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Disruption of Gja8 (8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation

¹ Department of Cell Biology, The Scripps Research Institute, La Jolla, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
² Institut Jacques Monod, University Paris VII, Paris, France

View larger version (69K): [in a new window]   Fig. 1. Homozygous 8 knockout mice developed microphthalmia with mild nuclear cataracts. (A) Physical maps of the wild-type Gja8 allele, the 8 targeting vector, and the disrupted Gja8 allele. The DNA probe used for Southern blotting is indicated on the corresponding region of the wild-type and knockout Gja8 alleles. A 7.5 kb band corresponded to the knockout allele, and a 3.8 kb band for the wild-type allele. (B) The upper panel shows a blot of an embryonic stem cell clone with a disrupted Gja8 allele (+/–), in comparison with the wild-type control (+/+). This was verified by Southern blotting using a DNA probe, labeled in A. The lower panel shows the PCR results from homozygous 8 knockout (–/–), heterozygous 8 knockout (+/–) and wild-type (+/+) mice. The left arrow indicates a 320 bp band from the wild-type allele and the right arrow for a 450 bp band from the knockout allele. (C) Phenotypic comparison of the eyes and lenses from homozygous 8 knockout (–/–), heterozygous 8 knockout (+/–), and wild-type (+/+) sibs. Scale bar, 1 mm.

View larger version (67K): [in a new window]   Fig. 2. Western blots from the lens homogenates of homozygous 8 knockout (–/–), heterozygous 8 knockout (+/–), and wild-type (+/+) littermates. Equal amounts of the lens homogenates (50 µg per lane) were loaded and separated by SDS-PAGE, then detected by polyclonal antibodies against 8 connexin, 3 connexin, and MP26. Molecular masses are listed on the right-hand side.

View larger version (65K): [in a new window]   Fig. 3. (A) Representative histological sections from the bow region of wild-type (+/+) and 8 homozygous knockout (–/–) lenses. The arrows indicate the lens bow region where epithelial cells differentiate to become fiber cells. Scale bar, 50 µm. (B) Double immunolabeling of 8 (red) and 3 (green) connexins in the cortical fibers of the frozen sections from wild-type (+/+) and 8 homozygous knockout (–/–) lenses. Scale bar, 5 µm.

View larger version (178K): [in a new window]   Fig. 4. Gap junctions are smaller in 8–/– lenses. Gap junctions in the lens cortical fibers from wild-type (+/+), 3 (–/–), and 8 (–/–) mice were examined by thin-section (upper panels) and freeze-fracture (lower panels) electron microscopy. Gap junctions are indicated by arrows. Scale bar, 60 nm.

View larger version (66K): [in a new window]   Fig. 5. Western blots of lens cortical homogenates (C) and lens nuclear homogenates (N) from wild-type, 3 homozygous knockout (–/–), and 8 homozygous knockout (–/–) mice. The left panel shows the results obtained using an anti-8 connexin antibody. The upper arrow indicates the intact 8 connexin protein band, while the lower arrow points to the cleaved bands of 8 connexin. The right panel shows the results obtained using an anti-3 connexin antibody. The arrow indicates the cleaved forms of 3 connexin. The molecular markers (in kDa) are listed on the right-hand side.

View larger version (63K): [in a new window]   Fig. 6. lacZ expression patterns in the lenses of 3 and 8 knockout mice. (A) A side view of an eye, stained for ß-gal, from an 8 heterozygous (+/–) knockout mouse. The lens equator is indicated by an arrow and anterior monolayer epithelium is blue. C, cornea; R, retina; O, optic nerve. B and C show lenses from adult 8+/– and 3+/– sibs, respectively, stained for ß-gal; anterior view. (D) An equatorial view of the 3+/– lens in C; an arrow indicates the lens equator. (E-G) 8+/–, wild-type (+/+) and 3+/– embryos at 14 dpc, respectively. (H-J) Magnified views of eyes of the embryos in E, F and G, respectively. Scale bars (A-G) 1 mm; (H-J) 250 µm.

View larger version (123K): [in a new window]   Fig. 7. A comparison of the lacZ expression patterns in the lenses of 8+/– and 8–/– sibs. ß-gal staining (A) in embryonic lenses from 8+/– and 8–/– sibs at 16.5 dpc; posterior view; (B) in 4-week old 8+/– and 8–/– sibs; anterior view (upper panels) and from the equatorial view (lower panels); (C) in sections of lenses from postnatal day 17 8+/– (left-hand panel) and 8–/– (right-hand panel) sibs. Arrowheads indicate the lens epithelium and arrows point to the bow region of the lenses. Scale bars (A) 0.2 mm; (B) 1 mm; (C) 100 µm.

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