Ocular phenotype of Fbn2-null mice

Abstract

Purpose: Fibrillin-2 (Fbn2) is the dominant fibrillin isoform expressed during development of the mouse eye. To test its role in morphogenesis, we examined the ocular phenotype of Fbn2(-/-) mice.

Methods: Ocular morphology was assessed by confocal microscopy using antibodies against microfibril components.

Results: Fbn2(-/-) mice had a high incidence of anterior segment dysgenesis. The iris was the most commonly affected tissue. Complete iridal coloboma was present in 37% of eyes. Dyscoria, corectopia and pseudopolycoria were also common (43% combined incidence). In wild-type (WT) mice, fibrillin-2-rich microfibrils are prominent in the pupillary membrane (PM) during development. In Fbn2-null mice, the absence of Fbn2 was partially compensated for by increased expression of fibrillin-1, although the resulting PM microfibrils were disorganized, compared with WTs. In colobomatous adult Fbn2(-/-) eyes, the PM failed to regress normally, especially beneath the notched region of the iris. Segments of the ciliary body were hypoplastic, and zonular fibers, although relatively plentiful, were unevenly distributed around the lens equator. In regions where the zonular fibers were particularly disturbed, the synchronous differentiation of the underlying lens fiber cells was affected.

Conclusions: Fbn2 has an indispensable role in ocular morphogenesis in mice. The high incidence of iris coloboma in Fbn2-null animals implies a previously unsuspected role in optic fissure closure. The observation that fiber cell differentiation was disturbed in Fbn2(-/-) mice raises the possibility that the attachment of zonular fibers to the lens surface may help specify the equatorial margin of the lens epithelium.

Keywords: ciliary epithelium; ciliary zonule; coloboma; fibrillin; lens; optic fissure; pupillary membrane; tunica vasculosa lentis.

Figure 1.

Iris malformations in Fbn2-null mice. In WT mice, the pupil is circular and properly centered (A). Fbn2-null mice exhibited a range of iris defects including corectopia (B), dyscoria (C), iris hypoplasia/pseudopolycoria (arrowed in [D]), and typical coloboma (E). Mice deficient in both Fbn2 and Mfap2 generally exhibited more severe iris phenotypes (F). The mice shown are 1 to 2 months old except for (B) and (D), which are 10 months old.

Figure 2.

Failure to sustain light-induced pupillary constriction in 2-month-old Fbn2^−/− mice. In Fbn2^−/− mice, corectopic pupils constrict initially in response to bright light (A). After 5 minutes in constant bright light, however, the pupil dilates (B), becoming unresponsive to further fluctuations in light intensity. Under the same illumination conditions, constriction is maintained in a WT mouse (C, D). Images are representative of six independent experiments.

Figure 3.

Arrangement of microfibrils and capillaries in the PM of WT and Fbn2^−/− mice at P1. (A) In WT mice, microfibrils (visualized using anti-Magp1; light blue) are abundant in the PM and the anastomosing pattern of vessels in the associated capillary bed is revealed by gaps in the Magp1 immunofluorescence. In contrast, microfibrils in the Fbn2^−/− PM are disorganized and capillaries are not visible. The position of a coloboma is indicated by the arrow. (B) The spatial relationship between microfibrils and capillaries is disturbed in the PM of Fbn2^−/− animals. In WT mice, evenly spaced microfibrils (visualized with anti-Magp1; light blue) are plentiful in the spaces between adjacent capillaries (visualized with anti-CD31; yellow). In contrast, microfibrils are disorganized and aggregated in the Fbn2^−/− PM and often project over the capillary elements. (C) Fbn1 expression in the PM is enhanced in the absence of Fbn2. Microfibrils in the PM were visualized using anti-Magp1 (red) or anti-Fbn1 (green). In the absence of Fbn2, Fbn1 immunofluorescence is markedly increased. In each case, images are representative of at least six independent experiments. Scale bars: (A) 250 μm, (B) 50 μm, (C) 25 μm.

Figure 4.

Ocular aperture is significantly increased in P1 Fbn2^−/− mice. The ocular aperture is the diametric distance from the ciliary process on one side of the eye to the other (arrowed in [B]). The aperture was consistently smaller in WT eyes (A) than in Fbn2^−/− eyes (B). Quantitative data in (C) represent mean ± SD (wild type n = 10; Fbn2^−/− n = 8). ***P < 0.001 (two-tailed t-test).

Figure 5.

Persistent PM in adult Fbn2^−/− mice with complete (A) or partial (B) iridal coloboma. Microfibrils (green) are labeled with anti-Magp1. Nuclear DNA (red) is labeled with Draq5. A persistent PM is associated with notched region of the iris (A). Within the PM, Magp1-positive capillaries are evident (arrow). In the partial coloboma (B), Magp1-positive adhesions link the margin of the iris to the underlying lens capsule. Scale bar: 500 μm.

Figure 6.

Anatomy of the ciliary body in 24-week-old WT and Fbn2^−/− mice. Anatomy of intact ciliary bodies was reconstructed in three dimensions to examine the complex folding pattern of the ciliary epithelium. In WT mice (A), the ciliary epithelium is elaborately folded and the folds are predominantly radially oriented (see higher-magnification central inset). In Fbn2^−/− animals (B), some regions of the ciliary body were folded normally, but in other areas (see central inset) folds were underdeveloped and circumferentially rather than radially oriented. Images are representative of four independent experiments. Scale bars: 100 μm.

Figure 7.

Zonular organization in 1-month-old WT and Fbn2^−/− mice imaged from the posterior aspect. In WT mice (A), Magp1-positive zonular fibers (red) extend from the ciliary body (CB) to the lens equator. There are two prominent sets of zonular fibers (anterior zonular fibers [AZF] and posterior zonular fibers [PZF]) which attach to the lens on either side of the equator. The arrangement of the AZF and PZF in WT mice is best appreciated in xz projections of the data stack (C). The PZFs attach to the lens surface at the fibrillar girdle (arrow in [A]). The edge of the lens epithelium is defined by the absence of Draq5 nuclear staining (light blue). The fibrillar girdle is located at the edge of the lens epithelium, where epithelial cells (ep) differentiate into fiber cells (fi). In Fbn2^−/− eyes (B, D–F), the arrangement of the zonular fibers is disturbed. In mildly affected cases (B), the AZFs are depleted in number (see also xz view shown in [D]) and many PZFs terminate posterior to the fibrillar girdle. In some cases, the zonules are profoundly disturbed (E, F). This is associated with de-synchronization of lens fiber differentiation, as indicated by the undulations in the epithelial border. In some regions the epithelium bulges beyond the equator (*), signifying a delay in fiber cell differentiation. Images are representative of six independent experiments. Scale bar: 200 μm.

Figure 8.

A comparison of lens size and shape in WT and Fbn2^−/− mice. The equatorial and polar dimensions of the lens are significantly reduced in comparison to age-matched Fbn2^−/− animals (A). In WT mice, the radius of curvature of the anterior lens face is significantly greater than the posterior face (B). In contrast, in Fbn2^−/− mice, the radii of the two faces are statistically indistinguishable at all ages (C). Comparative data are shown in (D), illustrating a significant decrease in the anterior radius of curvature of WT in comparison to age-matched Fbn2^−/− lenses. Data represent mean ± SEM for n = 4 animals (eight eyes) of each genotype at each age. Error bars fall within the data symbols. *P < 0.05; **P < 0.01; ***P < 0.001.