X-linked anhidrotic ectodermal dysplasia disruption yields a mouse model for ocular surface disease and resultant blindness

Abstract

X-linked anhidrotic/hypohidrotic ectodermal dysplasia (EDA) is caused by mutations in the (EDA) gene, which is required for the morphogenesis of ectoderm-derived tissues. Although EDA function in skin appendage development has been studied in Eda mutant "Tabby" mice, we have recently identified characteristic abnormalities in the ocular surface, an ectoderm-derived tissue. Histology of eyes of Tabby males revealed that 1) as previously reported, mice lacked meibomian glands; 2) >80% developed corneal lesions such as neovascularization, keratitis, ulceration, and keratinization identifiable from 9 weeks of age; and 3) > 80% showed ocular surface inflammation (blepharitis and conjunctivitis) when housed in a standard environment. Strikingly, both corneal defects and inflammation were prevented in Tabby mice bearing a transgene for the Eda-A1 isoform, but meibomian glands were restored little if at all. These findings suggest that intact ocular surface health is EDA dependent and that Tabby corneal abnormalities are not solely dependent on meibomian gland lipid secretion. Alternatively, susceptibility to inflammation and other phenotypes could result from failure of the usual EDA receptor to activate nuclear factor-kappaB transcription factors. This can be further tested in Tabby and Tabby-EDA transgenic mice, which provide unique models of severe ocular surface disease.

Figure 1

Phenotype, genotype, and mRNA expression of transgenic mice. A: Gross eye phenotype. Narrow palpebral fissure (blepharophimosis), thickened eyelid margins, and missing hair around eyelashes, but normal eyelashes in Tabby male mice (TA). Note white (arrow) and red corneal opacities (insert, also mucus) in Tabby (TA). mEDA-A1 transgene (TATG) almost fully rescued Tabby phenotype. Note “yellow hair ring” around eyelids in TATG and wild-type transgenic (WTTG) mice. B: Genotyping of transgenic mice. Lane 1, 390-bp PCR product from endogenous genomic EDA; lane 2, 330 bp of PCR product only from transgene; lane 3, SSCP for genetic background detection. PCR product from Tabby is 1 bp shorter than wild type, so that it moves faster on gel electrophoresis. C: Expression level of EDA in mice. Note lower EDA expression in Tabby (TA) and very high expression in transgenic mice (TATG and WTTG).

Figure 2

Photomicrographs showing corneal pathology in Tabby males from the pathogen-free facility. A: Normal histology of wild-type cornea at 30 weeks. B to F: Tabby corneas. B: Superficial corneal epithelial defect (arrow). C: Bullous keratopathy; separation between epithelium and stroma (arrow). D: Keratitis characterized by inflammatory cells (arrow) infiltrating the cornea. E: Ulceration, keratitis, hemorrhage, and neovascularization (arrow). F: Keratinization (arrow) and neovascularization (arrowhead). Epi, epithelium, Str, stroma. Numbers in parentheses represent age of mice (H&E staining at an original magnification of ×200).

Figure 3

Photomicrographs showing rescue of corneal lesions by EDA-A1 transgene in Tabby mice. A: Age-matched Tabby control shows acanthosis (arrow) and neovascularization (arrowhead) in cornea. B: Cornea lesions were recovered to wild type in EDA-A1 transgenic Tabby mice. Wild-type mouse with normal cornea (C) and EDA-A1 transgenic wild-type mice (D) show normal cornea morphology. H&E staining at an original magnification of ×200.

Figure 4

Summary scores of ocular phenotypes of Tabby mice. A: Corneal lesions in 80% of Tabby males housed in the pathogen free and 83% in the conventional facility (TA). One of four TATG mice showed minimal corneal keratinization (TATG), and no lesions were found in wild-type (WT) or transgenic mice in wild-type background (WTTG). B: Majority of the Tabby males housed in the conventional facility developed ocular surface inflammation (TA). Inflammation was decreased in transgenic Tabby (TATG) mice housed in the same facility. C: Tabby males lack meibomian glands (TA). EDA-A1 transgene failed to rescue meibomian glands in Tabby mice (TATG). BA, pathogen-free facility; CN, conventional facility.

Figure 5

Photomicrographs showing ocular surface inflammation in Tabby mice. A: Thickened eyelids (double-headed white arrows), absence of meibomian glands (single-headed white arrow), blepharitis (single-headed black arrows), and sparse hair follicles in Tabby eyelids (upper skin). B: Nearly normal eyelids, with partially rescued meibomian gland and fully restored hair follicles (upper skin) in EDA-A1 transgenic Tabby mice. C: Conjunctivitis (arrow) in Tabby. D: Follicular conjunctivitis in the fornix (arrow). E: Limbitis showing inflammatory cells (arrow) infiltrating the junction of the cornea and sclera. F: Normal conjunctiva, limbus, and cornea in transgenic wild-type mice. H&E staining; original magnifications in A and B, ×100; in C to F, ×200.

Figure 6

Photomicrographs showing prevention of inflammation by EDA-A1 transgene in Tabby mice hosted in the conventional facility. Tabby (A), transgenic Tabby (B), and wild-type (C) mice are littermates housed in the same cage. A: Moderate conjunctivitis (arrow) in Tabby. B to D: No inflammation in transgenic Tabby (B), wild-type (C) or transgenic wild-type mice (D). H&E staining at an original magnification of ×100.

Figure 7

Quantitative real-time PCR showing expression of EDA and EDAR. Relative expression level of EDA and its receptor, EDAR, in wild-type cornea (WTCR) and conjunctiva (WTCN) compared with wild-type (WTBS) and Tabby (TABS) back skin. Real-time PCR expression levels of EDA (A) and EDAR (B). Data normalized to GAPDH.