Genetic network regulating visual acuity makes limited contribution to visually guided eye emmetropization.

During postnatal development, the eye undergoes a refinement process whereby optical defocus guides eye growth towards sharp vision in a process of emmetropization. Optical defocus activates a signaling cascade originating in the retina and propagating across the back of the eye to the sclera. Several observations suggest that visual acuity might be important for optical defocus detection and processing in the retina; however, direct experimental evidence supporting or refuting the role of visual acuity in refractive eye development is lacking. Here, we used genome-wide transcriptomics to determine the relative contribution of the retinal genetic network regulating visual acuity to the signaling cascade underlying visually guided eye emmetropization. Our results provide evidence that visual acuity is regulated at the level of molecular signaling in the retina by an extensive genetic network. The genetic network regulating visual acuity makes relatively small contribution to the signaling cascade underlying refractive eye development. This genetic network primarily affects baseline refractive eye development and this influence is primarily facilitated by the biological processes related to melatonin signaling, nitric oxide signaling, phototransduction, synaptic transmission, and dopamine signaling. We also observed that the visual-acuity-related genes associated with the development of human myopia are chiefly involved in light perception and phototransduction. Our results suggest that the visual-acuity-related genetic network primarily contributes to the signaling underlying baseline refractive eye development, whereas its impact on visually guided eye emmetropization is modest.

Functional integration of eye tissues and refractive eye development: Mechanisms and pathways.

Refractive eye development is a tightly coordinated developmental process. The general layout of the eye and its various components are established during embryonic development, which involves a complex cross-tissue signaling. The eye then undergoes a refinement process during the postnatal emmetropization process, which relies heavily on the integration of environmental and genetic factors and is controlled by an elaborate genetic network. This genetic network encodes a multilayered signaling cascade, which converts visual stimuli into molecular signals that guide the postnatal growth of the eye. The signaling cascade underlying refractive eye development spans across all ocular tissues and comprises multiple signaling pathways. Notably, tissue-tissue interaction plays a key role in both embryonic eye development and postnatal eye emmetropization. Recent advances in eye biometry, physiological optics and systems genetics of refractive error have significantly advanced our understanding of the biological processes involved in refractive eye development and provided a framework for the development of new treatment options for myopia. In this review, we summarize the recent data on the mechanisms and signaling pathways underlying refractive eye development and discuss new evidence suggesting a wide-spread signal integration across different tissues and ocular components involved in visually guided eye growth.

Gene expression in response to optical defocus of opposite signs reveals bidirectional mechanism of visually guided eye growth.

Myopia (nearsightedness) is the most common eye disorder, which is rapidly becoming one of the leading causes of vision loss in several parts of the world because of a recent sharp increase in prevalence. Nearwork, which produces hyperopic optical defocus on the retina, has been implicated as one of the environmental risk factors causing myopia in humans. Experimental studies have shown that hyperopic defocus imposed by negative power lenses placed in front of the eye accelerates eye growth and causes myopia, whereas myopic defocus imposed by positive lenses slows eye growth and produces a compensatory hyperopic shift in refractive state. The balance between these two optical signals is thought to regulate refractive eye development; however, the ability of the retina to recognize the sign of optical defocus and the composition of molecular signaling pathways guiding emmetropization are the subjects of intense investigation and debate. We found that the retina can readily distinguish between imposed myopic and hyperopic defocus, and identified key signaling pathways underlying retinal response to the defocus of different signs. Comparison of retinal transcriptomes in common marmosets exposed to either myopic or hyperopic defocus for 10 days or 5 weeks revealed that the primate retina responds to defocus of different signs by activation or suppression of largely distinct pathways. We also found that 29 genes differentially expressed in the marmoset retina in response to imposed defocus are localized within human myopia quantitative trait loci (QTLs), suggesting functional overlap between genes differentially expressed in the marmoset retina upon exposure to optical defocus and genes causing myopia in humans. These findings identify retinal pathways involved in the development of myopia, as well as potential new strategies for its treatment.

APLP2 Regulates Refractive Error and Myopia Development in Mice and Humans.

Myopia is the most common vision disorder and the leading cause of visual impairment worldwide. However, gene variants identified to date explain less than 10% of the variance in refractive error, leaving the majority of heritability unexplained ("missing heritability"). Previously, we reported that expression of APLP2 was strongly associated with myopia in a primate model. Here, we found that low-frequency variants near the 5'-end of APLP2 were associated with refractive error in a prospective UK birth cohort (n = 3,819 children; top SNP rs188663068, p = 5.0 × 10-4) and a CREAM consortium panel (n = 45,756 adults; top SNP rs7127037, p = 6.6 × 10-3). These variants showed evidence of differential effect on childhood longitudinal refractive error trajectories depending on time spent reading (gene x time spent reading x age interaction, p = 4.0 × 10-3). Furthermore, Aplp2 knockout mice developed high degrees of hyperopia (+11.5 ± 2.2 D, p < 1.0 × 10-4) compared to both heterozygous (-0.8 ± 2.0 D, p < 1.0 × 10-4) and wild-type (+0.3 ± 2.2 D, p < 1.0 × 10-4) littermates and exhibited a dose-dependent reduction in susceptibility to environmentally induced myopia (F(2, 33) = 191.0, p < 1.0 × 10-4). This phenotype was associated with reduced contrast sensitivity (F(12, 120) = 3.6, p = 1.5 × 10-4) and changes in the electrophysiological properties of retinal amacrine cells, which expressed Aplp2. This work identifies APLP2 as one of the "missing" myopia genes, demonstrating the importance of a low-frequency gene variant in the development of human myopia. It also demonstrates an important role for APLP2 in refractive development in mice and humans, suggesting a high level of evolutionary conservation of the signaling pathways underlying refractive eye development.

A Review of Current Concepts of the Etiology and Treatment of Myopia.

Myopia occurs in more than 50% of the population in many industrialized countries and is expected to increase; complications associated with axial elongation from myopia are the sixth leading cause of blindness. Thus, understanding its etiology, epidemiology, and the results of various treatment regiments may modify current care and result in a reduction in morbidity from progressive myopia. This rapid increase cannot be explained by genetics alone. Current animal and human research demonstrates that myopia development is a result of the interplay between genetic and the environmental factors. The prevalence of myopia is higher in individuals whose both parents are myopic, suggesting that genetic factors are clearly involved in myopia development. At the same time, population studies suggest that development of myopia is associated with education and the amount time spent doing near work; hence, activities increase the exposure to optical blur. Recently, there has been an increase in efforts to slow the progression of myopia because of its relationship to the development of serious pathological conditions such as macular degeneration, retinal detachments, glaucoma, and cataracts. We reviewed meta-analysis and other of current treatments that include: atropine, progressive addition spectacle lenses, orthokeratology, and multifocal contact lenses.

Photopic visual input is necessary for emmetropization in mice.

It was recently demonstrated that refractive errors in mice stabilize around emmetropic values during early postnatal development, and that they develop experimental myopia in response to both visual form deprivation and imposed optical defocus similar to other vertebrate species. Animal studies also suggest that photopic vision plays critical role in emmetropization in diurnal species; however, it is unknown whether refractive eye development is guided by photopic vision in the mouse, which is a nocturnal species. We used an infrared mouse photorefractor and a high-resolution MRI to clarify the role of photopic visual input in refractive eye development in the mouse. Refractive eye development and form-deprivation myopia in P21-P89 C57BL/6J mice were analyzed under 12:12 h light-dark cycle, constant light and constant darkness regimens. Animals in all experimental groups were myopic at P21 (-13.2 ± 1.6 D, light-dark cycle; -12.5 ± 0.9 D, constant light; -12.5 ± 2.0 D, constant dark). The mean refractive error in the light-dark-cycle-reared animals was -0.5 ± 1.3 D at P32 and, and did not change significantly until P40 (+0.3 ± 0.6 D, P40). Animals in this group became progressively hyperopic between P40 and P89 (+2.2 ± 0.6 D, P67; +3.7 ± 2.0 D, P89). The mean refractive error in the constant-light-reared mice was -1.0 ± 0.7 D at P32 and remained stable until P89 (+0.1 ± 0.6 D, P40; +0.3 ± 0.6 D, P67; 0.0 ± 0.4 D, P89). Dark-reared animals exhibited highly hyperopic refractive errors at P32 (+5.2 ± 1.8 D) and became progressively more hyperopic with age (+8.7 ± 1.9 D, P40; +11.2 ± 1.4 D, P67). MRI analysis revealed that emmetropization in the P40-P89 constant-light-reared animals was associated with larger eyes, a longer axial length and a larger vitreous chamber compared to the light-dark-cycle-reared mice. Constant-light-reared mice also developed 4 times higher degrees of form-deprivation myopia on average compared to light-dark-cycle-reared animals (-12.0 ± 1.4 D, constant light; -2.7 ± 0.7 D, light-dark cycle). Dark-rearing completely prevented the development of form-deprivation myopia (-0.3 ± 0.5 D). Thus, photopic vision plays important role in normal refractive eye development and ocular response to visual form deprivation in the mouse.

Shedding light on myopia by studying complete congenital stationary night blindness.

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.

Genome-wide analysis of retinal transcriptome reveals common genetic network underlying perception of contrast and optical defocus detection.

BACKGROUND: Refractive eye development is regulated by optical defocus in a process of emmetropization. Excessive exposure to negative optical defocus often leads to the development of myopia. However, it is still largely unknown how optical defocus is detected by the retina. METHODS: Here, we used genome-wide RNA-sequencing to conduct analysis of the retinal gene expression network underlying contrast perception and refractive eye development. RESULTS: We report that the genetic network subserving contrast perception plays an important role in optical defocus detection and emmetropization. Our results demonstrate an interaction between contrast perception, the retinal circadian clock pathway and the signaling pathway underlying optical defocus detection. We also observe that the relative majority of genes causing human myopia are involved in the processing of optical defocus. CONCLUSIONS: Together, our results support the hypothesis that optical defocus is perceived by the retina using contrast as a proxy and provide new insights into molecular signaling underlying refractive eye development.

Lack of periostin leads to suppression of Notch1 signaling and calcific aortic valve disease.

The Postn gene encodes protein periostin. During embryonic development, it is highly expressed in the outflow tract (OFT) endocardial cushions of the developing heart, which give rise to several structures of the mature heart including the aortic valve. Periostin was previously implicated in osteoblast differentiation, cancer metastasis, and tooth and bone development, but its role in cardiac OFT development is unclear. To elucidate the role that periostin plays in the developing heart we analyzed cardiac OFT phenotype in mice after deletion of the Postn gene. We found that lack of periostin in the embryonic OFT leads to ectopic expression of the proosteogenic growth factor pleiotrophin (Ptn) and overexpression of delta-like 1 homolog (Dlk1), a negative regulator of Notch1, in the distal (prevalvular) cushions of the OFT. This resulted in suppression of Notch1 signaling, strong induction of the central transcriptional regulator of osteoblast cell fate Runx2, upregulation of osteopontin and osteocalcin expression, and subsequent calcification of the aortic valve. Our data suggest that periostin represses a default osteogenic program in the OFT cushion mesenchyme and promotes differentiation along a fibrogenic lineage. Lack of periostin causes derepression of the osteogenic potential of OFT mesenchymal cells, calcium deposition, and calcific aortic valve disease. These results establish periostin as a key regulator of OFT endocardial cushion mesenchymal cell fate during embryonic development.

Pharmacogenomic Approach to Antimyopia Drug Development: Pathways Lead the Way.

Myopia is the most common eye disorder in the world which is caused by a mismatch between the optical power of the eye and its excessively long axial length. Recent studies revealed that the regulation of the axial length of the eye occurs via a complex signaling cascade, which originates in the retina and propagates across all ocular tissues to the sclera. The complexity of this regulatory cascade has made it particularly difficult to develop effective antimyopia drugs. The current pharmacological treatment options for myopia are limited to atropine and 7-methylxanthine, which have either significant adverse effects or low efficacy. In this review, we focus on the recent advances in genome-wide studies of the signaling pathways underlying myopia development and discuss the potential of systems genetics and pharmacogenomic approaches for the development of antimyopia drugs.

Analysis of genetic networks regulating refractive eye development in collaborative cross progenitor strain mice reveals new genes and pathways underlying human myopia.

BACKGROUND: Population studies suggest that genetic factors play an important role in refractive error development; however, the precise role of genetic background and the composition of the signaling pathways underlying refractive eye development remain poorly understood. METHODS: Here, we analyzed normal refractive development and susceptibility to form-deprivation myopia in the eight progenitor mouse strains of the Collaborative Cross (CC). We used RNA-seq to analyze gene expression in the retinae of these mice and reconstruct genetic networks and signaling pathways underlying refractive eye development. We also utilized genome-wide gene-based association analysis to identify mouse genes and pathways associated with myopia in humans. RESULTS: Genetic background strongly influenced both baseline refractive development and susceptibility to environmentally-induced myopia. Baseline refractive errors ranged from - 21.2 diopters (D) in 129S1/svlmj mice to + 22.0 D in CAST/EiJ mice and represented a continuous distribution typical of a quantitative genetic trait. The extent of induced form-deprivation myopia ranged from - 5.6 D in NZO/HILtJ mice to - 20.0 D in CAST/EiJ mice and also followed a continuous distribution. Whole-genome (RNA-seq) gene expression profiling in retinae from CC progenitor strains identified genes whose expression level correlated with either baseline refractive error or susceptibility to myopia. Expression levels of 2,302 genes correlated with the baseline refractive state of the eye, whereas 1,917 genes correlated with susceptibility to induced myopia. Genome-wide gene-based association analysis in the CREAM and UK Biobank human cohorts revealed that 985 of the above genes were associated with myopia in humans, including 847 genes which were implicated in the development of human myopia for the first time. Although the gene sets controlling baseline refractive development and those regulating susceptibility to myopia overlapped, these two processes appeared to be controlled by largely distinct sets of genes. CONCLUSIONS: Comparison with data for other animal models of myopia revealed that the genes identified in this study comprise a well-defined set of retinal signaling pathways, which are highly conserved across different vertebrate species. These results identify major signaling pathways involved in refractive eye development and provide attractive targets for the development of anti-myopia drugs.

Large-Scale microRNA Expression Profiling Identifies Putative Retinal miRNA-mRNA Signaling Pathways Underlying Form-Deprivation Myopia in Mice.

Development of myopia is associated with large-scale changes in ocular tissue gene expression. Although differential expression of coding genes underlying development of myopia has been a subject of intense investigation, the role of non-coding genes such as microRNAs in the development of myopia is largely unknown. In this study, we explored myopia-associated miRNA expression profiles in the retina and sclera of C57Bl/6J mice with experimentally induced myopia using microarray technology. We found a total of 53 differentially expressed miRNAs in the retina and no differences in miRNA expression in the sclera of C57BL/6J mice after 10 days of visual form deprivation, which induced -6.93 ± 2.44 D (p < 0.000001, n = 12) of myopia. We also identified their putative mRNA targets among mRNAs found to be differentially expressed in myopic retina and potential signaling pathways involved in the development of form-deprivation myopia using miRNA-mRNA interaction network analysis. Analysis of myopia-associated signaling pathways revealed that myopic response to visual form deprivation in the retina is regulated by a small number of highly integrated signaling pathways. Our findings highlighted that changes in microRNA expression are involved in the regulation of refractive eye development and predicted how they may be involved in the development of myopia by regulating retinal gene expression.

Variational analysis of the mouse and rat eye optical parameters.

Rodent models are increasingly used to study refractive eye development and development of refractive errors; however, there is still some uncertainty regarding the accuracy of the optical models of the rat and mouse eye primarily due to high variability in reported ocular parameters. In this work, we have systematically evaluated the contribution of various ocular parameters, such as radii of curvature of ocular surfaces, thicknesses of ocular components, and refractive indices of ocular refractive media, using variational analysis and a computational model of the rodent eye. Variational analysis revealed that not all variation in ocular parameters has critical impact on the refractive status of the eye. Variation in the depth of the vitreous chamber, thickness of the lens, radius of the anterior surface of the cornea, radius of the anterior surface of the lens, as well as refractive indices for the lens and vitreous, appears to have the largest impact on the refractive error. The radii of the posterior surfaces of the cornea and lens have much smaller contributions to the refractive state. These data provide the framework for further refinement of the optical models of the rat and mouse eye and suggest that extra efforts should be directed towards increasing the linear resolution of the rodent eye biometry and obtaining more accurate data for the refractive indices of the lens and vitreous.

Ketamine-xylazine anesthesia causes hyperopic refractive shift in mice.

Mice have increasingly been used as a model for studies of myopia. The key to successful use of mice for myopia research is the ability to obtain accurate measurements of refractive status of their eyes. In order to obtain accurate measurements of refractive errors in mice, the refraction needs to be performed along the optical axis of the eye. This represents a particular challenge, because mice are very difficult to immobilize. Recently, ketamine-xylazine anesthesia has been used to immobilize mice before measuring refractive errors, in combination with tropicamide ophthalmic solution to induce mydriasis. Although these drugs have increasingly been used while refracting mice, their effects on the refractive state of the mouse eye have not yet been investigated. Therefore, we have analyzed the effects of tropicamide eye drops and ketamine-xylazine anesthesia on refraction in P40 C57BL/6J mice. We have also explored two alternative methods to immobilize mice, i.e. the use of a restraining platform and pentobarbital anesthesia. We found that tropicamide caused a very small, but statistically significant, hyperopic shift in refraction. Pentobarbital did not have any substantial effect on refractive status, whereas ketamine-xylazine caused a large and highly significant hyperopic shift in refraction. We also found that the use of a restraining platform represents good alternative for immobilization of mice prior to refraction. Thus, our data suggest that ketamine-xylazine anesthesia should be avoided in studies of refractive development in mice and underscore the importance of providing appropriate experimental conditions when measuring refractive errors in mice.

Analysis of postnatal eye development in the mouse with high-resolution small animal magnetic resonance imaging.

PURPOSE: Studies of myopia in mice have been complicated by the difficulty in obtaining accurate measurements of small changes observed in the growing mouse eye in vivo and the lack of data on refractive eye development. The purpose of this study was to carry out an in vivo high-resolution analysis of mouse eye growth and refractive development. METHODS: High-resolution small animal magnetic resonance imaging and high-resolution infrared photorefraction were used to analyze refractive development in postnatal day (P)21 to P89 C57BL/6J mice. RESULTS: The growth of the mouse eye decelerated after P40. The eye maintained a slightly prolate shape during growth. The anterior chamber growth exhibited a similar pattern, whereas the corneal radius of curvature (CRC) increased linearly. The growth rate of the lens remained constant until P89. The lens "overgrew" the eye at P40, resulting in a decline in vitreous chamber depth. Mice showed myopic refractive errors at a younger age (-13.2 +/- 2.0 D; mean +/- SD, P21). The refractive errors stabilized around emmetropic values by P32 and remained emmetropic until P40. Mice became progressively hyperopic with age (+1.2 +/- 1.7 D, P67; +3.6 +/- 2.3 D, P89). CONCLUSIONS: Development of ocular components in the mouse is similar to that of the tree shrew but different from that of higher primates and humans. Primary differences can be attributed to the age-related changes of the crystalline lens and CRC. In spite of these differences, mice appear to be able to achieve and maintain emmetropic refractive status at P32 to P40.

Mouse experimental myopia has features of primate myopia.

PURPOSE: Several recent studies have suggested that experimental myopia can be induced in mice. However, it is not clear what role the photopic visual input plays in this process and whether mouse myopia is similar to human myopia. The purpose of this study was to carry out an in vivo high-resolution analysis of changes in ocular components and refractive state of the eye upon induction of experimental myopia in mice. METHODS: A high-resolution small animal MRI system and a high-resolution automated eccentric infrared photorefractor were used to analyze changes of the refractive state and ocular components in C57BL/6J mice associated with experimental myopia induced by diffusers and -25 D lenses under photopic conditions. RESULTS: The authors found that both diffusers and -25 D lenses induce myopia in C57BL/6J mice under photopic conditions (continuous light, 200 +/- 15 lux). The extent of myopic shift induced by -25 D lenses was greater than the shift induced by diffusers (-15.2 +/- 0.7 D, lenses; -12.0 +/- 1.4 D, diffusers). Myopia in mice is attributed to an increase in size of the postequatorial segment of the eye. Experimental myopia in mice can be induced only during the susceptible period in postnatal development, which ends around postnatal day 67. CONCLUSIONS: Both diffusers and spectacle lenses induce myopia in mice under photopic conditions, during the susceptible period in postnatal development. Myopia in mice is associated with elongation of the vitreous chamber of the eye, as in humans and nonhuman primates.

Form deprivation modulates retinal neurogenesis in primate experimental myopia.

Juvenile primates develop myopia when their visual experience is degraded by lid fusion. In response to this abnormal visual input, retinal neural networks cause an excessive growth of the postequatorial segment of the eye, but the mechanism underlying this axial elongation is unknown. After fusion of the lids in one eye of juvenile rhesus macaques and green monkeys, we combined cDNA subtractions, microarray profiling, and real-time PCR to compare gene expression in the retinas of the closed and open eyes. This molecular analysis showed up-regulation of a number of genes associated with cell division in the retina of the closed eye and differential expression of six genes localized to chromosomal loci linked to forms of human hereditary myopia. In addition, it substantiated a previous observation, based on immunocytochemistry, that synthesis of vasoactive intestinal polypeptide was increased upon lid fusion. Injection of 5-bromo-2'-deoxyuridine and immunocytochemistry showed that the primate retinal periphery harbors mitotically active neuroprogenitor cells that increase in number when the visual experience is altered by lid fusion. Furthermore, the number of dividing cells is highly correlated with axial elongation of the eye and the resulting myopic refractive error. Thus, the retina undergoes active growth during the postnatal development of the primate eye. This growth is modulated by the visual input and accelerates considerably when the eye develops axial myopia. Vasoactive intestinal polypeptide may be the molecule that stimulates retinal growth.

Krtap16, characterization of a new hair keratin-associated protein (KAP) gene complex on mouse chromosome 16 and evidence for regulation by Hoxc13.

Intermediate filament (IF) keratins and keratin-associated proteins (KAPs) are principal structural components of hair and encoded by members of multiple gene families. The severe hair growth defects observed upon aberrant expression of certain keratin and KAP genes in both mouse and man suggest that proper hair growth requires their spatio-temporally coordinated activation. An essential prerequisite for studying these cis-regulatory mechanisms is to define corresponding gene families, their genomic organization, and expression patterns. This work characterizes eight recently identified high glycine/tyrosine (HGT)-type KAP genes collectively designated Krtap16-n. These genes are shown to be integrated into a larger KAP gene domain on mouse chromosome 16 (MMU16) that is orthologous to a recently described HGT- and high sulfur (HS)-type KAP gene complex on human chromosome 21q22.11. All Krtap16 genes exhibit strong expression in a narrowly defined pattern restricted to the lower and middle cortical region of the hair shaft in both developing and cycling hair. During hair follicle regression (catagen), expression levels decrease until expression is no longer detectable in follicles at resting stage (telogen). Since isolation of the Krtap16 genes was based on their differential expression in transgenic mice overexpressing the Hoxc13 transcriptional regulator in hair, we examined whether bona fide Hoxc13 binding sites associated with these genes might be functionally relevant by performing electrophoretic mobility shift assays (EMSAs). The data provide evidence for sequence-specific interaction between Hoxc13 and Krtap16 genes, thus supporting the concept of a regulatory relationship between Hoxc13 and these KAP genes.