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First published online 12 April 2006
doi: 10.1242/dev.02361

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Diverse gap junctions modulate distinct mechanisms for fiber cell formation during lens development and cataractogenesis

¹ School of Optometry and Vision Science Program, University of California at Berkeley, Berkeley, CA 94720, USA.
² UC Berkeley/UCSF Joint Bioengineering Graduate Program, University of California at Berkeley, Berkeley, CA 94720, USA.
³ Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.
⁴ Department of Anatomy and Neurobiology, Morehouse School of Medicine, Atlanta, GA 30310, USA.

View larger version (31K): [in a new window]   Fig. 1. A missense mutation of Gja8 (8-connexin or Cx50) leads to whole cataracts and microphthalmia in the L1 mutant mice. (A) A slit-lamp photo shows the severe cataract of a heterozygous mutant (L1/+) mouse at the age of 1 month. (B) The eyeballs of heterozygous mutant (L1/+) mice are about 30% of the size of wild-type (+/+) controls at the age of 3 weeks. (C) Genome-wide linkage analysis shows that the L1 mutation is mapped to chromosome 3 near marker D3Mit98 with a Lod score of 6.8, which is in the vicinity of Gja8 gene. (D) DNA sequencing of homozygous L1 mouse genomic DNA shows a missense mutation in the 8 connexin gene. A change of T to C at position 148 causes a substitution of the serine residue at codon 50 by a proline residue (S50P).

View larger version (85K): [in a new window]   Fig. 2. Both heterozygous 8S50P/+ 3+/+ and homozygous 8S50P/S50P 3+/+ lenses display lens rupture and severely altered fiber cells at weaning age. (A) Lens from an 8S50P/+ 3+/+ mouse at P21. (B) Lens from a P21 8S50P/S50P 3+/+ mouse. (C,D) Toluidine Blue-stained lens sections show posterior rupture and severely altered fiber cells in the 8S50P/+ 3+/+ (C) and 8S50P/S50P 3+/+ (D) lenses of P21 mice. Scale bars: 0.5 mm in the upper panels; 100 µm in the lower panels.

View larger version (67K): [in a new window]   Fig. 3. Primary fiber cells fail to fully elongate to contact the anterior epithelium only in 8S50P/+ 3+/+ embryonic lenses even though typical gap junctions are still present. (A,B,D) Wild-type, 8-/- knockout and 8S50P/S50P 3+/+ embryonic lenses do not have any space between the epithelium and the underlying fiber cells at E15.5. (C) An E15.5 8S50P/+ 3+/+ embryonic lens shows a large cystic lumen between posterior primary fiber cells and the anterior epithelium. All images were taken from Toluidine Blue-stained embryonic sections. (E,F) Triple-labeled images of 8 connexin (green), F-actin (red) and nuclei (blue) of frozen sections from wild-type (E) and 8S50P/+ 3+/+ (F) embryonic lenses, respectively. The punctate green fluorescent spots represent typical staining of 8 connexins in lens fiber cells. A large cystic lumen is present only in the 8S50P/+ 3+/+ embryonic lens in F. White arrowheads indicate the anterior ends of posterior fiber cells and a large white arrow indicates the anterior epithelium. (G) A high magnification image of the selected area in F shows punctate fluorescent spots (indicated by white arrows). (H) TEM shows bona fide gap junctions (indicated by white arrows) between posterior fiber cells of the E15.5 8S50P/+ 3+/+ lens. Scale bars: 50 µm for A-D; 100 µm for E,F; 5 µm for G; 100 nm for H.

View larger version (57K): [in a new window]   Fig. 4. The generation and characterization of 8S50P/+ 3-/- and 8S50P/- 3+/+ compound mutant mice. (A) Wild-type 3 connexins were eliminated by breeding the 8S50P/+ 3+/+ mice with the 3-/- knockout mice. Toluidine Blue-stained section of an E15.5 8S50P/+ 3-/- embryonic lens shows a large lumen between posterior primary fiber cells and the anterior epithelium, similar to the 8S50P/+ 3+/+ embryonic lens section in Fig. 3C. (B) Wild-type 8 connexins were eliminated by breeding the 8S50P/+ 3+/+ mice with the 8-/- knockout mice. An E15.5 8S50P/- 3+/+ lens section shows no space between the epithelium and posterior primary fiber cells. (C) A P14 8S50P/+ 3-/- lens section shows degenerated primary fibers in the lens nucleus with relatively normal peripheral secondary fibers. (D) A Toluidine Blue-stained P14 8S50P/- 3+/+ lens section shows severe disruption of fiber cells and ruptured posterior capsule. (E) Lens from an 8S50P/+ 3-/- mouse at P21 shows a nuclear cataract but a relatively normal cortex. (F) A lens from a P21 8S50P/- 3+/+ mouse shows a small and ruptured lens with cataract. Scale bars: 50 µm for E15.5 sections; 100 µm for P14 sections; 0.5 mm for P21 lenses.

View larger version (63K): [in a new window]   Fig. 5. A comparison of 8S50P/- 3-/- and 8-/- 3-/- lenses. (A) Toluidine Blue-stained E15.5 8S50P/- 3-/- and 8-/- 3-/- embryonic lens sections show no space between the epithelium and posterior primary fiber cells (upper panels). Photos of P21 8S50P/- 3-/- and 8-/- 3-/- lenses display large nuclear cataracts with transparent cortex (lower panels). (B) A Toluidine Blue-stained P14 8S50P/- 3-/- lens section shows degenerating inner fiber cells and normal peripheral secondary fibers. Scale bars: 50 µm for E15.5 sections; 0.5 mm for P21 lenses; 100 µm for P14 sections.

View larger version (37K): [in a new window]   Fig. 6. A model for the cataract formation produced by 8-S50P mutation. Normal embryonic and postnatal lenses are formed in the presence of wild-type 8 (8-WT) and 3 (3-WT) connexins (following the black arrows). In the absence of 3-WT, the interaction between 8-S50P mutant connexin (8-S50P) and 8-WT suppresses the elongation of primary fiber cells in embryonic lenses but does not affect the formation of secondary fiber cells in postnatal lenses (following the red arrows). In the absence of 8-WT, the 8-S50P mutant connexin interacts with endogenous 3-WT to disrupt the formation of secondary fiber cells in postnatal lenses but does not affect the elongation of primary fiber cells (following the green arrows).

View larger version (28K): [in a new window]   Fig. 7. An illustration of 8 connexin (Cx50) protein topology. Known human mutations are indicated by green squares (R23T, E48K, P88S, I247M) and an orange oval (V64G) (Shiels et al., 1998; Berry et al., 1999; Polyakov et al., 2001; Willoughby et al., 2003; Zheng et al., 2005), and known mouse mutations are indicated by red circles (G22R, D47A, and S50P) and an orange oval (V64A) (Chang et al., 2002; Graw et al., 2001; Steele et al., 1998). An arrow indicates the S50P mutation.