Fig. 1. Deletion of exon 3 in the perlecan (Hspg2) gene. (A) Region of the perlecan gene containing exons 2–5 (top), the targeting construct (middle) and the region after homologous recombination. Exons are depicted as black boxes and numbered. A 130 bp KpnI–BamHI fragment containing exon 3 was replaced by the PGK-neo selection cassette. The probe containing exons 4 and 5 that was used for Southern blots is depicted as a bar (top). E, EcoRI; B, BamHI; H, HindIII; K, KpnI. (B) Southern blot analysis of EcoRI-digested DNA from ES cell clones. M, λ DNA digested with HindIII, upper band 19.9 kb, lower band 9.5 kb. (C) Products of RT–PCR reactions performed with primers coding for the N-terminal region of the perlecan protein (for sequences, see text) separated in an agarose gel. Sources of RNA were whole E16 embryos and primary fibroblasts isolated from newborn wild-type(+/+) or Hspg2^Δ3/Δ3 mice. M, 100 bp ladder, uppermost band 1000 bp. The PCR products are 650 bp in wild-type and 605 bp in mutant samples. (D) Immunoblot analysis of proteoglycan-enriched fractions isolated from the culture medium of wild-type and Hspg2^Δ3/Δ3 fibroblasts. Samples were treated with (+) or without (–) chondroitinase (CSase) and heparitinase (HSase), subjected to SDS–PAGE in a 5% gel, and resolved by immunoblotting with an antibody against perlecan. Lane 1, wild-type sample treated with chondroitinase and heparitinase; lane 2, untreated mutant sample; lane 3, mutant sample treated with hepari tinase only; lane 4, mutant sample treated with chondroitinase only; lane 5, mutant sample treated with chondroitinase and heparitinase. The perlecan core protein produced by the mutant cells was of the same size as the wild-type perlecan, and core protein was observed after chondroitinase treatment, indicating that the major portion of the mutant perlecan carries chondroitin sulfate chains only.

Fig. 2. Early alterations in the eyes of Hspg2^Δ3/Δ3 mutant mice. (A) The eye from a newborn Hspg2^Δ3/Δ3 mutant mouse is well developed, but tiny drops of lens material (hardly visible at this magnification) are leaking from the lens (arrow). PAS staining. (B) Lateral region of a newborn mutant lens with two leakage sites (one indicated with an arrow) stained with an antibody against γ crystallin. (C–F) Sections of 14-day-old wild-type (C and E) and Hspg2^Δ3/Δ3 mutant (D and F) eyes immunostained for N-cadherin, which is localized in the cell membranes (red color). Nuclei are counterstained with hematoxylin. (C and D) Lateral region of the lens. The dotted line indicates the approximate border of the proliferation and differentiation zones. Gaps and irregularities in lens fiber organization are detected in the mutant lens (D). (E and F) The attachment of lens fibers to the lens capsule in the posterior region of the lens is altered in the mutant lens (F). Arrows point to irregularities in the lens fiber organization. Bar: 100 µm in (A), 50 µm in (B–F). CB, ciliary body; D, differentiation zone; L, lens; N, optic nerve; PZ, proliferation zone; R, retina.

Fig. 3. Degeneration of lenses in the Hspg2^Δ3/Δ3 mutant mice. All sections were stained by the PAS method, by which the lens capsule stains red. (A) Lens of a 20-day-old wild-type mouse. The lens is contained within the lens capsule and is composed of fiber cells and a monolayer of epithelial cells at the anterior surface. (B) The lateral region of the lens in a 6-month-old wild-type mouse. The lens capsule is compact and uniformly stained. (C and D) Lens of a 20-day-old Hspg2^Δ3/Δ3 mutant mouse. The fibers have lost their ordered pattern and have started to liquefy. The lens capsule is wrinkled and drops of lens material (arrow) are visible in the posterior chamber of the eye. (D) Boxed area in (C) in higher magnification. Vacuoles (arrows) are seen in the anterior and equatorial regions. (E and F) Degeneration of the lens capsule in perlecan^Δ3/Δ3 mutant mice. (E) Anterior region of the lens in a 6-month-old Hspg2^Δ3/Δ3 mutant mouse. Small cracks (thin arrows) and a degenerated cell within the lens capsule (faint hematoxylin staining, arrow) are visible. (F) A split capsule in the posterior region of a lens in an 11-month-old Hspg2^Δ3/Δ3 mutant mouse. (G) Degenerated lens material leaking out of the lens in a 6-month-old Hspg2^Δ3/Δ3 mutant mouse. Bar: 100 µm in (A) and (C), 20 µm in (B) and (D–G). C, cornea; LC, lens capsule; R, retina.

Fig. 4. Immunohistochemical detection of lens capsule components. (A, C and E) Frozen sections of eyes from 14-day-old wild-type and (B, D and F) Hspg2^Δ3/Δ3 mutant mice were stained for laminin (A and B), perlecan (C and D) and collagen IV (E and F). All three components are present in the mutant lens, but the lens capsule is more compact in appearance in the wild-type eye. Bar: 20 µm. CB, ciliary body; L, lens.

Fig. 5. Transmission electron micrographs of lenses from wild-type and Hspg2^Δ3/Δ3 mutant mice. (A) Anterior region of a wild-type lens. Lens epithelial cells form a monolayer covered by the lens capsule (LC). (B) Lens epithelial cells in the Hspg2^Δ3/Δ3 lens have a dilated endoplasmic reticulum, increased number of ribosomes and mitochondria, and filopodia-like protrusions reaching into the lens capsule. Lens fibers below the epithelial cell layer are deteriorating. (C) Lens capsule component aggregates on the surface of the wild-type lens capsule have the appearance of a layer of loose filaments. (D) Loose amorphous material is seen on the surface of the Hspg2^Δ3/Δ3 lens capsule. (E) The basolateral surface of the wild-type lens epithelial cell is distinct, and the lens capsule has a layered structure. (F) The surface of the Hspg2^Δ3/Δ3 lens epithelial cell is fuzzy in appearance and the lens capsule lacks the layered structure. (G) The basolateral surface of a Hspg2^Δ3/Δ3 lens epithelial cell with a protrusion. A dilated endoplasmic reticulum and loose contact with the lens capsule are discernible. (H) The fiber cells in the inner region of the wild-type lens are densely packed. Arrows point to gap junctions. (I) The fibers in the Hspg2^Δ3/Δ3 lens look swollen, and dense packing is lost. Gap junctions (arrows) are loosened. (J) A necrotic epithelial cell within the lens capsule of an Hspg2^Δ3/Δ3 lens. Neighbouring epithelial cells have extended protrusions into the lens capsule but have remained in the monolayer. Samples are from 20-day-old mice except (G), which is from a 14-day-old mouse. Bar: 1 µm in (A) and (B), 0.1 µm in (C–F), 0.5 µm in (G–I), 5 µm in (J).

Fig. 6. Abnormal development of lenses in Col18a1^–/– × Hspg2^Δ3/Δ3 mice. (A) Section of a newborn eye showing leakage of differentiating fiber cells (arrows) towards the ciliary body and iris. Lens external cytoplasmic material is marked with X. (B) Section of the lens of a 2-week-old mouse showing a mass of external lens cells (marked with X) between the disrupted iris and the ciliary body. Degeneration of the iris is typical of Col18a1^–/– mice. (C) The anterior region of a lens in a 2-week-old mouse, with a cluster of proliferating epithelial cells depositing red-stained lens capsule-like extracellular matrix. (D) Invasion of lens epithelial cells towards the iris/ciliary body in a lens of a 2-week-old mouse. (E) The external cells in the 10-week-old eye have been degraded, and remnants of lens capsule material connect the ciliary body and the deteriorated lens. (F) The capillary (arrow) connecting the ciliary body and the lens capsule is still present in the 1-week-old mutant eye. (G) The capillary has regressed in the 2-week-old eye, but a bridge-like structure (arrow) has formed between the lens and the ciliary body. The iris is tightly attached to the lens capsule. All sections were stained with PAS method. Bar: 50 µm. C, cornea; CB, ciliary body; LEC, lens epithelial cells; R, retina.

Fig. 7. Detection of apoptotic nuclei in the lens. (A–C) Apoptosis in the eyes of newborn wild-type (A), Hspg2^Δ3/Δ3 (B) and Col18a1^–/– × Hspg2^Δ3/Δ3 mice (C) was analyzed by the TUNEL assay. TUNEL-positive nuclei are stained red (arrows). Only nuclei in the epithelial layer (black arrows) were counted for statistical analysis. (D) A cluster of apoptotic cells in the epithelial layer of an 11-day-old Col18a1^–/– × Hspg2^Δ3/Δ3 mouse. These clusters were not observed in neonatal samples. Nuclei were counterstained with hematoxylin. Bar: 50 µm.