Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts

Abstract

In the ocular lens, gap junctional communication is a key component of homeostatic mechanisms preventing cataract formation. Gap junctions in rodent lens fibers contain two known intercellular channel-forming proteins, connexin50 (Cx50) and Cx46. Since targeted ablation of Cx46 has been shown to cause senile-type nuclear opacities, it appears that Cx50 alone cannot meet homeostatic requirements. To determine if lens pathology arises from a reduction in levels of communication or the loss of a connexin-specific function, we have generated mice with a targeted deletion of the Cx50 gene. Cx50-null mice exhibited microphthalmia and nuclear cataracts. At postnatal day 14 (P14), Cx50-knockout eyes weighed 32% less than controls, whereas lens mass was reduced by 46%. Cx50-knockout lenses also developed zonular pulverulent cataracts, and lens abnormalities were detected by P7. Deletion of Cx50 did not alter the amounts or distributions of Cx46 or Cx43, a component of lens epithelial junctions. In addition, intercellular passage of tracers revealed the persistence of communication between all cell types in the Cx50-knockout lens. These results demonstrate that Cx50 is required not only for maintenance of lens transparency but also for normal eye growth. Furthermore, these data indicate that unique functional properties of both Cx46 and Cx50 are required for proper lens development.

Figure 1

Targeting strategy for inactivation of the Cx50 gene and genotyping of generated mice. (a) The genomic structure near Cx50, whose coding region is contained within a single exon. A replacement targeting vector containing a pgk-neo cassette was flanked by 5′ and 3′ homology regions of 1.3 and 7.1 kb, respectively, and included a pgk-TK cassette downstream of the 5′ homology region. (b) For genotyping, a 5′ flanking primer (pcr 1) was paired with either a 3′ primer derived from specific sequences in the replacement cassette (pcr 2) or a primer derived from the Cx50 coding region (pcr 3). Primers 1 + 2 amplified a 1,370-bp band from knockout chromosomes. Amplification of wild-type chromosomes with primers 1 + 3 produced a 1,600-bp band. Genomic structure was confirmed by probing Southern blots of Nco1-digested ES cell or tail DNA with a fragment from the 5′ homology region common to both alleles. A 5.5-kb band was expected for wild-type alleles, and a 3.8-kb band was diagnostic of knockout alleles. Only relevant restriction enzyme sites are shown: N, Nco1; S, Sac1; M, Msc1; B, BamH1.

Figure 2

Immunohistochemical analysis of connexin expression in control and knockout lenses. (a) No signal was detected in knockout lenses with an anti-Cx50 antibody, whereas control littermates (b) showed a typical macular labeling of fiber cell membranes. (c) Cx46 continued to be expressed in the Cx50-knockout lenses with a fiber cell distribution similar to that seen in control lenses (d). Therefore, the loss of Cx50 did not grossly affect the distribution of Cx46 in lens fibers.

Figure 3

Cx43 and Cx46 expression levels in Cx50-knockout lens. Aliquots of membrane preparations of wild-type (lanes 1, 3, and 5) and knockout (lanes 2, 4, and 6) lenses containing equal protein concentrations were Western blotted with antibodies to Cx43 (lanes 1 and 2), Cx46 (lanes 3 and 4), and Cx50 (lanes 5 and 6). Both Cx43 and Cx46 continued to be expressed in knockout lenses at similar levels to those detected in the wild-type samples. As expected, Cx50 was not detected in the knockout lens.

Figure 4

Cx50-knockout mice are microphthalmic. Gross anatomical examination revealed that the eyes and lenses of Cx50-knockout mice were abnormally small in comparison to those from control littermates. To quantitate these differences, eyes, lenses, and other control organs were dissected from adult male control (n = 17) or Cx50-knockout (n = 19) mice and weighed. No significant differences were observed in the average mass of the animals, their kidneys, or their testes. In contrast, the eyes dissected from Cx50-knockout mice were 32% smaller, and the lenses were 46% smaller when compared with control littermates. Thus, deletion of the Cx50 gene resulted in an ocular growth deficiency. Values are the mean ± SD.

Figure 5

Lens and eye growth in Cx50-knockout mice. The mass of animals, eyes, and lenses were recorded as a function of age. (a) Overall postnatal growth was identical for Cx50-knockout and control mice. (b and c) Both eyes and lenses in the knockout animals grew over time, but the growth lagged that of control mice. (d) At P0, knockout eyes were identical to controls. At P7, knockout eyes weighed 82% as much as wild type, and by P14 the mass of knockout eyes was only 68% of control, a ratio which then remained constant throughout adulthood. Reduction of lens growth was even more striking. Again, masses were identical at P0, but by P7 the mass ratio was 56%, which remained constant thereafter. Thus, a transient inhibition of lens growth occurs in the first postnatal week in Cx50 knockouts. n = 6–20 at each time point. Values are the mean ± SEM.

Figure 6

Electron microscopy of lens fibers. Coronal sections through the lens equator of Cx50^−/− (KO) and Cx50^+/+ (WT) lenses revealed cross sections of synthetically active lens fibers with polyribosomes and mitochondria. Measurements of fiber diameter in these specimens revealed no significant differences between wild-type and knockout lenses. Bar, 1 μm.

Figure 7

Nuclear cataracts in Cx50-knockout mice. (a) Lenses dissected from Cx50-knockout mice at P14 were smaller than those of control littermates (b) and exhibited an irregular and diffuse nuclear opacity (a, arrow) not present in control lenses. By 6 mo of age (c), knockout lenses had a fine particulate precipitate confined to the nucleus (c, arrow), characteristic of zonular pulverulent cataracts. In addition, a sharply defined line in the inner cortical region of the lens was present (arrowhead), the nature of which is not clear. (d) 6-mo-old control lenses had no light scattering opacities. Bar, 1 mm.

Figure 8

Growth deficiencies and pathological changes in Cx50-knockout lenses occur postnatally. Tissues were fixed, serially sectioned, and stained with hematoxylin and eosin. At E16.5, there were no differences in the size, or integrity of lenses from control (a) and Cx50-knockout (b) embryos. At P7, control lenses had grown considerably, and the lens nuclei were stained with eosin throughout the lens (c), whereas knockout lenses were much smaller and exhibited a large clear zone in their nuclear region (d). At higher magnification (e), this clear zone was composed of fiber cells lacking the normal eosinophilic staining.

Figure 9

The effect of the loss of Cx50 on the solubility and stability of crystallin proteins. Wild-type (lanes 1–5) and knockout (lanes 6–10) lenses were homogenized and separated into soluble supernatants (lanes 1 and 6) and pellets by centrifugation. The pellets were then sequentially washed (lanes 2 and 7), extracted with NaOH (lanes 3 and 8), washed again (lanes 4 and 9), and the final insoluble pellets were resuspended (lanes 5 and 10). Equal volumes of all steps of the fractionation were Western blotted with anti-crystallin antibodies. αA-crystallin (a) and αB-crystallin (b) were detected in the insoluble fraction of Cx50-knockout lenses, but not wild-type lenses. (c) Differences in the solubility of γ-crystallin were also detected and knockout lenses consistently showed a higher molecular weight aggregate of this protein. In contrast to the Cx46 knockout, no lower molecular weight proteolytic fragments of γ-crystallin were detected with the γ-crystallin antibody in the Cx50-knockout lens. (d). Silver-stained gel of the extracted lens fractions. The majority of insoluble proteins in the knockout lens were in the molecular weight range of the crystallins, although additional minor bands were also present.

Figure 10

Targeted ablation of Cx50 did not eliminate gap junctional communication between epithelial cells, between fiber cells, or between the epithelium and the fibers. Intercellular communication in E15.5 embryonic lenses was assessed by intracellular injection of low molecular mass tracers. Single lens fibers were impaled with microelectrodes containing Lucifer yellow and neurobiotin. During the injections into whole lenses, the spread of Lucifer yellow (green) between fibers was readily detected in both knockout (a) and control (b) lenses. After injection, lenses were fixed and paraffin sections were stained with rhodamine-avidin to label neurobiotin (red). Extensive transfer of neurobiotin to many neighboring fiber cells was observed in both the knockout (c) and control (d) lenses. Epithelial to fiber communication was tested by microinjection of single epithelial cells with Lucifer yellow and neurobiotin. (e) Injection of a single epithelial cell in a knockout lens resulted in a weak transfer of Lucifer yellow to neighboring epithelial cells, but transfer to the underlying fibers was below the threshold of detection. In contrast, the neurobiotin dispersed widely through the epithelial cell layer and was easily detected in the underlying lens fibers (f).

Figure 11

Electron microscopy of the epithelium/fiber interface in a Cx50^+/+ lens. Occasional heptalaminar gap junctions (arrowhead) were found joining epithelial cells and lens fibers. Epithelial cell apical surfaces were characterized by a terminal web of actin filaments (TW) and the fiber (F) by its dense protein-rich cytoplasm and lack of cellular organelles. Bar, 60 nm.