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
*Author for correspondence (e-mail: gong{at}scripps.edu)
Accepted 3 October 2001
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SUMMARY |
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Key words: Connexin, Lens, Microphthalmia, Cataract, Mouse
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INTRODUCTION |
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The differentiating fibers eventually lose all their intracellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, etc., to become mature fibers in the deep cortical region of the lens (Bassnett and Beebe, 1992). This maturation of the fibers is one of the critical properties of the lens in its function of transmitting a light image onto the retina. If there are intracellular organelles in the interior region of the lens, this will cause light scattering, or so-called cataracts. Because the interior mature fibers lose all their intracellular organelles, they have an extremely low metabolic activity and depend mainly on the epithelium and peripheral differential fibers for maintenance. Therefore, out of necessity, the lens has developed a sophisticated cell-cell communication network that includes gap junction channels, which facilitate both an active metabolism and the transport of small metabolites, such as ions, water and secondary messengers, in order to maintain its transparency (Goodenough, 1992; Mathias et al., 1997).
The development and function of the vertebrate lens utilizes gap junction channels that are formed by the products of at least three connexin genes: 1 (Cx43),
3 (Cx46) and
8 (Cx50) (Beyer et al., 1987; Kistler et al., 1988; Paul et al., 1991; White et al., 1992). Connexin
1 is restrictively expressed in the lens epithelial cells, and its protein can also be detected in the newly differentiating cells in the bow region of the lens. The lens fibers mainly utilize
3 and
8 connexins, which are two connexin isoforms colocalized in the same gap junction plaque in the plasma membrane of fiber cells (Gong et al., 1997; Benedetti et al., 2000).
8 connexin has also been detected in lens epithelial cells (Dahm et al., 1999), and this suggests that different regulatory machinery may control the expression of
3 and
8 connexin genes in lens epithelial cells, despite the fact that the same factors may control their expression in fiber cells.
In one of our previous studies, we reported that Gja3tm1 (3/) mutant mice developed nuclear cataracts to varying degrees, depending on the genetic background of the strain (Gong et al., 1997; Gong et al., 1999). In order to compare the functional roles of
3 and
8 connexin in vivo, in this study we have generated
8 knockout mice with a knock-in lacZ reporter gene, similar to that in our previous studies on
3 knockout mice with a knock-in lacZ reporter gene (Gong et al., 1997). In this way, we were able to: (1) compare the phenotypic changes between
8 and
3 knockout mice; (2) examine the expression pattern of the knock-in lacZ reporter gene during lens development in the two knockout mice in parallel; and (3) try to find distinctive morphological and biochemical alterations in the two knockout mice. This study provides some important insights into the utilization of both
8 and
3 connexins in the lens.
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MATERIALS AND METHODS |
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Histological, immunohistochemical and lacZ expression analysis
Standard histological methods were used for analyzing different mutant mice (Lovicu and Overbeek, 1998). The ß-galactosidase staining method was carried out as previously described (Bonnerot and Nicholas, 1993).
Frozen sections of lenses were prepared and used for the antibody staining, following the procedure described in our previous paper (Gong et al., 1997). We used a mouse monoclonal antibody (6-4-B2-C6, provided by Dr Kistler) (Bond et al., 1996) for 8 connexin and a rabbit polyclonal antibody against the intracellular loop region of the
3 connexin, the same antibody used in a previous study (Gong et al., 1997) for double immunolabeling. A deconvolution light microscope (Delta Vision Optical Sectioning Microscope Model 283) was used to examine the fluorescence of the indirect immunostaining.
Electron microscopic analysis
For freeze-fracture electron microscopic analysis, the lenses were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 5 days, then processed and analyzed as described previously (Benedetti et al., 2000; Risek et al., 1994). For transmission electron microscopic analysis, the lenses were postfixed in 1% OsO4, stained with 0.5% tannic acid/0.05 M cacodylate buffer, and neutralized with 1% Na2SO4 in 0.1 M cacodylate buffer. The lenses were then stained en bloc with 1% uranyl acetate/10% ethanol, and further dehydrated in a standard ethanol series (the ethanol was exchanged with HPMA). Thereafter, the lenses were infiltrated overnight in a 1:1 HPMA/LX112 (Ladd Scientific) mixture while rotating, immersed in 100% LX112 for 4 hours, then embedded in LX112. They were polymerized for 24-36 hours at 60°C. A standard method was used for thin sectioning and they were examined with a Philips CM100 electron microscope.
Western blotting
Different lens homogenates were prepared as described previously (Gong et al., 1997; Gong et al., 1999). For quantitative western blotting, the lens proteins were dissolved in lysis buffer (20 mM Tris (pH7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM PMSF), and the protein concentration was measured using a MicroBCA protein assay kit from Pierce Chemicals. Equal amounts of these lens protein samples were loaded and run onto SDS-PAGE, then proteins were transferred onto nitrocellulose membranes and detected using specific antibodies with standard protocols. Rabbit polyclonal antibodies against 8 connexin (generously provided by Dr Thomas White),
3 connexin and MP26 were used for western blotting.
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RESULTS |
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The 8+/ mice appeared to have normal eyes and lenses, while the
8/ mice developed microphthalmia with smaller lenses (Fig. 1C). The size and weight of the lenses of the adult
8/ mice were around 60% that of the lenses of their wild-type (+/+) littermates (n=20). Very mild nuclear cataracts were observed in the
8/ lenses of the adult mice (Fig. 1C).
No 8 connexin proteins were detected in the lens homogenates of adult
8/ mice, while half the amount of
8 connexin proteins, as determined by densitometric analysis (n>3), was detected in the lens homogenates of the
8+/ sibs, when compared to their wild-type counterparts (Fig. 2). According to western blot analysis, there were no obvious changes in the
3 connexin or the major lens membrane protein (MP26 or aquaporin-0) in the lens homogenates of
8/ mice, as compared to the
8+/+ and
8+/ mice (Fig. 2). Moreover, no additional changes in the
-, ß-, or
-crystallins were detected in the 3-week-old lenses using their specific antibodies (data not shown), aside from a reduction in the amount of total crystallins due to the small size of the
8/ lenses. Substantial degradation of the crystallins such as the cleavage of
-crystallin in the
3/ lenses (Gong et al., 1997) was not found in the
8/ lenses.
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Consistent with western blot results, no 8 protein was detected in the frozen lens sections from the
8/ mice by a double immunolabeling for
3 and
8, using their specific antibodies (Fig. 3B, right panel). Interestingly, the fluorescent spots for
3 connexin, detected in the
8/ sections, were similar in size to the small
3 connexin spots (green) in wild-type lens sections (Fig. 3B, left panel), but there was additional
3 connexin (orange) which was colocalized with the
8 connexin to form larger fluorescent spots.
Gap junctions in the 8/ lenses were further examined by both thin-section and freeze-fracture electron microscopy (Fig. 4). A much smaller gap junction was observed in the cortical fibers of the
8/ lenses than in those of wild-type lenses by EM. The largest gap junction that we observed from more than eight
8/ samples was around 60 nm in length while most of gap junctions in wild-type or
3/ lenses were 4 times longer (Gong et al., 1997).
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DISCUSSION |
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The different phenotypes of the lenses and eyes in 3 and
8 knockout mice suggest distinctive functional role(s) for
3 and
8 connexins in vivo. Connexin
3 is essential for maintaining lens transparency, while
8 is critical for the growth of the lens. Different expression patterns of
3 and
8 connexin genes were also reflected by the expression of the knock-in lacZ reporter gene in the lens epithelium of the knockout mice. More importantly, the loss of
8 connexin was associated with a delayed maturation process of the lens interior fibers, indicated by the retention of the cell nuclei. However, this was not observed in the
3-deficient lenses (Gong et al., 1997). The presence of the
8 connexin in the lens nucleus was dependent on the existence of
3 connexin. Interestingly, the large size of the gap junction plaques in the cortical fibers was dependent on the presence of
8 connexin. It is quite clear, then, that this study has demonstrated the importance of both
3 and
8 connexin in the lens to ensure its growth and the maintenance of its transparency.
The co-existence of 3 and
8 connexins in the lens has been verified in chick (Jiang et al., 1995), sheep (Yang et al., 2000), cattle (Gupta et al., 1994), mouse (Gong et al., 1997), rat (Paul et al., 1991), primate (Lo et al., 1996) and human (Church et al., 1995). Results from both the
3 and
8 knockout mice showed that the regulatory machinery of
3 and
8 connexin genes operate independently of each other, since there was no compensation in the protein levels of the
8 connexin when there was a loss of
3, and vice versa. Moreover, there was no compensation in the expression of the wild-type allele of
3 or
8 connexin in the heterozygous mice, since a 50% reduction in the amount of
8 or
3 connexin proteins was detected in the lens homogenates of their heterozygous knockout mice compared with the wild type counterpart. These results suggest that a precise regulatory mechanism exists to control the expression of
3 and
8 connexin genes in the lens.
It is very likely that the interactions and regulations between 3 and
8 connexins are at the posttranslational level in vivo. Both
3 and
8 connexin were detected in the same gap junctional plaques in fiber cells by indirect immunohistochemical staining of frozen sections (Gong et al., 1997) and immuno-gold labeling with an EM replica (Benedetti et al., 2000). The segregation of
3 connexin from
8 connexin in lens fibers, which was observed by deconvolution microscopy, supports the notion that lens gap junction plaques consist of a mixture of homomeric
3 and
8 channels as well as heteromeric channels. Our recent electrophysiological studies indicated that interior fibers are mainly coupled by homomeric
3 channels (Baldo et al., 2001). Moreover, the mixing of homomeric gap junction channels, which consist of different connexin isoforms in the same plaque, has been reported in a cultured cell system (Falk, 2000). According to freeze-fracture graphics and electrophysiological data, both types of knockout mice showed that the
3 and
8 connexin were able to form homomeric functional channels in vivo (Gong et al., 1997; Gong et al., 1998; Baldo et al., 2001). Heteromeric connexons have also been demonstrated biochemically in the lens (Konig et al., 1995; Jiang et al., 1996). In addition, homomeric, heteromeric and heterotypic channels formed by
3 and/or
8 connexins were characterized in paired Xenopus oocytes (Ebihara et al., 1999), as well as in a communication-deficient neuroblastoma (N2A) cell line, using an electrophysiological assay (Hopperstad et al., 2000). These results have indicated that diversified gap junction channels can be formed by the mixing of
3 and
8 connexins in vitro.
It is interesting to note that both the 3 and
8 connexin demonstrated dependence on one another in a variety of different ways in vivo. For example, the presence of
8 connexin in the lens nucleus was dependent on the existence of
3 connexin. This is supported by the fact that no
8 connexins were detected in the lens nucleus homogenate of the
3/ lenses (Fig. 4), even though the
8 connexin was able to form functional gap junction channels in the cortical fibers of those lenses (Gong et al., 1998). It is clear that the homomeric
8 gap junction channel was somehow eliminated in the interior fibers of the
3/ lenses. The mechanism for this is unknown. A simple explanation would be that only the
8 proteins formed heteromeric channels with the
3 connexin, or that the homomeric
8 channels that mixed with the homomeric
3 channels in the same junctional plaque (or domain) remained in the normal lens nucleus. It is also possible that the loss of
8 connexin in the
3/ lens nucleus was due to cataract formation in these lenses. Interestingly, the presence of
3 connexin in the lens nucleus was independent from
8 connexin (Fig. 4). Moreover, the
3 connexin in the
8/ lenses was able to form only the small gap junction plaques, which were much smaller than the gap junction plaques in either the wild-type (Fig. 5) or
3/ lenses (Gong et al., 1997). This suggests that a regulatory mechanism must be present to control the size of gap junction domains (plaques) formed by
3 and
8 connexins. As of yet, the mechanism that controls the size of gap junction plaques and the packing of gap junction particles in the plasma membrane has not been elucidated. Studies on either
3 or
8 gene knockout mice can verify only the functional role of the remaining homomeric gap junction channels formed by either
3 or
8 subunits, and not the heteromeric and heterotypic interactions between the two isoforms. Further studies are, therefore, required to verify the existence of these types of interactions in vivo, as well as to determine the physiological and biological importance of these interactions in the lens.
Surprisingly, we observed a delayed denucleation in the interior fibers of the 8/ lenses. The elimination of the cell nucleus of the interior fibers, as well as the other intracellular organelles, is an essential process for generating lens transparency, and the process is precisely regulated in the lens (Bassnett and Mataic, 1997; Dahm et al., 1998). It has been reported that the denucleation was a part of the apoptotic processes (Wride, 2000). Members of the bcl-2 and caspase families have been reported to be involved in the regulation of nuclear degeneration (Wride et al., 1999), but, the question still remains as to whether this was a simple correlative phenomenon, since the degradation process of the lens nucleus requires days to be completed, while a typical apoptosis only takes minutes to hours. So far, we do not know the molecular interactions linking the loss of
8 connexin and the denucleation process. It will be crucial to investigate the molecular changes associated with this delayed denucleation in the
8/) lenses. The
8 knockout mouse will undoubtedly provide a useful system for elucidating the mechanism that controls the maturation of lens fiber cells.
Genetic studies have shown that 3 and
8 gene mutations in humans (Shiels et al., 1998; Berry et al., 1999; Mackay et al., 1999; Rees et al., 2000), as well as one point mutant of
8 in mice (Steele et al., 1998), were linked to semi-dominant cataracts. This is inconsistent with the fact that both
3 and
8 knockout mice showed recessive cataractous phenotypes (Gong et al., 1997; White et al., 1998). Therefore, it will be important to generate or identify
3 and
8 point mutants with dominant cataracts so that we will be able to try to elucidate the molecular basis for the cataractogenesis linked to connexin mutations in both mice and humans.
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ACKNOWLEDGMENTS |
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