Fig 1.
Experimental design and effect of positive and negative lenses on refractive eye development in marmosets.
(A) Two groups of marmosets were treated with +5D lenses (red) for 10 days or 5 weeks; +5D lenses were applied to the right eyes, while contralateral left eyes served as controls and were fitted with plano lenses. (B) Positive lenses shift focal point in front of the retina and produce myopic defocus, which inhibits eye growth (dashed line). (C) Two groups of marmosets were treated with −5D lenses (green) for 10 days or 5 weeks; −5D lenses were applied to the right eyes, while contralateral left eyes served as controls and were fitted with plano lenses. (D) Negative lenses shift the focal point behind the retina and produce hyperopic defocus, which stimulates eye growth (dashed line).
Table 1.
Summary of biometric data for animals in the four experimental groups.
Fig 2.
Hyperopic optical defocus induces large-scale changes in gene expression and signaling pathways in the retina.
RNA-seq analysis of differential gene expression in two groups of marmosets exposed to −5D-lens-imposed hyperopic defocus for 10 days or 5 weeks. (A and E) Volcano plots showing differentially expressed genes. (B and F) Hierarchical clustering results showing that differentially expressed genes are organized into two clusters, i.e., genes that are down-regulated and genes that are up-regulated in the retina exposed to defocus. Right, −5D-lens-treated eye. Left, plano-lens-treated control eye. Letters and numbers above each column of the hierarchical clustering results indicate animal IDs. (C and G) Gene ontology analysis results showing biological processes affected by the differentially expressed genes. Vertical yellow line indicates P = 0.05. (D and H) Canonical signaling pathways affected by the differentially expressed genes. Vertical yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways. See S1, S2, S5, S6, S9, S10, S13, and S14 Tables for details. AKT, Serine and Threonine kinase AKT; BRCA1, breast cancer 1 early onset; cAMP, cyclic adenosine monophosphate; DARPP32, dopamine- and cAMP-regulated phosphoprotein 32 kDa; HIPPO, protein kinase Hippo; nNOS, neuronal nitric oxide synthase; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; RAN, Ras-related nuclear protein; RNA-seq, massive parallel RNA sequencing; TNFR1, tumor necrosis factor receptor 1; tRNA, transfer RNA; Wnt, Wingless-integrated.
Fig 3.
Myopic optical defocus induces large-scale changes in gene expression and signaling pathways in the retina.
RNA-seq analysis of differential gene expression in two groups of marmosets exposed to +5D-lens-imposed myopic defocus for 10 days or 5 weeks. (A and E) Volcano plots showing differentially expressed genes. (B and F) Hierarchical clustering results showing that differentially expressed genes are organized into two clusters, i.e., genes that are down-regulated and genes that are up-regulated in the retina exposed to defocus. Right, +5D-lens-treated eye. Left, plano-lens-treated control eye. Letters and numbers above each column of the hierarchical clustering results indicate animal IDs. (C and G) Gene ontology analysis results showing biological processes affected by the differentially expressed genes. Vertical yellow line indicates P = 0.05. (D and H) Canonical signaling pathways affected by the differentially expressed genes. Vertical yellow line indicates P = 0.05. Z-score shows activation or suppression of the corresponding pathways. See S3, S4, S7, S8, S11, S12, S15, and S16 Tables for details. CNTF, ciliary neurotrophic factor; CREB, cAMP responsive element binding protein; EIF2, eukaryotic translation initiation factor 2; eIF4, eukaryotic initiation factor 4; ERK, extracellular signal-regulated kinase; iNOS, inducible nitric oxide synthase; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NAD, nicotinamide adenine dinucleotide; NGF, nerve growth factor; p70S6K, ribosomal protein S6 kinase; RANK, receptor activator of nuclear factor kappa-B; RNA-seq, massive parallel RNA sequencing; SAPK, stress-activated protein kinase; Stat, signal transducer and activator of transcription protein.
Fig 4.
Optical defocus affects different genes and signaling pathways in the retina after 10 days and 5 weeks of exposure.
Comparison of differential genes and pathways affected by imposed hyperopic or myopic defocus after 10 days and 5 weeks of exposure. (A and C) Venn diagrams showing very little overlap between genes differentially expressed after 10 days or 5 weeks of exposure to defocus. (B and D) Graphs showing expression of genes differentially expressed both after 10 days and after 5 weeks of exposure to defocus. Error bars, SD. R, +5D- or −5D-lens-treated eye. L, plano-lens-treated control eye. (E and F) Comparison of biological processes affected by either positive or negative defocus after 10 days or 5 weeks of exposure. Vertical yellow line indicates P = 0.05. (G and H) Comparison of canonical signaling pathways affected by either positive or negative defocus after 10 days or 5 weeks of exposure. Vertical yellow line indicates P = 0.05. Colors indicate experimental marmoset groups and correspond to the colors in Venn diagrams. See S9–S18 Tables for details. AKT, Serine and Threonine kinase AKT; BRCA1, breast cancer 1 early onset; cAMP, cyclic adenosine monophosphate; CNTF, ciliary neurotrophic factor; CREB, cAMP responsive element binding protein; DARPP32, dopamine- and cAMP-regulated phosphoprotein 32 kDa; EIF2, eukaryotic translation initiation factor 2; eIF4, eukaryotic initiation factor 4; ERK, extracellular signal-regulated kinase; HIPPO, protein kinase Hippo; iNOS, inducible nitric oxide synthase; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; L, left; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NAD, nicotinamide adenine dinucleotide; NGF, nerve growth factor; nNOS, neuronal nitric oxide synthase; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; p70S6K, ribosomal protein S6 kinase; R, right; RAN, Ras-related nuclear protein; RANK, receptor activator of nuclear factor kappa-B; SAPK, stress-activated protein kinase; Stat, signal transducer and activator of transcription protein; TNFR1, tumor necrosis factor receptor 1; tRNA, transfer RNA; Wnt, Wingless-integrated.
Fig 5.
Myopic and hyperopic defocus affect different genes and signaling pathways in the retina.
Comparison of differential genes and signaling pathways affected by either +5D-lens-imposed myopic defocus or −5D-lens-imposed hyperopic defocus. (A and C) Venn diagrams showing very little overlap between genes differentially expressed after exposure to −5D or +5D lenses. (B and D) Graphs showing expression of genes differentially expressed both after exposure to −5D and +5D lenses. Error bars, SD. R, −5D- or +5D-lens-treated eye. L, plano-lens-treated control eye. (E and F) Comparison of biological processes affected by either −5D or +5D lenses. Vertical yellow line indicates P = 0.05. (G and H) Comparison of canonical signaling pathways affected by either −5D or +5D lenses. Vertical yellow line indicates P = 0.05. Colors indicate experimental marmoset groups and correspond to the colors in Venn diagrams. See S9–S16, S19, and S20 Tables for details. AKT, Serine and Threonine kinase AKT; BRCA1, breast cancer 1 early onset; cAMP, cyclic adenosine monophosphate; CNTF, ciliary neurotrophic factor; CREB, cAMP responsive element binding protein; DARPP32, dopamine- and cAMP-regulated phosphoprotein 32 kDa; EIF2, eukaryotic translation initiation factor 2; eIF4, eukaryotic initiation factor 4; ERK, extracellular signal-regulated kinase; HIPPO, protein kinase Hippo; iNOS, inducible nitric oxide synthase; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; L, left; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NAD, nicotinamide adenine dinucleotide; NGF, nerve growth factor; nNOS, neuronal nitric oxide synthase; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; p70S6K, ribosomal protein S6 kinase; R, right; RAN, Ras-related nuclear protein; RANK, receptor activator of nuclear factor kappa-B; SAPK, stress-activated protein kinase; Stat, signal transducer and activator of transcription protein; TNFR1, tumor necrosis factor receptor 1; tRNA, transfer RNA; Wnt, Wingless-integrated.
Fig 6.
Genes differentially expressed in the retina of marmosets exposed to optical defocus are localized within human QTLs associated with myopia.
(A) Venn diagram showing overlap between genes localized within human myopia QTLs and genes differentially expressed in the retina of marmosets exposed to hyperopic defocus imposed by −5D lenses. (B) Venn diagram showing overlap between genes localized within human myopia QTLs and genes differentially expressed in the retina of marmosets exposed to myopic defocus imposed by +5D lenses. (C) Heatmap depicting genes, odds ratios, and corresponding P values for the overlaps between specific QTLs and genes differentially expressed in the retina of marmosets exposed to optical defocus. Colors indicate odds ratios and numbers within each cell indicate P values. See S21–S24 Tables for details. no, no overlap; OR, odds ratio; QTL, quantitative trait locus.
Fig 7.
Summary of the key genes and pathways underlying BESOD.
Our findings support the view that emmetropization is regulated by a bidirectional growth control mechanism, which involves active stimulation of eye growth by hyperopic defocus and active suppression of eye growth by myopic defocus (first column). We find that these two signals propagate in the retina via largely distinct (possibly even independent) pathways. Early response to optical defocus involves different sets of genes, with no genes exhibiting sign-of-defocus–sensitive expression. Although the sustained response also involves largely different sets of genes, several genes (listed in the second column) exhibit sign-of-defocus–sensitive expression. We speculate that they may act as switches that trigger largely distinct sign-of-defocus–specific signaling cascades underlying response to the defocus of opposite signs. We identified a total of 819 genes differentially expressed in the retina exposed to myopic defocus and 428 genes differentially expressed in the retina exposed to hyperopic defocus. The top five up- and down-regulated genes in each condition are shown in the third column. Some of over 100 canonical pathways involved in the regulation of the retinal response to defocus of opposite signs are listed in the fourth column. Three pathways have been previously targeted pharmacologically for myopia control (fifth column): (1) dopamine signaling was suggested to be a target for apomorphine, reserpine, and 6-OHDA [52]; (2) nitric oxide signaling was implicated in the anti-myopic atropine effect [53]; and (3) α-adrenergic signaling was suggested to be a target of atropine, oxyphenonium, and pirenzepine [54]. BESOD, Bidirectional Emmetropization by the Sign of Optical Defocus; cAMP, cyclic adenosine monophosphate; CREB, cAMP responsive element binding protein; CNTF, ciliary neurotrophic factor; DARPP32, dopamine- and cAMP-regulated phosphoprotein 32 kDa; EIF2, eukaryotic translation initiation factor 2; eIF4, eukaryotic initiation factor 4; HIPPO, protein kinase Hippo; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; mTOR, mammalian target of rapamycin; NAD, nicotinamide adenine dinucleotide; NGF, nerve growth factor; nNOS, neuronal nitric oxide synthase; PTEN, phosphatase and tensin homolog; p70S6K, ribosomal protein S6 kinase; RAN, Ras-related nuclear protein; RANK, receptor activator of nuclear factor kappa-B; SAPK, stress-activated protein kinase; Stat, signal transducer and activator of transcription protein; TNFR1, tumor necrosis factor receptor 1; Wnt, Wingless-integrated; 6-OHDA, 6-hydroxydopamine.