Targeted deletion of fibrillin-1 in the mouse eye results in ectopia lentis and other ocular phenotypes associated with Marfan syndrome

Figures

• 

  Fig. 1.. Expression of genes encoding zonular proteins in ocular tissues during development and aging. (A) An example of multiplexed fluorescence in situ hybridization in the eye of a 1-day-old (P1) wild-type mouse. Fbn2 transcripts (red puncta) are restricted largely to the non-pigmented ciliary epithelium (NPCE). In contrast, Mfap2 (green puncta) is expressed in the NPCE and the lens epithelium (LE) at comparable levels. (B) Representative image from a P30 eye probed for Fbn1 and Ltbp2 expression. Note that, for both transcripts, the signal is stronger in the pars plana region of the ciliary epithelium than in the pars plicata region. (C) Expression of zonule genes shows marked spatial and temporal fluctuations. Fbn2, Fbn1 and Ltbp2 are expressed predominantly in the NPCE, Mfap2 has approximately equal expression levels in NPCE and lens, and Adamtsl4 is expressed predominantly in the LE. Non-specific binding, assessed using a probe set to a bacterial gene DapB, was negligible (data not shown). Data represent mean±s.d. NPCE, non-pigmented ciliary epithelium; PCE, pigmented ciliary epithelium; LE, lens epithelium. Scale bar: 50 µm.

• 

  Fig. 2.. Conditional deletion of Fbn1 in the nasal/temporal NPCE causes a reduction in fibrillin-1 immunofluorescence in the adjacent zonule and a corresponding increase in fibrillin-2 immunofluorescence. In this and other images, the interior of the eye is viewed from the posterior aspect (the retina and posterior sclera having been removed). (A) In 1-month-old control mice, the large, centrally located lens (L) is suspended from the non-pigmented ciliary epithelium (NPCE) by the ciliary zonule (light blue and arrow). Cre expression (green) is restricted to the nasal (N) and temporal (T) segments of the NPCE. Cre-negative regions (red) are located superiorly (S) and inferiorly (I). Fibrillin-1 immunofluorescence is equally strong in zonular fibers projecting from the Cre-positive and Cre-negative regions of the NPCE. Fibers attach at the lens equator with some elements (posterior extensions) running a short distance along the posterior lens surface before terminating (arrowhead). (B) In Fbn1-NPCE mice, fibrillin-1 immunofluorescence is markedly reduced in fibers projecting from the Cre-positive nasal/temporal segments of the NPCE, although residual immunofluorescence is present near the…

• 

  Fig. 3.. Onset of EL in Fbn1-NPCE mice. (A) Anterior segment is visualized by optical coherence tomography (OCT) in living mice. The cornea (C), iris (I) and lens (L) are visible. The distance between the inner surface of the cornea and the anterior surface of the lens is the anterior chamber depth (ACD; double-headed arrow). The position of the lens is stable in control mice or Fbn1-lens mice. In Fbn1-NPCE mice, the lens is initially in the correct position. However, between 6 and 8 weeks of age the lens dislocates backward in the eye (lower right panel). As a result, the ACD increases sharply and the iris, which is normally convex because of the support of the underlying lens, flattens. (B) In Fbn1-NPCE mice, there is a sharp increase in ACD beginning at 6 weeks of age. This reflects the onset of EL, a condition that becomes 100% penetrant by 3 months. Note that EL did not develop in control, heterozygous Fbn1-NPCE mice or conditional lens knockouts. N≥10 eyes at all ages. Data represent mean±s.d.

• 

  Fig. 4.. Breakage of zonular fibers in Cre-positive (green) regions precedes lens dislocation. (A) Eye from an 8-week-old Fbn1-NPCE mouse. The zonular fibers (light blue) are visualized using MFAP-2 immunofluorescence. Note the breakage of a stretch of zonular fibers (between the arrows) projecting from the nasal (N) region of the NPCE. (B) The fibers break near their midpoints, with fiber remnants visible on the surface of the lens and NPCE. N, nasal; T, temporal; S, superior; I, inferior.

• 

  Fig. 5.. Biomechanical properties of the ciliary zonule in wild-type, control and Fbn1-NPCE mice. Tensile strength and breaking displacement were determined using a pull-up assay (see Materials and Methods for details). In wild-type mice, the tensile strength (A) and breaking displacement distance (B) of the zonule increases with age. (C) Compared to 1-month-old control mice, the force required to break the zonule in age-matched Fbn1-NPCE mice is significantly decreased. (D) Displacement distance does not differ significantly between control and Fbn1-NPCE mice. (E) Tensile strength of the Fbn1-NPCE zonule in 2-month-old mice is reduced by >90% compared to controls but the displacement distance is unaffected (F). In Fbn1-NPCE mice, fibrillin-1-rich zonular fibers coexist with fibrillin-depleted fibers. To parse the contribution of fibrillin-1-rich and -deficient fibers to the mechanical properties of the zonule, subsets of fibers were cut in 1-month-old control or Fbn1-NPCE mice. (G) In control mice (gray bars), the tensile strength is reduced by ≈55% if the nasal/temporal (N/T) or superior/inferior (S/I) fibers are cut (P<0.01). In Fbn1-NPCE mice (orange bars), the tensile strength of the…

• 

  Fig. 6.. Ultrastructure of control and Fbn1-depleted zonular fibers, imaged at midspan. The images are oriented such that the ciliary epithelium is at the top and lens at the bottom (although neither tissue is visible in these high-magnification images). (A,C) Control and (B,D) Fbn1-depleted zonular fibers. (A) As they course toward the lens, control fibers coalesce to form thicker bundles (arrows). (B) In contrast, fibrillin-1-depleted fibers are generally thinner and do not converge to form bundles. (C) At higher magnification, the individual microfibrils that comprise the fibers are visible. Each microfibril is 10-12 nm in diameter. (D) Although thinner on average, zonular fibers in Fbn1-NPCE mice are otherwise indistinguishable from control fibers.

• 

  Fig. 7.. Zonular fiber diameter is reduced in Fbn1-depleted regions. Fiber diameter was measured by SEM in randomly selected fibers from the superior and nasal/temporal quadrants of control and Fbn1-NPCE mice. The resulting size distributions were evaluated using the two-sample Kolmogorov–Smirnov test. (A) Histogram of fiber size distributions in the three samples. Note that all distributions are positively skewed and that a higher proportion of small-diameter fibers is observed in the nasal/temporal (Cre-positive) region of Fbn1-NPCE eyes. (B) Cumulative frequency plots of fiber diameters in the superior and nasal/temporal regions of control eyes. There is no significant difference in fiber diameters between the two regions. (C) Similarly, there is no significant difference in diameter between superficial fibers in control eyes and Fbn1-NPCE eyes. (D) However, fibers in the nasal/temporal region of Fbn1-NPCE eyes are significantly thinner than fibers in the superior region of the same eyes. S, superior; N, nasal; T, temporal; D, Kolmogorov–Smirnov statistic (i.e. maximum difference between the cumulative distributions).

• 

  Fig. 8.. Ectopic lenses become cataractous. (A) In control mice, lenses retain their clarity, even in aged animals (see also lower right panel in D). (B) In aged Fbn1-NPCE animals, ectopic lenses often lose their transparency. (C) In some cases, the lens cortex disintegrates and the lens nucleus is expressed through the pupil into the anterior chamber. (D) Gallery showing the effect of age on the prevalence of cataract in Fbn1-NPCE mice. Cataracts are rare in young animals but increasingly common in aged Fbn1-NPCE mice. M, month; WT, wild type.

• 

  Fig. 9.. Eyes from aged Fbn1-NPCE mice have a larger volume than age-matched controls. (A) Representative examples of eyes from 10-month-old Fbn1-NPCE or control mice showing increased axial length and corneal hazing in the knockout animals. (B) Aggregated data show a difference in ocular volume that is evident at 8 months and statistically significant (*P<0.05) by 10 months (data represent mean±s.d.; n≥6 eyes at each time point). (C) For both types of mice, IOP declines with age but differences between genotypes are not significant (n≥6 eyes at each time point).