Lrit3 -/-mouse strains (12) (Figure 5A). The 52 DEGs present in at least two mouse strains could be sorted into 10 clusters (Figure 5B). The largest cluster consisted of genes downregulated in the cCSNB models (cluster 1, Figure 5B). Quantitative RT-PCR (RT-qPCR) performed for 24 of those genes confirmed that all 24 were differentially expressed in the retina of knock-out mouse strains compared to the corresponding wild-type mice (Figure 6 shows some of the validated genes, Supplementary data). Of note, some genes were found to be differentially expressed only in mice lacking either Lrit3 or Gpr179 genes by RNA-Seq, but were found to be significantly differentially expressed in all three knock-outs by RT-qPCR (e.g. Prkca and Tpbg, Figure 6). Although not all differences observed with RNA-Seq were validated by RT-qPCR, which may be explained by the lesser sensitivity of RT-qPCR experiments [START_REF] Costa | Comprehensive molecular screening: from the RT-PCR to the RNA-seq[END_REF], the trend was the same for the majority of the 24 DEGs, thus validating the transcriptome sequencing approach.

Known genes implicated in CSNB

Mutations in NYX, GRM6, GPR179, TRPM1 and LRIT3 that cause cCSNB affect protein localization in retinal ON-BCs [START_REF] Audo | Whole-Exome Sequencing Identifies Mutations in GPR179 Leading to Autosomal-Recessive Complete Congenital Stationary Night Blindness[END_REF][START_REF] Bech-Hansen | Mutations in NYX, encoding the leucinerich proteoglycan nyctalopin, cause X-linked complete congenital stationary night blindness[END_REF][START_REF] Dryja | Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6[END_REF][START_REF] Li | Recessive mutations of the gene TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness in humans[END_REF][START_REF] Peachey | GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Pusch | The complete form of X-linked congenital stationary night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein[END_REF][START_REF] Van Genderen | Mutations in TRPM1 are a common cause of complete congenital stationary night blindness[END_REF][START_REF] Varin | Better understanding of the ON-bipolar cells signaling cascade and development of therapies for Congenital Stationary Night Blindness[END_REF][START_REF] Zeitz | Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Zeitz | Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz flicker electroretinogram[END_REF]. The proteins localize in the outer plexiform layer (OPL) and in most cases in the dendritic tips of ON-BCs [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Similar to human subjects, ON-BC function is affected in cCSNB mouse models [START_REF] Gregg | Identification of the gene and the mutation responsible for the mouse nob phenotype[END_REF][START_REF] Koike | TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade[END_REF][START_REF] Masu | Specific deficit of the ON response in visual transmission by targeted disruption of the mGluR6 gene[END_REF][START_REF] Morgans | TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells[END_REF][START_REF] Neuille | Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB)[END_REF][START_REF] Peachey | GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Proteins involved in cCSNB often show an interdependency in their localization, and may or may not show correlated changes in expression levels [START_REF] Neuille | LRIT3 is essential to localize TRPM1 to the dendritic tips of depolarizing bipolar cells and may play a role in cone synapse formation[END_REF]. For example, nyctalopin (encoded by Nyx) and TRPM1 interact. Localization of TRPM1 at the ON-BC dendritic tips is dependent on the presence of nyctalopin, mGluR6 and LRIT3 [START_REF] Cao | TRPM1 forms complexes with nyctalopin in vivo and accumulates in postsynaptic compartment of ON-bipolar neurons in mGluR6dependent manner[END_REF][START_REF] Hasan | LRIT3 is Required for Nyctalopin Expression and Normal ON and OFF Pathway Signaling in the Retina[END_REF][START_REF] Neuille | LRIT3 is essential to localize TRPM1 to the dendritic tips of depolarizing bipolar cells and may play a role in cone synapse formation[END_REF][START_REF] Pearring | A role for nyctalopin, a small leucine-rich repeat protein, in localizing the TRP melastatin 1 channel to retinal depolarizing bipolar cell dendrites[END_REF]Zeitz et al., 2015). Using RNA-Seq, we showed that Trpm1 mRNA levels are dependent on the expression of Gpr179, Lrit3, and Grm6, and that expression levels of Grm6 are dependent on the expression of Lrit3 (Figure 7). However, although Trpm1 expression levels seems to be dependent on Gpr179 expression, the localization of TRPM1 at the dendritic tips of BCs does not appear to be affected in Gpr179 -/-mice [START_REF] Orhan | A New Mouse Model for Complete Congenital Stationary Night Blindness Due to Gpr179 Deficiency[END_REF][START_REF] Orlandi | GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes[END_REF]. Aside from the targeted gene in each knock-out strain, most of the other known genes underlying cCSNB, are not strongly differentially expressed (Supplementary Tables S1 andS6), suggesting that the 52 DEGs in the cCSNB mouse models are not necessarily candidate genes for cCSNB.

Transcriptome analyses of DEGs in cCSNB

All 52 DEGs that we identified are expressed in the human retina (Supplementary Table S6, Figure 8) [START_REF] Farkas | Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes[END_REF]. Most of them were already reported in retina transcriptome databases [START_REF] Blond | Functional Genomics of the Retina to Elucidate its Construction and Deconstruction[END_REF][START_REF] Farkas | Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes[END_REF][START_REF] Kalathur | RETINOBASE: a web database, data mining and analysis platform for gene expression data on retina[END_REF][START_REF] Macosko | Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets[END_REF][START_REF] Shekhar | Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics[END_REF][START_REF] Siegert | Transcriptional code and disease map for adult retinal cell types[END_REF][START_REF] Siegert | Genetic address book for retinal cell types[END_REF]. Here, we mainly focus either on genes expressed in ON-BCs or in downstream ACs and RGCs. Twenty genes (5730419F03Rik, Cxcr7, Ampd3, Arc, Bdnf, Cacna1s, Car8, Lect1, Edn2, Eomes, Gabrr1, Ldb2, Lrp2bp, Olfm3, Plekhf1, Rgs16, Slco5a1, Syn2, Tgbf2 and Tpbg) showed an agedependent relative increase in expression in the rd1-transcriptome database, as a result of the age-dependent photoreceptor degeneration [START_REF] Blond | Functional Genomics of the Retina to Elucidate its Construction and Deconstruction[END_REF][START_REF] Kalathur | RETINOBASE: a web database, data mining and analysis platform for gene expression data on retina[END_REF]. These genes are expressed in the INL or ganglion cell layer (GCL) and they seem to be important for BC, AC and RGC function and maintenance (Supplementary Table S6). In addition, a number of novel candidate genes absent in the current transcriptome databases were also identified. Several of these genes were found in rod (Col5a1 and Vgll4), cone Interestingly, 5730419F03Rik, Ampd3, Car8, Casp7, Ccnjl, Lect1, Dusp4, Gabrr1, Gm20754, Lrp2bp, Prkca, Ptprr, Rgs16, Slco5a1 and Tpbg were also shown to be differentially expressed in mice lacking the BC-specific basic helix-loop-helix (bHLH) transcription factor, Bhlhe23, alias Bhlhb4 [START_REF] Bramblett | The transcription factor Bhlhb4 is required for rod bipolar cell maturation[END_REF][START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF]. This transcription factor was found to be expressed in rod bipolar cells (RB). ERGs in the adult Bhlhb4 knockout mice, in a C57Bl6 strain, showed that the loss of BHLBH4 resulted in disrupted rod signaling and profound retinal dysfunction resembling CSNB, characterized by the loss of the scotopic ERG b-wave. A depletion of INL cells in the adult Bhlhb4 knockout has been ascribed to the abolishment of the RB cell population during postnatal development.

Other retinal cell populations including photoreceptors were unaltered. In the absence of information from these databases, our data was completed using expression data available from the literature and single cell RNA-Seq analyses (https://www.proteinatlas.org) (Supplementary Table S6, Figure 8).

Strikingly, Lect1 (Cnmd) was up-regulated in Gpr179 -/-and Lrit3 -/-mice and slightly downregulated in Grm6 -/-mice compared to wild-type mice. These findings were validated by RT-qPCR (Figure 5B, cluster 2, Figure 6). In contrast, Etv4 was down-regulated in Gpr179 -/-and Lrit3 -/-mice and up-regulated in Grm6 -/-mice compared to wild-type animals (Figure 5B, cluster 9). LECT1 (MIM# 605147), coding for the leukocyte cell-derived chemotaxin 1 protein (also called chondromodulin 1) is an angiogenesis inhibitor first isolated from fetal bovine cartilage. Although confined to cartilage and the eye at later stages of development, fetal whole-mount in situ hybridization revealed expression of Lect1 mRNA in somites, heart, bronchial arches, roof plate, retina and limb buds. LECT1 expression pattern suggested a role in morphogenesis during embryonic development [START_REF] Shukunami | Generation of multiple transcripts from the chicken chondromodulin-I gene and their expression during embryonic development[END_REF]. In adult retina, LECT1 was found in the GCL and INL as revealed by RNA in situ hybridization and immunohistochemistry studies [START_REF] Funaki | Expression and localization of angiogenic inhibitory factor, chondromodulin-I, in adult rat eye[END_REF]. A knock-out mouse model showed no overt abnormality during embryogenesis [START_REF] Brandau | Chondromodulin I is dispensable during enchondral ossification and eye development[END_REF][START_REF] Nakamichi | Chondromodulin I is a bone remodeling factor[END_REF].

Analysis of expression databases revealed that LECT1 is expressed in the inner retina, photoreceptors, BCs, ACs and RGCs and that its expression appears to be reduced in Bhlhe23 -/-mice [START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF].

Single cell analyses of DEGs in subpopulation of bipolar cells

To investigate in which BCs (rod BCs, cone ON-or OFF-BCs) the more abundant DEGs were expressed, our data was compared to a single-cell BC-specific transcriptome database [START_REF] Shekhar | Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics[END_REF]. We found that most genes were enriched in rod BCs and cone ON-BCs, but some were also expressed in OFF-BCs (BC1-4 = OFF-BCs, BC5-BC7 = ON-BCs (Figure 9). These data are further explored below for genes where additional functional information has been described previously.

Pathway analyses of DEGs involved in CSNB and correlation with myopia

In rare cases, subjects with mutations in NYX [START_REF] Yip | A novel missense mutation in the NYX gene associated with high myopia[END_REF][START_REF] Zhang | Mutations in NYX of individuals with high myopia, but without night blindness[END_REF][START_REF] Zhou | NYX mutations in four families with high myopia with or without CSNB1[END_REF], TRPM1 [START_REF] Zhou | Novel TRPM1 mutations in two Chinese families with early-onset high myopia, with or without complete congenital stationary night blindness[END_REF] and GRM6 [START_REF] Jin | Trio-based exome sequencing arrests de novo mutations in early-onset high myopia[END_REF][START_REF] Xu | Sequence variations of GRM6 in patients with high myopia[END_REF] present with early-onset high myopia despite the absence of documented cCSNB diagnosis. However, it is believed that most mutations in NYX, LRIT3, TRPM1, GRM6 and GPR179 are not directly responsible for high myopia. Instead, it is hypothesized that myopia in cCSNB is caused by a disruption of the ON-pathway [START_REF] Chakraborty | ON pathway mutations increase susceptibility to form-deprivation myopia[END_REF]. Our analysis revealed that mitogenactivated protein kinase (MAPK) pathways, synaptic signaling, G protein-coupled receptor ligand binding pathways, and proteins implicated in eye, endoderm and connective tissue development were affected in cCSNB (Figure 10A). Some of the genes appeared in multiple pathways (Figure 10B). In the following paragraphs we detail these genes and pathways and describe correlations between cCSNB and associated ocular alterations, focusing on myopia. When genes appeared in multiple pathways, they are described only once. In addition, we focus on genes with additional data available in the literature. These genes are highlighted in bold. A detailed description of all DEGs, their expression pattern in different cell types of the retina, their roles and putative involvement in myopia development can be found in the supplementary data and supplementary Tables S6 andS7).

MAPK pathway

To our knowledge, the MAPK signaling pathway has not previously been implicated in cCSNB. In the normal retina, phosphorylated MAPKs are barely detectable. However, subjects and mice lacking functional retinoschisin (RS1) exhibit decreased ERG b-wave amplitudes and increased MAPK activation [START_REF] Gehrig | Genome-wide expression profiling of the retinoschisin-deficient retina in early postnatal mouse development[END_REF][START_REF] Plossl | The X-linked juvenile retinoschisis protein retinoschisin is a novel regulator of mitogen-activated protein kinase signalling and apoptosis in the retina[END_REF]. In addition, under certain pathological conditions, such as induced retinal ischemia, MAPKs are activated by phosphorylation in several retinal layers, including in the INL with a concomitant reduction of the b-wave amplitude [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Interestingly, inhibitors of MAPK phosphorylation significantly normalized the b-wave amplitude after ischemia [START_REF] Roth | Mitogenactivated protein kinases and retinal ischemia[END_REF]. Although MAPK signaling in the retina has been investigated, its role in cCSNB and accompanying ocular manifestations are poorly understood. Fifteen DEGs associated with the MAPK pathway were identified: Ackr3/Cxcr7, Bdnf, Cacna1s, Casp7, Ccnjl, Cdc25c, Dusp4, Ldb2, Prkca, Ptprr, Spred3, Synj2, Tgfb2, Tpbg and Vgll4 (Figures 10 and11). LRIT3 (MIM# 615004) encoding leucine-rich repeat, immunoglobulin-like, and transmembrane domain-containing protein 3 (LRIT3) is mutated in human subjects with cCSNB (MIM# 615058) and mice [START_REF] Neuille | Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB)[END_REF]. Interestingly, LRIT3 is itself a modulator of the fibroblast growth factor receptor 1 (FGFR1) [START_REF] Kim | Leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) is a modulator of FGFR1[END_REF], which also plays an important role in the MAPK signaling pathway, though not identified as such in pathway analyses. Phosphorylated FGFR1 regulates different signaling pathways including

MAPK.

TGFB2 (MIM# 190220) encodes the transforming growth factor beta 2 (Supplementary Table S6 and S7,. Mice lacking Tgfb2 exhibit perinatal mortality and a wide range of developmental defects, including congenital heart defects, skeletal defects and eye defects [START_REF] Sanford | TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes[END_REF], which suggests that TGFB2 also plays a role in eye development, albeit with a different outcome dependent on the species tested. Studies in tree shrews revealed that expression of Tgfb2 was reduced in animals with experimentally induced myopia [START_REF] Jobling | Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression[END_REF] but increased in the experimentally-induced myopia guinea pig model [START_REF] Chen | Altered TGF-beta2 and bFGF expression in scleral desmocytes from an experimentally-induced myopia guinea pig model[END_REF]. Studies of human aqueous humor also found that levels of TGFB2 positively correlated with axial length in cases with myopia [START_REF] Jia | Human aqueous humor levels of TGF-beta2: relationship with axial length[END_REF]. In addition, a genetic variant in TGFB2 [START_REF] Lin | Sclera-related gene polymorphisms in high myopia[END_REF] and the myopia Harmonize GAD database [START_REF] Rouillard | The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins[END_REF] linked TGFB2 to myopia (Supplementary Tables S6 andS7). To our knowledge, a connection between down-regulation of TGFB2 in inner retinal cells, including rod BCs and cone ON-BCs, and myopia has not been previously established.

BDNF (MIM# 113505) encoding brain-derived neurotrophic factor (B)DNF (Supplementary

Table S6 and S7, serves as a survival, mitogenic, and differentiation factor in both the developing and adult central and peripheral nervous systems (reviewed in [START_REF] Klocker | Brainderived neurotrophic factor-mediated neuroprotection of adult rat retinal ganglion cells in vivo does not exclusively depend on phosphatidyl-inositol-3'-kinase/protein kinase B signaling[END_REF]). The cellular effects of neurotrophins are mediated by the activation of Trk tyrosine kinase receptors and the p75 receptor. Upon binding to these receptors, neurotrophins are known to activate various intracellular signal transduction pathways, including the MAPK pathway (Figure 11), which affect cell survival and cell death mechanisms. BDNF is expressed in the inner retina [START_REF] Herzog | Contributions of the optic tectum and the retina as sources of brain-derived neurotrophic factor for retinal ganglion cells in the chick embryo[END_REF] including all bipolar cells (Supplementary Table S6, Figures 8 and9). Its neuroprotective and healing properties in these cells are well known [START_REF] Afarid | Neuroprotective and restorative effects of the brain-derived neurotrophic factor in retinal diseases[END_REF]. Several polymorphisms in BDNF have been associated with myopia [START_REF] Musolf | Caucasian Families Exhibit Significant Linkage of Myopia to Chromosome 11p[END_REF]. In addition, BDNF appeared in the myopia Harmonize GAD database [START_REF] Rouillard | The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins[END_REF] (Supplementary Table S6 and S7, Figure 8). ACKR3 (MIM# 610376), also known as CXCR7, and located on chromosome 2q37.3, encodes the atypical chemokine receptor 3. It is expressed in several regions of the CNS, in endothelial and various immune cells [START_REF] Salazar | The chemokine receptor CXCR7 interacts with EGFR to promote breast cancer cell proliferation[END_REF], as well as in retina (Supplementary Table S6, Figures 891011). ACKR3 is able to heterodimerize with CXCR4, which increases ligand stimulated membrane recruitment of β-arrestin, thus causing sustained activation of the ERK1/2 and p38 MAPK signaling pathways [START_REF] Quinn | Emerging roles of atypical chemokine receptor 3 (ACKR3) in normal development and physiology[END_REF] (Figure 11). Interestingly, ACKR3/ CXCR7 was linked to myopia in a mapped region on chromosome 2q37 (Figure 8) [START_REF] Schache | Fine mapping linkage analysis identifies a novel susceptibility locus for myopia on chromosome 2q37 adjacent to but not overlapping MYP12[END_REF]. A few mutations in ACKR3 have been shown to cause oculomotor synkinesis, characterized by involuntary eye movements in mouse and humans [START_REF] Whitman | Decreased ACKR3 (CXCR7) function causes oculomotor synkinesis in mice and humans[END_REF]. CACNA1S (MIM# 114208), coding for the L-type voltage-dependent calcium channel, alpha-1S subunit, is required for normal skeletal and cardiac muscle function [START_REF] Elbaz | Hypokalemic periodic paralysis and the dihydropyridine receptor (CACNL1A3): genotype/phenotype correlations for two predominant mutations and evidence for the absence of a founder effect in 16 caucasian families[END_REF]).

CACNA1S is homologous to other calcium channel alpha-1 subunits, such as CACNA1F (Cav1.4), which is mutated in subjects with incomplete CSNB [START_REF] Bech-Hansen | Loss-of-function mutations in a calciumchannel alpha1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness[END_REF][START_REF] Strom | An L-type calcium-channel gene mutated in incomplete Xlinked congenital stationary night blindness[END_REF]. In the retina, Cacna1s was found to be expressed in BCs and more recently in cones and rods (Supplementary Table S6, Figures 891011). Initially, it was thought to localize in dendritic tips of ON-BCs, similar to other genes implicated in cCSNB [START_REF] Specht | Effects of presynaptic mutations on a postsynaptic Cacna1s calcium channel colocalized with mGluR6 at mouse photoreceptor ribbon synapses[END_REF][START_REF] Tummala | Localization of Cacna1s to ON Bipolar Dendritic Tips Requires mGluR6-Related Cascade Elements[END_REF] but its exact protein localization in the retina remains unclear, since commercially available CACNA1S antibodies have been shown to cross-react with GPR179 [START_REF] Hasan | CACNA1S expression in mouse retina: Novel isoforms and antibody cross-reactivity with GPR179[END_REF], which is localized at the dendritic tips of ON-BCs.

Calcium channels and their subunits in general have been linked to MAPK signaling [START_REF] Ikeda | Signal transduction. Calcium channels--link locally, act globally[END_REF]) (Figure 11). L-type voltage-gated calcium channels in both retinal photoreceptors and bipolar neurons are under circadian control [START_REF] Hull | Diurnal changes in exocytosis and the number of synaptic ribbons at active zones of an ON-type bipolar cell terminal[END_REF][START_REF] Ko | Circadian regulation of ion channels and their functions[END_REF][START_REF] Ko | The expression of L-type voltage-gated calcium channels in retinal photoreceptors is under circadian control[END_REF].

PRKCA (MIM# 176960) encodes protein kinase C, alpha (PKCalpha, PRKCA), which is a calcium-activated, phospholipid-and diacylglycerol (DAG)-dependent serine/threonineprotein kinase (Supplementary Table S6, Figures 891011). It is involved in regulation of cell proliferation, apoptosis, differentiation, migration and adhesion, tumorigenesis, cardiac hypertrophy, angiogenesis, platelet function and inflammation, by directly phosphorylating targets or activating signaling cascades involving ERK1/2 (Figure 11). In the retina, PRKCA present at glutamatergic rod-BC synapses could act as a molecular switch to trigger intracellular signaling cascades in response to the activation of G-protein-coupled receptors [START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF]. Its role in activating MAPK pathways or in cCSNB have not been studied. We found reduced expression of Prkca in cCSNB models by RNA-Seq and validated it by RT-qPCR (Figure 6). In addition, we showed a trend for reduced PRKCA protein levels in Gpr179 -/-and Lrit3 -/-mice (Figure 12). PRKCA expression at both transcript and protein levels in Grm6 -/-mice was more variable (compare Figure 6 and Figures 12). A reduced PRKCA immunofluorescent labeling was also seen in Gpr179 -/-and Lrit3 -/-mice (Figure 13). PTPRR (MIM: 602853) encoding protein-tyrosine phosphatase receptor type R, (PTPRR) was found to be expressed in many different retinal cells including in rod BCs and cone . PTPRR has been implicated in MAPK deactivation in the brain (for review [START_REF] Chirivi | Altered MAP kinase phosphorylation and impaired motor coordination in PTPRR deficient mice[END_REF]). Mice lacking Ptprr showed increased phosphorylation of ERK1/2 in Purkinje cells and impaired motor coordination [START_REF] Chirivi | Altered MAP kinase phosphorylation and impaired motor coordination in PTPRR deficient mice[END_REF]. In the retina, PTPRR was suggested to play an important role in neuronal development and maintenance of the mature retina [START_REF] Horvat-Brocker | Receptor protein tyrosine phosphatases are expressed by cycling retinal progenitor cells and involved in neuronal development of mouse retina[END_REF].

Mutations in PTPRR could be involved in the development of myopia by a change in the enzyme's ability to bind to, or dephosphorylate MAPK, leading to increased MAPK growth signals. Increased expression of PTPRR in rapidly growing fetal retina and choroid may be the result of normal developmental or growth regulation, suggesting that PTPRR plays a role in controlling ocular growth. Disruption of this protein's function may account for the rapid eye growth seen in highly myopic individuals during early childhood, which slows in adulthood. As growth decreases, the need for this protein would be reduced [START_REF] Hawthorne | Association mapping of the high-grade myopia MYP3 locus reveals novel candidates UHRF1BP1L, PTPRR, and PPFIA2[END_REF]. In addition, PTPRR was identified by GWAS as linked to myopia (Supplementary Table S6 andS7).

TPBG (MIM# 190920), encoding trophoblast glycoprotein (Supplementary Table S6 andS7 ,, was found to activate MAPK signaling in smooth muscle cells and adventitial pericyte-like cells [START_REF] Spencer | Role of TPBG (Trophoblast Glycoprotein) Antigen in Human Pericyte Migratory and Angiogenic Activity[END_REF]. TPBG is directly connected to ACKR3 (CXCR7; see above). Indeed, TPBG silencing downregulated phosphorylated ERK and ACKR3. In the retina, TPBG is highly expressed by rod-BCs, where it has been shown to exhibit PRKCAdependent phosphorylation [START_REF] Wakeham | Expression and distribution of trophoblast glycoprotein in the mouse retina[END_REF][START_REF] Wakeham | Identification of PKCalpha-dependent phosphoproteins in mouse retina[END_REF]. TPBG phosphorylation was decreased in Prkca -/-compared to wild-type mice. Our studies revealed lower expression of Tpbg at the mRNA level, and a trend of lower protein levels in all cCSNB mouse strains (Figures 7 and12). In addition, TPBG immunofluorescence was severely reduced in the OPL and inner part of the IPL representing the dendritic tips and synaptic terminals of rod-BCs, in mice lacking Lrit3 and Gpr179 (Figure 13). VGLL4 (MIM# 6186962) encodes vestigial-like family member 4 (Supplementary Table S6 and Figures 891011). Studies in zebrafish [START_REF] Xue | The expression patterns of vestigial like family member 4 genes in zebrafish embryogenesis[END_REF], xenopus [START_REF] Barrionuevo | Two different vestigial like 4 genes are differentially expressed during Xenopus laevis development[END_REF][START_REF] Faucheux | Vestigial like gene family expression in Xenopus: common and divergent features with other vertebrates[END_REF] and mice suggested that VGLL4 takes part in early embryonic development, including eye development. Interestingly, this gene is a part of the Hippo signaling pathway, which has also been implicated in myopia [START_REF] Zhu | YAP and TAZ, the conductors that orchestrate eye development, homeostasis, and disease[END_REF]. DUSP4 (MIM# 602747 alias MKP-2), located on chromosome 8p12, codes for dualspecificity phosphatase 4 [START_REF] Guan | Isolation and characterization of a novel dual specific phosphatase, HVH2, which selectively dephosphorylates the mitogen-activated protein kinase[END_REF]. It belongs to the DUSP protein family that inactivates their target kinases, namely MAPKs, by dephosphorylating both phosphoserine/threonine and phosphotyrosine residues. Expression of DUSP proteins is induced by various extracellular stimuli, and has diverse tissue distribution and subcellular localization [START_REF] Dickinson | Diverse physiological functions for dual-specificity MAP kinase phosphatases[END_REF][START_REF] Patterson | Dual-specificity phosphatases: critical regulators with diverse cellular targets[END_REF]. In melanocytes, DUSP4 expression may be regulated by the microphthalmia-associated transcription factor (MITF), which is a melanocytic lineage-specific transcription factor with a role in the development of malignant melanoma. Similarly, MITF regulates TRPM1 expression, which is implicated in both cCSNB and melanoma-associated retinopathy (MAR) [START_REF] Hoek | Novel MITF targets identified using a two-step DNA microarray strategy[END_REF][START_REF] Li | Recessive mutations of the gene TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness in humans[END_REF][START_REF] Van Genderen | Mutations in TRPM1 are a common cause of complete congenital stationary night blindness[END_REF][START_REF] Varin | Identification and characterization of novel TRPM1 autoantibodies from serum of patients with melanoma-associated retinopathy[END_REF][START_REF] Xiong | Serum TRPM1 autoantibodies from melanoma associated retinopathy patients enter retinal on-bipolar cells and attenuate the electroretinogram in mice[END_REF]. Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. It has been recently

shown that the genetic inactivation of DUSP4 reduced the proliferation of melanoma cells, thus making DUSP4 an interesting therapeutic target [START_REF] Christodoulou | Analysis of CRISPR-Cas9 screens identifies genetic dependencies in melanoma[END_REF]. DUSP4 is expressed in different tissues, including the eye, and more importantly in retinal BCs (Supplementary Table S6, Figures 891011). In tissues other than the retina, DUSP4 was mainly localized to the nucleus and found to be induced by multiple agents, including serum, growth factors, UV-light, and oxidative stress. Depending on the tissue, it dephosphorylates MAPKs, such as ERK, JNK or p38 (Figure 11). In addition, DUSP4 was shown to play a role in innate and adaptive immunity, in cancer, and in brain function [START_REF] Lawan | MKP-2: out of the DUSP-bin and back into the limelight[END_REF][START_REF] Seternes | Dual-specificity MAP kinase phosphatases in health and disease[END_REF]. Supporting our RNA-Seq findings, a significant downregulation of Dusp4 expression was found in Lrit3 -/-and Gpr179 -/-mice by RT-qPCR and a trend was noted in Grm6 -/-mice (Supplementary Figure 1). In Trpm1 -/-mice, significantly reduced expression of Dusp4 was also observed (Supplementary Figure 1). These findings were confirmed at the protein level, but with more variability (Figure 12). Western blot analyses of retinal extracts from wild-type mice revealed a band at 43 kDa, the calculated molecular weight of DUSP4.

This band was absent in Dusp4 -/-mice (data not shown). The role of DUSP4 in the retina is largely unknown. Transcriptional profiling of endogenous germ layer precursor cells identified Dusp4 as an essential gene in zebrafish endoderm specification [START_REF] Brown | Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification[END_REF]. Although most of the vertebrate eye is derived from the ectoderm [START_REF] Bassnett | The lens growth process[END_REF], zebrafish Dusp4 morphants had small necrotic heads, suggesting an essential role of DUSP4 in anterior development [START_REF] Brown | Transcriptional profiling of endogenous germ layer precursor cells identifies dusp4 as an essential gene in zebrafish endoderm specification[END_REF]. However, the role of DUSP4 in eye development was not further described. Interestingly, in well-established models of refractive error induction, Dusp4 appeared to be down-regulated in the eye upon inductions of both FDM and LIM in chicks and mice [START_REF] Mcglinn | Form-deprivation myopia in chick induces limited changes in retinal gene expression[END_REF][START_REF] Stone | Image defocus and altered retinal gene expression in chick: clues to the pathogenesis of ametropia[END_REF][START_REF] Tkatchenko | Large-Scale microRNA Expression Profiling Identifies Putative Retinal miRNA-mRNA Signaling Pathways Underlying Form-Deprivation Myopia in Mice[END_REF]. Subjects with an 8p deletion, which includes the 8p12 DUSP4, often present with eye defects including nystagmus, strabismus and myopia, among other clinical syndromes (https://www.rarechromo.org/media/information/Chromosome%20%208/8p%20interstitial%2 0deletions%20including%208p12%20FTNP.pdf) (e.g. [START_REF] Beighle | Small structural changes of chromosome 8. Two cases with evidence for deletion[END_REF][START_REF] Chilcote | Association of red cell spherocytosis with deletion of the short arm of chromosome 8[END_REF][START_REF] Devriendt | Delineation of the critical deletion region for congenital heart defects, on chromosome 8p23.1[END_REF][START_REF] Klopocki | A novel 8 Mb interstitial deletion of chromosome 8p12-p21.2[END_REF]). Such defects are also found in cCSNB cases. Together, these findings suggest that DUPS4 may be one of the genes that underlies cCSNB-related high myopia (Supplementary Table S6, Figure 8).

Synapse

Several signaling pathways involved in synaptic transmission were strongly associated with cCSNB (Figure 10A), including glutamatergic, serotonergic, cholinergic and GABAergic (gamma-aminobutyric acid) signaling. GABA is one of the major inhibitory neurotransmitters of the retina and central nervous system [START_REF] Cheng | rho1 GABAC receptors are expressed in fibrous and cartilaginous layers of chick sclera and located on sclera fibroblasts and chondrocytes[END_REF]. Twelve DEGs were associated with GABAergic signaling: Ackr3/Cxcr7, Cacna1s, Car8, Edn2, Gabrr1, Gng4, Grm6, Prkca, Ptprr, Rgs16, Tac2 and Trpc1 (Figure 10A). A detailed description of their expression in the different cell types of the retina, focusing on the inner retina, as well as their function and possible involvement in myopia can be found in the supplementary data (Supplementary Tables S6 andS7) and Figure 8). GABRR1 (MIM# 137161), coding for the gamma-aminobutyric acid receptor, rho-1 (GABRR1) (alternatively named GABA-C Receptor, rho-1 subunit), is a member of a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the central nervous system [START_REF] Cutting | Cloning of the gamma-aminobutyric acid (GABA) rho 1 cDNA: a GABA receptor subunit highly expressed in the retina[END_REF]. It represents one of the subunits of the GABA-C receptor (rho1-rho3) (e.g. [START_REF] Enz | GABA(C) receptors: a molecular view[END_REF]) expressed in the inner retina in cone photoreceptors, bipolar cells and horizontal cells (Supplementary Table S6, Figures 8 and9).

In addition, GABRR1 was found to be differentially expressed in bipolar cell-deficient Bhlhe23 -/-mice [START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF]. Mice deficient for the Gabrr1 subunit lacked GABA-C receptor-mediated GABA-evoked responses, and signaling from rod BCs to third order cells was altered [START_REF] Mccall | Elimination of the rho1 subunit abolishes GABA(C) receptor expression and alters visual processing in the mouse retina[END_REF]. The elimination of GABRR1 was demonstrated to result in the absence of GABA-C receptors in the retina. While global retinal morphology was not affected and the scotopic electroretinogram revealed a normal shape of the a-wave (rodfunction) and b-wave (mainly rod-BC function), the implicit time of the b-wave was shorter and more oscillatory potentials (OPs) were found in mice lacking Gabrr1 [START_REF] Mccall | Elimination of the rho1 subunit abolishes GABA(C) receptor expression and alters visual processing in the mouse retina[END_REF]. It has been suggested that some of the OPs are generated by a negative feedback loop in the IPL between the GABAergic ACs and the axon terminals of rod BCs [START_REF] Kolb | Amacrine cells of the cat retina[END_REF], which may indicate that the altered ERG is due to a transmission defect between ACs and rod BCs. Together, these findings confirm that GABRR1 has an important function in normal visual processing. Several studies identified an association between GABRR1 and myopia (Supplementary Table S6 and Figure 8). A genome-wide association study identified GABRR1 as one of the genes associated with refractive error in Asian populations (Fan et al., 2016;[START_REF] Lin | Genome-Wide Association of Genetic Variants With Refraction, Axial Length, and Corneal Curvature: A Longitudinal Study of Chinese Schoolchildren[END_REF]. TPMPA ((1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid)), a GABA-C antagonist has been shown to inhibit synaptic transmission in rat spinal cord [START_REF] Rozzo | Antagonism by (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid of synaptic transmission in the neonatal rat spinal cord in vitro: an electrophysiological study[END_REF], modulate sleep-waking behavior in rats [START_REF] Arnaud | Study of a GABAC receptor antagonist on sleep-waking behavior in rats[END_REF], enhance memory in chicks [START_REF] Gibbs | Opposing roles for GABAA and GABAC receptors in short-term memory formation in young chicks[END_REF] and most importantly, inhibit myopia development in chicks and guinea pigs [START_REF] Chebib | Novel, potent, and selective GABAC antagonists inhibit myopia development and facilitate learning and memory[END_REF][START_REF] Cheng | Inhibition of form-deprivation myopia by a GABAAOr receptor antagonist, (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA), in guinea pigs[END_REF][START_REF] Stone | GABA, experimental myopia, and ocular growth in chick[END_REF], indicating possible roles for GABA-C receptors in memory, circadian rhythms, and myopia [START_REF] Cheng | rho1 GABAC receptors are expressed in fibrous and cartilaginous layers of chick sclera and located on sclera fibroblasts and chondrocytes[END_REF]. Although GABA-C receptors are pentameric structures formed from three possible subunits, the rho1 subunit, encoded by Gabrr1, was found to be the primary target of GABA-C antagonists (cis-and trans-(3-aminocyclopentanyl)butylphosphinic acid (cis-and trans-3-ACPBPA) inhibiting myopia [START_REF] Chebib | Novel, potent, and selective GABAC antagonists inhibit myopia development and facilitate learning and memory[END_REF]. In addition to its expression in the retina, Gabrr1 is also expressed in the fibrous and cartilaginous layers of the chick sclera, where it is localized to the sclera fibroblasts and chondrocytes, which have been previously connected to myopia [START_REF] Cheng | rho1 GABAC receptors are expressed in fibrous and cartilaginous layers of chick sclera and located on sclera fibroblasts and chondrocytes[END_REF]. Together, these findings are consistent with reduced levels of Gabrr1 expression in cCSNB (Figure 6), as a possible cause for the development of high myopia in cCSNB. CA8 (MIM# 114815) and its mouse orthologue Car8, encoding carbonic anhydrase VIII (CA8), has been found to be important for GABAergic synaptic transmission. Car8 is mainly expressed in the inner retina, with some expression in cone photoreceptors (Supplementary Table S6, Figures 8 and9). In addition, it was found to be differentially expressed in Bhlhe23 -/-mice [START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF]. Car8 has been mostly studied in the brain, where it is highly expressed in cerebellar Purkinje neurons [START_REF] Jiao | Carbonic anhydrase-related protein VIII deficiency is associated with a distinctive lifelong gait disorder in waddles mice[END_REF][START_REF] Kato | Sequence of a novel carbonic anhydrase-related polypeptide and its exclusive presence in Purkinje cells[END_REF] and other parts of the brain [START_REF] Lakkis | Expression of the acatalytic carbonic anhydrase VIII gene, Car8, during mouse embryonic development[END_REF]. Mice lacking functional Car8 [START_REF] Jiao | Carbonic anhydrase-related protein VIII deficiency is associated with a distinctive lifelong gait disorder in waddles mice[END_REF] show gait ataxia and dystonia with some ultrastructural differences in synapses between Purkinje cell dendritic spines, indicating a role for carbonic anhydrase VIII in synapse formation and/or maintenance. Similarly, subjects with mutations in CA8 exhibit ataxia and mild mental retardation [START_REF] Turkmen | CA8 mutations cause a novel syndrome characterized by ataxia and mild mental retardation with predisposition to quadrupedal gait[END_REF]. Recent studies in mouse retina showed CAR8 immunolabelling in rod BCs co-labelled for PRKCA, with particular bright staining in the axon terminal regions and weaker staining in the cytoplasmic compartments [START_REF] Puthussery | Carbonic anhydrase-related protein VIII is expressed in rod bipolar cells and alters signaling at the rod bipolar to AII-amacrine cell synapse in the mammalian retina[END_REF]. In addition, some labelling was observed in a subset of cone BCs and GABAergic ACs [START_REF] Puthussery | Carbonic anhydrase-related protein VIII is expressed in rod bipolar cells and alters signaling at the rod bipolar to AII-amacrine cell synapse in the mammalian retina[END_REF]. Mice lacking Car8 do not have a CSNB phenotype, as measured by electroretinography, but light-evoked current responses from AII-ACs were significantly larger and more prolonged than in control mice, suggesting that CAR8 is important for modulating retinal neurotransmission [START_REF] Puthussery | Carbonic anhydrase-related protein VIII is expressed in rod bipolar cells and alters signaling at the rod bipolar to AII-amacrine cell synapse in the mammalian retina[END_REF]. Interestingly, genome-wide association studies found a potential association between CA8 and myopia [START_REF] Kiefer | Genome-wide analysis points to roles for extracellular matrix remodeling, the visual cycle, and neuronal development in myopia[END_REF] (Supplementary Table S6 and Figure 8). The reduced expression of Car8 in Gpr179 -/-and Lrit3 -/-mice, as identified by RNA-Seq studies, was confirmed by RT-qPCR and at the protein level (Figures 6 and12). GRM6 (MIM# 604096) encodes metabotropic glutamate receptor 6 (GRM6 or mGluR6), which has been well described as causing cCSNB when mutated [START_REF] Dryja | Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6[END_REF][START_REF] Zeitz | Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz flicker electroretinogram[END_REF] and which we used here as a mouse model [START_REF] Masu | Specific deficit of the ON response in visual transmission by targeted disruption of the mGluR6 gene[END_REF] to identify DEGs in cCSNB. Grm6 seems to be the only "cCSNB gene" with a significantly reduced expression in other cCSNB models (here Lrit3 -/-), indicating that the expression of Lritr3 may influence Grm6 transcript levels. Grm6 is uniquely expressed in ON-BCs, where the ON-visual pathway originates (Supplementary Table S6) [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. As mentioned above, several studies have described mutations in GRM6 [START_REF] Jin | Trio-based exome sequencing arrests de novo mutations in early-onset high myopia[END_REF][START_REF] Xu | Sequence variations of GRM6 in patients with high myopia[END_REF] that have been associated with early-onset high myopia despite undocumented cCSNB (Supplementary Table S7, Figure 8). RGS16 (MIM# 602514) encodes regulator of G-protein signaling 16, which is expressed in the inner retina [START_REF] Dhingra | Probing neurochemical structure and function of retinal ON bipolar cells with a transgenic mouse[END_REF] (Supplementary Table S6 and Figures 8 and9) and differentially expressed in Bhlhe23 -/-mice [START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF]. While other regulatory proteins such as GNB5 (GTP-binding protein beta 5), RGS9 and RGS11 are known to be implicated in ON-BC signal transduction, the role of RGS16 in this signaling cascade remains to be elucidated. Further, we are not aware of a link between RGS16 and retinal disorders, including myopia. Genome-wide association studies revealed a variant in this gene associated with circadian rhythms in individuals claiming to be morning persons [START_REF] Hu | GWAS of 89,283 individuals identifies genetic variants associated with self-reporting of being a morning person[END_REF].

Interestingly, Rgs16 -/-mice were shown to have a longer circadian period [START_REF] Doi | Circadian regulation of intracellular G-protein signalling mediates intercellular synchrony and rhythmicity in the suprachiasmatic nucleus[END_REF].

EDN2 (MIM# 131241) encodes endothelin2 (EDN2

). Edn2 is one the few genes expressed at higher levels in cCSNB mouse strains compared to the wild-type mice (Supplementary Table S6, Figures 5 and10A). It was found to be a potent vasoconstrictor that binds to two G protein-coupled receptors, i.e., EDNRA (which resides on bipolar dendrites) and EDNRB (which is present on Mueller and horizontal cells). Both receptors are also present on choroidal vessels [START_REF] Prasanna | Eyeing endothelins: a cellular perspective[END_REF]. Interestingly, it has been shown that Edn2 was also up-regulated in the retina of several models of photoreceptor degeneration [START_REF] Chen | Light damage induced changes in mouse retinal gene expression[END_REF][START_REF] Cottet | Biological characterization of gene response in Rpe65-/-mouse model of Leber's congenital amaurosis during progression of the disease[END_REF][START_REF] Rattner | The genomic response to retinal disease and injury: evidence for endothelin signaling from photoreceptors to glia[END_REF][START_REF] Van Schil | Autosomal recessive retinitis pigmentosa with homozygous rhodopsin mutation E150K and non-coding cis-regulatory variants in CRX-binding regions of SAMD7[END_REF], as well as following retinal damage, such as retinal detachment [START_REF] Rattner | The genomic response to retinal disease and injury: evidence for endothelin signaling from photoreceptors to glia[END_REF][START_REF] Zacks | Activation of signaling pathways and stress-response genes in an experimental model of retinal detachment[END_REF].

These observations suggest that increased expression of Edn2 may be a general response to retinal insult (reviewed in [START_REF] Bramall | Endothelin-2-mediated protection of mutant photoreceptors in inherited photoreceptor degeneration[END_REF]). EDN2 was linked to myopia by a recent genome-wide association study (Tedja et al., 2018), suggesting its increased expression as a potential cause of cCSNB-related myopia (Supplementary Table S6 and Figure 8).

LRP2BP (MIM# 619020) encodes low density lipoprotein receptor-related protein 2b

(LRP2B). We are including it here among BC synaptic proteins because of its apparent retinal localization [START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF] (Supplementary Table S6 and Figure 8). LRP2BP is a scaffold protein, known to bind and recruit proteins to the megalin receptor (LRP2), which participates in endocytosis and signal transduction [START_REF] Petersen | Functional interaction of megalin with the megalinbinding protein (MegBP), a novel tetratrico peptide repeat-containing adaptor molecule[END_REF]. Interestingly, mutations in LRP2 cause the Donnai-Barrow/Facio-Oculo-Acoustico-Renal syndrome, which is partially characterized by high-grade myopia [START_REF] Kantarci | Mutations in LRP2, which encodes the multiligand receptor megalin, cause Donnai-Barrow and facio-oculo-acoustico-renal syndromes[END_REF][START_REF] Stora | A 56-year-old female patient with facio-oculo-acoustico-renal syndrome (FOAR) syndrome. Report on the natural history and of a novel mutation[END_REF].

Histological investigations of ocular mouse tissues also identified a severe myopia phenotype in megalin-deficient mice [START_REF] Storm | Megalin-deficiency causes high myopia, retinal pigment epithelium-macromelanosomes and abnormal development of the ciliary body in mice[END_REF] and suggested that LRP2 is required for normal eye growth [START_REF] Cases | Foxg1-Cre Mediated Lrp2 Inactivation in the Developing Mouse Neural Retina, Ciliary and Retinal Pigment Epithelia Models Congenital High Myopia[END_REF]. Similarly, a zebrafish model for Lrp2 developed myopia [START_REF] Veth | Mutations in zebrafish lrp2 result in adult-onset ocular pathogenesis that models myopia and other risk factors for glaucoma[END_REF]. Even more importantly, LRP2BP is located on chromosome 4q35.1, a region deleted in cases with high myopia, among other disorders [START_REF] Manolakos | Prenatal diagnosis of two de novo 4q35-qter deletions characterized by array-CGH[END_REF][START_REF] Rossi | Clinical and genomic characterization of distal duplications and deletions of chromosome 4q: study of two cases and review of the literature[END_REF] (Figure 8). SLCO5A1 (MIM# 613543) encoding solute carrier organic anion transport family, member 5A1 (SLCO5A1), was also included in this section, because of a recent finding suggesting that this protein is an important component of retinal synapses and appears to be localized in the OPL [START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF]. It is not clear if it is also located elsewhere in the retina (Supplementary Tables S6 andFigure 8). Subjects with the mesomelia-synostoses syndrome have many extraocular disorders but may also present with myopia. This syndrome is due to a non-recurrent microdeletion on chromosome 8q13 encompassing SULF1 and SLCO5A1 [START_REF] Isidor | Mesomelia-synostoses syndrome results from deletion of SULF1 and SLCO5A1 genes at 8q13[END_REF] (Figure 8).

GPCR ligand binding

Signaling related to G protein-coupled receptor ligand binding was also overrepresented in our database. Twelve different genes were associated with G protein-coupled receptor ligand binding, i.e., Ackr3/Cxcr7, Bdnf,Edn2,Gng4,Grm6,Plekhf1,Prkca,Tac2,Tgfb2,Tpbg,Trpc1 and Umodl1 (Figure 10). A description of their expression patterns in different cell types of the retina, focusing on the inner retina, and their putative role in myopia can be found in the supplementary data (Supplementary Tables S6 andS7 and Figure 8).

One of these genes, UMODL1 (MIM# 613859) encodes an extracellular matrix adhesion and cellular migration uromodulin-like 1 protein (UMODL1) [START_REF] Di Schiavi | UMODL1/Olfactorin is an extracellular membrane-bound molecule with a restricted spatial expression in olfactory and vomeronasal neurons[END_REF][START_REF] Shibuya | Initial characterization of an uromodulin-like 1 gene on human chromosome 21q22.3[END_REF]. It is known to be expressed in olfactory and vomeronasal sensory neurons [START_REF] Di Schiavi | UMODL1/Olfactorin is an extracellular membrane-bound molecule with a restricted spatial expression in olfactory and vomeronasal neurons[END_REF]. It is also present in retina gene expression databases, but the specific cell types expressing Umodl1 remain unknown. UMODL1 was found to be associated with myopia in a genome-wide association study [START_REF] Nishizaki | New susceptibility locus for high myopia is linked to the uromodulinlike 1 (UMODL1) gene region on chromosome 21q22.3[END_REF] (Supplementary Tables S6, S7 and Figure 8).

Eye, endoderm, and connective tissue development

The vertebrate eye is a complex organ. Its development is guided by highly organized processes that take place during embryonic development, and mutations in key genes involved in these processes lead to severe congenital disorders as well as myopia [START_REF] Graw | Eye development[END_REF]. Several DEGs identified in cCSNB mouse models were associated with eye, endoderm and connective tissue development, including Arc, Bdnf,Cacna1s,Cnmd/Lect1,Col5a1,Eomes,Etv4,Dusp4,Grm6,Olfm3,Prkca,Spred3,Tgfb2,Unmodl1 and Vgll4 (Supplementary Tables S6 andS7, Figures 7 and Figure 10).

ARC (MIM# 612461) encodes the activity-regulated cytoskeleton-associated protein (ARC)

(Supplementary Table S6 and Figure 8). ARC mRNA is rapidly produced and delivered into dendrites following synaptic activity in neurons [START_REF] Dynes | Dynamics of bidirectional transport of Arc mRNA in neuronal dendrites[END_REF]. Interestingly, its expression in rodent brains is robustly induced by prolonged wakefulness [START_REF] Cirelli | Differential expression of plasticity-related genes in waking and sleep and their regulation by the noradrenergic system[END_REF]. Converging evidence has led to the hypothesis that wakefulness potentiates, and sleep reduces, synaptic strength. Recently, ARC was demonstrated to be important for the induction of multiple behavioral and molecular responses associated with sleep homeostasis [START_REF] Suzuki | Loss of Arc attenuates the behavioral and molecular responses for sleep homeostasis in mice[END_REF]. COL5A1 (MIM# 120215) encodes the type V collagen alpha-1 (COL5A1) protein, which belongs to group V and XI collagens. These collagens control fibrillogenesis, probably by forming a core within the fibrils. Another characteristic of these collagens is the partial retention of their N-propeptide extensions [START_REF] Fichard | Another look at collagen V and XI molecules[END_REF]. Collagen V was found to be expressed in the human placenta and skin, but later studies showed that it is also present in other organs. Our transcriptome meta-analyses revealed that Col5a1 is expressed in rods and in BCs in the retina (Supplementary Table S6 andFigure 8). Mutations in COL5A1 were found in subjects with the classic type 1 Ehlers-Danlos syndrome (MIM#130000). Ehlers-Danlos syndromes (EDS) are a group of heritable connective tissue disorders that share the common features of skin hyperextensibility, articular hypermobility, and tissue fragility. The main features of classic Ehlers-Danlos syndrome are loose-jointedness and fragile, bruisable skin that heals with peculiar 'cigarette-paper' scars [START_REF] Beighton | Ehlers-Danlos syndrome[END_REF]. Ophthalmic clinical features have also been described, including retinal detachment [START_REF] Pemberton | Familial retinal detachment and the Ehlers-Danlos syndrome[END_REF] and high pathologic myopia [START_REF] Gharbiya | Ocular features in joint hypermobility syndrome/ehlersdanlos syndrome hypermobility type: a clinical and in vivo confocal microscopy study[END_REF][START_REF] Perez-Roustit | Ocular manifestations in Ehlers-Danlos Syndromes: Clinical study of 21 patients[END_REF]. EOMES (MIM# 604615) encodes eomesodermin (EOMES), also called Tbr2. It is directly regulated by Brn3b and is important for RGC development [START_REF] Mao | Eomesodermin, a target gene of Pou4f2, is required for retinal ganglion cell and optic nerve development in the mouse[END_REF]. In agreement with these observations, our gene expression data revealed that Eomes is mainly expressed in the inner retina and more specifically, in RGCs (Supplementary Table S6 and Figure 8).

Homozygous silencing of EOMES leads to microcephaly with polymicrogyria and corpus callosum agenesis [START_REF] Baala | Homozygous silencing of T-box transcription factor EOMES leads to microcephaly with polymicrogyria and corpus callosum agenesis[END_REF]. We are not aware of a retinal phenotype in subjects with this gene defect; however, mice lacking Eomes (alias Trpb2) have reduced retinal projections to non-image-forming brain nuclei and an attenuated pupillary light reflex [START_REF] Sweeney | Tbr2 is required to generate a neural circuit mediating the pupillary light reflex[END_REF]. Interestingly, subjects with CSNB have a reduced redilation and smaller baseline pupil diameters, suggesting a disinhibition of intrinsically photosensitive RGCs due to affected post-photoreceptor transduction via BCs [START_REF] Schatz | Disinhibition of intrinsic photosensitive retinal ganglion cells in patients with X-linked congenital stationary night blindness[END_REF]. OLFM3 (MIM# 607567), encoding olfactomedin 3, is a downstream target of PAX6, which plays a critical role in eye development [START_REF] Grinchuk | The Optimedin gene is a downstream target of Pax6[END_REF]. Although the function of OLFM3 is not yet understood, it may be involved in cell-cell adhesion and cell attachment to the extracellular matrix, and affect cell migration and axon growth [START_REF] Grinchuk | The Optimedin gene is a downstream target of Pax6[END_REF].

Our gene expression data revealed that Olfm3 is expressed in ACs in the retina (Supplementary Table S6 and Figure 9). OLFM3 was also implicated in glaucoma and myopia (Supplementary Table S6 and Figure 8) [START_REF] Podkolodnaya | Application of the ANDCell Computer System to Reconstruction and Analysis of Associative Networks Describing Potential Relationships between Myopia and Glaucoma[END_REF].

Genes not associated with any pathway, unknown function and limited amount of expression in the retina

Several not confirmed but annotated mouse genes (5730419F03Rik, Dbhos (dopamine beta hydroxylase, opposite strand, long coding RNA), Gm20754, Gm40477, Gm49760 and Gm5478) of unknown function and only limited information about their retinal expression patterns were differentially expressed in several cCSNB mouse lines (Supplementary Table S6). Two genes (5730419F03Rik and Gm20754) appeared to be also differentially expressed in mice lacking Bhlhe23. In addition, 5730419F03Rik is most likely expressed in the inner retina, suggesting that it may have a role in inner retinal signaling. Interestingly, Dbhos was also differentially expressed in mice exposed to blue light [START_REF] Ouyang | Whole transcriptome analysis on blue light-induced eye damage[END_REF]. Future studies aimed at investigating the exact cellular expression patterns of these genes and their roles in the retina, as well as identification of the human orthologs will shed light on their contribution to retinal pathophysiology.

Although some information is available about the expression in the retina of the following DEGs, AFAP1L1 (MIMI# 614410), Pdzph1, PLEKHF1 (MIM# 615200), ST6GALNAC5 (MIMI# 610134) and MAST2 (MIM# 612257) (Supplementary Table S6, supporting material), their roles in retinal physiology and possible pathologies remains to be elucidated.

Other genes from myopia databases that are differentially regulated in at least one mouse model with cCSNB

Several other genes identified in myopia databases appear to be significantly differentially expressed as determined by RNA-Seq analyses in at least one mouse model of cCSNB (Supplementary Tables S6 andS7 and Figure 8).

Cause of reduced expression of many DEGs in cCSNB

Reduced expression of DEGs and their respective proteins in cCSNB may be due to reduced expression levels or a lower number of cells expressing the DEGs. Genes implicated in cCSNB share a common phenotype, namely an ON-BC signaling defect. Although the morphology of the retina in human subjects and animal models with cCSNB is believed to be non-degenerative and stable, subtle cellular changes have been described in mice lacking Lrit3 and Trpm1 [START_REF] Neuille | Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB)[END_REF][START_REF] Takeuchi | Different Activity Patterns in Retinal Ganglion Cells of TRPM1 and mGluR6 Knockout Mice[END_REF]. SD-OCT revealed thinning of the INL, IPL, GCL, and nerve fiber layer in Lrit3 -/-mice [START_REF] Neuille | Lrit3 deficient mouse (nob6): a novel model of complete congenital stationary night blindness (cCSNB)[END_REF]. This was not found in Gpr179 -/-mice [START_REF] Orhan | A New Mouse Model for Complete Congenital Stationary Night Blindness Due to Gpr179 Deficiency[END_REF]. Interestingly, rod BC terminals were significantly smaller in Trpm1 -/-retinas than in Grm6 -/-retinas, suggesting that deficiency of TRPM1, but not GRM6 affects rod BC synaptic terminal maturation and alter signaling between rod BCs and ACs [START_REF] Kozuka | The TRPM1 Channel Is Required for Development of the Rod ON Bipolar Cell-AII Amacrine Cell Pathway in the Retinal Circuit[END_REF].

Notably, we found that Prkca transcripts were reduced in cCSNB retinas, and that, similar to TPBG, the level of PRKCA protein, which is specifically localized to rod BCs, was greatly reduced in Gpr179 -/-and Lrit3 -/-mice, but not in Grm6 -/-mice (Figure 13), while the numbers of nuclei present in the INL were unaffected (Figure 14). Visualizing synapses with a proteinindependent marker needs to be performed to evaluate if the lower amounts of PRKCA and TPBG are due to morphological changes in rod BCs, particularly their synaptic terminals.

Similarly, we found that levels of DA and DOPAC were also reduced in cCSNB retinas.

Since the number of nuclei in the INL (Figure 14), remained unchanged in the cCSNB mouse models, and reduction of TH-positive dopaminergic amacrine cells was not significantly different in cCSNB compared to wild-type mice (tested for Gpr179 -/-versus Gpr179 +/+ ) (Figure 15), we conclude that the reduced expression of some genes in cCSNB, as well as the reduction in DA and DOPAC levels are not due to degeneration of retinal cells, but may be linked to altered synapse morphology and/or lower RNA expression and/or reduced protein levels. We speculate that the absence of cCSNB genes such as Gpr179 and Lrit3, leads to the classical ERG-defect but that other differentially regulated genes may contribute to myopia in those subjects with cCSNB. Similarly, exposure to reduced light levels in genetically unaffected individuals may also lead to lower expression of these genes and thus to myopia.

6. Future perspectives 6.1. Identification of differentially expressed genes in cCSNB models using a whole transcriptomic approach may yield novel proteins important for retinal signaling, as well as novel myopia-related genes Mutations in NYX, GRM6, GPR179, TRPM1 and LRIT3 likely lead to cCSNB because the encoded proteins are necessary for ON-BC signaling [START_REF] Audo | Whole-Exome Sequencing Identifies Mutations in GPR179 Leading to Autosomal-Recessive Complete Congenital Stationary Night Blindness[END_REF][START_REF] Bech-Hansen | Mutations in NYX, encoding the leucinerich proteoglycan nyctalopin, cause X-linked complete congenital stationary night blindness[END_REF][START_REF] Dryja | Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6[END_REF][START_REF] Li | Recessive mutations of the gene TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness in humans[END_REF][START_REF] Peachey | GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Pusch | The complete form of X-linked congenital stationary night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein[END_REF][START_REF] Van Genderen | Mutations in TRPM1 are a common cause of complete congenital stationary night blindness[END_REF][START_REF] Varin | Better understanding of the ON-bipolar cells signaling cascade and development of therapies for Congenital Stationary Night Blindness[END_REF][START_REF] Zeitz | Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Zeitz | Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz flicker electroretinogram[END_REF]. The retina of cCSNB human subjects is generally well preserved and the disease course is non-progressive. The proteins are localized in the outer plexiform layer and, in most cases, in the dendritic tips of rod BCs and cone ON-BCs [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Mouse models of cCSNB show a similar ON-BC transmission defect as human cases. The diagnosis is confirmed by an electronegative waveform at the full-field electroretinogram with severely reduced or absent b-wave under scotopic and photopic conditions, reflecting defective ON-pathway signaling [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Candidate genes causing this disorder have been identified by linkage analyses in humans, using animal models, and more recently, by next-generation sequencing approaches (e.g. [START_REF] Audo | Whole-Exome Sequencing Identifies Mutations in GPR179 Leading to Autosomal-Recessive Complete Congenital Stationary Night Blindness[END_REF][START_REF] Pusch | The complete form of X-linked congenital stationary night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein[END_REF][START_REF] Varin | Better understanding of the ON-bipolar cells signaling cascade and development of therapies for Congenital Stationary Night Blindness[END_REF][START_REF] Zeitz | Whole-exome sequencing identifies LRIT3 mutations as a cause of autosomal-recessive complete congenital stationary night blindness[END_REF][START_REF] Zeitz | Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz flicker electroretinogram[END_REF]), with the help of transcriptomic data-bases to reinforce candidate genes [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF].

Recently, Woods et al. used comparative transcriptomic analyses in a mouse model lacking the rod BC transcription factor Bhlhe23 (also known as BHLHB4 or basic helix-loop-helix family member, b4), to identify potential genes implicated in CSNB [START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF]. The phenotype of Bhlhe23 -/-mice is slightly different than that of cCSNB. Although both phenotypes are stationary, rod BCs are almost completely absent in mice lacking Bhlhe23, resulting in a thinner INL but the cone pathway is not affected; thus Bhlhe23 -/-mice have an isolated rod BC defect, while both, rod BC and cone ON-BC functions are affected in cCSNB. This is also reflected by the b-wave amplitude only being affected under scotopic, but not photopic conditions in Bhlhe23 -/-mice [START_REF] Bramblett | The transcription factor Bhlhb4 is required for rod bipolar cell maturation[END_REF]. While the phenotype of Bhlhe23 -/-mice is different from cCSNB, night vision may indeed be affected. To date, however subjects with mutations in BHLHE23 and a similar phenotype have not been described [START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF]. This might be due to the fact that only scotopic vision is affected, and ERG recordings are required to detect such a phenotype in affected subjects.

Because of the absence of rod BCs, one might expect that some of the genes differentially expressed in Bhlhe23 -/-mice may be important for night vision. However, expression of only a few of the "cCSNB-genes" was significantly reduced in Bhlhe23 -/-mice compared to wildtype mice. Furthermore, mice deficient for genes expressed at lower levels, such as mice lacking Car8, did not exhibit a typical cCSNB phenotype [START_REF] Puthussery | Carbonic anhydrase-related protein VIII is expressed in rod bipolar cells and alters signaling at the rod bipolar to AII-amacrine cell synapse in the mammalian retina[END_REF]. This is consistent with our findings, indicating that while these approaches may identify novel candidates for cCSNB this is not necessarily the case.

It is important to note that cCSNB is characterized not only by night blindness, but also by high myopia, nystagmus and often strabismus. Strikingly, since cCSNB is present from birth and young subjects are often not aware of their vision defects in dim light conditions, they are often first diagnosed due to their high myopia and/or infantile nystagmus. Thus, a main goal of this work was to identify other genes implicated in the same pathway as proteins underlying cCSNB and in that way identify additional candidates for the other associated ocular abnormalities associated with cCSNB. Since myopia represents one of the most common eye diseases worldwide, with increasing numbers of affected persons expected in coming years, we focused our study on DEGs implicated in cCSNB and associated with myopia.

Retinal expression of DEGs, pathway analyses and correlation to eye phenotypes

To understand in which retinal cell types the 52 DEGs are expressed, we queried several retina expression databases, including single cell transcriptome databases, as well as PubMed (https://pubmed.ncbi.nlm.nih.gov/) and search tools such as "Google"

(https://www.google.com/) [START_REF] Macosko | Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets[END_REF][START_REF] Shekhar | Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics[END_REF][START_REF] Siegert | Transcriptional code and disease map for adult retinal cell types[END_REF][START_REF] Siegert | Genetic address book for retinal cell types[END_REF][START_REF] Woods | A comparative analysis of rod bipolar cell transcriptomes identifies novel genes implicated in night vision[END_REF]. These data showed that almost all 52 DEGs are expressed in the human retina, including in photoreceptors, BCs, ACs and RGCs, but for only a few of them was their retinal function described in the scientific literature (Supplementary Table S6 and Figure 8). Several DEGs, expressed in various retinal cell types were found to be associated with retinal disorders. For example, genes expressed in rods photoreceptors have been implicated in rod-cone dystrophies. Another example are genes expressed in ON-

BCs, for which the encoded proteins are localized at the dendritic tips, which have been implicated in cCSNB [START_REF] Zeitz | Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF]. Variants in FRMD7 (MIM # 300628), coding for Ferm domain-containing protein 7, which is expressed in ACs [START_REF] Yonehara | Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity[END_REF], are associated with idiopathic nystagmus in mice and human subjects (MIM #310700) [START_REF] Tarpey | Mutations in FRMD7, a newly identified member of the FERM family, cause X-linked idiopathic congenital nystagmus[END_REF]. Thus, other genes expressed in ACs may also be linked to nystagmus. The contribution of genes expressed in ACs to nystagmus was further substantiated in a study with cCSNB humans and mice lacking functional nyctalopin [START_REF] Winkelman | Nystagmus in patients with congenital stationary night blindness (CSNB) originates from synchronously firing retinal ganglion cells[END_REF]. Thus, DEGs described herein and expressed in ACs may contribute to nystagmus observed in subjects with cCSNB (Figure 8). Recently, Bui Quoc and Milleret suggested that an abnormal routing of the RGC axons at the level of the optic chiasm might cause an imbalance between contra-lateral and ipsi-lateral projections and thus lead to strabismus [START_REF] Bui Quoc | Origins of strabismus and loss of binocular vision[END_REF]. This might occur due to an abnormal expression of genes controlling RGCs axonal projections, and which may be differentially expressed in cCSNB. Further experiments and database analyses will be needed to determine if DEGs expressed in ACs and RGCs (Figure 9) contribute to nystagmus and strabismus observed in human subjects with cCSNB. In addition, these genes may also represent candidate genes in cases of isolated nystagmus and strabismus.

Our meta-analysis examined the expression of DEGs and their correlation with myopia using published literature and database analyses. About 50% of the 52 DEGs expressed in the retina, have been already linked to myopia (Figure 8), reinforcing our hypothesis that genes differentially expressed in cCSNB may cause the myopia phenotype observed in cCSNB.

Indeed, GWAS studies showed that several genes expressed in various retinal cell types contribute to the development of myopia (Verhoeven et al., 2013). Some of the genes implicated in myopia are functionally involved in processes that facilitate communication between cells in the retina or control the ability of photoreceptors to respond to light. Some others are involved in pre-natal eye growth and development (Verhoeven et al., 2013). There is some overlap between genes previously described as being involved in myopia and those described here. We assume that some of the others may represent novel myopia-related genes [START_REF] Hysi | Meta-analysis of 542,934 subjects of European ancestry identifies new genes and mechanisms predisposing to refractive error and myopia[END_REF]Tedja et al., 2018;Verhoeven et al., 2013). Interestingly, many DEGs described here, when deleted or part of deleted chromosomal regions, lead to severe phenotypes that often include myopia. Our hypothesis is that the expression levels of these genes are associated with high myopia observed in cCSNB which makes cCSNB a more complex disorder than previously recognized. In addition, DEGs may also produce the cCSNB-associated ocular alterations nystagmus and strabismus. Our results combined with GWAS findings highlight the light-dependent retina-to-sclera signaling cascade that can cause myopia if disrupted and outline potential pathobiological molecular drivers (Tedja et al., 2018). It is unlikely that signals from the retina control scleral growth directly. Instead, it is suggested that retinal signals are relayed by the RPE, choroid and ciliary body, which in turn release signaling molecules to directly affect scleral growth [START_REF] Wallman | Homeostasis of eye growth and the question of myopia[END_REF].

Our findings using cCSNB models suggest that due to an altered ON-pathway the expression of several genes becomes insufficient, which may result in myopia. We hypothesize, that exposure to insufficient light levels, as found in children with not enough outdoor activities similarly leads to alterations of the ON pathway and myopia.

Not surprisingly, the cCSNB DEGs included genes that are important for synapse formation and signaling. In addition, MAPK pathways and developmental genes were identified by our study, and to our knowledge, their association with cCSNB is novel. These same genes, however, are well known to be involved in the development of myopia [START_REF] Li | Insight into the molecular genetics of myopia[END_REF], further reinforcing our predictions that genes differentially expressed in cCSNB are indeed good candidates for cCSNB-associated high myopia, as well as serving as candidates for nonsyndromic myopia. Other DEGs identified here were not associated with specific myopiarelated search terms or pathways, but, because of their expression in the retina and described physiological function, may be nevertheless be associated with myopia. Interestingly, a few DEGs have been implicated in circadian rhythms. Circadian clocks are cellular oscillators that generate daily rhythms even in the absence of external timing cues [START_REF] Dunlap | Molecular bases for circadian clocks[END_REF][START_REF] Pittendrigh | Temporal organization: reflections of a Darwinian clock-watcher[END_REF] and have also been associated with eye growth and refractive error development [START_REF] Chakraborty | Circadian rhythms, refractive development, and myopia[END_REF].

Novel candidate genes important for vision

The following DEGs represent genes not previously associated with myopia and may represent novel candidate genes underlying high myopia in cCSNB or other retinal disorders:

Adgra3/Gpr125, Afap1l1, Ampd3, Arc, Cacna1s, Ccnjl, Cdc25c, Dbhos, Eomes, Etv4, Fbox32, Frem3, Gm20754, Gm40477, Gm49760, Gm5478/Krt6c, Gng4, Insyn2b, Ldb2, Mast2, Mir670hg, Myh7b, Olfm3, Pdzph1, Plekhf1, Prkca, Rgs16, Shroom4, Spred3, St6galnac5, Synj2, Tac2, Tpbg, Trpc1 and Umodl1. More analyses are needed to validate their putative functional roles in vison. For a few of them, highlighted in bold, animal models or more functional analyses are available and may thus represent the best candidates to be analyzed in the future.

Treatment of cCSNB and myopia

Although cCSNB is believed to be a relatively mild non-degenerative stationary retinal disease, this study shows that gene defects causing cCSNB impact the expression of other genes important for the correct development of the retina and eye. Treatment efficacy may also be impacted. Indeed, at least in mice, cCSNB treatment was more successful in immature retina, and only under scotopic conditions [START_REF] Hasan | Presynaptic Expression of LRIT3 Transsynaptically Organizes the Postsynaptic Glutamate Signaling Complex Containing TRPM1[END_REF][START_REF] Scalabrino | Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness[END_REF]Varin et al., 2021a;Varin et al., 2021b), suggesting that the development of the retinal structure, e.g., synapse formation, is irreversibly impaired in cCSNB. These subtle changes of the retina may only be visible with sensitive methods such as immunohistochemistry or electron microscopy. Such developmental defects may be regulated by the genes differentially expressed in cCSNB identified in this study. Replacing the missing gene implicated in cCSNB, such as GRM6 or LRIT3, by an AAV-approach may restore the correct localization of the corresponding protein and its function, but it is not clear if this will also restore the normal expression of the cCSNB-associated DEGs. Defects in developmental processes, such as synapse formation, may lead to the observed failure to rescue photopic vision in adult mice.

Furthermore, whether an AAV-based therapeutic approach can cure myopia remains unclear.

Although refractive errors associated with myopia can be corrected using optical correction, myopic subjects may still develop a number of other vision-threatening complications such as myopic macular degeneration, retinal tears, retinal detachment and glaucoma [START_REF] Hornbeak | Myopia genetics: a review of current research and emerging trends[END_REF]. Therefore, identifying therapeutic approaches to prevent the development of myopia would be beneficial. Pharmacological treatments may become important means to prevent and/or slow the progression of myopia, but they are unlikely to be able to revert or cure established myopia.

Pharmacological myopia treatment

A signaling cascade, which begins in the retina, via photoreceptors, BCs and dopaminergic, GABAergic, and glycinergic ACs, and propagate across all ocular tissues to the sclera is likely to contribute to human myopia development and progression (reviewed by (Tkatchenko and Tkatchenko, 2019)). This signaling cascade is triggered by optical blur, which plays a critical role in this process. Here, we suggest that cCSNB is an additional valuable model to study myopia, because the disruption of inner retinal signaling in cCSNB has a significant impact on refractive eye development, potentially similar to a low light environment. As discussed above, the neurotransmitter DA and its metabolite DOPAC have been proposed to regulate refractive eye development and their retinal concentrations are reduced in myopia models [START_REF] Chakraborty | ON pathway mutations increase susceptibility to form-deprivation myopia[END_REF][START_REF] Pardue | High susceptibility to experimental myopia in a mouse model with a retinal on pathway defect[END_REF][START_REF] Stone | Retinal dopamine and formdeprivation myopia[END_REF][START_REF] Zhou | Dopamine signaling and myopia development: What are the key challenges[END_REF].

Here, we have shown that DA and DOPAC levels are also reduced in the retina of cCSNB mice. Intravitreal injections of DA in rabbits [START_REF] Gao | Effects of direct intravitreal dopamine injections on the development of lid-suture induced myopia in rabbits[END_REF] and intraperitoneal injections of DA in guinea pigs [START_REF] Mao | Levodopa inhibits the development of form-deprivation myopia in guinea pigs[END_REF] and mice [START_REF] Landis | Increased endogenous dopamine prevents myopia in mice[END_REF] inhibit FDM myopia induction. Enhancing DA signaling by daily intraperitoneal injections of non-selective DA receptor agonists prevented FDM in chicken, guinea pig, monkey and mice (reviewed by [START_REF] Zhou | Dopamine signaling and myopia development: What are the key challenges[END_REF]. More studies are needed, however, to define the cellular mechanisms of DA action, a requirement for the development of an effective myopia treatment. Bright light has also been shown to protect against the development of myopia and to increase DA release in a number of studies with animal models and in children (reviewed by [START_REF] Zhou | Dopamine signaling and myopia development: What are the key challenges[END_REF].

It has been suggested that retinal DA may induce choroidal thickening and inhibit ocular growth by triggering the release of other transmitters such as nitric oxide (NO) from the retina or choroid (reviewed by [START_REF] Zhou | Dopamine signaling and myopia development: What are the key challenges[END_REF]. Clinical trials testing the use of dopaminergic agents to treat amblyopia, myopia and attention deficit disorders revealed negative side effects of systemic activation of DA signaling (reviewed by [START_REF] Zhou | Dopamine signaling and myopia development: What are the key challenges[END_REF]). In contrast, local delivery of DA by APO eye drops given to infant rhesus monkeys reduced development of FDM in the absence of systemic toxicity and other negative side effects [START_REF] Iuvone | Effects of apomorphine, a dopamine receptor agonist, on ocular refraction and axial elongation in a primate model of myopia[END_REF]. Despite these promising findings, we are not aware of further studies in non-human primates or clinical trials using dopaminergic compounds to inhibit myopia progression.

A few other pharmacological agents targeting neurotransmitter receptors have been shown to suppress the development of experimental myopia in animals, such as the muscarinic acetylcholine receptor (mAChR) antagonists pirenzepine and atropine, which are expected to act on muscarinic receptors, expressed in the sclera and in the inner retina. Further longterm clinical studies with large samples sizes are needed to validate the efficacy of this potential anti-myopia therapy (reviewed (Tkatchenko and Tkatchenko, 2019;[START_REF] Wang | Pharmacotherapeutic candidates for myopia: A review[END_REF]). Today, however, atropine, a nonselective muscarinic receptor antagonist, is the most widely used anti-myopia drug, although the exact mechanism of atropine's inhibitory effect on myopia is not well understood. Several clinical trials using atropine eye drops in combination with optical devices such as multifocal lenses that impose a peripheral positive defocus, showed positive outcomes, albeit with low efficacy (reviewed (Tkatchenko and Tkatchenko, 2019)). It would be interesting to investigate if atropine, potentially together with optical devices would also inhibit myopia development in CSNB patients and animal models.

The recent identification of signaling pathways, identified by us here and others (Tkatchenko and Tkatchenko, 2019)), that control the response of the eye to optical defocus and susceptibility to myopia may be helpful for the development of new myopia therapies.

Interestingly, many of those pathways play important roles in multiple retinal cell types and in multiple ocular tissues. For example, the MAPK pathway and GABA receptor signaling influence myopia development via multiple retinal cell types as well as the RPE/choroid (reviewed (Tkatchenko and Tkatchenko, 2019)). Our study found that genes implicated in the MAPK pathway are differentially regulated in cCSNB, suggesting they may contribute to myopia in these subjects. Inhibitors of MAPK phosphorylation have been shown to inhibit myopia [START_REF] She | Modulation of the ERK1/2-MMP-2 pathway in the sclera of guinea pigs following induction of myopia by flickering light[END_REF]), but we are not aware of clinical trials testing this target.

Inhibition of GABA activity, such as with GABA receptor antagonists, may prevent excessive myopia (reviewed [START_REF] Wang | Pharmacotherapeutic candidates for myopia: A review[END_REF]). Schmid et al. suggested that the GABAergic pathway may interact with the dopaminergic pathway and that the balance between excitatory and inhibitory signaling is important for normal eye growth [START_REF] Schmid | The effects and interactions of GABAergic and dopaminergic agents in the prevention of form deprivation myopia by brief periods of normal vision[END_REF]. This is in line with our findings. Interestingly, we found that Gabrr1, coding for the rho-1 GABA receptor subunit, and expressed in rod BC synaptic terminals [START_REF] Mccall | Elimination of the rho1 subunit abolishes GABA(C) receptor expression and alters visual processing in the mouse retina[END_REF] is significantly downregulated in mice lacking Gpr179 and Lrit3. It is possible that this decrease in GABA receptors is a consequence of an increased release of GABA from amacrine cells, leading in turn to myopia in cCSNB. Moreover, abnormal visual processing was observed in mice lacking Gabrr1 [START_REF] Mccall | Elimination of the rho1 subunit abolishes GABA(C) receptor expression and alters visual processing in the mouse retina[END_REF] and several studies linked the human orthologue with myopia (Supplementary Table S6 and Figure 9) (Fan et al., 2016;[START_REF] Lin | Genome-Wide Association of Genetic Variants With Refraction, Axial Length, and Corneal Curvature: A Longitudinal Study of Chinese Schoolchildren[END_REF]. We are not aware of clinical trials testing GABA receptor antagonists for myopia prevention, most likely because such drugs would be expected to have dangerous side effects.

Further understanding of additional signaling pathways and ocular tissues implicated in myopia and their interactions with various environmental factors will lead to the development of anti-myopia therapies (Tkatchenko and Tkatchenko, 2019)). Considering the complexities of the signaling cascades underlying myopia development, an ideal anti-myopia drug (or a drug combination) would target multiple pathways. Although cCSNB appears to be a good myopia model, refractive errors are only measured following experimental myopia inductions.

Alternatively, treatment efficacy could be evaluated by measuring the restoration of neurotransmitter and other metabolite levels or by experimentally assessing the normalization of gene expression within the gene regulatory networks involved in myopia development.

Together, we and others showed that myopia is a complex disorder influenced by many genetic and environmental factors. Future studies are needed to develop therapies targeting these signaling cascades and determine the efficacy of potential treatment approaches [START_REF] Brennan | Efficacy in myopia control[END_REF]. Myopia can be induced in mice using in-house-made goggles with negative spectacle lenses and induced refractive error can be measured using an automated photorefractor [START_REF] Schaeffel | Measurement of refractive state and deprivation myopia in two strains of mice[END_REF]. Wild-type mice (n=12) were goggled at P21 for three weeks. The goggles were fitted with -25.00 D lenses for the right eyes (RE) and no lens for the left eyes (LE), which

were used as controls. The interocular differences between treated and control eyes was The largest cluster consists of genes less expressed in the cCSNB models (cluster 1). BC1-4 = OFF-BCs, BC5-BC7 = ON-BCs (generated using the data from [START_REF] Shekhar | Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics[END_REF][START_REF] Shekhar | Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics[END_REF] .

(

  Cxcr7, Car8, Casp7, Gabrr1, Ptprr, Spred3, Tac2 and Tpbg), rod-cone (Lect1, Gng4, Tgfb2 and Trpc1), BC (Cxcr7, Bdnf, Cacna1s, Car8, Casp7, Ccnjl, Cdc25c, Lect1, Col5a1, Dusp4, Gabrr1, Gng4, Grm6, Ldb2, Lrp2bp, Plekhf1, Prkca, Ptprr, Rgs16, Slco5a1, Spred3, St6galnac5, Tac2, Tpbg, Tgfb2), AC (Arc, Cxcr7, Car8, Lect1, Fbxo32, Gabrr1, Gng4, Ldb2, Olfm3, Plekhf1, Ptprr, Tac2, Tgfb2, Tpbg and Vgll4) and RGC (Cxcr7, Car8, Lect1, Eomes, Gabrr1, Gng4, Plekhf1, Ptprr, Tgfb2, Trpc1, Tpbg and Vgll4) databases (Figure 8).

Figure 1 :

 1 Figures legends:

Figure 2 :

 2 Figure 2: Example of scotopic electroretinograms in mice lacking Aplp2, a gene associated

Figure 3 :

 3 Figure 3: Lens-induced myopia in wild-type mice measured after three weeks of goggling.

  measured at P21 before goggle application and after goggle application at P42. Statistical significance was obtained with p-values, where p ≤ 0.05=*, p≤0.01 = ** and p. ≤0.001 = ***.

Figure 4 :

 4 Figure 4: Reduced retinal DOPAC and DA in light adapted cCSNB mouse models lacking

Figure 5 :

 5 Figure 5: A, Venn diagram of differentially expressed genes (DEGs) in the retina of three

Figure 6 :

 6 Figure 6: Examples of genes found to be differentially expressed in three (red) and two

Figure 7 :

 7 Figure 7: Interdependence of expression between genes implicated in cCSNB at the

Figure 8 :

 8 Figure 8: Schematic drawing (created with BioRender.com) of differentially expressed genes

Figure 9 :

 9 Figure 9: Schematic representation of the DEG expression in the different types of BCs.

Figure 10 :

 10 Figure 10: A, DEGs were enriched in different pathways, including the MAPK signaling

Figure 11 :

 11 Figure 11: Schematic drawing of DEGs (shown in brown) enriched in the MAPK signaling

Figure 12 :

 12 Figure12: Quantification of western blot analyses for proteins for which RNA-Seq studies showed differential expression by RNA-Seq and RT-qPCR studies in Gpr179 -/-, Lrit3 -/-and Grm6 -/-cCSNB mouse models (n ≥ 4). The protein amounts were normalized against

Figure 13 :

 13 Figure13: Immunostaining in Gpr179 -/-, Lrit3 -/-and Grm6 -/-cCSNB mouse models showed a

Figure 14 :

 14 Figure 14: Quantification of all cell nuclei stained with DAPI revealed an equal number of

Figure 15 :

 15 Figure15: Quantification of all TH-positive cells revealed no significant differences between Gpr179 +/+ and Gpr179 -/-(n=4 mice).

  Figure 1

Acknowledgements

We are thankful to Manuel Simonutti, Julie Degardin, Quenol Cesar and Ruben Goulet for their support on animal phenotyping (Institut de la Vision platform) and to the platform of