Alteration of a developmentally regulated, heat-stable polypeptide in the lens of the Philly mouse. Implications for cataract formation

The beta-crystallin basic principal polypeptide (beta Bp) appears to be altered in the lens of the Philly mouse and may be the main defect in this hereditary cataract. Northern blot analysis showed that an mRNA encoding for beta Bp is present in the Philly mouse lens, but normal beta Bp could not be detected. Instead, a different protein related to beta Bp has been observed. Western blot analysis with antibodies against specific beta Bp peptide sequences showed that the Philly protein shares the same amino-terminal residue as beta Bp but lacks a part of the carboxyl-terminal half of normal beta Bp. The altered protein is slightly smaller than beta Bp and has a more acidic isoelectric point by two-dimensional gel electrophoresis. It also lacks the property of heat stability characteristic of normal beta Bp. The mapping of the alteration in beta Bp may give insight into the nature of the heat stability of this protein as well as some indication of the structural components that are necessary to maintain optical clarity in the lens.     

Deletion mutation in an eye lens beta-crystallin. An animal model for inherited cataracts.

The most prevalent proteins in the lens of the eye are called crystallins, and it is thought that aberrant crystallins may cause opacification of lens tissue. The Philly mouse, a strain with an inherited cataract, has an abnormal beta B2-crystallin, the principal beta-crystallin in the mouse. The cDNA that codes for the beta B2-crystallin protein has been cloned and sequenced from both the normal and the cataractous Philly mouse. The normal mouse beta B2 cDNA is 756 nucleotides in length with 618 nucleotides of open reading frame. An in-frame deletion of 12 nucleotides has occurred in the Philly mouse cDNA, which results in the loss of 4 amino acids. The sequence of the mutant beta B2 was analyzed against the reported structure of the normal bovine beta B2-crystallin determined by x-ray crystallography. The region, in which the deletion of the amino acids occurs near the COOH terminus, is essential for the formation of the tertiary structure of the beta B2-crystallin. The loss of these residues could explain the alterations that are seen with the Philly beta B2 protein and may account for the instability of the Philly beta B2 protein. This abnormal beta B2-crystallin may be the cause of the cataract in this animal.     

CD44 expression is developmentally regulated in the mouse lens and increases in the lens epithelium after injury

Hyaluronan is an oligosaccharide found in the pericellular matrix of numerous cell types and hyaluronan-induced signaling is known to facilitate fibrosis and cancer progression in some tissues. Hyaluronan is also commonly instilled into the eye during cataract surgery to protect the corneal endothelium from damage. Despite this, little is known about the distribution of hyaluronan or its receptors in the normal ocular lens. In this study, hyaluronan was found throughout the mouse lens, with apparently higher concentrations in the lens epithelium. CD44, a major cellular receptor for hyaluronan, is expressed predominately in mouse secondary lens fiber cells born from late embryogenesis into adulthood. Surgical removal of lens fiber cells from adult mice resulted in a robust upregulation of CD44 protein, which preceded the upregulation of α-smooth muscle actin expression typically used as a marker of epithelial–mesenchyma transition in this model of lens epithelial cell fibrosis. Mice lacking the CD44 gene had morphologically normal lenses with a response to lens fiber cell removal similar to wildtype, although they exhibited an increase in cell-associated hyaluronan. Overall, these data suggest that lens cells have a hyaluronan-containing pericellular matrix whose structure is partially regulated by CD44. Further, these data indicate that CD44 upregulation in the lens epithelium may be an earlier marker of lens injury responses in the mouse lens than the upregulation of α-smooth muscle actin.     

The asynchrony of gamma- and beta-chain synthesis during human erythroid cell maturation. III. gamma- and beta-mRNA in immature and mature erythroid clones

The synthesis of the β-crystallin polypeptides has been studied in different regions of the embryonic chicken lens. Seven β-crystallin polypeptides ranging in molecular weight from approximately 19,000 (19K) to 35,000 (35K) daltons were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each polypeptide was synthesized in a rabbit reticulocyte cell-free system supplemented with RNA from the embryonic lens fiber cells suggesting that each is encoded by a separate mRNA. Analysis of the cell-free translation products of the RNAs from 6-, 15-, and 19-day-old embryonic chicken lens fibers demonstrated that all seven polypeptides are translated at each of the stages and that the proportion of β-crystallin mRNAs increases as the chicken embryo matures. Fingerprints of methionine-containing tryptic peptides indicated that the three predominant β-crystallin polypeptides synthesized in the reticulocyte lysate (20K, 26K, and 35K) have related but distinct primary structures. Surprisingly, both the 35K β-crystallin polypeptide and its mRNA were selectively absent from the cells in the central region of the epithelium. Synthesis of this polypeptide from extracted RNAs was detected in the elongating cells of the equatorial region of the epithelium and from the fiber cells. In contrast to the 35K polypeptide, the six lower-molecular-weight β-crystallin polypeptides were synthesized in a reticulocyte lysate directed by RNAs extracted from all three regions of the lens. These data indicate that lens cell elongation and fiber cell differentiation in the embryonic chicken are accompanied by the appearance of the mRNA for the 35K polypeptide.     

Mechanism of small heat shock protein function in vivo: a knock-in mouse model demonstrates that the R49C mutation in alpha A-crystallin enhances protein insolubility and cell death

alphaA-crystallin (Cryaa/HSPB4) is a small heat shock protein and molecular chaperone that prevents nonspecific aggregation of denaturing proteins. Several point mutations in the alphaA-crystallin gene cause congenital human cataracts by unknown mechanisms. We took a novel approach to investigate the molecular mechanism of cataract formation in vivo by creating gene knock-in mice expressing the arginine 49 to cysteine mutation (R49C) in alphaA-crystallin (alphaA-R49C). This mutation has been linked with autosomal dominant hereditary cataracts in a four-generation Caucasian family. Homologous recombination in embryonic stem cells was performed using a plasmid containing the C to T transition in exon 1 of the cryaa gene. alphaA-R49C heterozygosity led to early cataracts characterized by nuclear opacities. Unexpectedly, alphaA-R49C homozygosity led to small eye phenotype and severe cataracts at birth. Wild type littermates did not show these abnormalities. Lens fiber cells of alphaA-R49C homozygous mice displayed an increase in cell death by apoptosis mediated by a 5-fold decrease in phosphorylated Bad, an anti-apoptotic protein, but an increase in Bcl-2 expression. However, proliferation measured by in vivo bromodeoxyuridine labeling did not decline. The alphaA-R49C heterozygous and homozygous knock-in lenses demonstrated an increase in insoluble alphaA-crystallin and alphaB-crystallin and a surprising increase in expression of cytoplasmic gamma-crystallin, whereas no changes in beta-crystallin were observed. Co-immunoprecipitation analysis showed increased interaction between alphaA-crystallin and lens substrate proteins in the heterozygous knock-in lenses. To our knowledge this is the first knock-in mouse model for a crystallin mutation causing hereditary human cataract and establishes that alphaA-R49C promotes protein insolubility and cell death in vivo.     

Change in potassium ion homeostasis of the crystalline lens in mice with hereditary cataracts (the CatFr line)

Fraeser mouse lens morphology and potassium homeostasis were studied. It was shown that just at the age of one month mouse lens exposed "bull" cells which could be observed in patients with senile cataract as well. Nucleated fusiform extended cells were found 4 months later in the central part of the lens which was not typical for this part of the whole normal lens. Studied homogenates of 34 mice with hereditary cataract demonstrated statistically significant increase (1.5-fold about) in K+-content estimated for the whole lens weight as compared to the control group (38 lens). The difference in potassium content in aqueous humor between affected and control animals was statistically indistinguishable. The role of potassium ions in Fraeser cataract pathogenesis is discussed.     

Jagged 1 is necessary for normal mouse lens formation

In mammals, two spatially and temporally distinct waves of fiber cell differentiation are crucial steps for normal lens development. In between these phases, an anterior growth zone forms in which progenitor cells migrate circumferentially, terminally exit the cell cycle and initiate differentiation at the lens equator. Much remains unknown about the molecular pathways orchestrating these processes. Previously, the Notch signal transduction pathway was shown to be critical for anterior lens progenitor cell growth and differentiation. However, the ligand or ligand(s) that direct these events are unknown. Using conditional gene targeting, we show that Jagged1 is required for lens fiber cell genesis, particularly that of secondary fiber cells. In the absence of Jagged1, the anterior growth and equatorial transition zones fail to develop fully, with only a handful of differentiated fiber cells present at birth. Adult Jagged1 conditional mutants completely lack lenses, along with severe anterior chamber deformities. Our data support the hypothesis that Jagged1-Notch signaling conveys a lateral inductive signal, which is indispensable for lens progenitor cell proliferation and differentiation.     

Two interactive genes responsible for a new inherited cataract (RCT) in the mouse

We discovered a mutant mouse, RCT (Rinshoken cataract), with a new congenital cataract in strain SJL/J. The opacity of the lens associated with microphthalmia could be observed visually at 3 to 3.5 months of age. Marked degeneration of the lens, including loss of the fine structure of the lens fibers and swelling of epithelial cells with vacuoles of various sizes in the cortex, but no other defects except photoreceptor degeneration in the retina, was detected. Histological change in the lens was first observed at 2 days after birth. No sex-related differences were detected, and normal phenotypes in the F₁ progeny of RCT and normal mice indicated that the cataract was recessive. The chromosomal location of the causative gene was determined by interval mapping by using intersubspecific backcross progeny of RCT and MSM/Ms, an inbred strain from the Japanese wild mouse Mus musculus molossinus. Backcross progeny were divided into three groups according to phenotype: mice (1) with an early-onset cataract, which can be detected visually as in RCT mice, (2) with a late-onset cataract, which can be detected histologically but not visually, and (3) with a normal lens. Three phenotypes were found to be expressed by allele combinations of two recessive genes, rct and mrct (a modifier of rct). The rct locus essential for the onset of the cataract was tightly linked to D4Mit278 on Chromosome (Chr) 4 with no recombination. The mrct locus was closely linked to D5Mit239 (χ²= 66.3, P << 0.00001) on Chr 5. Received: 5 September 2000 / Accepted: 4 December 2000     

delta- and beta-Crystallin mRNA levels in the embryonic and posthatched chicken lens: temporal and spatial changes during development

The levels of delta- and beta-crystallin mRNAs were examined by cDNA hybridization in the embryonic and posthatched chicken eye lens. Four different cloned beta-crystallin cDNAs were used, allowing discrimination among different members of the beta-crystallin family. Each crystallin mRNA displayed a characteristic temporal and spatial pattern in the developing lens. delta-Crystallin mRNA accumulated rapidly during early embryonic development; by contrast, the beta-crystallin mRNAs began to accumulate rapidly near the end of embryogenesis. Both delta- and beta-crystallin mRNAs increased in the lens for the first month after hatching and began to decrease 3 months after hatching. The levels of the delta- and the different beta-crystallin mRNAs were also differentially regulated in cultured embryonic lens epithelia. The most fiber cell specific crystallin gene product in the differentiating lens was the beta 35 mRNA. These experiments provide a quantitative basis for exploring the differential expression of the delta- and beta-crystallin gene families in the chicken lens.     

Fine localization of a new cataract locus, Kec, on mouse chromosome 14 and exclusion of candidate genes as the gene that causes cataract in the Kec mouse

A mouse with cataract, Kec, was generated from N-ethyl-N-nitrosourea (ENU) mutagenesis. Cataract in the Kec mouse was observable at about 5 weeks after birth and this gradually progressed to become completely opaque by 12 weeks. Dissection microscopy revealed that vacuoles with a radial or irregular shape were located primarily in the cortex of the posterior and equatorial regions of the lens. At the late stage, the lens structure was distorted, but not ruptured. This cataract phenotype was inherited in an autosomal recessive manner. We performed a genetic linkage analysis using 133 mutant and 67 normal mice produced by mating Kec mutant (BALB/c) and F1 (C57BL/6 x Kec) mice. The Kec locus was mapped to the 3 cM region encompassed by D14Mit34 and D14Mit69. In addition we excluded coding sequences of 9 genes including Rcbtb2, P2ry5, Itm2b, Med4, Nudt15, Esd, Lcp1, Slc25a30, and 2810032E02Rik as the candidate gene that causes cataract in the Kec mouse.     

Differentiation potential of germ line stem cells derived from the postnatal mouse ovary

General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics.     

Diverse roles of Eph/ephrin signaling in the mouse lens

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout (-/-) mice often display anterior polar cataracts while EphA2(-/-) lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5(-/-) lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5(-/-) lenses. Moreover, the disruption of membrane-associated β-catenin and E-cadherin junctions is observed in ephrin-A5(-/-) lens central epithelial cells. In contrast, EphA2(-/-) lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates β-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells.     

GROWTH OF THE CHICKEN EMBRYONIC LENS TRANSPLANTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

The influence of neural retina on the growth of chicken embryonic lens was studied by comparing the growth pattern of the lens transplanted onto chorio-allantoic membrane (CAM) with that of the normal lens. The lens from 6-day embryo, transplanted onto CAM after labeled with ³H-thymidine, continued to grow in the absence of neural retina at least for 12 days of incubation, although its growth rate was reduced. In the transplanted lens, no ³H-labeled epithelial cell differentiated into fiber at least for 2 days of incubation and ³H-labeled nuclei first appeared in the fiber cells on the fourth day of incubation, while, in the normal lens of 6-day embryo labeled with ³H-thymidine in situ, ³H-labeled epithelial cells differentiated into fibers within 24 hours. On the other hand, the fiber cells differentiated before transplantation maintained the nearly normal growth rate on CAM. The neural retina transplanted onto CAM together with lens induced the new fibers from the lens epithelium. These observations suggest that the neural retina initiates and promotes the fiber differentiation in the chicken lens, but its continued influence is not always necessary for the successive differentiation of epithelial cell into fiber and especially for the growth of the differentiated fiber cells.     

DNA methyltransferase polypeptides in mouse and human cells

DNA methyltransferase was isolated as a single polypeptide of 190 kDa from mouse P815 mastocytoma cells by immunoaffinity chromatography. This polypeptide seems to be highly susceptible to proteolytic degradation resulting in additional polypeptides in the size range of 150 to 190 kDa. A polypeptide of 190 kDa was immunoprecipitated by monoclonal anti-DNA methyltransferase antibodies from extracts of two different human cell lines, Raji and K562. The 190 kDa polypeptide was synthesized in rapidly proliferating cells and, albeit at a much lower rate, also in cells grown to saturating density. DNA methyltransferase polypeptides smaller than 190 kDa were synthesized neither in log phase nor in stationary phase cells.     

Differentiation and oncogenesis: phenotypically distinct lens tumors in transgenic mice

We report on two lines of transgenic mice that express a murine alpha A-crystallin/SV40 tumor antigen fusion gene in the eye lens. The alpha T1 line develops fast growing, poorly differentiated lens tumors, whereas the alpha T2 line produces lens tumors that are slow growing and well differentiated. There is a striking difference between these two lines in the temporal and spatial patterns of tumor antigen expression during initial lens development. In the alpha T1 line, the transgene is expressed very early in development in most lens cells, and no primary fiber differentiation takes place. In the alpha T2 line, transgene expression occurs after primary fiber formation has been initiated, and is restricted to differentiating fiber cells. The anterior epithelium from both alpha T lines undergoes normal development and remains morphologically normal until after birth, although in alpha T1 mice, these anterior cells produce considerable amounts of SV40 tumor antigens. This suggests that the state of differentiation of the lens cell plays an important role in its response to oncogene products.     

Investigation of the mechanisms of crystallin aggregation induced by pulsed laser UV irradiation at 308 nm

The results of the investigations of photoaggregation of the main eye lens proteins alpha-, beta- and gamma-crystallins and the model protein carbonic anhydrase in response to pulsed irradiation by a XeCI laser at 308 nm in the wide range of pulse energy densities (w) and pulse repetition rates (F) have been reviewed. A nonlinear dependence of aggregation efficiency on the values of w, F, and the concentration of protein solution was found. A theoretical model that qualitatively describes the experimental results was developed. The aggregation of N-amino-arm truncated beta A3-crystallin was analyzed. It was found that the loss of the N-amino-arm as a result of mutation or eye lens aging increases the probability of UV-induced beta-crystallin aggregation, thereby increasing the predisposition of eye lens to senile cataract. The influence of some short-chain peptides on the aggregation efficiency of beta-crystallin and beta-crystallin in solution with alpha-crystallin was investigated. Based on the results obtained, a combination of peptides (called "a new preparation") was found that most effectively delays the crystallin aggregation. The preparation has been probed on experimental animals. The trials showed that the preparation increases the delay in the development of UV-induced cataract in rats. The possibility of designing a drug for the prophylaxis of the development of cataract in humans based on this preparation is discussed.     

Bilateral congenital cataracts result from a gain-of-function mutation in the gene for aquaporin-0 in mice

Cataract Tohoku (Cat(Tohm)) is a dominant cataract mutation that leads to severe degeneration of lens fiber cells. Linkage analysis showed that the Cat(Tohm) mutation is located on mouse chromosome 10, close to the gene for aquaporin-0 (Aqp0), which encodes a membrane protein that is expressed specifically in lens fiber cells. Sequence analysis of Aqp0 revealed a 12-bp deletion without any change in the reading frame, which resulted in a deletion of four amino acids within the second transmembrane region of the AQP0 protein. Targeted expression of the mutated Aqp0 caused lens opacity in transgenic mice, the pathological severity of which depended on the expression level of the transgene. The mutated AQP0 protein was localized to the intracellular and perinuclear spaces rather than to the plasma membranes of the lens fiber cells. The cataract phenotype of Cat(Tohm) is caused by a gain-of-function mutation in the mutated AQP0 protein and not by a loss-of-function mutation.     

Major intrinsic polypeptide (MIP26K) of the lens membrane: covalent change in an internal sequence during human senile cataractogenesis

Polyclonal antisera to three synthetic peptides of bovine MIP26K have been used in combination with Western blot analysis to probe for changes of the MIP26K molecule during human senile cataractogenesis. Anti-MIP26K229-237 binds well to the 26K component from cataractous lens membranes, but binds poorly to the same component from normal lens. In contrast, antisera to two other sequences of MIP26K (anti-MIP26K252-259 and anti-MIP26K256-263) bind approximately equally well to the 26K component from either cataractous or normal lens. Together, these results demonstrate that during cataract development there is a selective covalent change in a region of the MIP26K molecule that may have profound effects upon the ability of this molecule to facilitate intercellular communication between lens fiber cells.     

Rise and fall of crystallin gene messenger levels during fibroblast growth factor induced terminal differentiation of lens cells.

Explanted rat lens epithelial cells differentiate synchronously in vitro to lens fiber cells in the presence of basic fibroblast growth factor (bFGF). We have monitored the expression of the three rat crystallin gene families, the alpha-, beta-, and gamma-crystallin genes, during this process. The expression of these gene families is sequentially activated, first the alpha-crystallin genes at Day 1, then the beta-crystallin genes at Day 3, and finally the gamma-crystallin genes at Day 8. The steady state levels of alpha- and beta-crystallin mRNA are not affected by incubation with actinomycin D, suggesting that these mRNAs are stable. Nevertheless, all crystallin mRNAs disappear from the differentiated explants between Days 10 and 11, a process signaled by bFGF. At this time a novel abundant mRNA appears. Cloning and sequencing showed that this mRNA encoded aldose reductase. Our results suggest a novel model for the regulation of crystallin synthesis during lens cell differentiation: a gene pulse delivers a certain amount of stable mRNA, this mRNA is removed at a later stage of differentiation by a stage-specific breakdown mechanism. Each of these regulatory steps requires a signal from bFGF.