Molecular organization of protein-lipid components of the crystalline lens

Lens transparency is primarily a physical phenomenon and is a manifestation of the lens structural organization. Traditionally the lens is considered as a "sac filled with proteins uniformly". Such studies have described overall average properties of the lens but have dealt with neither structural nor functional inhomogeneities in the lens tissue. All morphological, biochemical and physiological processes of the lens are aimed at the maintenance of transparency and refractive index. Minimizing of the lens light scatter is created in the lens by the processes that organize regularity at two structural levels: the fiber cytoplasmic matrix (cytoskeleton and soluble protein) and the fiber cell plasma membrane. Biochemical fractions of the lens are considered that are responsible for the physical basis of lens transparency.     

The lens: a classical model of embryonic induction providing new insights into cell determination in early development

The lens was the first tissue in which the concept of embryonic induction was demonstrated. For many years lens induction was thought to occur at the time the optic vesicle and lens placode came in contact. Since then, studies have revealed that lens placodal progenitor cells are specified already at gastrula stages, much earlier than previously believed, and independent of optic vesicle interactions. In this review, I will focus on how individual signalling molecules, in particular BMP, FGF, Wnt and Shh, regulate the initial specification of lens placodal cells and the progressive development of lens cells. I will discuss recent work that has shed light on the combination of signalling molecules and the molecular interactions that affect lens specification and proper lens formation. I will also discuss proposed tissue interactions important for lens development. A greater knowledge of the molecular interactions during lens induction is likely to have practical benefits in understanding the causes and consequences of lens diseases. Moreover, knowledge regarding lens induction is providing fundamental important insights into inductive processes in development in general.     

Molecular aging of lens crystallins and the life expectancy of the animal. Age-related protein structural changes studied in situ by Raman spectroscopy.

In order to investigate the relationship of molecular aging of lens crystallins to an animal's life expectancy or to the type of the lens, Raman spectra have been measured in situ for rabbit and guinea-pig lens nuclei at various stages of aging; these spectra have been compared with those of rat and mouse lens nuclei previously reported. Lens aging results in pronounced differences among the Raman spectra of the lens nuclei of the four species. It is shown that the rates of dehydration, inter- and intramolecular disulfide bond formation, and microenvironmental changes in the tryptophan residues of lens crystallins are different among the four species. Much faster changes occur for rat and mouse, which have a shorter life expectancy (2 years) and give rise to hard lens nuclei while slower changes occur for rabbit and guinea-pig, which have a longer life expectancy (5-7 years), and give soft lens nuclei. In addition, the Raman data reveal, for all the species investigated, that there are correlations among the rates of the dehydration, the inter- and intramolecular disulfide bond formation, and the microenvironmental changes in the tryptophan residues. Therefore, there seems to be a common mechanism for molecular aging of lens crystallins among the four species, although the rate of the molecular aging strongly depends upon the life expectancy of the animal and the type of the lens. The most important factor determining the rate of the molecular aging is probably the dehydration which decreases free water in the lens nucleus.     

Lactate Dehydrogenase Like Crystallin: A Potentially Protective Shield for Indian Spiny-Tailed Lizard (Uromastyx hardwickii) Lens Against Environmental Stress?

Taxon specific lens crystallins in vertebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uromastyx hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in U. hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure–function relationship of these proteins.     

Requirement for TGFbeta receptor signaling during terminal lens fiber differentiation

Several families of growth factors have been identified as regulators of cell fate in the developing lens. Members of the fibroblast growth factor family are potent inducers of lens fiber differentiation. Members of the transforming growth factor beta (TGFbeta) family, particularly bone morphogenetic proteins, have also been implicated in various stages of lens and ocular development, including lens induction and lens placode formation. However, at later stages of lens development, TGFbeta family members have been shown to induce pathological changes in lens epithelial cells similar to those seen in forms of human subcapsular cataract. Previous studies have shown that type I and type II TGFbeta receptors, in addition to being expressed in the epithelium, are also expressed in patterns consistent with a role in lens fiber differentiation. In this study we have investigated the consequences of disrupting TGFbeta signaling during lens fiber differentiation by using the mouse alphaA-crystallin promoter to overexpress mutant (kinase deficient), dominant-negative forms of either type I or type II TGFbeta receptors in the lens fibers of transgenic mice. Mice expressing these transgenes had pronounced bilateral nuclear cataracts. The phenotype was characterized by attenuated lens fiber elongation in the cortex and disruption of fiber differentiation, culminating in fiber cell apoptosis and degeneration in the lens nucleus. Inhibition of TGFbeta signaling resulted in altered expression patterns of the fiber-specific proteins, alpha-crystallin, filensin, phakinin and MIP. In addition, in an in vitro assay of cell migration, explanted lens cells from transgenic mice showed impaired migration on laminin and a lack of actin filament assembly, compared with cells from wild-type mice. These results indicate that TGFbeta signaling is a key event during fiber differentiation and is required for completion of terminal differentiation.     

The lens epithelium: focus on the expression and function of the alpha-crystallin chaperones

Lens epithelial cells are the parental cells responsible for growth and development of the transparent ocular lens. Many elegant investigations into their biology have focused on the factors that initiate and regulate lens epithelial cell differentiation. Because they serve key transport and cell maintenance functions throughout life, and are the primary source of metabolic activity in the lens, mechanisms to maintain lens epithelial cell integrity and survival are critical for lens transparency. The molecular chaperones alpha-crystallins are abundant proteins synthesized in the differentiated lens fiber cell cytoplasm. However, their expression in lens epithelial cells has only been appreciated very recently. Besides their important roles in the refractive and light focusing properties of the lens, alpha-crystallins have been implicated in a number of non-refractive pathways including those involving stress response, apoptosis and cell survival. The most convincing evidence for their importance in the lens epithelium has been shown by studies on the properties of lens epithelial cells from alphaA and alphaB-crystallin gene knockout mice. Novel combination of genetics, cell and molecular biology should lead to a greater understanding of how lens epithelial cells proliferate, differentiate and survive.     

Comparative study of lens proteins of gray squirrel and human

1. The four crystallins of the gray squirrel lens have been characterized using gel filtration chromatography, polyacrylamide gel electrophoresis, and immunoblotting. Alpha, beta-heavy, beta-light, and gamma crystallins of squirrel lenses have been identified immunologically, and they cross-react strongly with rabbit polyclonal antibodies. The gamma-24 crystallin of the squirrel lens also reacts strongly with monoclonal anti-human lens gamma-24, as shown by its inhibition of the ELISA reaction by 85%. 2. The water-insoluble urea soluble proteins represent non-covalently associated species of soluble crystallins and the lens cytoskeletal proteins. The membrane intrinsic protein in the urea insoluble pellet has a mol. wt of 27,000 but other lower and higher mol. wt components are also present, which were removed by washing with 0.1 NaOH. The N-terminal 30 amino acid of squirrel lens gamma crystallin was found to be identical to that of the bovine (and human) lens. 3. Measurements of the distribution and state of SH and SS compounds in the squirrel lens have shown greater similarities to those of primates than those of rodents. The findings show that on the basis of both protein and sulfur chemistry the squirrel lens is a representative model for studies of oxidative lens changes in diurnal animals, including man.     

A Focus on the Optical Properties of the Regenerated Newt Lens

Lens regeneration studies in the adult newt suggest that molecular aspects of lens regeneration are complete within 5 weeks of lentectomy. However, very little is known about the optical properties of the regenerated lens. In an aquatic environment, the lens accounts for almost all of the refractive power of the eye, and thus, a fully functional lens is critical. We compared the optical properties of 9- and 26-week regenerated lenses in the red spotted newt, Notophthalmus viridescens, with the original lenses removed from the same eyes. At 9 weeks, the regenerated lenses are smaller than the original lenses and are histologically immature, with a lower density of lens proteins. The 9 week lenses have greater light transmission, but significantly reduced focal length and refractive index than the original lenses. This suggests that following 9 weeks of regeneration, the lenses have not recovered the functionality of the original lens. By 26 weeks, the transmission of light in the more mature lens is reduced, but the optical parameters of the lens have recovered enough to allow functional vision.     

A novel role of the hedgehog pathway in lens regeneration

Lens regeneration in the adult newt is a classic example of replacing a lost organ by the process of transdifferentiation. After lens removal, the pigmented epithelial cells of the dorsal iris proliferate and dedifferentiate to form a lens vesicle, which subsequently differentiates to form a new lens. In searching for factors that control this remarkable process, we investigated the expression and role of hedgehog pathway members. These molecules are known to affect retina and pigment epithelium morphogenesis and have been recently shown to be involved in repair processes. Here we show that Shh, Ihh, ptc-1, and ptc-2 are expressed during lens regeneration. The expression of Shh and Ihh is quite unique since these genes have never been detected in lens. Interestingly, both Shh and Ihh are only expressed in the regenerating and developing lens, but not in the intact lens. Interfering with the hedgehog pathway results in considerable inhibition of the process of lens regeneration, including decreased cell proliferation as well as interference with lens fiber differentiation in the regenerating lens vesicle. Down-regulation of ptc-1 was also observed when inhibiting the pathway. These results provide the first evidence of a novel role for the hedgehog pathway in specific regulation of the regenerating lens.     

Apoptosis in lens development and pathology

The ocular lens is a distinct system to study cell death for the following reasons. First, during animal development, the ocular lens is crafted into its unique shape. The crafting processes include cell proliferation, cell migration, and apoptosis. Moreover, the lens epithelial cells differentiate into lens fiber cells through a process, which utilizes the same regulators as those in apoptosis at multiple signaling steps. In addition, introduction of exogenous wild-type or mutant genes or knock-out of the endogenous genes leads to apoptosis of the lens epithelial cells followed by absence of the ocular lens or formation of abnormal lens. Finally, both in vitro and in vivo studies have shown that treatment of adult lens with stress factors induces apoptosis of lens epithelial cells, which is followed by cataractogenesis. The present review summarizes the current knowledge on apoptosis in the ocular lens with emphasis on its role in lens development and pathology.     

AlphaA-crystallin expression prevents gamma-crystallin insolubility and cataract formation in the zebrafish cloche mutant lens

Cataracts, the loss of lens transparency, are the leading cause of human blindness. The zebrafish embryo, with its transparency and relatively large eyes, is an excellent model for studying ocular disease in vivo. We found that the zebrafish cloche mutant, both the cloche(m39) and cloche(S5) alleles, which have defects in hematopoiesis and blood vessel development, also have lens cataracts. Quantitative examination of the living zebrafish lens by confocal microscopy showed significant increases in lens reflectance. Histological analysis revealed retention of lens fiber cell nuclei owing to impeded terminal differentiation. Proteomics identified gamma-crystallin as a protein that was substantially diminished in cloche mutants. Crystallins are the major structural proteins in mouse, human and zebrafish lens. Defects in crystallins have previously been shown in mice and humans to contribute to cataracts. The loss of gamma-crystallin protein in cloche was not due to lowered mRNA levels but rather to gamma-crystallin protein insolubility. AlphaA-crystallin is a chaperone that protects proteins from misfolding and becoming insoluble. The cloche lens is deficient in both alphaA-crystallin mRNA and protein during development from 2-5 dpf. Overexpression of exogenous alphaA-crystallin rescued the cloche lens phenotype, including solubilization of gamma-crystallin, increased lens transparency and induction of lens fiber cell differentiation. Taken together, these results indicate that alphaA-crystallin expression is required for normal lens development and demonstrate that cataract formation can be prevented in vivo. In addition, these results show that proteomics is a valuable tool for detecting protein alterations in zebrafish.     

Chordate betagamma-crystallins and the evolutionary developmental biology of the vertebrate lens

Several animal lineages, including the vertebrates, have evolved sophisticated eyes with lenses that refract light to generate an image. The nearest invertebrate relatives of the vertebrates, such as the ascidians (sea squirts) and amphioxus, have only basic light detecting organs, leading to the widely-held view that the vertebrate lens is an innovation that evolved in early vertebrates. From an embryological perspective the lens is different from the rest of the eye, in that the eye is primarily of neural origin while the lens derives from a non-neural ectodermal placode which invaginates into the developing eye. How such an organ could have evolved has attracted much speculation. Recently, however, molecular developmental studies of sea squirts have started to suggest a possible evolutionary origin for the lens. First, studies of the Pax, Six, Eya and other gene families have indicated that sea squirts have areas of non-neural ectoderm homologous to placodes, suggesting an origin for the embryological characteristics of the lens. Second, the evolution and regulation of the betagamma-crystallins has been studied. These form one of the key crystallin gene families responsible for the transparency of the lens, and regulatory conservation between the betagamma-crystallin gene in the sea squirt Ciona intestinalis and the vertebrate visual system has been experimentally demonstrated. These data, together with knowledge of the morphological, physiological and gene expression similarities between the C. intestinalis ocellus and vertebrate retina, have led us to propose a hypothesis for the evolution of the vertebrate lens and integrated vertebrate eye via the co-option and combination of ancient gene regulatory networks; one controlling morphogenetic aspects of lens development and one controlling the expression of a gene family responsible for the biophysical properties of the lens, with the components of the retina having evolved from an ancestral photoreceptive organ derived from the anterior central nervous system.     

Extracellular matrix and integrin signaling in lens development and cataract

During development of the vertebrate lens there are dynamic interactions between the extracellular matrix (ECM) of the lens capsule and lens cells. Disruption of the ECM causes perturbation of lens development and cataract. Similarly, changes in cell signaling can result in abnormal ECM and cataract. Integrins are key mediators of ECM signals and recent studies have documented distinct repertoires of integrin expression during lens development, and in anterior subcapsular cataract (ASC) and posterior caspsule opacification (PCO). Increasingly, studies are being directed to investigating the signaling pathways that integrins modulate and have identified Src, focal adhesion kinase (FAK) and integrin-linked kinase (ILK) as downstream kinases that mediate proliferation, differentiation and morphological changes in the lens during development and cataract formation.     

The mechanism of cell elongation during lens fiber cell differentiation

Lens fiber formation is characterized by extensive cell elongation. Earlier studies have shown that lens cell elongation in vitro can occur in the absence of microtubules and is associated with a proportional increase in cell volume. We have previously suggested that lens fiber cell elongation is directly caused by an increase in cell volume. In this report, lenses from 3- and 6-day-old chicken embryos were three-dimensionally reconstructed from serial sections to provide a measure of cell volume and length during various stages of primary and secondary lens fiber formation. In both cases, cell volume was highly correlated with cell length during lens cell elongation. In addition, during primary lens fiber formation, large intercellular spaces between lens vesicle cells disappeared as these cells began to elongate to form lens fibers. Loss of intercellular spaces would be expected if increasing cell volume were responsible for cell elongation. Finally, results of experiments in which the lens capsule was cut with a fine tungsten needle suggested that the capsule was elastic and normally under tension. These findings were used to formulate a model which accounts for the major events in lens morphogenesis based on (1) the regulation of cell volume, (2) the junctions present between lens cells, and (3) the constraint provided by the elasticity of the lens capsule.     

干细胞与晶状体再生

白内障摘除联合人工晶状体植入术是目前治疗白内障的唯一有效措施。然而,人工晶状体作为替代材料,仍然存在一些如屈光调节力差以及术后眩光等未能克服的缺陷。寻找更理想的晶状体替代物及低等两栖类动物（如蝾螈）强大的晶状体再生能力,为晶状体再生的研究提供了原动力和依据。近年来,人们已探索出将胚胎干细胞/诱导的多能干细胞在体外诱导分化为类晶状体样结构的培养方法,为白内障的治疗开辟了新的思路。晶状体再生的研究为探索晶状体正常发育机制及晶状体疾病的发生和防治提供了新的平台。晶状体再生的成功也将为白内障的防治带来里程碑性的突破。本文拟总结晶状体正常发育过程及其调控机制,回顾国内外对晶状体体内再生能力的研究成果,并对目前人们探索利用胚胎干细胞和诱导的多能干细胞再造晶状体的研究进展作一概述,希望对干细胞与晶状体再生的后续相关研究提供一定的借鉴。     

Expression of Frizzleds and secreted frizzled-related proteins (Sfrps) during mammalian lens development

Recent studies indicate a role for Wnt signaling in regulating lens cell differentiation (Stump et al., 2003). Here we investigated expression patterns of Wnt receptors, the Frizzleds (Fzs) and the Wnt signaling regulators, the secreted frizzled-related proteins (Sfrps), during rodent lens development. RT-PCR showed that Fz receptors, Fz1-Fz8 are expressed in lens. In situ hybridization showed that all the Fz genes examined have similar expression patterns. Fzs are expressed throughout the early lens primordium. At embryonic day 14.5 (E14.5), Fz gene expression is predominantly localized to the epithelium and elongating cells at the lens equator. Fz expression is absent from lens fibers. This pattern of Fz gene expression continues throughout early postnatal development. Immunolocalization studies showed that Fz protein distribution closely follows that of the mRNAs. In addition, epithelial cells in FGF-treated explants show strongest Fz reactivity in cellular protrusions as they migrate and elongate. Sfrp1- Sfrp5 are expressed and all, except Sfrp2, have similar patterns of expression to each other and to the Fzs during lens development. Sfrp2 is strongly expressed in all lens pit cells but becomes restricted to the presumptive epithelial cells of the lens vesicle. By E14.5, Sfrp2 is only present in a few cells above the lens equator. Sfrp2 is not detected in the lens at E18.5 or at later stages. This study shows that multiple Fz and Sfrp genes are expressed during lens morphogenesis and differentiation. This is consistent with a role for Wnt-Fz signaling during both embryonic and postnatal lens development.     

The lens equator: A platform for molecular machinery that regulates the switch from cell proliferation to differentiation in the vertebrate lens

The vertebrate lens is a transparent, spheroidal tissue, located in the anterior region of the eye that focuses visual images on the retina. During development, surface ectoderm associated with the neural retina invaginates to form the lens vesicle. Cells in the posterior half of the lens vesicle differentiate into primary lens fiber cells, which form the lens fiber core, while cells in the anterior half maintain a proliferative state as a monolayer lens epithelium. After formation of the primary fiber core, lens epithelial cells start to differentiate into lens fiber cells at the interface between the lens epithelium and the primary lens fiber core, which is called the equator. Differentiating lens fiber cells elongate and cover the old lens fiber core, resulting in growth of the lens during development. Thus, lens fiber differentiation is spatially regulated and the equator functions as a platform that regulates the switch from cell proliferation to cell differentiation. Since the 1970s, the mechanism underlying lens fiber cell differentiation has been intensively studied, and several regulatory factors that regulate lens fiber cell differentiation have been identified. In this review, we focus on the lens equator, where these regulatory factors crosstalk and cooperate to regulate lens fiber differentiation. Normally, lens epithelial cells must pass through the equator to start lens fiber differentiation. However, there are reports that when the lens epithelium structure is collapsed, lens fiber cell differentiation occurs without passing the equator. We also discuss a possible mechanism that represses lens fiber cell differentiation in lens epithelium.     

大白鼠糖致白内障的激光喇曼光谱研究

作者用激光喇曼光谱法分析半乳糖导致大白鼠晶状体混浊过程中构象的变化。通过SPEX 1403型激光喇曼光谱仪得到了正常及不同混浊度晶状体的喇曼光谱。结果表明晶状体可溶性蛋白质二级结构的光谱未见异常,其残基酪氨酸及色氨酸微环境起了变化。随着晶状体混浊度的增加,SH谱峰强度变小而S-S键谱峰增强,同时观察到荧光背景逐渐加强。经分析认为晶状体混浊是与蛋白质分子的聚集有关。