Embryonic Lens Research Articles

Expression patterns of β1-related α integrin subunits in murine lens during embryonic development and wound healing

Purpose. To study the expression patterns of β1-related α integrin subunits in murine lens epithelial cells, comparing embryonic fiber differentiation with injury-induced epithelial mesenchymal transition (EMT).Methods. Adult mice type C57BL/6, pregnant as well as with an eye injured, were sacrificed at different time-course intervals. The embryonic and the injured eyes were obtained and deparaffinized sections of these eyes were processed for immunohistochemistry staining for detection of integrin α subunits.Results. Embryonic lens epithelial cells expressed primarily α3 and α5 subunits, whereas embryonic fiber cells expressed α2, α5, and α6 subunits.Adult lens epithelial cells expressed α3, and α6 subunits, whereas injured lens cells expressed α2, α3, and α6 integrin subunits.Conclusions. The phenotypic changes of lens epithelial cells during embryonic fiber differentiation and EMT are characterized by different expression of integrin subunits as a result both of the altered extracellular matrix conditions and of the altered cell signaling pathways recruited in each process.

Ca2+-Mobilization and Cell Contraction after Muscarinic Cholinergic Stimulation of the Chick Embryo Lens

In the embryonic lens, cells of the anterior epithelium proliferate, migrate through the equatorial zone, and elongate to form primary lens fibers at the posterior pole. During this stage of development, cholinesterase (ChE) activity has been described as in other embryonic tissues implicated in morphogenesis. The purpose of the study was to demonstrate in addition to ChE the presence of muscarinic acetylcholine receptors (mAChR) and choline acetyltransferase (ChAT) and to test whether the muscarinic cholinergic system is involved in the regulation of cellular movements. In the chick embryo lens (Hamburger-Hamilton [HH] stage 21), the expression of mAChR and ChAT were demonstrated by immunohistochemistry. Isolated whole embryonic lenses were loaded with fura-2/AM, and changes of cytosolic calcium were measured after muscarinic stimulation. Size changes were assessed by morphometry with the use of time-lapse videos. mAChR was present in the equatorial zone of the lens and in the elongating primary lens fibers. Anti-ChAT immunoreactivity was restricted to the primary lens fibers. Addition of carbachol induced an intracellular Ca2+ peak followed by a plateau phase of extracellular Ca2+ influx. The plateau phase was reversed by addition of atropine. Concomitant with the calcium release, a contraction of the lens and an increase in opacity was observed. The localization of ChAT in the differentiating fibers of the lens indicates that acetylcholine is synthesized during differentiation and modulates morphogenesis and elongation of mAChR-positive progenitor cells in the equatorial zone. The carbachol-induced contraction indicates that the embryonic muscarinic system may be involved in the regulation of cellular movements.

Expression of zebrafish Lc3 synthase gene in embryonic lens requires hedgehog signaling.

Glycosyltransferases are involved in synthesis of various glycolipids and glycoproteins that play important roles in many biological processes. We have identified a zebrafish gene encoding a member of beta1,3-N-acetylglucosaminyltransferase family, the Lc3 synthase/bGn-T5. Whole-mount in situ hybridization reveals that the Lc3 synthase gene is expressed in two distinct phases during the zebrafish embryogenesis. The early phase extends from late blastulation to the completion of epiboly, during which the expression occurs in the superficial layer of the embryos. The second phase of expression starts during mid-segmentation and persists until day 3, during which the expression occurs prominently in the developing lens. The expression of the Lc3 synthase gene in the lens is inhibited in you-too (yot) mutant embryos that are defective in Hedgehog signaling. The expression in the lens also decreases in cyclops (cyc) and one-eyed-pinhead (oep) mutant embryos and lefty1-injected embryos, which are deficient in Nodal signaling and lack Hedgehog activity in the ventral brain. These results suggest that Hedgehog signaling is required for the Lc3 synthase expression in embryonic lens.

Retroviral overexpression of bcl-2 in the embryonic chick lens influences denucleation in differentiating lens fiber cells

During the course of their differentiation, embryonic lens fibers undergo loss of their cytoplasmic organelles and nuclei. The denucleation process bears similarities to the nuclear breakdown that occurs during apoptosis. This has given rise to the hypothesis that this denucleation is analogous to apoptosis, but without the plasma membrane changes characteristic of apoptotic cell death. Previous work has shown that several members of the apoptotic cascade are active during denucleation. Here, we have overexpressed the anti-apoptotic molecule bcl-2 in developing lenses of the 8-day-old chick embryo in ovo, using the replication-competent retrovirus RCAS. We find that lenses overexpressing bcl-2 show varying degrees of distortion in comparison with untreated and negative insert controls, including a more spherical shape and disorganized fiber cells. All overexpressing lenses showed significantly higher numbers of smaller nuclei in the lens core, where denucleation begins. There was no change in cell size or pattern of proliferation. These in vivo results were confirmed in vitro using lens epithelial cell cultures, which differentiate into lentoids. The lentoids in treated cultures showed the same effect on nuclear number and size. We further found that in lenses overexpressing bcl-2 there was a reduction in the activation of caspase-9 and the cleavage of the caspase substrate DFF45, and, in the lens core, a failure of the nuclear chromatin to condense. These results provide strong support for the view that embryonic lens fiber cell denucleation is analogous to the nuclear degradation that occurs during apoptosis, and that similar control pathways are involved in both these phenomena.

Protein expression patterns for ubiquitous and tissue specific calpains in the developing mouse lens

Calcium activated proteases (calpains) have been implicated in the processing of lens crystallins during lens maturation and cataract formation. Ubiquitous type calpain 2 and calpain 10 and lens specific Lp82 and Lp85 protein distribution were determined using immunohistochemistry and immunoblotting in embryonic and post-natal mouse eyes. Calpain 2 was first expressed late in embryonic development and localized to the lens epithelium and transition zone. Lp82 was expressed at E9·5 in the lens placode, head ectoderm, and throughout the fiber cells during embryonic lens maturation. Lp82 co-localized at sites of crystallin modification in the juvenile lens. In the adult lens, Lp82 protein was maintained in cortical fibers but could not be detected in the lens nucleus. Lp85, the slightly larger splice variant of Lp82, was first observed at E9·5 and throughout early embryonic lens development. Abundant localization of this enzyme was observed in the cell nuclei of lens epithelium, elongating fibers, and undifferentiated mesoderm. Robust peri-nuclear localization of calpain 10 was observed in the head ectoderm, lens placode, and optic vesicle during early eye induction. Further, calpain 10 protein was maintained in the lens epithelium of pre- and post-natal lens. These data support the hypothesis that Lp82 in rodent lens has an important role in crystallin proteolysis during normal lens maturation. In contrast, calpain 2, Lp85, and calpain 10 may have roles in cell signaling pathways.

The Cellular and Molecular Bases of Vertebrate Lens Regeneration

Lens regeneration takes place in some vertebrates through processes of cellular dedifferentiation and transdifferentiation, processes by which certain differentiated cell types can give rise to others. This review describes the principal forms of lens regeneration that occur in vivo as well as related in vitro systems of transdifferentiation. Classic experimental studies are reviewed that define the tissue interactions that trigger these events in vivo. Recent molecular analyses have begun to identify the genes associated with these processes. These latter studies generally reveal tremendous similarities between embryonic lens development and lens regeneration. Different models are proposed to describe basic molecular pathways that define the processes of lens regeneration and transdifferentiation. Finally, studies are discussed suggesting that fibroblast growth factors play key roles in supporting the process of lens regeneration. Retinoids, such as retinoic acid, may also play important roles in this process.

Equarin, a novel soluble molecule expressed with polarity at chick embryonic lens equator, is involved in eye formation

The lens plays an important role in eye development. To investigate the molecular mechanisms involved, we used signal sequence trap screens with a chicken lens cDNA library and identified a novel secreted molecule, equarin. Equarin encodes consensus repeat domains conserved in human SRPX and mouse Urb. In the embryonic eye, equarin transcript is detected exclusively in the lens, and persists in the lens equatorial region in a high-dorsal-to-low-ventral gradient. In vitro analysis of equarin protein indicated that after translation, it is modified, cleaved, and secreted to extracellular locations. Microinjection of equarin mRNA into Xenopus embryos induced abnormal eye development. These data suggest that equarin is involved in eye formation.

Uncleaved and Matrix-Free

The α subunit of most integrin α/β heterodimers undergoes an endoproteolytic cleavage, yielding a heavy and a light chain, which are linked by a disulfide bond. However, uncleaved α subunits are also present in some functional, extracellular matrix-binding integrins, although their physiological significance has not been clear. Walker et al. show that the cleavage state of the α6 integrin is regulated during chicken embryogenesis. Both cleaved and uncleaved forms of α6 are expressed at the surface of embryonic lens cells, but a switch to the uncleaved form occurs during differentiation. The uncleaved form is underglycosylated and becomes localized to the lateral cell interface of differentiating lens cells. Uncleaved integrins associated with Shc and Grb2, two molecules known to mediate integrin-initiated signaling and cell survival. The cell-cell interface is devoid of matrix proteins, suggesting ligand-independent signaling by the uncleaved integrin subunit at the time of differentiation. Underglycosylated integrins are thought to localize to lipid microdomains that are enriched in signaling molecules. The authors propose that uncleaved integrins may provide a cell survival signal during lens cell differentiation, a period when the lens becomes progressively transparent through caspase-mediated degradation of nuclei and organelles in an apoptosis-like process. J. L. Walker, L. Zhang, A. S. Menko, A signaling role for the uncleaved form of α6 integrin in differentiating lens fiber cells. Dev. Biol. 251 , 195-202 (2002). [Online Journal]

A Signaling Role for the Uncleaved Form of α6 Integrin in Differentiating Lens Fiber Cells

Many α integrin subunits are cleaved during their processing to yield heavy and light chains, which remain associated by disulfide bonds. While uncleaved α integrin subunits can form functional receptors that sometimes have distinct signaling roles from their better-characterized endoproteolytically cleaved counterparts, their expression at the cell surface and their association with signaling complexes have yet to be determined in vivo. In this study, we demonstrate that, in differentiating lens fiber cells, the uncleaved form of α6 integrin was expressed at the cell surface. This form of α6 integrin coimmunoprecipitated with both the signaling adaptor molecule Shc and its downstream effector Grb2, suggesting that, in lens fiber cells, uncleaved α6 integrin was associated with a Shc-mediated signaling complex. We show that expression of the cleaved form of α6 integrin progressively decreased relative to uncleaved α6 integrin as the state of lens cell differentiation increased, resulting in the predominance of uncleaved α6 integrin in the lens fiber cell zones. Interestingly, we previously have shown that α6 integrin is localized principally along the extensive cell–cell interfaces of these lens fiber cells, in the absence of its extracellular matrix ligand laminin. While we found that the cleaved form of α6 integrin contained both high mannose and complex sugars, the uncleaved form of α6 integrin contained only high mannose sugars. These properties suggest that the uncleaved form of α6 integrin may have a unique role in the embryonic lens. Its high association with Shc and Grb2 in the differentiating cortical fiber cell zone indicates that α6 integrin may provide a cell survival signal in the presence of the apoptotic-like processes that are initiated in this region of the embryonic lens to clear the lens cells of their organelles.

Localization of Two Conserved Cis -acting Enhancer Regions for the Filensin Gene Promoter That Direct Lens-specific Expression

Filensin is a unique eye lens intermediate filament protein, and a major component of ‘beaded filament’ cytoskeletal network. Expression is restricted to lens fiber cells and is believed to be functionally important for lens development by maintaining lens fiber cell shape conformation and lens transparency. The mouse filensin gene promoter core was identified by the promoter activity analysis and found to cover the region from −56 to +4. A reporter vector driven by the filensin promoter core was constructed and DNA fragments derived from the 5′-flanking regions of the gene were cloned into the vector to identify putative cis -acting promoter enhancing activities. The activity of these constructs was monitored by transfection into the chicken embryonic lens cell culture system. Two fragments were identified with cis -acting enhancer activity that re-conferred the lens fiber cell specific expression of the promoter. The first was a 1.7-kbp region located 7.5 kbp upstream from the transcriptional start point while a second 2.1-kbp region was found adjacent to the promoter core. Both fragments specifically directed the lens cell specific expression of only the filensin promoter and not other heterologous promoters. Comparison of 10 kbp of human and mouse 5′-upstream sequences of the filensin promoter revealed two highly conserved sequences that corresponded in their spacing to the putative enhancer regions. These studies have identified the first conserved regulatory sequences important in the lens specific regulation of the filensin gene.

Meis homeoproteins directly regulate Pax6 during vertebrate lens morphogenesis.

Pax6 is a pivotal regulator of eye development throughout Metazoa, but the direct upstream regulators of vertebrate Pax6 expression are unknown. In vertebrates, Pax6 is required for formation of the lens placode, an ectodermal thickening that precedes lens development. Here we show that the Meis1 and Meis2 homeoproteins are direct regulators of Pax6 expression in prospective lens ectoderm. In mice, Meis1 and Meis2 are developmentally expressed in a pattern remarkably similar to Pax6 and their expression is Pax6-independent. Biochemical and transgenic experiments reveal that Meis1 and Meis2 bind a specific sequence in the Pax6 lens placode enhancer that is required for its activity. Furthermore, Pax6 and Meis2 exhibit a strong genetic interaction in lens development, and Pax6 expression is elevated in lenses of Meis2-overexpressing transgenic mice. When expressed in embryonic lens ectoderm, dominant-negative forms of Meis down-regulate endogenous Pax6. These results contrast with those in Drosophila, where the single Meis homolog, Homothorax, has been shown to negatively regulate eye formation. Therefore, despite the striking evolutionary conservation of Pax6 function, Pax6 expression in the vertebrate lens is uniquely regulated.

The role of mitochondria, cytochrome c and caspase-9 in embryonic lens fibre cell denucleation.

During the differentiation of secondary lens fibre cells from the lens epithelium, the fibre cells lose all of their cytoplasmic organelles as well as their nuclei. The fibre cells, containing crystallins, which confer optical clarity, then persist in the adult lens. The process of denucleation of these cells has been likened to an apoptotic event which is not followed by the plasma membrane changes that are characteristic of apoptosis. We have examined the expression and subcellular translocation of molecules of the apoptotic cascade in differentiating lens epithelial cells in culture. In this culture system, the epithelial cells differentiate into lentoids composed of lens fibre cells. We find that caspase-9, which is expressed and activated before embryonic day 12 in intact lenses, is localized in the cytosol outside mitochondria in non-differentiating cultured cells. In lentoid cells, caspase-9 migrates into mitochondria after the latter undergo a membrane permeability transition that is characteristic of apoptotic cells. At the same time, caspase-9 co-localizes with cytochrome c in the cytosol. The cytochrome c is apparently released from the mitochondria in lentoid cells after the mitochondrial membrane permeability transition and during the period of nuclear shrinkage. Also during this time, the mitochondria aggregate around the degenerating nuclei. Cytochrome c disappears rapidly, while mitochondrial breakdown occurs approximately coincident with the disappearance of the nuclei, but mitochondrial remnants persist together with cytochrome c oxidase, which is a mitochondrial marker protein. Apaf-1, another cytosolic protein of the apoptotic cascade, also migrates to the permeabilized mitochondria and also co-localizes with caspase-9 and cytochrome c in the cytosol or mitochondria of denucleating cells, thus providing evidence for the formation of an 'apoptosome' in these cells, as in apoptotic cells. At no time did we observe the translocation of molecules between cytoplasmic compartments and the nucleus in differentiating lentoid cells. We suggest that the uncoupling of nuclear and membrane apoptotic events in these cells may be due to the early permeability changes in the mitochondria, resulting in the loss of mitochondrial signalling molecules, or to the failure of molecules to migrate to the nucleus in these cells, thus failing to activate nuclear-plasma membrane signalling pathways.

Cloning and sequencing of complete tau-crystallin cDNA from embryonic lens of Crocodylus palustris.

tau-Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete tau-crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the a-enolase gene from non-lenticular tissues. Quantitatively, the tau-crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile tau-crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5'- and 3'-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete tau-crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5'- and 3'-ends respectively. Further, it was found to be identical to its putative counterpart enzyme a-enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of tau-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.

Characterizing gene expression during lens formation in Xenopus laevis: evaluating the model for embryonic lens induction.

Few directed searches have been undertaken to identify the genes involved in vertebrate lens formation. In the frog Xenopus, the larval cornea can undergo a process of transdifferentiation to form a new lens once the original lens is removed. Based on preliminary evidence, we have shown that this process shares many elements of a common molecular/genetic pathway to that involved in embryonic lens development. A subtracted cDNA library, enriched for genes expressed during cornea-lens transdifferentiation, was prepared. The similarities/identities of specific clones isolated from the subtracted cDNA library define an expression profile of cells undergoing cornea-lens transdifferentiation ("lens regeneration") and corneal wound healing (the latter representing a consequence of the surgery required to trigger transdifferentiation). Screens were undertaken to search for genes expressed during both transdifferentiation and embryonic lens development. Significantly, new genes were recovered that are also expressed during embryonic lens development. The expression of these genes, as well as others known to be expressed during embryonic development in Xenopus, can be correlated with different periods of embryonic lens induction and development, in an attempt to define these events in a molecular context. This information is considered in light of our current working model of embryonic lens induction, in which specific tissue properties and phases of induction have been previously defined in an experimental context. Expression data reveal the existence of further levels of complexity in this process and suggests that individual phases of lens induction and specific tissue properties are not strictly characterized or defined by expression of individual genes.

Disruption of Gja8 (alpha8 connexin) in mice leads to microphthalmia associated with retardation of lens growth and lens fiber maturation.

The development of the vertebrate lens utilizes a sophisticated cell-cell communication network via gap junction channels, which are made up of at least three connexin isoforms, alpha8 (Cx50), alpha3 (Cx46) and alpha1 (Cx43), and which are encoded by three different genes. In a previous study, we reported that, with a disruption of Gja3 (alpha3 connexin), mice developed nuclear cataracts with a normal sized lens. We show that Gja8tm1 (alpha8-/-) mice develop microphthalmia with small lenses and nuclear cataracts, while the alpha8 heterozygous (+/-) mice have relatively normal eyes and lenses. A comparative study of these alpha3 and alpha8 knockout mice showed that the protein levels of both alpha3 and alpha8 were independently regulated and there was no compensation for either the alpha3 or alpha8 protein from the wild-type allele when the other allele was disrupted. More interestingly, western blotting data indicated that the presence of alpha8 in the lens nucleus is dependent on alpha3 connexin, but not vice versa. The staining of the knock-in lacZ reporter gene showed the promoter activity of alpha8 connexin is much higher than that of alpha3 connexin in embryonic lenses and in adult lens epithelium. More importantly, a delayed denucleation process was observed in the interior fibers of the alpha8-/- lenses. Therefore, alpha8 connexin is required for proper fiber cell maturation and control of lens size.

Heat shock proteins of adult and embryonic human ocular lenses

Heat shock proteins of adult and embryonic human ocular lenses

Role for alpha 6 integrin during lens development: Evidence for signaling through IGF-1R and ERK.

We show that alpha 6 integrin function was required for normal lens cell differentiation by using an antisense construct to suppress alpha 6 integrin expression. To elucidate the mechanism by which this integrin functions in the regulation of the lens cell differentiation process, we determined the molecular composition of alpha 6 integrin signaling complexes at distinct stages of differentiation in vivo. Because both alpha 6 integrin and insulin-like growth factor-1 (IGF-1) have been implicated in signaling lens cell differentiation, we examined the possibility that they formed a signaling complex in the embryonic lens. Coprecipitation analysis revealed that alpha 6 integrin/IGF-1 receptor complexes were present and that their association was greatest in the equatorial zone, the region of the embryonic lens in which lens cells proliferate and then initiate their differentiation. These results provide in vivo support for the formation of integrin/growth factor receptor signaling complexes. We also found that extracellular signal-regulated kinase (ERK), a downstream effector of both integrin and growth factor receptor signaling pathways, was associated with the alpha 6 integrin signaling complexes in the embryonic lens. This result was supported by our findings that activated ERK, in addition to its nuclear location, localized to lens cell membranes in specific regions of cell-matrix and cell-cell contact. A connection between integrin ligand engagement and ERK activation was shown in vitro after lens cell attachment to laminin. These results demonstrate that alpha 6 integrin function is required for the early stages of lens cell differentiation most likely through its association with the IGF-1 receptor and the activation of ERK.

Regulation of lens connexin 45.6 by apoptotic protease, caspase-3.

Gap junctions are important in maintaining lens homeostasis. Here we report that connexin 45.6 (Cx45.6) was partially truncated to a 46 kDa fragment during chicken lens development. This specific truncation initiated during embryonic days and the truncated fragment accumulated towards the later developmental stages. When membranes of the embryonic lens were subjected to caspase-3 treatment, the 46 kDa fragment of Cx45.6 was reproduced, suggesting apoptotic protease caspase-3 is a potential protease involved. The COOH-terminus of Cx45.6 in GST-fusion protein was also cleaved by caspase-3, confirming that Cx45.6 is a direct substrate of caspase-3. Induction of apoptosis in lens primary cultures regenerated the 46 kDa fragment and this cleavage was blocked by a caspase-3 inhibitor. Alteration of amino acid residue Asp364 or Glu367 to Ala prevented Cx45.6 from cleavage by caspase-3, suggesting that the cleavage site of Cx45.6 is likely to be between Glu367 and Gly361. Phosphorylation of Ser363, a known substrate for casein kinase II (CKII) in vivo, inhibited the cleavage of Cx45.6 by caspase-3. Thus, this study demonstrates that a lens connexin can be a direct target of caspase-3 and the cleavage by caspase-3 leads to the development-associated truncation of Cx45.6. Finally, caspase-3 mediated truncation can be modulated by the specific connexin phosphorylation.

Microphthalmia due to p53-mediated apoptosis of anterior lens epithelial cells in mice lacking the CREB-2 transcription factor

CREB-2 (also called ATF4, TAXREB67, or C/ATF) is an evolutionarily conserved member of the CREB/ATF family of basic-leucine zipper transcription factors. CREB-2 is expressed ubiquitously in the adult mouse and can function as both a transcriptional activator and a repressor. However, little was understood about the normal function of CREB-2 in mammalian development or organ physiology. In this report we have used gene targeting to produce CREB-2-deficient (CREB-2-/-) mice. Adult CREB-2-/- mice displayed microphthalmia due to the complete absence of a lens. Early embryonic lens development including formation of the optic vesicle, primary lens fibers, and proliferating anterior epithelial cells occurred normally in these mice. However, beginning at ED 14.5 the CREB-2-deficient anterior epithelial lens cells underwent massive and synchronous apoptosis. This was followed by the complete resorption of the developing lens. Consistent with this defect in anterior epithelial cell survival, in situ hybridization studies showed that CREB-2 is expressed at high levels in wild-type anterior epithelial lens cells at ED 14.5. The defect in lens formation seen in the CREB-2-/- mice was not associated with qualitative defects in the expression of Pax-6, alphaA-crystallin, c-maf, or PDGF-R alpha. However, apoptosis of the anterior epithelial cells was mediated by a p53-dependent cell death pathway because ablation of the p53 gene rescued anterior epithelial cell death and allowed the formation of a lens in the absence of CREB-2. Taken together, these results identify CREB-2 as an important regulator of mammalian lens development.

Casein Kinase II Phosphorylates Lens Connexin 45.6 and Is Involved in Its Degradation

Connexin (Cx) 45.6, an avian counterpart of rodent Cx50, is phosphorylated in vivo, but the sites and function of the phosphorylation have not been elucidated. Our peptide mapping experiments showed that the Ser(363) site in the carboxyl (COOH) terminus of Cx45.6 was phosphorylated and that this site is within casein kinase (CK) II consensus sequence, although showing some similarity to CKI sequence. The peptide containing Ser(363) could be phosphorylated in vitro by CKII, but not by CKI. Furthermore, CKII phosphorylated Cx45.6 in embryonic lens membrane and the fusion protein containing the COOH terminus of Cx45.6. Two-dimensional peptide mapping experiments showed that one of the Cx45.6 peptides phosphorylated in vivo migrated to the same spot as one of those phosphorylated by CKII in vitro. Furthermore, CKII activity could be detected in lens lysates. To assess the function of this phosphorylation event, exogenous wild type and mutant Cx45.6 (Ser(363) --> Ala) were expressed in lens primary cultures by retroviral infection. The mutant Cx45.6 was shown to be more stable having a longer half-life compared with wild type Cx45.6. Together, the evidence suggests that CKII is likely a kinase responsible for the Ser(363) phosphorylation, leading to the destablization and degradation of Cx45.6. The connexin degradation induced by phosphorylation has a broad functional significance in the regulation of gap junctions in vivo.