Connexin50 Is Essential for Normal Postnatal Lens Cell Proliferation

Abstract

purpose. Connexin50 (Cx50) is absolutely essential for normal postnatal lens growth. Deletion of Cx50 or replacement with Cx46 by knockin resulted in smaller lenses containing fewer cells. To determine why Cx50-deficient lenses fail to grow normally, cell proliferation was assayed during the period of growth failure.

methods. Wild-type, Cx50-knockout, and Cx50KI46 mice were injected with 5′-bromo-2′-deoxyuridine (BrdU) and lenses were dissected and fixed after 1 hour or 24 hours. BrdU incorporation was visualized by immunocytochemical staining, and the mitotic index (MI) was determined between postnatal day (P)0 and P6. Levels of total ERK and phospo-ERK were determined by Western blot analysis.

results. On P2 to P3, wild-type lenses displayed a significantly increased MI not evident in knockout lenses, and knockin lenses only partially rescued the growth deficit. Reductions in the number of mitotic cells did not reflect a decrease in the rate of cell division and temporally correlated with reduction in lens mass. Levels of phosphorylated ERK1/2 were identical in wild-type and Cx50-deficient lens epithelia.

conclusions. These results demonstrate that Cx50-mediated communication is necessary to achieve peak mitosis. In addition, they suggest a novel mitogenic role for gap junctional coupling that is connexin specific and independent of MAPK signaling.

The ocular 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 poles. Throughout life, epithelial cells undergo mitosis, migrate to the equator, and differentiate to give rise to new fibers ^{1 2 3} producing radially symmetric lens growth. These processes of epithelial cell division and fiber differentiation do not proceed at a constant rate. In the rodent lens, maximum growth occurs during the first postnatal week, resulting in increases of lens weight of up to 87% in a single day, and growth rates oscillate in cycles that can be replicated in vitro by pulsatile administration of growth factors. ⁴ Although roles for growth factor signaling in the regulation of both proliferation and differentiation have been identified, ^{5 6 7 8} additional mechanisms of intercellular communication may also influence lens growth. 

We have identified a requirement for connexin (Cx)-mediated intercellular communication in postnatal lens growth. The lens expresses three connexins: Cx43, Cx46, and Cx50. ^{9 10 11} In mammalian lenses, Cx43 is present only at low levels in the epithelial cells, and disappears in the lens fibers, ¹² whereas Cx46 is abundantly expressed during fiber differentiation. ¹³ Cx50 is highly transcribed and is translated within the epithelium, and its expression persists and increases in the differentiated fiber cells. ^{14 15} Knockout of Cx50 produced lenses that were significantly smaller than wild-type, and this growth failure was manifested during the first postnatal week of life. ^{15 16 17} Replacement of Cx50 with Cx46 by genetic knockin failed to restore normal lens growth, even though Cx46 protein expression was shifted into the epithelial cell population, ^{18 19} demonstrating that Cx50-specific junctional communication was absolutely required. The growth defect was less severe in knockin mice (34% reduction) compared with knockout mice (46% reduction), but in both cases growth failed in the early postnatal period and the lenses remained smaller throughout life. 

Because the lens is a solid cellular cyst, reduced organ size could result only from either a decrease in the total number of cells or a reduction in the unit cell size. Morphologic and histologic studies of Cx50-knockout and -knockin mice have established that cell dimensions in these lenses are not significantly different from those in wild-type, ^{16 18} excluding decreased cell size as an explanation for the growth deficit. Reduced lens cell numbers could occur through increased apoptosis, as has been reported for the αA-crystallin knockout mice, ²⁰ which exhibit a degree of microphthalmia similar to that in the Cx50-deficient mice. Alternatively, a reduction in mitosis would also produce fewer lens cells, and epithelial proliferation is controlled by growth factors whose receptors activate the mitogen-activated protein kinase (MAPK) pathway. ^{1 5 6 8 21} Genetically engineered mice in which this pathway had been positively ²² or negatively ²³ regulated produce larger or smaller lenses, respectively. 

Gap junctional communication has been historically linked to the suppression of aberrant growth in pathologic conditions such as cancer. ^{24 25 26 27} Thus, it was surprising that loss of Cx50 resulted in a growth deficiency, when other connexin knockouts have produced increased susceptibility to tumorigenesis. ²⁸ In the current study, we investigated the mechanism whereby Cx50 facilitates normal lens growth. We showed that the mitotic index (MI) in wild-type lenses undergoes a large transient increase on P2 and P3, which is not manifested in Cx50-knockout lenses. Knockin lenses also exhibited a decreased MI on P2 and P3. These statistically significant reductions in the number of dividing cells did not reflect a decrease in the rate of cell division, and temporally correlated with the lens mass reductions that were subsequently maintained throughout life. Activation of the classic MAPK pathway, evaluated by the phosphorylation status of ERK, was not significantly different in wild-type, knockout, and knockin lenses during the first postnatal week. Taken together, these data suggest a positive mitogenic role for gap junctional communication that is connexin specific and distinct from growth factor signaling. 

Material and Methods

Mice

This study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The generation and phenotype of both Cx50-knockout and -knockin mice have been previously described. ^{16 18 19 29} For this study, both colonies were backcrossed into the C57Bl/6NTac genetic background of the wild-type control mice (Taconic, Germantown, NY). 

BrdU Injection and Histology

P0 to P6 mouse pups were injected intraperitoneally with 100 μg/g body weight of 5′-bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO). BrdU at 10 mg/mL was dissolved in phosphate-buffered saline (PBS) at 37°C just before use. Injected pups were returned to their mothers for either 1 hour or 24 hours of exposure to BrdU. Eyes were then dissected, cut open at the posterior pole and fixed overnight in 4% formaldehyde, made fresh from paraformaldehyde in PBS. After dehydration through an ethanol series and xylene, eyes were embedded in paraffin. Serial sagittal sections of ∼3 μm were cut on a diamond knife and collected on coated slides (Vectabond; Vector Laboratories, Burlingame, CA). BrdU incorporation was immunolabeled with a BrdU in situ detection Kit (BD Pharmigen, San Diego, CA) according to the manufacturer’s instructions, except endogenous peroxidase was quenched with 3% hydrogen peroxide diluted in absolute methanol, and all antibody incubations were performed 37°C. BrdU-negative nuclei were counterstained with hematoxylin. Stained sections were viewed with 20× and 40× objectives (model BX51 microscope; Olympus, Tokyo, Japan) and photographed with a digital camera (MagnaFire; Optronics, Goleta, CA). 

Quantitation of MIs

BrdU-positive and unlabeled (counterstained blue with hematoxylin) nuclei were counted in serial 3-μm sections from the central 300-μm region of each lens. The average distance between sections counted was ∼15 μm. The MI was calculated for each section, and mean MIs were plotted as a function of postnatal age. Data were collected from four to five lenses at each time point on each day for each genotype. Statistical analyses between wild-type, knockout, and knockin lenses were performed using the one-way ANOVA with a significance level of 0.01. 

Analysis of Early Postnatal Lens Growth

P0 to P6 mouse pups of each genotype were directly weighed and euthanatized. Lenses were dissected and photographed with a microscope (model SZX9; Olympus) and a digital camera (DP12; Olympus). P0 to P6 lenses were too small to be accurately weighed, but their diameter could be precisely measured, and their volume was calculated based on lens radii and assuming a spherical shape. Volumes were converted to mass by multiplying by the density assuming ρ = 1, as previously described. ¹⁶  

Western Blot Analysis

For biochemical analysis of epithelial cell populations in the absence of the differentiating fiber cell mass, lens capsules were stripped from freshly dissected lenses on P2 or P6, pooled, and collected in sample buffer. Because protein concentrations in these samples were low and could not be easily determined by standard methods, the final volumes of samples were adjusted and normalized to the total lens mass in each pool, calculated from the number of lenses used multiplied by the average lens mass for the postnatal day analyzed. Equal volumes of capsule protein samples from each genotype were electrophoresed on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Keene, NH). Equal protein loading and efficiency of protein transfer for the three genotypes was verified by staining filters with ponceau S (Sigma-Aldrich). Blots were then immunostained with antibodies specific for total ERK, or phospho-ERK (Cell Signaling Technology, Beverly, MA), followed by alkaline phosphatase–conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) using CDP-star as a chemiluminescent substrate (Applied Biosystems, Foster City, CA). Protein standards (MagicMark; Invitrogen, Carlsbad, CA) were used as molecular weight markers. Blots were digitized, and band intensities were quantified with image-analysis software (Kodak 1D; Eastman Kodak, Rochester, NY). Values were normalized to the mean value of band intensity in the wild-type sample for each postnatal day analyzed. Statistical analyses between wild-type, knockout, and knockin total-ERK and phospho-ERK levels were performed with the one-way ANOVA with a significance level of 0.01. 

Results

Pattern of Postnatal Lens Mitosis

Rodent lenses do not grow at a constant rate during the first postnatal week. ⁴ To determine the index and spatial pattern of epithelial cell division during this period, we labeled dividing cells in wild-type, knockin, and knockout mouse lenses with 5′-bromo-2′-deoxyuridine (BrdU). Figure 1 demonstrates changes in the wild-type pattern of postnatal mitosis revealed after a 1-hour exposure to BrdU. On P1, mitotically active cells were detected in both the germinative zone and across the central anterior epithelium (Figs. 1A 1B 1C) . By P3, the number of dividing cells had increased across the entire lens epithelium, and the BrdU-positive nuclei were frequently clustered in small groups of three to four neighboring cells (Figs. 1D 1E 1F) . On P5, there was a dramatic decrease in the number of labeled cells, and they were predominantly found in the germinative zones near the lens equator (Figs. 1G 1H 1I) . Thus, the location and number of mitotically active lens epithelial cells changed dramatically during the first postnatal week. 

Quantification of Mitotic Indices

Cx50-deficient lenses were smaller because they had fewer cells than wild-type lenses, ^{16 18} which could have resulted from a decrease in the number of cells undergoing mitosis. To quantify and compare the number of cells undergoing cell division in wild-type, knockin, and knockout mice, MIs were calculated after a 1-hour exposure to BrdU and plotted as a function of postnatal age (Fig. 2) . After birth, the wild-type MI rose dramatically, reaching a peak on days P2 and P3 and was lowest on P6. In contrast, knockin and knockout MIs were lower than wild-type from P0 to P4, and were equivalent to wild-type on days P5 to P6. The most dramatic difference was evident on P2 to P3: Knockin animals displayed a small increase on P2, but not P3, and knockout mice exhibited no increase in mitosis on either day (Fig. 2A) . These results suggest that Cx50-deficient lenses are smaller than wild-type because they failed to increase their MI transiently on P2 and P3. In addition, there was a transient difference in mitotic deficit between Cx50-knockout and knockin lenses. On P2, knockin lenses had a MI that was lower than wild-type, yet higher than knockout. Comparison of the three data sets using one-way analysis of variance (ANOVA) confirmed that there were statistically significant differences (P < 0.01) in the mean values between all three groups on P2, and that on P3 knockout and knockin means were not statistically different from each other, but both were significantly lower than wild-type. 

The diminished number of mitotic cells on P2 in the Cx50-deficient lenses could have resulted from an intrinsically slower mitotic rate, or a reduction in the number of cells responding to mitogenic factors. To determine whether mitotic rates were different among the three lens genotypes, postnatal lenses were exposed to BrdU for 24 hours and MIs were calculated, plotted as a function of postnatal age, and compared to the 1-hour data. After longer exposure to BrdU, the MI patterns for wild-type, knockin, and knockout lenses were similar to those seen after a 1-hour labeling. The wild-type MI rose on days P2 and P3, whereas the knockin and knockout MIs were significantly lower (P < 0.01) on these days and were equivalent to wild-type by the end of the first postnatal week (Fig. 2B) . Comparison of the ratio of MIs at 24 hours and 1 hour showed that the rates of entry into the S-phase of mitosis were similar for each genotype and relatively constant during the first postnatal week (Fig. 2C) . For example, the average number of labeled wild-type cells increased 1.6-fold between a 1-hour and 24-hour labeling, and the 1.8-fold average increase in knockout lenses was not significantly different (P > 0.05). Therefore, Cx50-deficient lenses were smaller because fewer cells were recruited into the cell cycle during the period between P2 and P3, when wild-type lenses underwent a large transient increase in MI. 

Altered Mitosis Patterns in Cx50-Deficient Lenses

To determine whether the spatial distribution of mitotic cells in Cx50-deficient lenses was also altered, MIs in the germinative zone and across the central anterior epithelium were individually quantitated and compared in wild-type, knockin, and knockout lenses on P2. As observed for P3 lenses (Fig. 1) , in wild-type P2 lenses numerous BrdU-positive nuclei were clustered in small groups of neighboring cells in both the anterior epithelium and in the germinative zone (Figs. 3A 3B) . In knockin and knockout lenses, fewer nuclei were labeled, and clusters of cells were not evident (Figs. 3C 3D and 3E 3F , respectively). To quantify regional differences the epithelial arc was divided into four equal quadrants, with the two central quadrants representing the central anterior epithelium and the two peripheral quadrants comprising the germinative zone. 

Calculation of MIs in these two zones revealed differences in the growth failures between knockin and knockout animals (Fig. 3G) . Wild-type mice displayed a 14% lower MI in the central epithelium than in the germinative zone. Knockin lenses had lower indices in both zones than did the wild-type, but retained the same relative change, with a 15% reduction in the central epithelium. In contrast, knockout mice had the lowest MI in the germinative zone and further exhibited a 54% reduction in the central epithelial cells. Thus, both Cx50-deficient models displayed lower MIs than wild-type, but there were both temporal and spatial differences in the reductions between knockin and knockout animals. 

Lens Growth Failure and Mitotic Deficiency

We have shown that Cx50-deficient lenses were normally sized at birth but become significantly smaller when weighed 1 to 2 weeks later. ^{16 18} To determine whether the observed differences in MIs on P2 and P3 temporally correlates with the growth failure, lenses from wild-type, knockin, and knockout mice were measured during the first postnatal week. Between P0 and P6, pups of all three genotypes grew identically, increasing their body mass threefold (Fig. 4A) . In contrast, lens growth diverged significantly (P < 0.01) between wild-type and knockin or knockout on P3, with knockin lens mass reduced 31% and knockout reduced 37% compared with wild-type. A further significant difference (P < 0.01) in lens mass between knockin and knockout was evident by P5. By P6, lenses had already established the same mass differences that were displayed later throughout adult life, ^{16 18} with knockin lenses being one third smaller and knockout lens mass reduced by half. This quantitative difference between knockin and knockout lens mass suggests different degrees of growth deficiency in these two animal models and could be related to the slightly elevated value of MI for knockin lenses compared with knockout lenses on P2. These results suggest that differences in adult lens size were determined by events during the first postnatal week and were consistent with the reductions in MI on P2 and P3 that cause the Cx50-dependent growth failure. 

ERK Signaling in Cx50-Deficient Lenses

Lens cell proliferation is modulated by growth factors, ^{4 5 6 7} and after receptor activation, many of these mitogenic signal transduction processes involve activation of the extracellular signal regulated kinase (ERK) pathway. ^{1 21} To determine whether the reduced MIs in Cx50-deficient lenses results from a reduced transduction of mitogenic signals, we evaluated the phosphorylation status of ERK1/2 in postnatal wild-type, knockout, and knockin lenses on P2, a day on which the differences in MIs were at a maximum and on P6 when MIs were identical. 

On either P2 or P6, there were no obvious differences in levels of total ERK1/2 between wild-type, knockout, or knockin lenses (Fig. 5B) . Similarly, the fraction of total ERK1/2 recognized by phospho-ERK specific antibodies was equivalent in wild-type, knockout, and knockin lenses on both days (Fig. 5C) . Plotting the mean densitometry values of replicate blots (n = 3) failed to reveal any significant differences (P > 0.01) in the levels of total ERK1/2, or the fraction of phosphorylated ERK1/2 between the three lens types on P2 or P6 (Fig. 5D) . Taken together, these data suggest that the growth defect in Cx50-deficient lenses is not manifested at the level of growth factor mitogenic signal input. 

Discussion

We identified a unique role for Cx50 specific gap junctional communication in stimulating mitosis in the postnatal lens. Loss or replacement of Cx50 resulted in fewer dividing cells only during the days of maximum lens growth. Stimulated lens mitosis has been linked to growth factors, ^{4 5 6} which in turn activate the ERK subfamily of MAPK signaling. ^{8 30} Loss of Cx50 did not diminish ERK phosphorylation, suggesting that it played a role in mitotic recruitment downstream of the signal transduction induced by growth factor receptor activation. Expression of Cx46 in place of Cx50 by genetic knockin was not able to restore mitosis fully, suggesting that more than simple ionic coupling, which would be provided by either connexin, was required for propagation of mitotic stimulation. We speculate that Cx50, but not Cx46, selectively mediates intercellular propagation of the second messengers of growth factor signal transduction in lens epithelia, resulting in greater recruitment of cells into mitosis. 

Lens growth results from the regulated proliferation of epithelial cells that migrate toward the equatorial region where they differentiate into new fiber cells. ³¹ We have shown that homozygous knock-in of Cx46 fail to rescue growth, ¹⁸ whereas heterozygous knockin lenses grow normally, despite having severe cataracts. ¹⁹ Normal lens growth was also observed in heterozygous Cx50 knockouts, ^{15 16} implying that one allele of Cx50 provided sufficient coupling to maintain a high MI. Finally, the growth deficit in Cx50-knockout lenses was not affected by genetic background, whereas the cataract severity was influenced by genetic modifiers. ¹⁷ Taken together, these data suggest that normal lens growth requires only one copy of Cx50 and is independent of lens clarity. 

The pulsatile pattern of MI we observed in wild-type mice during the first postnatal week is remarkably consistent with the discontinuous increase in mass previously reported for rat lenses. ⁴ However, Brewitt and Clark ⁴ found that lens growth and transparency were linked, and maintenance of both properties required pulsatile delivery of platelet-derived growth factor (PDGF). In addition, they subsequently found that the observed oscillations in lens mass corresponded to the length of stages of the lens epithelial cell cycle. ³² Thus, although it is tempting to speculate that PDGF signaling may be driving the alternating MI, the relationship of these data to Cx50 is complicated by the lack of correspondence between growth defects and cataracts in Cx50-deficient lenses. 

More experimentation is needed to determine the exact mechanism whereby Cx50 facilitates maximal mitosis. Future studies might examine whether Cx50-deficient epithelia are capable of responding to growth factors in vitro ^{4 6} by increasing levels of phosphorylated of ERK and MEK. ⁸ Growth rates of lens epithelial cells from the different genotypes can be assayed in primary culture ³³ to determine whether differences in the in vivo MI can be replicated. Our findings have identified a novel function for gap junctional communication in the promotion of coordinated mitosis in the lens, and further molecular dissection of these results will open up new avenues of research in the contributions of intercellular communication to ocular growth control. 

 Supported by National Eye Institute Grant EY13163.

 Submitted for publication February 23, 2004; revised April 14, 2004; accepted April 20, 2004.

 Disclosure: C. Sellitto, None; L. Li, None; T.W. White, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Distribution of mitotic nuclei in wild-type postnatal lenses. (A) At P1, mitotically active cells were detected in the central anterior epithelium (B) and the germinative zone (C). At P3 (D–F), the number of dividing cells had increased significantly, and mitotic nuclei were often clustered in groups of three or more neighboring cells (arrowheads). At P5 (G–I), there was a significant decrease in BrdU-labeled cells, and they predominantly localized in the germinative zones. Thus, the location and number of mitotically active lens epithelial cells changed dramatically during the first postnatal week.

Figure 2.

 

Quantification of MIs. (A) Mean MIs (±SD) for wild-type, knockin, and knockout lenses after a 1-hour exposure to BrdU. In wild-type mice, cell division increased significantly (P < 0.01) on P2 and P3, compared with P0 and P1, and P4 and P6. In knockin mice, the MI was initially lower than in wild-type and showed only a moderate increase on P2, but not on P3. In knockout lenses, the dramatic increase in cell division in wild-type animals on P2 and P3 was absent, and the rate of mitosis was essentially constant from P0 to P6. (B) Mean MIs (±SD) after 24 hours of exposure to BrdU. The MI patterns for all three genotypes were similar to those seen after a 1-hour labeling. The wild-type MI rose on days P2 and P3, and the knockin and knockout MIs were significantly lower (P < 0.01). (C) Comparison of the ratio of MIs at 24 hours and 1 hour showed that the rates of entry into the S-phase were not significantly different between genotypes, but were essentially constant during the first postnatal week. Thus, Cx50-deficient lenses had fewer dividing cells on P2 and P3.

Figure 3.

 

Differences in the extent and spatial localization of mitosis in Cx50-deficient P2 lenses. Wild-type lenses had numerous BrdU-positive nuclei clustered in small groups of neighboring cells in both the anterior epithelium (A) and the germinative zone (B). In knockin (C, D) and knockout (E, F) lenses, fewer nuclei were labeled and clusters of cells were not evident. Comparison of MIs in the central epithelium and the germinative zone (G) revealed differences in the growth failures between knockin and knockout animals. Wild-type mice had a slightly lower MI in the central epithelium than in the germinative zone. A similar pattern was seen in knockin lenses, although the MI was reduced compared with wild-type. Knockout animals had a significantly lower MI in the germinative zone and a larger reduction in the central epithelial cells. Thus, there were both temporal and spatial differences between the reduced MI in knockin and knockout lenses.

Figure 4.

 

Cx50-deficient lens growth was temporally correlated with reduced mitosis. (A) From P0 to P6, no difference in overall growth was observed between the three genotypes. (B) Lens growth diverged significantly (P < 0.01) between genotypes by P3, with knockin lens mass reduced 31% and knockout reduced 37% compared with wild-type. At P6, the mass differences between Cx50-deficient and wild-type lenses displayed throughout adult life ^{16 18} had already been established. Thus, differences in adult lens size were determined by events during the first postnatal week and correlate temporally with the reduction in MI on P2 and P3.

Figure 5.

 

MAPK signaling is normally activated in Cx50-knockout lenses. (A) Protein loadings based on total lens mass were equivalent on the Western blots when visualized by ponceau S staining. (B). There were no apparent differences in total ERK1/2 between wild-type, knockout, and knockin lenses between P2 and P6. (C) The fraction of phospho-ERK1/2 was also equivalent in wild-type, knockout, and knockin lenses on both days. (D). Quantitation of ERK in wild-type, knockin, and knockout lenses. Relative amounts of total- and phospho-ERK in the lens capsule fractions were not statistically different (P > 0.01) from wild-type on either P2 or P6. Data are expressed as the mean ± SD of results in three independent experiments. Thus, the growth defect in Cx50-deficient lenses is not manifested at the level of ERK signaling.