Smarce1 and Tensin 4 Are Putative Modulators of Corneoscleral Stiffness

Abstract

The biomechanical properties of the cornea and sclera are important in the onset and progression of multiple ocular pathologies and vary substantially between individuals, yet the source of this variation remains unknown. Here we identify genes putatively regulating corneoscleral biomechanical tissue properties by conducting high-fidelity ocular compliance measurements across the BXD recombinant inbred mouse set and performing quantitative trait analysis. We find seven cis-eQTLs and non-synonymous SNPs associating with ocular compliance, and show by RT-qPCR and immunolabeling that only two of the candidate genes, Smarce1 and Tns4, showed significant expression in corneal and scleral tissues. Both have mechanistic potential to influence the development and/or regulation of tissue material properties. This work motivates further study of Smarce1 and Tns4 for their role(s) in ocular pathology involving the corneoscleral envelope as well as the development of novel mouse models of ocular pathophysiology, such as myopia and glaucoma.

Keywords: Smarce1; Tensin 4; corneal stiffness; glaucoma; mechanical properties; myopia; ocular compliance; scleral stiffness.

Figure 1

Volume-normalized ocular compliance from BXD mice. Bars mark the mean and limits of the 95% confidence interval for each strain, with each point representing one eye. Number of eyes is shown for each strain. (A) Compliance calculated using the Volume Filling method (see text), n = 129 eyes across 17 BXD strains. (B) Compliance calculated using the Step Response method (see text), n = 179 eyes across 22 BXD strains. The color scheme from strains included in the Volume Filling data set (A) is preserved in (B), with additional strains shown in black.

Figure 2

Interval map of normalized ocular compliance (${\overline{\varphi }}_{norm}$) across the mouse genome, as determined with the Volume Filling data analysis method. The blue line in each panel represents the likelihood ratio statistic (LRS), a measure of the linkage between differences in the measured trait (${\overline{\varphi }}_{norm}$) and genotype markers. Thresholds for significant (p < 0.05) and “suggestive” (p < 0.63) peaks are indicated with horizontal pink and gray lines (Abiola et al., 2003). Red and green peaks indicate the contributions of the B6 and D2 alleles, respectively. In (A), a suggestive peak is observed on Chr11. In (B), a magnified view of the region of interest is shown, accompanied by a haplotype map. B6 and D2 alleles are denoted by red and green bars, respectively, and are ordered from high to low ${\overline{\varphi }}_{norm}$ values. Unmapped regions are shown in gray. Genomic markers used for interval mapping are labeled on the haplotype map and indicated with black vertical lines. Yellow vertical lines along the bottom of subplot (B) mark SNP locations.

Figure 3

Interval map of normalized ocular compliance (${\overline{\varphi }}_{norm}$) across the mouse genome, as determined with the Step Response data analysis method. Interpretation of Figure is as described in Figure 2.

Figure 4

Corneal and scleral mRNA expression levels of Smarce1 (A) and Tns4 (B) normalized to whole eye expression levels (indicated by dashed red line), as measured by PCR. Asterisks indicate significant difference from whole eye expression levels.

Figure 5

Staining patterns for Smarce1 and Tns4 protein in the mouse eye. Sections through the mouse eye were stained for Smarce1 (A,D,G) and Tns4 (B,E,H), and the staining pattern was compared to similar sections stained with the secondary antibody only (C,F,I). In the cornea (A–C) the epithelium and keratinocytes (arrows) were positive for Smarce1 (A) and Tns4 (B). These structures were not stained in the secondary control. In the retina, both antibodies labeled cellular components, with positive staining of the external limiting membrane (arrowhead), suggesting that one cellular component recognized by the antibodies are Müller cells. In higher magnifications of the sclera (G–I), staining of scleral keratinocytes can be observed (arrows) for both Smarce1 and Tns4. The locations of the photographs of the sclera (G–I) are shown by boxes in (D–F). The images in (A–C) are at the same magnification and the scale bar in (C) represents 25 μm. The images in (D–F) are at the same magnification and the scale bar in (C) represents 50 μm. The legend to the right indicates specific structures: corneal epithelium (Epi), corneal endothelium (Stroma, Endo), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), inner segments/outer segments (IS/OS), Choroid, Sclera, and Muscle.