Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1 -/-mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1 -/-mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow-derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis.

We applied surface-enhanced Raman spectroscopy (SERS) to immunolabeled endothelial cells to derive enhanced spectra of the biomolecular makeup of the cellular surface. A two-step immunolabeling protocol with gold-conjugated antibodies coupled with silver enhancement to attach silver nanoparticles to the cell surface was employed. This approach generated ∼50-fold SERS enhancement of spectral signals. The SERS spectra exhibited several SERS-enhanced peaks associated with cell membrane components. The SERS detection of silver nanoparticles proved more far more sensitive than conventional light microscopy techniques. The SERS enhancement allowed us to carry out spectral mapping using wavenumbers associated with membrane components that correlated directly with the distribution of silver nanoparticles. SERS has the potential to detect immunolabeling at lower levels than is possible using conventional immunolabeling methods while simultaneously providing unique, spatially defined, biochemical information.

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BackgroundAltered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6 +/− heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6+/Sey-Neu (Pax6+/−) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77Tg/− transgenics, which over-express Pax6 and model human PAX6 duplication.Methodology/Principal FindingsWe used electron microscopy to investigate ocular defects in Pax6+/− heterozygotes (low Pax6 levels) and PAX77Tg/− transgenics (high Pax6 levels). As well as the well-documented epithelial defects, aberrant Pax6 dosage had profound effects on the corneal stroma and endothelium in both genotypes, including cellular vacuolation, similar to that reported for human macular corneal dystrophy. We used mosaic expression of an X-linked LacZ transgene in X-inactivation mosaic female (XLacZTg/−) mice to investigate corneal epithelial maintenance by LESC clones in Pax6+/− and PAX77Tg/− mosaic mice. PAX77Tg/− mosaics, over-expressing Pax6, produced normal corneal epithelial radial striped patterns (despite other corneal defects), suggesting that centripetal cell movement was unaffected. Moderately disrupted patterns in Pax6+/− mosaics were corrected by introducing the PAX77 transgene (in Pax6+/−, PAX77Tg/− mosaics). Pax6Leca4/+, XLacZTg/− mosaic mice (heterozygous for the Pax6Leca4 missense mutation) showed more severely disrupted mosaic patterns. Corrected corneal epithelial stripe numbers (an indirect estimate of active LESC clone numbers) declined with age (between 15 and 30 weeks) in wild-type XLacZTg/− mosaics. In contrast, corrected stripe numbers were already low at 15 weeks in Pax6+/− and PAX77Tg/− mosaic corneas, suggesting Pax6 under- and over-expression both affect LESC clones.Conclusions/Significance Pax6+/− and PAX77Tg/− genotypes have only relatively minor effects on LESC clone numbers but cause more severe corneal endothelial and stromal defects. This should prompt further investigations of the pathophysiology underlying human aniridia and ARK.

SR-FTIR microspectroscopy coupled with PCA appears to enable the identification of SC, TA cell, and TD cell populations. The results also suggest that a small subpopulation of cells in the corneal epithelial SC niche possess TA cell-like characteristics. The most significant spectral characteristics associated with the SCs appear to relate to differences in nucleic acid conformation. This finding is consistent with recent theories suggesting that the control of differentiation is related to major changes in chromatin structure.