June 2003
Volume 44, Issue 6
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Lens  |   June 2003
Genetic Background Influences Cataractogenesis, but Not Lens Growth Deficiency, in Cx50-Knockout Mice
Author Affiliations
  • Dwan A. Gerido
    From the Department of Physiology and Biophysics, State University of New York, Stony Brook, New York.
  • Caterina Sellitto
    From the Department of Physiology and Biophysics, State University of New York, Stony Brook, New York.
  • Leping Li
    From the Department of Physiology and Biophysics, State University of New York, Stony Brook, New York.
  • Thomas W. White
    From the Department of Physiology and Biophysics, State University of New York, Stony Brook, New York.
Investigative Ophthalmology & Visual Science June 2003, Vol.44, 2669-2674. doi:10.1167/iovs.02-1311
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      Dwan A. Gerido, Caterina Sellitto, Leping Li, Thomas W. White; Genetic Background Influences Cataractogenesis, but Not Lens Growth Deficiency, in Cx50-Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2003;44(6):2669-2674. doi: 10.1167/iovs.02-1311.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. Deletion of connexin (Cx)50 produces microphthalmia with nuclear cataracts. To determine whether these two traits are influenced by genetic background and are dependent on each other, mice carrying the Cx50 deletion in two different strains were generated, and the growth defect and severity of cataracts were analyzed.

methods. Cx50-knockout mice were generated in the 129S6 strain, and back-crossed into the C57BL/6J genetic background. To analyze the influence of genetic background on the observed phenotype, postnatal lens growth, lens clarity, lens histology and crystallin solubility were determined and compared between the two strains of Cx50-knockout mice.

results. The growth deficiency persisted, regardless of genetic background, but genetic modifiers that differentially altered the solubility of crystallin proteins influenced the severity of cataracts. Expression levels of Cx46 were similar in all animals, regardless of genetic background, indicating that the differences were not due to a compensatory upregulation of Cx46.

conclusions. Taken together, these data indicate that the two components of the Cx50 phenotype are independent of each other and that cataractogenesis is under the influence of an unidentified genetic modifier.

The mammalian lens is a multicellular organ in which the cells act as a functional syncytium. The lens is composed of a single layer of epithelial cells located at the anterior surface and a solid mass of elongated fiber cells that extend from the anterior to posterior pole. During differentiation, lens epithelial cells undergo a process of maturation and elongation during which they become new lens fiber cells. 1 2 The mature lens fiber cells are unique in that they have no intracellular organelles and lack a vascular supply of nutrients. The absence of blood supply and intracellular organelles is necessary for transparency, but presents serious homeostatic and metabolic challenges to the cells within the lens. Lens fiber cells have overcome these challenges through gap-junction-mediated intercellular communication, which allows the metabolically active epithelial cells to share ions, second messengers, and metabolites with the lens fiber cells. 3  
Structural proteins belonging to the connexin (Cx) family comprise the intercellular channels present in gap junctions. Functional channels are formed when connexons on adjacent cells align, giving rise to a direct pathway for cell-to-cell communication. Each connexin forms channels with distinctly different physiological properties of permeation, gating, and selective interaction with other members of the connexin family. 4 5 The connexin gene family encodes at least 20 different proteins, of which three are expressed in the lens. 6 7 The lens epithelium predominantly expresses Cx43, 8 9 but during epithelium-to-fiber cell differentiation, Cx43 expression is downregulated and replaced by two different connexins, Cx46 and Cx50. 10 11 12 13  
Studies involving the deletion of connexin proteins expressed in the lens have revealed their diverse roles in lens homeostasis. Knockout of Cx43 leads to cardiac malformation and neonatal death. 14 Thus, analysis of a lens phenotype in the absence of Cx43 was limited to prenatal development, which was found to proceed normally, at least until birth. 15 16 Deletion of Cx46 results in a severe, senile-type cataract, with otherwise normal ocular development, whereas deletion of Cx50 produces much milder nuclear pulverulent cataracts, along with a significant reduction in ocular growth. 17 18 19  
The difference in cataract severity between Cx46- and Cx50-knockout mice has been attributed to abnormal proteolytic cleavage of crystallins. Crystallin proteins comprise the major cytoplasmic component of lens fiber cells, and loss of crystallin solubility leads to cataractogenesis. 20 Deletion of Cx46 resulted in an aberrant cleavage of γ-crystallin, which was not observed in the Cx50-knockout. 17 18 This cleavage resulted from an increase in calcium accumulation and activity of the calcium-dependent protease Lp82 in Cx46-knockout lenses. 21  
Although these studies clearly support different roles for Cx46 and Cx50 in lens development, they do not address the question of whether other genetic factors are able to exert an influence on the phenotype in animals where these genes have been deleted. It has been shown that the deletion of Cx46 in animals of different genetic backgrounds resulted in differences in cataract severity that directly correlated with the relative levels of cleaved γ-crystallin. This finding demonstrated that other genetic factors could profoundly influence the phenotype of connexin-knockout animals. In the current study, we explored whether genetic factors also influence the Cx50-knockout phenotype. We found that the growth deficiency persisted regardless of genetic background, but that the process of the cataractogenesis was markedly different between the two animal strains tested. 
Methods
Cx50-Deficient Mice
The study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The generation of Cx50-deficient mice in the 129S6 and C57BL/6J mouse strains (Taconic Farms Inc., Germantown, NY) has been described. 16 18 Animals were genotyped by PCR screening by a three-primer protocol for the Cx50 allele. A common 5′ flanking primer (pcr 1; 5′-GCCCCCTCCTGCTTATTTCTG-3′) was paired with either a 3′ primer derived from vector sequences unique to the Cx50 replacement cassette (pcr 2; 5′-CGGGCCTCTTCGCTATTACG-3′), or a third primer derived from the Cx50-coding region (pcr 3; 5′-CTCCATGCGAACGTGGTGTAC-3′). Primers 1+2 amplified a 1370-bp band from Cx50-knockout chromosomes. Amplification of wild-type chromosomes with primers 1+3 produced a 1600-bp band. DNAs isolated from tail biopsy samples were amplified in a thermal cycler (DNA Engine Dyad; MJ Research, Waltham, MA), and amplified products were resolved by agarose gel electrophoresis. 
Protein Analysis
For protein expression analysis, lenses were dissected from adult Cx50 wild-type and Cx50-knockout mice from each genetic background. Equal wet weights of wild-type and knockout lenses were homogenized in 1 mL of 0.1 M NaCl, 0.1 M Na2HPO4, 10 mM ascorbic acid, and protease inhibitors (10 μg/mL each of chymostatin, leupeptin, and pepstatin). The homogenized tissue was centrifuged at 14,000g for 20 minutes at room temperature, after which the soluble fraction was recovered and stored at −80°C. The insoluble fraction was subjected to a second wash with 1 mL of 0.1 M NaCl and 0.1 M Na2HPO4, followed by a subsequent wash with 1 mL of 20 mM NaOH. After centrifugation at 14,000g for 20 minutes, the insoluble fraction was finally washed in 1 mL of 1 mM Na2CO3, after which the pellets were resuspended in 0.1 mL of sample buffer and stored at −80°C. 
Equal aliquots of samples from each animal strain and genotype were electrophoresed on 15% polyacrylamide gels, transferred to nitrocellulose membranes, and incubated with antibodies specific for Cx50 12 ; Cx46 11 ; major intrinsic polypeptide (MIP); and αA-, αB-, β-, and γ-crystallin proteins (αA-, αB- and β-crystallin antibodies were purchased from Stressgen, Victoria, British Columbia, Canada; MIP and γ-crystallin antibodies were generously provided by Joseph Horwitz, Jules Stein Eye Institute, University of California Los Angeles). Primary antibodies were detected with alkaline phosphatase-conjugated goat anti-rabbit IgG (Roche Molecular Biochemicals, Indianapolis, IN) with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate used as substrates (Sigma Chemical Co., St. Louis, MO). Blots were digitized, and band intensities were quantified with image-analysis software (1D; Eastman Kodak, Rochester, NY). Values were normalized to the mean band intensity in the 129S6 strain for each protein analyzed and multiplied by 100 to express a percentage. 
Growth and Cataract Analysis
Lens growth in Cx50 wild-type and knockout animals from each mouse strain was studied at 2, 4, and 6 weeks of age. Animals were killed and weighed to determine their overall growth. Eye and lens growth was examined by sequentially dissecting each organ before weighing. Lenses were dissected from wild-type and knockout animals in each mouse strain on a 37°C stage in M199 medium supplemented with Hanks’ salts and 10 mM HEPES [pH 7.4]. Cataracts were visualized using a microscope (model SZX9; Olympus Corporation of America, Lake Success, NY) and photographed with a digital camera (model C3030; Olympus). 
Histology
Mouse eyes were dissected and fixed in 4% formaldehyde, freshly prepared from paraformaldehyde, in phosphate-buffered saline (PBS) for 16 to 24 hours at room temperature. Fixed eyes were rinsed in PBS, dehydrated through an ethanol series and embedded in paraffin. Sections of 2 to 3 μm were cut on a diamond knife, deparaffinized, and stained with hematoxylin and eosin. 16 18 22 Histologic sections were viewed with 10× and 40× objectives on a microscope (model BX51; Olympus) and photographed with an digital camera (MagnaFire; Optronics, Goleta, CA). 
Results
Ocular Growth of Cx50-Knockout Mice
Initial characterization of mice without the Cx50 gene in a mixed genetic background demonstrated deficient eye and lens growth. 18 19 To determine whether these growth abnormalities are influenced by genetic background, the mass of the animals, eyes, and lenses from knockout mice in the 129S6 and C57BL/6J backgrounds were recorded at 2, 4, and 6 weeks of age and compared with wild-type animals. There were no statistically significant differences in the overall postnatal growth of wild-type and Cx50-knockout mice in either the 129S6 or C57BL/6J genetic backgrounds (Fig. 1A) . Although the overall growth of knockout mice was similar to the control, growth of both the eyes and lenses of Cx50 knockouts was significantly reduced (Student’s t-test, P < 0.05) when compared with wild-type animals (Figs. 1B 1C) . The reductions in both eye and lens mass were similar in either the 129S6 or the C57BL/6J knockout mice at all ages tested, with knockout lenses displaying an approximate 42% reduction in growth compared with wild-type animals. Thus, genetic background did not influence the growth-deficient aspect of the Cx50-knockout phenotype. 
Comparison of Lens Clarity
In animals of mixed genetic background, disruption of the Cx50 allele produced cataracts. 18 To determine whether the degree of lens opacity was influenced by genetic background, lenses from 8-week-old wild-type and knockout 129S6 and C57BL/6J animals were dissected and photographed using dark-field microscopy. Wild-type lenses were clear when viewed through the anterior epithelium (Fig. 2A) or through the equatorial edge (Fig. 2B) . Disruption of the Cx50 allele produced cataracts in both genetic backgrounds, with distinct differences in the character of lens opacity. Cx50-knockout mice from the 129S6 genetic background displayed pulverulent nuclear cataracts with a prominent lamellar opacity (Figs. 2C 2D) . In contrast, Cx50-knockout mice from the C57BL/6J genetic background contained an irregular nuclear opacity with a greatly reduced, or absent, lamellar cataract (Figs. 2E 2F) . To determine whether a lamellar cataract develops in the C57BL/6J knockout lenses later in life, lenses from 6-month-old mice were also examined. We found that the cataract phenotypes of older 129S6 (Figs. 3A 3B) and C57BL/6J (Figs. 3C 3D) lenses were quite comparable to those of 8-week-old animals and that the differences in opacities did not simply result from a similar process operating with different kinetics. These results indicate that the cataract phenotype of Cx50-knockout mice was influenced by genetic factors that produced differences in the degree of lens opacity and that the differences in lens opacity were constant at least up to 6 months of age. 
Analysis of Crystallin Solubility
Deletion of either Cx46 or Cx50, results in the loss of crystallin solubility and produces cataracts. 17 18 19 In the case of Cx46, it was subsequently shown that cataract severity is influenced by genetic background, with 129SvJ (129S4) Cx46-knockout mice having more severe cataracts than C57BL/6J Cx46-knockout mice. 23 To ascertain whether crystallin precipitation in Cx50-knockout mice is influenced by genetic background, the solubility of crystallin proteins was examined in 129S6 and C57BL/6J animals. Control and knockout lenses of adult mice in both backgrounds were separated into soluble and insoluble components, 18 and equal volumes of these fractions were electrophoresed, analyzed by Western blot, and probed with antibodies to different crystallins. All the crystallin proteins examined were abundant in the soluble fractions from either wild-type or knockout lenses in both backgrounds (Figs. 4A 4B 4C 4D , left). Consistent with the presence of opacities, αA-, αB-, β-, and γ-crystallins were present in the insoluble fraction of Cx50-knockout lenses, but not in wild-type lenses (Figs. 4A 4B 4C 4D , right). There also appeared to be clear differences in the relative amounts of precipitated crystallin proteins in the two backgrounds, with more crystallins being present in the insoluble fraction of 129S6 than in that of C57BL/6J Cx50-knockout mice. In contrast, MIP was absent from the soluble fractions of all animals and equally abundant in the insoluble fractions of wild-type and Cx50-knockout lenses from both genetic backgrounds (Fig. 4E)
Differences in band intensity were quantified to determine the relative amounts of precipitated crystallin proteins in the two strains (Fig. 5) . Cx50-knockout animals from the 129S6 genetic background displayed significantly increased amounts of αA-, αB-, and β-crystallin proteins when compared with Cx50-knockout mice from the C57BL/6J background (Student’s t-test, P < 0.05). In a similar fashion, γ-crystallin was qualitatively more abundant in the 129S6 background, but statistical significance could not be ascertained because of the lack of sufficient antibody to perform multiple analyses. As a loading control for the insoluble fractions, we analyzed levels of MIP, and they not differ significantly between 129S6 and C57BL/6J lenses (P = 0.75). Thus, 129S6 Cx50-knockout mice displayed a significant increase in the amount of precipitated crystallins when compared with levels in the C57BL/6J animals. This finding is consistent with the more prominent opacities that were observed in the 129S6 knockout lenses, and supports the notion that a genetic modifier results in milder cataracts in the C57BL/6J mice. 
Analysis of Connexin Synthesis
One possible explanation for the difference in cataractogenesis between Cx50-knockout mice in the 129S6 and C57BL/6J backgrounds is that different levels of Cx46 are normally present in the two strains. To test whether Cx46 was differentially expressed in the Cx50-knockout mice, the insoluble fractions used for crystallin analysis were probed with antibodies to Cx50 and Cx46. Western blot analysis of the insoluble fractions confirmed that Cx50 was absent in knockout animals from both genetic backgrounds, whereas wild-type animals continued to synthesize Cx50 protein (Fig. 6A) . The expression of Cx46 was qualitatively similar in Cx50-knockout animals of both genetic backgrounds and comparable to that in wild-type mice (Fig. 6B) . Consistent with many prior studies, there was no apparent upregulation of Cx46 in the absence of Cx50, and there were no differences in Cx46 expression between the C57BL/6J and 129S6 mice. 16 18 19 24 Thus, differential levels of Cx46 cannot explain the variation in Cx50-knockout-induced cataracts in the two mouse strains. 
Lens Histology
The differences in lens opacities due to genetic background suggest that different cellular pathologic disorders may have occurred in the two genetic backgrounds. To determine pathologic differences, histologic sections of mouse eyes were stained with hematoxylin and eosin. Sections of postnatal day-7 wild-type eyes revealed normal ocular development without cellular damage (Fig. 7A) , particularly in the core of the lens (Fig. 7D) , where cataracts occurred in the Cx50-knockout mice. In contrast, sections of Cx50-knockout eyes displayed disruption of the normally uniform staining in the lens core (Figs. 7B 7C , arrows) and increased cellular damage in the nuclear region with apparent differences between the two genetic backgrounds. In the 129S6 mice there was an abrupt transition between healthy fiber tissue and the disrupted core (Figs. 7B 7E) , whereas C57BL/6J lenses displayed a gradual transition into the zone of cellular damage (Figs. 7C 7F) . These histologic differences in the Cx50-knockout lenses from 129S6 or C57BL/6J backgrounds support the idea that a genetic modifier influences the cataract phenotype in Cx50-deficient mice. 
Discussion
Studies have shown that genetically altered mice display diverse phenotypes, which can be dependent on the genetic background of the knockout animals. 23 25 Deletion of Cx50 in mice produces mild nuclear cataracts and an approximately 42% reduction in lens size. 18 19 In the current study, Cx50-dependent growth deficiency persisted, regardless of genetic background, whereas cataracts were different in the 129S6 and C57BL/6J mice. These results suggest the two elements of the Cx50-knockout phenotype are independent of each other, and a genetic modifier influences cataractogenesis, whereas growth is strictly dependent on Cx50-mediated intercellular communication. 
A genetic modifier has also been shown to influence cataractogenesis in mice with a deletion of Cx46, 23 raising the question of whether a common mechanism could be responsible, regardless of which lens connexin is deleted. Studies with Cx46-knockout mice from the 129S4 and C57BL/6J backgrounds have revealed that cataracts are also more severe in the 129S4 mice than in the C57BL/6J mice. Cataract severity also correlates well with increased cleavage of γ-crystallin in 129S4 Cx46 knockouts compared with C57BL/6J mice, suggesting that the modifier may have influenced crystallin stability. 23 However, deletion of Cx50 produces cataracts without the cleavage of γ-crystallin, 18 19 and the observed differences in cataractogenesis between strains appear to be independent of crystallin proteolysis. Therefore, if a common genetic modifier influences cataractogenesis resulting from deficient gap junctional communication, crystallin proteolysis must be a secondary consequence of its activity, because the mechanism of cataractogenesis is clearly different in Cx46- and Cx50-knockout mice. 
The γ-crystallin cleavage in the 129S4 Cx46-knockouts results from an increased calcium concentration in the lens core and activity of the calcium-dependent protease Lp82. 21 The two mouse strains may differ in their ability to buffer increased concentrations of cytoplasmic calcium with different consequences in the two knockout models. In Cx50-knockouts, higher Ca2+ could lead to γ-crystallin precipitation without cleavage, whereas in Cx46-knockouts elevated Ca2+ activates Ca-dependent proteases. These diverse effects could result from differences in the magnitude or spatial arrangement of coupling provided by these two distinct connexins. 24 Thus, it is possible that a common genetic modifier contributes to cataract severity in the two knockout models by influencing calcium homeostasis. This hypothesis could be tested by measuring cytoplasmic calcium in wild-type and knockout lenses from the two models in the two mouse strains. 
Although genetic modifier(s) that influence cataracts in mice with connexin deletions have been observed (Ref. 23 and the present work), their chromosomal location is not currently known. Recent evidence has shown that mutations in the Cx50 gene are also capable of generating cataracts in rats and mice, 26 27 28 29 and in one of these studies, a modifier gene was identified. Yamashita et al., 29 found that the presence of cataracts segregated with a Cx50 mutation on rat chromosome 2 (Uca), but that the age of onset of cataractogenesis significantly varied between the animals. This variation was mapped to a particular region on rat chromosome 5, thus identifying a genetic modifier (Ucad1) on a different chromosome than Cx50, which influenced the onset of cataractogenesis caused by a Cx50 mutation. These results clearly demonstrate the presence of a genetic modifier that influences the cataract phenotype resulting from deficient gap junctional communication. Although it is too early to tell whether the same genetic modifier influences cataract severity in the connexin-knockout animals, the corresponding Ucad1 locus on mouse chromosome 4 would be a good candidate region to examine. 
In contrast to the dissimilarity of the opacities in different strains, growth of Cx50-knockout lenses was equally impaired in either genetic background, suggesting that the growth defect was independent of cataractogenesis. In support of this view, we have recently shown that Cx46 can differentially rescue the two aspects of the Cx50-knockout phenotype. Replacement of the Cx50 coding region with Cx46 by gene knock-in restored optical clarity, but not lens growth. 22 The persistence of a growth deficiency in these mice, despite rescue of the cataract phenotype, further supports the hypothesis that although both of these defects depend on Cx50, they are independent of each other. In the present study, reductions in eye and lens size were identical in the 129S6 and C57BL/6J Cx50-knockouts at all ages examined, demonstrating that the kinetics, as well as magnitude of the growth defect were unaffected by genetic background. The precise mechanism whereby Cx50 influences lens growth remains to be elucidated; however, integration of the results of the present study with those in a growing body of work from several laboratories leads to the conclusion that Cx46 and Cx50 fulfill unique roles in lens physiology and that the lens can segregate the contributions of gap-junctional communication to the control of normal growth and to the maintenance of clarity. 
 
Figure 1.
 
Eye and lens growth in Cx50-knockout mice. The mass of the animals, eyes, and lenses were recorded as a function of age. (A) Overall postnatal growth was similar between wild-type and knockout animals in both genetic backgrounds. (B) Eye growth in Cx50-knockout mice, in each genetic background significantly lagged that of wild-type mice. (C) Lens growth was also severely impaired in Cx50-knockout mice in each genetic background. The mass of Cx50-knockout eyes and lenses were not significantly different between the two genetic backgrounds, in both cases knockout lenses displayed a ∼42% reduction in growth when compared with wild-type mice. Data are the mean ± SD (n = 4 to 8 animals at each time point).
Figure 1.
 
Eye and lens growth in Cx50-knockout mice. The mass of the animals, eyes, and lenses were recorded as a function of age. (A) Overall postnatal growth was similar between wild-type and knockout animals in both genetic backgrounds. (B) Eye growth in Cx50-knockout mice, in each genetic background significantly lagged that of wild-type mice. (C) Lens growth was also severely impaired in Cx50-knockout mice in each genetic background. The mass of Cx50-knockout eyes and lenses were not significantly different between the two genetic backgrounds, in both cases knockout lenses displayed a ∼42% reduction in growth when compared with wild-type mice. Data are the mean ± SD (n = 4 to 8 animals at each time point).
Figure 2.
 
Nuclear cataracts in young Cx50-knockout mice. Eight-week-old lenses from each genotype were dissected and photographed for analysis of opacities. Wild-type lenses displayed no opacities when viewed either through the anterior surface (A) or on equatorial edge (B). Cx50-knockout mice in the 129S6 genetic background (C, D) displayed pulverulent nuclear cataracts (arrows) with a profound lamellar opacity in the lens cortex (arrowheads). In contrast, Cx50-knockout mice from the C57BL/6J genetic background (E, F) showed dense nuclear opacities that were markedly different from the pulverulent cataracts in mice from the 129S6 genetic background (arrows) and also showed no cortical lamellar cataract.
Figure 2.
 
Nuclear cataracts in young Cx50-knockout mice. Eight-week-old lenses from each genotype were dissected and photographed for analysis of opacities. Wild-type lenses displayed no opacities when viewed either through the anterior surface (A) or on equatorial edge (B). Cx50-knockout mice in the 129S6 genetic background (C, D) displayed pulverulent nuclear cataracts (arrows) with a profound lamellar opacity in the lens cortex (arrowheads). In contrast, Cx50-knockout mice from the C57BL/6J genetic background (E, F) showed dense nuclear opacities that were markedly different from the pulverulent cataracts in mice from the 129S6 genetic background (arrows) and also showed no cortical lamellar cataract.
Figure 3.
 
Nuclear cataracts in older Cx50-knockout mice. Six-month-old lenses from Cx50-knockout animals in both genetic backgrounds were dissected and photographed for analysis. Lenses from 129S6 mice (A, B) showed little difference in the severity or pattern of crystallin precipitation from that observed at 8 weeks of age, displaying pulverulent nuclear cataracts (arrows) with a profound lamellar opacity in the lens cortex (arrowheads). Adult lenses from Cx50-knockout animals in the C57BL/6J genetic background differed from the 129S6 mice in the same manner as the younger lenses, displaying an irregular nuclear cataract (arrows) and an absence of lamellar opacity (C, D).
Figure 3.
 
Nuclear cataracts in older Cx50-knockout mice. Six-month-old lenses from Cx50-knockout animals in both genetic backgrounds were dissected and photographed for analysis. Lenses from 129S6 mice (A, B) showed little difference in the severity or pattern of crystallin precipitation from that observed at 8 weeks of age, displaying pulverulent nuclear cataracts (arrows) with a profound lamellar opacity in the lens cortex (arrowheads). Adult lenses from Cx50-knockout animals in the C57BL/6J genetic background differed from the 129S6 mice in the same manner as the younger lenses, displaying an irregular nuclear cataract (arrows) and an absence of lamellar opacity (C, D).
Figure 4.
 
Biochemical analysis of crystallin solubility. Lenses were separated into soluble and insoluble fractions and subjected to Western blot analysis. Equal volumes of lens homogenates were probed with antibodies specific for αA-(A), αB-(B), β-(C), and γ-crystallin (D). Crystallin proteins were abundant in the soluble fractions of wild-type and knockout animals, as expected. The insoluble fractions of wild-type mice contained little or no crystallin proteins, whereas the insoluble fractions of the 129Sv and C57BL/6J knockout mice contained precipitates of all three crystallin proteins. In addition, for each crystallin examined, there was qualitatively more present in the insoluble fraction from 129S6 than C57BL/6J knockout lenses. MIP expression levels (E) were unchanged in all mice, independent of the deletion of Cx50.
Figure 4.
 
Biochemical analysis of crystallin solubility. Lenses were separated into soluble and insoluble fractions and subjected to Western blot analysis. Equal volumes of lens homogenates were probed with antibodies specific for αA-(A), αB-(B), β-(C), and γ-crystallin (D). Crystallin proteins were abundant in the soluble fractions of wild-type and knockout animals, as expected. The insoluble fractions of wild-type mice contained little or no crystallin proteins, whereas the insoluble fractions of the 129Sv and C57BL/6J knockout mice contained precipitates of all three crystallin proteins. In addition, for each crystallin examined, there was qualitatively more present in the insoluble fraction from 129S6 than C57BL/6J knockout lenses. MIP expression levels (E) were unchanged in all mice, independent of the deletion of Cx50.
Figure 5.
 
Quantitation of crystallin precipitation in the 129S6 and C57BL/6J genetic backgrounds. Cx50-knockout mice from the 129S6 genetic background displayed significantly greater precipitation of αA-, αB-, and β crystallins that did Cx50-knockouts in the C57BL/6J genetic background (Student’s t-test, P < 0.05). Precipitation of γ-crystallin was also reduced in the C57BL/6J strain in one experiment. Relative amounts of MIP in the insoluble fractions were not significantly different. Data are the mean ± SD of the indicated number of experiments.
Figure 5.
 
Quantitation of crystallin precipitation in the 129S6 and C57BL/6J genetic backgrounds. Cx50-knockout mice from the 129S6 genetic background displayed significantly greater precipitation of αA-, αB-, and β crystallins that did Cx50-knockouts in the C57BL/6J genetic background (Student’s t-test, P < 0.05). Precipitation of γ-crystallin was also reduced in the C57BL/6J strain in one experiment. Relative amounts of MIP in the insoluble fractions were not significantly different. Data are the mean ± SD of the indicated number of experiments.
Figure 6.
 
Analysis of connexin synthesis. Equal volumes of the lens fractions used in Figure 3 were probed with antibodies specific for Cx50 (A) and Cx46 (B). Cx50 was present in the insoluble fraction of wild-type animals only, confirming that the Cx50 gene was deleted. Cx46 expression levels were unchanged in all mice, independent of the deletion of Cx50.
Figure 6.
 
Analysis of connexin synthesis. Equal volumes of the lens fractions used in Figure 3 were probed with antibodies specific for Cx50 (A) and Cx46 (B). Cx50 was present in the insoluble fraction of wild-type animals only, confirming that the Cx50 gene was deleted. Cx46 expression levels were unchanged in all mice, independent of the deletion of Cx50.
Figure 7.
 
Pathologic changes in postnatal Cx50-knockout lenses. Eyes were fixed, serially sectioned, and stained with hematoxylin and eosin. (A) One-week-old wild-type eyes had normal ocular development and the lenses were uniformly stained with eosin throughout the core region. In contrast, knockout lenses exhibited a large clear zone in their nuclear region (B, C, arrows). At higher magnification, wild-type lens fibers were homogeneously labeled (D), whereas the clear zone in knockout lenses was composed of fiber cells that had lost the eosinophilic staining (E, F). In addition, the transition between stained and unstained regions was abrupt in the 129S6 background (B, E) and more gradual in the C57BL/6J mice (C, F).
Figure 7.
 
Pathologic changes in postnatal Cx50-knockout lenses. Eyes were fixed, serially sectioned, and stained with hematoxylin and eosin. (A) One-week-old wild-type eyes had normal ocular development and the lenses were uniformly stained with eosin throughout the core region. In contrast, knockout lenses exhibited a large clear zone in their nuclear region (B, C, arrows). At higher magnification, wild-type lens fibers were homogeneously labeled (D), whereas the clear zone in knockout lenses was composed of fiber cells that had lost the eosinophilic staining (E, F). In addition, the transition between stained and unstained regions was abrupt in the 129S6 background (B, E) and more gradual in the C57BL/6J mice (C, F).
The authors thank Richard Mathias for critically reading the manuscript. 
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Figure 1.
 
Eye and lens growth in Cx50-knockout mice. The mass of the animals, eyes, and lenses were recorded as a function of age. (A) Overall postnatal growth was similar between wild-type and knockout animals in both genetic backgrounds. (B) Eye growth in Cx50-knockout mice, in each genetic background significantly lagged that of wild-type mice. (C) Lens growth was also severely impaired in Cx50-knockout mice in each genetic background. The mass of Cx50-knockout eyes and lenses were not significantly different between the two genetic backgrounds, in both cases knockout lenses displayed a ∼42% reduction in growth when compared with wild-type mice. Data are the mean ± SD (n = 4 to 8 animals at each time point).
Figure 1.
 
Eye and lens growth in Cx50-knockout mice. The mass of the animals, eyes, and lenses were recorded as a function of age. (A) Overall postnatal growth was similar between wild-type and knockout animals in both genetic backgrounds. (B) Eye growth in Cx50-knockout mice, in each genetic background significantly lagged that of wild-type mice. (C) Lens growth was also severely impaired in Cx50-knockout mice in each genetic background. The mass of Cx50-knockout eyes and lenses were not significantly different between the two genetic backgrounds, in both cases knockout lenses displayed a ∼42% reduction in growth when compared with wild-type mice. Data are the mean ± SD (n = 4 to 8 animals at each time point).
Figure 2.
 
Nuclear cataracts in young Cx50-knockout mice. Eight-week-old lenses from each genotype were dissected and photographed for analysis of opacities. Wild-type lenses displayed no opacities when viewed either through the anterior surface (A) or on equatorial edge (B). Cx50-knockout mice in the 129S6 genetic background (C, D) displayed pulverulent nuclear cataracts (arrows) with a profound lamellar opacity in the lens cortex (arrowheads). In contrast, Cx50-knockout mice from the C57BL/6J genetic background (E, F) showed dense nuclear opacities that were markedly different from the pulverulent cataracts in mice from the 129S6 genetic background (arrows) and also showed no cortical lamellar cataract.
Figure 2.
 
Nuclear cataracts in young Cx50-knockout mice. Eight-week-old lenses from each genotype were dissected and photographed for analysis of opacities. Wild-type lenses displayed no opacities when viewed either through the anterior surface (A) or on equatorial edge (B). Cx50-knockout mice in the 129S6 genetic background (C, D) displayed pulverulent nuclear cataracts (arrows) with a profound lamellar opacity in the lens cortex (arrowheads). In contrast, Cx50-knockout mice from the C57BL/6J genetic background (E, F) showed dense nuclear opacities that were markedly different from the pulverulent cataracts in mice from the 129S6 genetic background (arrows) and also showed no cortical lamellar cataract.
Figure 3.
 
Nuclear cataracts in older Cx50-knockout mice. Six-month-old lenses from Cx50-knockout animals in both genetic backgrounds were dissected and photographed for analysis. Lenses from 129S6 mice (A, B) showed little difference in the severity or pattern of crystallin precipitation from that observed at 8 weeks of age, displaying pulverulent nuclear cataracts (arrows) with a profound lamellar opacity in the lens cortex (arrowheads). Adult lenses from Cx50-knockout animals in the C57BL/6J genetic background differed from the 129S6 mice in the same manner as the younger lenses, displaying an irregular nuclear cataract (arrows) and an absence of lamellar opacity (C, D).
Figure 3.
 
Nuclear cataracts in older Cx50-knockout mice. Six-month-old lenses from Cx50-knockout animals in both genetic backgrounds were dissected and photographed for analysis. Lenses from 129S6 mice (A, B) showed little difference in the severity or pattern of crystallin precipitation from that observed at 8 weeks of age, displaying pulverulent nuclear cataracts (arrows) with a profound lamellar opacity in the lens cortex (arrowheads). Adult lenses from Cx50-knockout animals in the C57BL/6J genetic background differed from the 129S6 mice in the same manner as the younger lenses, displaying an irregular nuclear cataract (arrows) and an absence of lamellar opacity (C, D).
Figure 4.
 
Biochemical analysis of crystallin solubility. Lenses were separated into soluble and insoluble fractions and subjected to Western blot analysis. Equal volumes of lens homogenates were probed with antibodies specific for αA-(A), αB-(B), β-(C), and γ-crystallin (D). Crystallin proteins were abundant in the soluble fractions of wild-type and knockout animals, as expected. The insoluble fractions of wild-type mice contained little or no crystallin proteins, whereas the insoluble fractions of the 129Sv and C57BL/6J knockout mice contained precipitates of all three crystallin proteins. In addition, for each crystallin examined, there was qualitatively more present in the insoluble fraction from 129S6 than C57BL/6J knockout lenses. MIP expression levels (E) were unchanged in all mice, independent of the deletion of Cx50.
Figure 4.
 
Biochemical analysis of crystallin solubility. Lenses were separated into soluble and insoluble fractions and subjected to Western blot analysis. Equal volumes of lens homogenates were probed with antibodies specific for αA-(A), αB-(B), β-(C), and γ-crystallin (D). Crystallin proteins were abundant in the soluble fractions of wild-type and knockout animals, as expected. The insoluble fractions of wild-type mice contained little or no crystallin proteins, whereas the insoluble fractions of the 129Sv and C57BL/6J knockout mice contained precipitates of all three crystallin proteins. In addition, for each crystallin examined, there was qualitatively more present in the insoluble fraction from 129S6 than C57BL/6J knockout lenses. MIP expression levels (E) were unchanged in all mice, independent of the deletion of Cx50.
Figure 5.
 
Quantitation of crystallin precipitation in the 129S6 and C57BL/6J genetic backgrounds. Cx50-knockout mice from the 129S6 genetic background displayed significantly greater precipitation of αA-, αB-, and β crystallins that did Cx50-knockouts in the C57BL/6J genetic background (Student’s t-test, P < 0.05). Precipitation of γ-crystallin was also reduced in the C57BL/6J strain in one experiment. Relative amounts of MIP in the insoluble fractions were not significantly different. Data are the mean ± SD of the indicated number of experiments.
Figure 5.
 
Quantitation of crystallin precipitation in the 129S6 and C57BL/6J genetic backgrounds. Cx50-knockout mice from the 129S6 genetic background displayed significantly greater precipitation of αA-, αB-, and β crystallins that did Cx50-knockouts in the C57BL/6J genetic background (Student’s t-test, P < 0.05). Precipitation of γ-crystallin was also reduced in the C57BL/6J strain in one experiment. Relative amounts of MIP in the insoluble fractions were not significantly different. Data are the mean ± SD of the indicated number of experiments.
Figure 6.
 
Analysis of connexin synthesis. Equal volumes of the lens fractions used in Figure 3 were probed with antibodies specific for Cx50 (A) and Cx46 (B). Cx50 was present in the insoluble fraction of wild-type animals only, confirming that the Cx50 gene was deleted. Cx46 expression levels were unchanged in all mice, independent of the deletion of Cx50.
Figure 6.
 
Analysis of connexin synthesis. Equal volumes of the lens fractions used in Figure 3 were probed with antibodies specific for Cx50 (A) and Cx46 (B). Cx50 was present in the insoluble fraction of wild-type animals only, confirming that the Cx50 gene was deleted. Cx46 expression levels were unchanged in all mice, independent of the deletion of Cx50.
Figure 7.
 
Pathologic changes in postnatal Cx50-knockout lenses. Eyes were fixed, serially sectioned, and stained with hematoxylin and eosin. (A) One-week-old wild-type eyes had normal ocular development and the lenses were uniformly stained with eosin throughout the core region. In contrast, knockout lenses exhibited a large clear zone in their nuclear region (B, C, arrows). At higher magnification, wild-type lens fibers were homogeneously labeled (D), whereas the clear zone in knockout lenses was composed of fiber cells that had lost the eosinophilic staining (E, F). In addition, the transition between stained and unstained regions was abrupt in the 129S6 background (B, E) and more gradual in the C57BL/6J mice (C, F).
Figure 7.
 
Pathologic changes in postnatal Cx50-knockout lenses. Eyes were fixed, serially sectioned, and stained with hematoxylin and eosin. (A) One-week-old wild-type eyes had normal ocular development and the lenses were uniformly stained with eosin throughout the core region. In contrast, knockout lenses exhibited a large clear zone in their nuclear region (B, C, arrows). At higher magnification, wild-type lens fibers were homogeneously labeled (D), whereas the clear zone in knockout lenses was composed of fiber cells that had lost the eosinophilic staining (E, F). In addition, the transition between stained and unstained regions was abrupt in the 129S6 background (B, E) and more gradual in the C57BL/6J mice (C, F).
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