Altered gene expression in tree shrew retina and retinal pigment epithelium produced by short periods of minus-lens wear

Abstract

Hyperopic refractive error is detected by retinal neurons, which generate GO signals through a direct emmetropization signaling cascade: retinal pigment epithelium (RPE) into choroid and then into sclera, thereby increasing axial elongation. To examine signaling early in this cascade, we measured gene expression in the retina and RPE after short exposure to hyperopia produced by minus-lens wear. Gene expression in each tissue was compared with gene expression in combined retina + RPE. Starting 24 days after normal eye opening, three groups of juvenile tree shrews (n = 7 each) wore a monocular -5 D lens. The untreated fellow eye served as a control. The "6h" group wore the lens for 6 h; the "24h" group wore the lens for 24 h; each group provided separate retina and RPE tissues. Group "24hC" wore the lens for 24 h and provided combined retina + RPE tissue. Quantitative PCR was used to measure the relative differences (treated eye vs. control eye) in mRNA levels for 66 candidate genes. In the retina after 6 h, mRNA levels for seven genes were significantly regulated: EGR1 and FOS (early intermediate genes) were down-regulated in the treated eyes. Genes with secreted protein products, BMP2 and CTGF, were down-regulated, whilst FGF10, IL18, and SST were up-regulated. After 24 h the pattern changed; only one of the seven genes still showed differential expression; BMP2 was still down-regulated. Two new genes with secreted protein products, IGF2 and VIP, were up-regulated. In the RPE, consistent with its role in receiving, processing, and transmitting GO signaling, differential expression was found for genes whose protein products are at the cell surface, intracellular, in the nucleus, and are secreted. After 6 h, mRNA levels for 17 genes were down-regulated in the treated eyes, whilst four genes (GJA1, IGF2R, LRP2, and IL18) were up-regulated. After 24 h the pattern was similar; mRNA levels for 14 of the same genes were still down-regulated; only LRP2 remained up-regulated. mRNA levels for six genes no longer showed differential expression, whilst nine genes, not differentially expressed at 6 h, now showed differential expression. In the combined retina + RPE after 24 h, mRNA levels for only seven genes were differentially regulated despite the differential expression of many genes in the RPE. Four genes showed the same expression in combined tissue as in retina alone, including up-regulation of VIP despite significant VIP down-regulation in RPE. Thus, hyperopia-induced GO signaling, as measured by differential gene expression, differs in the retina and the RPE. Retinal gene expression changed between 6 h and 24 h of treatment, suggesting evolution of the retinal response. Gene expression in the RPE was similar at both time points, suggesting sustained signaling. The combined retina + RPE does not accurately represent gene expression in either retina or, especially, RPE. When gene expression signatures were compared with those in choroid and sclera, GO signaling, as encoded by differential gene expression, differs in each compartment of the direct emmetropization signaling cascade.

Keywords: Animal models; Axial elongation; Direct emmetropization signaling cascade; Gene expression; Myopia; Refractive error.

Figure 1

Compartments of the direct emmetropization pathway signaling cascade. Refractive hyperopia causes retinal neurons to generate GO signals that pass into, and are transformed in, each compartment. In the sclera, in mammals, the result is remodeling that increases the axial elongation rate of the eye.

Figure 2

Retina gene expression fold-differences (treated vs. control eyes) in the (A) 6h and (B) 24h groups. Negative values represent down-regulation in the treated eyes. Headings separated by vertical dashed lines indicate cellular location/functional category of the gene’s protein product. Filled bars represent statistically significant differences (p<0.05); bar color is arbitrary and intended to help in comparing the same gene in the different conditions; error bars = SEM.

Figure 3

Comparison of the gene expression differences (treated vs. control eye) in retina after 6 h (Figure 2A) and 24 h (Figure 2B) of minus-lens wear. Values near the dashed line indicate genes that responded similarly at the two time points. Stars = significant fold-differences for both the 6h and 24h groups; triangles = significant fold-differences only for the 6h group; squares = significant fold-differences only for the 24h group; open circles = expression not significantly different at either time point.

Figure 4

RPE gene expression fold-differences (treated vs. control eyes) in the (A) 6h and (B) 24h groups. Negative values represent down-regulation in the treated eyes. Headings separated by vertical dashed lines indicate cellular location/functional category of the gene’s protein product. Filled bars represent statistically significant differences (p<0.05); bar color is arbitrary and intended to help in comparing the same gene in the different conditions; error bars = SEM.

Figure 5

Comparison of the gene expression differences (treated vs. control eye) in RPE after 6 h (Figure 4A) and 24 h (Figure 4B) of minus-lens wear. Values near the dashed line indicate genes that responded similarly at the two time points. Stars = significant fold-differences for both 6h and 24h groups; triangles = significant fold-differences only for 6h; squares = significant fold-differences only for 24h; open circles = expression not significantly different at either time point.

Figure 6

Combined retina+RPE gene expression fold-differences (treated vs. control eyes) in the 24hC group. Negative values represent down-regulation in the treated eyes. Headings separated by vertical dashed lines indicate cellular location/functional category of the gene’s protein product. Filled bars represent statistically significant differences (p<0.05); error bars = SEM.

Figure 7

Comparison of the GO gene expression across compartments of the direct emmetropization signaling cascade. (A) Differential expression in retina and RPE after 6 h of minus-lens wear. (B) Differential expression in RPE after 24 h and choroid after 2 days of minus-lens wear. (C) Differential expression in choroid and sclera after 2 days of minus-lens wear. Values near the dashed line indicate genes that responded similarly in both compartments. Stars = significant fold-differences for both compartments; triangles = significant fold-differences only for the compartment plotted on the x-axis; squares = significant fold-differences only for the compartment plotted on the y-axis; open circles = expression not significantly different in either compartment. Choroid data from He et al. 2014a. Sclera data from Guo et al. 2013.