The role of crumbs genes in the vertebrate cornea

Abstract

Purpose: To evaluate the role of crumbs genes and related epithelial polarity loci in the vertebrate cornea.

Methods: The authors used histologic analysis and electron microscopy to evaluate the corneas of zebrafish mutant for a crumbs locus oko meduzy (ome) and in mutants of four other loci, nagie oko (nok), heart and soul (has), mosaic eyes (moe), and ncad (formerly glass onion), that function in the same or related genetic pathways. In parallel, they performed an evaluation of corneas in human carriers of a crumbs gene, CRB1, and mutations using topography and biomicroscopy. The expression of the CRB1 gene in the normal human cornea was examined by polymerase chain reaction (PCR) and immunohistochemical staining.

Results: The corneas of zebrafish mutants display severe abnormalities of the epithelial and stromal layers. The epithelial cells do not properly adhere to each other, and fluid-filled spaces form between them. In addition, the layering of the corneal stroma is poorly formed or absent. The corneas of human carriers of CRB1 mutations display shape deviations compared with what has been observed in normal individuals. A PCR product of the correct size was obtained from normal human corneal samples. Sequence analyses confirmed its identity to be the human CRB1 gene. Immunohistochemical staining using anti-CRB1 yielded positive brown deposits in the human cornea.

Conclusions: crumbs genes play a role in the differentiation of the vertebrate cornea. Corneal defects associated with crumbs gene mutations are very severe in the zebrafish model and, in comparison, appear clinically less pronounced in the human eye.

Figure 1.

Electron micrographs of transverse sections through eyes of wild-type and oko meduzy (ome) mutant zebrafish at 3 dpf. (A, B) Low-magnification view of the lens and the overlying cornea. Note that the surface of the mutant cornea features more protrusions than the wild-type cornea. Additionally, fluid-filled spaces are found between epithelial cells in the mutant. Arrowheads: corneal surface. (C, D) Higher-magnification images of the cornea. (D, arrows) Fluid-filled spaces between epithelial cells. (E, F) Higher magnifications of the corneal surface. Wild-type and mutant apical junctions (arrows) do not display any obvious differences. (G, H) Junctions are present on the interface between two layers of corneal epithelial cells. At least some of them persist in the mutant (arrows). L, lens.

Figure 2.

Electron micrographs of transverse sections through eyes of wild-type and mutant zebrafish at 3 dpf. Left: lens and overlying cornea (arrows). Right: higher magnifications of the corneal epithelium and stromal layer. Each row of images corresponds to a different genotype. Top to bottom: corneas of the wild-type and the following mutant strains: glo, has, nok, and moe. Right, arrows: fluid-filled spaces between epithelial cells of mutant corneas. L, lens.

Figure 3.

Pedigrees of two Pakistani families that carry defects in the CRB1 gene based on previously published data. Shaded boxes: family members who were tested for corneal defects. Asterisks: patients who show signs of corneal abnormalities based on KSS analysis. S, severe corneal scarring.

Figure 4.

Normal and CRB1 mutant corneas. (A, B) Corneal topography data from a 3330RP family control (A) and from a 010LCA family homozygous carrier of a CRB1 mutation (B). (A, B) Top: graphic representations of corneal curvature. Steeper areas are represented by warmer colors, and flatter regions are represented by cooler colors, as color-coded on the scale to the right. Bottom: raw numerical data of corneal curvature from the same two patients. The cornea of a crumbs mutation carrier is characterized by steeper curvatures and irregular astigmatism, most pronounced in the inferior temporal region. The control features much more homogeneous corneal curvatures. (C) Cornea of patient V-4 (010LCA pedigree; Fig. 3A). Severe corneal scarring, possibly a result of corneal ectasia, prevented topography measurement in this patient.

Figure 5.

Measurements of corneal curvature in controls (WT) and in homozygous carriers of CRB1 mutations (CRB). Measurements of the steepest point on the cornea (left), the flattest point (middle), and the difference between the two (right) are provided. The vertical axis shows dioptric values, and the horizontal axis shows genotypes. Averages and standard deviations are provided below each graph.

Figure 6.

Expression of CRB1 gene in the human cornea. (A) PCR amplification of the human CRB1 gene. A 516-bp CRB1 PCR product was amplified from normal human corneal and retinal cDNA templates but not from the water control. (B) Immunohistochemical staining for CRB1 in the normal human cornea (a, b). Immunoreactive products (brown) were noted in the epithelial layer. Negative control (c) in which primary antibody was omitted showed minimal staining. Brackets: the corneal epithelium.