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David C Beebe - One of the best experts on this subject based on the ideXlab platform.

  • The tumor suppressor gene Trp53 protects the mouse lens against posterior subcapsular cataracts and the BMP receptor Acvr1 acts as a tumor suppressor in the lens
    Disease Models & Mechanisms, 2011
    Co-Authors: Luke A. Wiley, Ramya Rajagopal, Lisa K. Dattilo, David C Beebe
    Abstract:

    We previously found that lenses lacking the Acvr1 gene, which encodes a bone morphogenetic protein (BMP) receptor, had abnormal proliferation and cell death in epithelial and cortical Fiber Cells. We tested whether the tumor suppressor protein p53 (encoded by Trp53) affected this phenotype. Acvr1 conditional knockout (Acvr1CKO) mouse Fiber Cells had increased numbers of nuclei that stained for p53 phosphorylated on serine 15, an indicator of p53 stabilization and activation. Deletion of Trp53 rescued the Acvr1CKO cell death phenotype in embryos and reduced Acvr1-dependent apoptosis in postnatal lenses. However, deletion of Trp53 alone increased the number of Fiber Cells that failed to withdraw from the cell cycle. Trp53CKO and Acvr1;Trp53DCKO (double conditional knockout), but not Acvr1CKO, lenses developed abnormal collections of Cells at the posterior of the lens that resembled posterior subcapsular cataracts. Cells from human posterior subcapsular cataracts had morphological and molecular characteristics similar to the Cells at the posterior of mouse lenses lacking Trp53. In Trp53CKO lenses, Cells in the posterior plaques did not proliferate but, in Acvr1;Trp53DCKO lenses, many Cells in the posterior plaques continued to proliferate, eventually forming vascularized tumor-like masses at the posterior of the lens. We conclude that p53 protects the lens against posterior subcapsular cataract formation by suppressing the proliferation of Fiber Cells and promoting the death of any Fiber Cells that enter the cell cycle. Acvr1 acts as a tumor suppressor in the lens. Enhancing p53 function in the lens could contribute to the prevention of steroid- and radiation-induced posterior subcapsular cataracts.

  • functions of the type 1 bmp receptor acvr1 alk2 in lens development cell proliferation terminal differentiation and survival
    Investigative Ophthalmology & Visual Science, 2008
    Co-Authors: Ramya Rajagopal, David C Beebe, Lisa K. Dattilo, Vesa Kaartinen, Chuxia Deng, Lieve Umans, An Zwijsen, Anita B Roberts, Erwin P Bottinger
    Abstract:

    Lens formation is one of the most widely studied examples of embryonic induction. 1,2 As a result of the precise localization of cell proliferation and differentiation in the developing lens, it has often been used to demonstrate the fundamental molecular mechanisms that control terminal cell differentiation and cell proliferation in all tissues.3-6 Lens morphogenesis begins on embryonic day (E) 9 of mouse development with the formation of the lens placode. After the lens placode invaginates and separates from the surface ectoderm to form the lens vesicle, Cells in the posterior part of the vesicle stop proliferating and form primary lens Fiber Cells. The anterior epithelial Cells continue to proliferate. Proliferation eventually becomes restricted to the epithelial Cells near the lens equator, which subsequently differentiate into secondary Fiber Cells, accounting for lens growth throughout life. Lens formation and its subsequent development are regulated by members of at least two major families of growth factors. Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β (TGFβ) superfamily, are essential for lens induction. Targeted deletion or inactivation of the BMP ligands Bmp4 and Bmp7 results in the failure of lens formation.2,7-9 Lenses lacking the type 1 BMP receptor Bmpr1a (Alk3) are small, have thin epithelia, and degenerate Fiber Cells.10 Fibroblast growth factors (FGFs) are the other family of growth factors important for lens formation. Deletion of Fgfr2 results in defects in Fiber cell terminal differentiation and reduced cell survival,11 and the deletion of Fgfr1, Fgfr2, and Fgfr3 in the lens vesicle prevents subsequent Fiber cell formation.12 Finally, there is genetic evidence of interactions between the FGF and BMP signaling pathways during early lens development.13 Although BMPs are essential for lens development, little is known about the cellular events initiated by BMP signaling and the downstream signaling molecules that mediate these events during lens induction and subsequent development. The BMP signaling cascade is initiated by type 2 and type 1 cell surface serine/threonine kinase receptors. Ligand-activated receptors phosphorylate downstream signaling molecules, the receptor-activated Smads or R-Smads. Activated R-Smads complex with the common mediator Smad (Co-Smad) known as Smad4 and translocate to the nucleus to regulate gene expression.14 To gain an understanding of the molecular events mediating BMP signaling in the lens, we used the Cre-loxP approach to inactivate the type 1 BMP receptor Acvr1 (Alk2) in the lens-forming head ectoderm of the mouse embryo. Acvr1 is expressed in the prospective ectoderm at the time of lens induction,15 but its function in lens development has not been examined. Acvr1CKO (conditional knockout) lenses formed but were smaller than wild-type lenses. Analysis of the cause of the smaller lens size revealed that Acvr1 promoted proliferation at early stages (Rajagopal R et al., manuscript submitted) but inhibited epithelial cell proliferation later in lens development. Inhibition of cell proliferation by Acvr1 was necessary for the proper regionalization of the lens epithelium and promoted the withdrawal of lens Fiber Cells from the cell cycle. Deletion of the downstream Smad effector proteins showed that Acvr1 inhibited proliferation and promoted cell cycle exit by engaging the BMP-specific R-Smads (Smad1 and Smad5) and the Co-Smad (Smad4). Although Acvr1 is required for Fiber Cells to withdraw from the cell cycle, it is not required for the expression of proteins that are characteristic of differentiated Fiber Cells. Acvr1 signaling promoted the survival of lens epithelial and Fiber Cells. The initial decrease in proliferation, along with an increase in cell death in the Acvr1CKO lens epithelia and Fiber Cells, appears to account for the overall decrease in lens size.

  • identification and expression of hop an atypical homeobox gene expressed late in lens Fiber cell terminal differentiation
    Molecular Vision, 2007
    Co-Authors: Oleg Vasiliev, Simon J Rhodes, David C Beebe
    Abstract:

    The lens is composed of two types of epithelial Cells: Asheet of cuboidal Cells, the lens epithelium, covers its anteriorsurface, and post-mitotic, elongated Fiber Cells comprise thebulk of the lens (Figure 1). Stimulation by factors present inthe vitreous body causes epithelial Cells near the lens equatorto withdraw from the cell cycle and differentiate into lens fi-ber Cells. Differentiating Fiber Cells elongate and initiate thetranscription of genes that encode a distinct array of abundantmembrane, cytoskeletal, and cytoplasmic proteins. The accu-mulation of high concentrations of cytoplasmic proteins(crystallins) in Fiber Cells is important for the transparencyand refractive power of the lens. Some crystallins, cytoskeletal,and membrane proteins are found primarily in lens Cells, orare present only at very low levels in non-lens tissues [1-7].Lens Fiber Cells undergo remarkable morphologicalchanges during their differentiation. Fiber Cells first elongateto many times their original length, extending to over 140 µmper day in the chicken embryo [8]. As they elongate, the ante-rior and posterior ends of the Fiber Cells extend beneath thelens epithelium and along the posterior lens capsule towardthe optical axis. When the ends of these Cells approach theanterior and posterior poles of the lens, they meet elongatingFiber Cells extending from the other side, resulting in the for-mation of the anterior and posterior sutures (Figure 1). Oncethe Cells stop elongating, they become buried beneath the nextgroup of elongating Fiber Cells. Soon after the Fiber Cells de-tach from the posterior capsule, the composition of their cell-cell adhesion proteins changes [9], their lateral membranesbecome interdigitated [8] and partially fuse with the mem-branes of neighboring Fiber Cells [8,10], and all intracellular,membrane-bound organelles are degraded [11-17]. Mature fi-ber Cells persist in this state for the life of the organism.

  • signaling through fgf receptor 2 is required for lens cell survival and for withdrawal from the cell cycle during lens Fiber cell differentiation
    Developmental Dynamics, 2005
    Co-Authors: C M Garcia, Michael L. Robinson, Kai Yu, Haotian Zhao, Ruth Asherypadan, David M Ornitz, David C Beebe
    Abstract:

    Fibroblast growth factors (FGFs) play important roles in many aspects of development, including lens development. The lens is derived from the surface ectoderm and consists of an anterior layer of epithelial Cells and elongated, terminally differentiated Fiber Cells that form the bulk of the tissue. FGF signaling has been implicated in lens induction, proliferation, and differentiation. To address the role of FGFs in lens development, we inactivated FGF receptor-2 (Fgfr2) using a Cre transgene that is expressed in all prospective lens Cells from embryonic day 9.0. Inactivation of Fgfr2 shows that signaling through this receptor is not required for lens induction or for the proliferation of lens epithelial Cells. However, Fgfr2 signaling is needed to drive lens Fiber Cells out of the cell cycle during their terminal differentiation. It also contributes to the normal elongation of primary lens Fiber Cells and to the survival of lens epithelial Cells. Developmental Dynamics 233:516 –527, 2005. © 2005 Wiley-Liss, Inc.

  • changes in adhesion complexes define stages in the differentiation of lens Fiber Cells
    Investigative Ophthalmology & Visual Science, 2001
    Co-Authors: David C Beebe, Oleg Vasiliev, Yingbo Shui, Steven Bassnett
    Abstract:

    PURPOSE. During their differentiation, lens Fiber Cells elongate, detach from the lens capsule, associate at the sutures, and degrade all cytoplasmic membrane-bound organelles. Changes in the expression or organization of cell adhesion and cytoskeleton-associated proteins were correlated with these events during Fiber cell differentiation in chicken embryos. METHODS. Fixed or living lenses were sliced with a tissue slicer, permeabilized or extracted with detergents, stained with antibodies or fluorescent-labeled phalloidin, and viewed with a confocal microscope. The distribution of N-cadherin in elongating and mature Fiber Cells was determined by Western blot analysis. Reverse transcription‐polymerase chain reaction (RTPCR) was used to determine the distribution of vinculin and paxillin transcripts. RESULTS. Staining for N-cadherin and band 4.1 protein decreased soon after Fiber Cells detached from the capsule. Detergent extraction of lens sections and Western blots of dissected lens regions showed that much of this decrease in staining was due to epitope masking. Vinculin immunoreactivity was barely detectable on the lateral membranes of elongating Fiber Cells but increased markedly once these Cells reached their maximum length and formed the sutures. Staining for paxillin was also low in elongating Fiber Cells but increased late in Fiber cell differentiation, just before the Cells destroyed their membrane-bound organelles. Spectrin and ankyrin immunoreactivity did not change when Fiber Cells reached the sutures. Staining for F-actin increased transiently in Cells that had just reached the sutures. Messenger RNAs for vinculin and paxillin were more abundant in maturing than in elongating Fiber Cells. CONCLUSIONS. The adhesion complexes of lens Fiber Cells change in organization and composition soon after these Cells finish elongating and detach from the capsule. Increased staining for vinculin and paxillin defines distinct stages of Fiber cell differentiation that are intermediate between the completion of cell elongation and the time when lens Fiber Cells degrade their membrane-bound organelles. Remodeling adhesion complexes during Fiber cell maturation may assure the stability of Fiber‐Fiber associations, once these Cells are no longer transcriptionally active. (Invest Ophthalmol Vis Sci. 2001;42: 727‐734) T

Paul J. Donaldson - One of the best experts on this subject based on the ideXlab platform.

  • confocal microscopy reveals zones of membrane remodeling in the outer cortex of the human lens
    Investigative Ophthalmology & Visual Science, 2009
    Co-Authors: Kerry L Walker, Paul J. Donaldson, Trevor Sherwin, Kevin L Schey
    Abstract:

    The transparency of the lens is linked to the unique structure and function of its Fiber Cells. These highly differentiated Cells are derived from equatorial epithelial Cells that exit the cell cycle and embark on a differentiation process that produces extensive cellular elongation, the loss of cellular organelles and nuclei, and the expression of Fiber-specific proteins.1,2 Because this process is continual, Fiber Cells become internalized, creating an inherent age gradient that encapsulates all stages of Fiber cell differentiation throughout the lifetime of a person. In human lenses, light, transmission, and scanning electron microscopy have described five distinct zones that correspond to different stages of human lens development.3–7 The cortex consists of elongating Fiber Cells undergoing differentiation, the adult nucleus is composed of differentiated Fiber Cells formed since puberty, the juvenile nucleus contains Fiber Cells formed from birth until the onset of puberty, the fetal nucleus consists of Fiber Cells formed from the seventh week of development until birth, and the embryonic nucleus encompasses primary Fiber Cells formed in the 6 weeks after fertilization.7 Within these five zones of the human lens, there are distinct differences in Fiber cell morphology, the extent of cell compaction, and the degree of membrane interdigitations. Fiber Cells of the deep cortex are arranged in radial cell columns, whereas in the adult nucleus, Cells are compacted and are irregularly shaped. In the juvenile nucleus, Fiber cell shape is similar shape to that of the adult nucleus, but the Cells are less compacted. In the fetal nucleus, Cells are organized in irregular rows and are rounded; in the embryonic nucleus, Cells are irregularly shaped, can be large or small, and are arranged in no evident pattern.7 Throughout these regions, ultrastructure studies have revealed numerous interdigitations (ball and socket joints,7 interlocking edge processes,7 tongue and groove junctions,8 gap junctions and square arrays9) that serve to bind together adjacent lens Fiber Cells and stabilize the lens structure during accommodation.7 Similar studies in primate lenses also reveal equivalent changes in Fiber cell morphology, a progression from smooth to furrowed membranes, and a higher degree of interdigitations with increasing distance into the lens.3,10 Cumulative data from morphologic studies conducted with electron microscopy have enabled investigators to visualize Fiber Cells at high resolution.3,5–10 However, with this approach, it is often difficult to obtain an overall idea of how these changes in Fiber cell morphology are related to the process of Fiber cell differentiation. Furthermore, it is difficult to determine how the expression patterns of the membrane proteins involved in the formation of these various membrane junctions also change during the course of Fiber cell differentiation because immunoelectron microscopy can often be problematic. In the rat lens, we have successfully developed an immunohistochemical approach with the use of confocal microscopy that enables us to acquire high-resolution data sets across large distances,11 allowing us to map the subcellular distribution of specific membrane proteins as a function of Fiber cell differentiation.12–15 In this study, we attempted to optimize our immunohistochemical mapping approaches developed in the rat to map the morphology of Fiber Cells throughout the human lens with confocal microscopy. By optimizing lens fixation and sectioning protocols, we have replicated previous morphologic results attained through light and electron microscopy. In addition, our protocols allow immunolabeling for the major lens protein AQPO in all five regions. Furthermore, unlike previous light and electron microscopy studies, our protocols routinely allow us to preserve the epithelium and outer cortex of the lens enabling us to characterize, for the first time, two unique zones of membrane remodeling within the outer cortex region. These regions appear to represent an area of extreme Fiber cell remodeling before internalization of these Cells into the adult nucleus.

  • whole cell patch clamping of isolated Fiber Cells confirms that spatially distinct cl influx and efflux pathways exist in the cortex of the rat lens
    Investigative Ophthalmology & Visual Science, 2009
    Co-Authors: Kevin F Webb, Paul J. Donaldson
    Abstract:

    PURPOSE. To test the hypothesis that lens Fiber Cells use different combinations of transport proteins to mediate Cl influx and efflux in order to regulate their steady state volume. METHODS. Cells were isolated from rat lenses by enzymatic dissociation in the presence of Gd 3 , and short and long Fiber Cells were assigned to peripheral efflux and deeper influx zones, respectively. Electrical properties were of isolated Cells, and whole lenses were analyzed by using whole-cell patch clamping and intracellular microelectrodes, respectively, before and after exposure to hyposmotic challenge and/or the addition of [(dihydronindenyl)oxy] alkanoic acid (DIOA). RESULTS. Cells from the influx zone were dominated by an outwardly rectifying Cl conductance, and exposure to hyposmotic challenge increased this conductance. Cells isolated from the efflux zone were dominated by K conductance(s) with only a minimal contribution from the Cl conductance. Exposure of Cells that exhibited a minimal baseline Cl conductance to hyposmotic challenge caused swelling and a transient increase in Cl current. In other Cells that initially lacked aC l conductance, hyposmotic challenge caused swelling, but no increase in outward current. However, the subsequent addition of DIOA exacerbated swelling and activated a Cl current. Under isosmotic conditions, addition of DIOA also induced cell swelling and the transient activation of a Cl current. In whole lenses, exposure to hyposmotic challenge increased the contribution of an anion conductance to the membrane potential. CONCLUSIONS. In peripheral Cells, Cl efflux is primarily mediated by potassium chloride cotransporters (KCCs) and its activity can be upregulated by hyposmotic challenge. In addition, these Cells also contain a Cl channel that exhibits a variable baseline activity level and that can be recruited to effect regulatory volume decrease if the KCC transporters are inhibited. (Invest Ophthalmol Vis Sci. 2009;50:3808‐3818) DOI:10.1167/iovs.08-2680

  • Roles for KCC transporters in the maintenance of lens transparency.
    Investigative Ophthalmology & Visual Science, 2006
    Co-Authors: Kaa-sandra N. Chee, Joerg Kistler, Paul J. Donaldson
    Abstract:

    PURPOSE. To determine whether the potassium chloride co-transporter (KCC) family is expressed in the rat lens and to ascertain whether the transporters are involved in the regulation of lens volume and transparency. METHODS. RT-PCR was performed on RNA extracted from Fiber Cells to identify members of the KCC family expressed in the lens. Western blot analysis and immunocytochemistry, using KCC isoform-specific antibodies, were used to verify expression at the protein level and to localize KCC isoform expression. Organ-cultured rat lenses were incubated in isotonic artificial aqueous humor (AAH) that contained either the KCC-specific inhibitor [(dihydronindenyl)oxy] alkanoic acid (DIOA), the KCC activator N-ethylmaleimide (NEM), or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) for up to 18 hours. Lens wet weight was monitored, and lens transparency and tissue morphology were recorded with dark-field and confocal microscopy, respectively. RESULTS. Molecular experiments to characterize KCC isoform expression showed that KCC1, -3, and -4 were all expressed in the lens at both the transcript and protein levels and that KCC2 was not. Immunocytochemistry indicated that the three KCC isoforms exhibited distinct differentiation-dependent expression patterns, with KCC1 and -3 being restricted to the lens cortex, whereas KCC4 was found throughout the entire lens, including the lens core. In the lens cortex, most of the labeling for all KCC isoforms was cytoplasmic, whereas in the lens core, KCC4 labeling was associated with the membrane. Incubation of lenses in 100 μM DIOA for 18 hours caused lenses to increase their wet weight and induced a cortical opacity that was caused by extensive damage to peripheral Fiber Cells located up to 150 μm in from the lens capsule, whereas deeper Fiber Cells appeared unaffected by DIOA exposure. Lower concentrations of DIOA (10 μM) revealed that this damage was initiated primarily by the swelling of peripheral Fiber Cells. In contrast, NPPB-treated lenses exhibited a deeper zone (>100 μm) of cell damage that was initiated by the dilation of the extracellular space between Fiber Cells. Exposure of lenses to the KCC activator NEM caused cell shrinkage in peripheral Fiber Cells but extensive cell swelling in deeper Fiber Cells. Peripheral cell swelling caused a differential recruitment of KCC isoforms from a cytoplasmic pool to the plasma membrane. DIOA-induced cell swelling increased the association of KCC4 with membrane, whereas hypotonic cell swelling dramatically increased the association of KCC1 with the membrane. CONCLUSIONS. The rat lens expresses three KCC transporter isoforms (KCC1, -3, and -4) in a differentiation-dependent manner. Modulation of transporter activity and subcellular localization suggests that multiple KCC transporters mediate KCl efflux in peripheral Fiber Cells in a dynamic fashion. These results indicate that, in addition to Cl - channels, KCC transporters play a role in mediating a circulating flux of Cl - ions, which contributes to the maintenance of lens transparency through controlling the steady state volume of lens Fiber Cells.

  • Expression Patterns for Glucose Transporters GLUT1 and GLUT3 in the Normal Rat Lens and in Models of Diabetic Cataract
    Investigative Opthalmology & Visual Science, 2003
    Co-Authors: B. Rachelle Merriman-smith, J. Kistler, Anatoly Krushinsky, Paul J. Donaldson
    Abstract:

    PURPOSE. To determine whether the expression levels and cellular distribution of the facilitative glucose transporters GLUTI and -3 undergo changes in the hyperglycemic lens. METHODS. Hyperglycemia was induced in vivo by injecting rats with streptozotocin or in vitro by culturing lenses in the presence of 50 mM glucose. Northern blot analysis and quantitative RT-PCR were used to detect changes in GLUT1 and -3 transcript levels, and Western blot analysis was used to monitor changes in GLUT3 protein expression levels in diabetic rats. Immunocytochemistry was used to map the cellular distribution of GLUT3 in normal and hyperglycemic lenses. RESULTS. GLUTI and -3 were found to be differentially expressed in the epithelial and Fiber Cells, respectively. In the Fiber Cells, the distribution of GLUT3 protein changed as a function of Fiber cell differentiation. In young differentiating Fiber Cells, GLUT3 was mainly found in the cytoplasm, but with increasing depth into the lens became inserted into the narrow sides of older Fiber Cells, before becoming completely dispersed around the entire membrane of the oldest Fiber Cells. Hyperglycemia had similar effects on tissue damage and transporter expression in both the in vitro and in vivo models. Tissue damage was characterized by an initial local cell swelling that with prolonged insult gradually spread and resulted in the creation of large areas of tissue liquefaction. Northern blot analysis and quantitative RT-PCR showed that transcript for GLUT3 but not GLUT1 was upregulated under hyperglycemic conditions. This increase in GLUT3 expression was confirmed at the protein level by both Western blot analysis and immunocytochemistry. In hyperglycemic lenses, GLUT3 antibody labeling was localized to the region of tissue liquefaction. CONCLUSIONS. GLUT3 in the lens exhibits dynamic changes in expression levels and cellular localization as a function of Fiber cell differentiation and hyperglycemia. In the lens cortex, regions of GLUT3 overexpression and hyperglycemic tissue damage overlap, suggesting a functional relationship.

  • Galectin-3 is associated with the plasma membrane of lens Fiber Cells.
    Investigative ophthalmology & visual science, 2000
    Co-Authors: Tamir Gonen, Paul J. Donaldson, Joerg Kistler
    Abstract:

    PURPOSE. To discover proteins that have the potential to contribute to the tight packing of Fiber Cells in the lens. METHODS. Crude Fiber cell membranes were isolated from ovine lens cortex. Proteins were separated by two-dimensional gel electrophoresis, and selected protein spots identified by microsequencing. The identification of galectin-3 was confirmed by immunoblotting with a specific antibody. The association of galectin-3 with the Fiber cell plasma membrane was investigated using immunofluorescence microscopy, solubilization trials with selected reagents, and immunoprecipitation to identify candidate ligands. RESULTS. A cluster of three protein spots with an apparent molecular weight of 31,000 and isoelectric points ranging between 7 and 8.5 were resolved and identified as galectin-3. This protein was associated peripherally with the Fiber cell plasma membrane and interacted with MP20, an abundant intrinsic membrane protein that had been identified previously as a component of membrane junctions between Fiber Cells. CONCLUSIONS. The detection of galectin-3 in the lens is a novel result and adds to the growing list of lens proteins with adhesive properties. Its location at the Fiber cell membrane and its association with the junction-forming MP20 is consistent with a potential role in the development or maintenance of the tightly packed lens tissue architecture. (Invest Ophthalmol Vis Sci. 2000;41:199 ‐203)

Michael L. Robinson - One of the best experts on this subject based on the ideXlab platform.

  • Comparative transcriptome analysis of epithelial and Fiber Cells in newborn mouse lenses with RNA sequencing.
    Molecular vision, 2014
    Co-Authors: Thanh Hoang, Praveen Kumar Raj Kumar, Sreeskandarajan Sutharzan, Panagiotis A. Tsonis, Chun Liang, Michael L. Robinson
    Abstract:

    Purpose: The ocular lens contains only two cell types: epithelial Cells and Fiber Cells. The epithelial Cells lining the anterior hemisphere have the capacity to continuously proliferate and differentiate into lens Fiber Cells that make up the large proportion of the lens mass. To understand the transcriptional changes that take place during the differentiation process, high-throughput RNA-Seq of newborn mouse lens epithelial Cells and lens Fiber Cells was conducted to com prehensively compare the transcriptomes of these two cell types. Methods: RNA from three biologic replicate samples of epithelial and Fiber Cells from newborn FVB/N mouse lenses was isolated and sequenced to yield more than 24 million reads per sample. Sequence reads that passed quality filtering were mapped to the reference genome using Genomic Short-read Nucleotide Alignment Program (GSNAP). Transcript abundance and differential gene expression were estimated using the Cufflinks and DESeq packages, respectively. Gene Ontology enrichment was analyzed using GOseq. RNA-Seq results were compared with previously published microarray data. The differential expression of several biologically important genes was confirmed using reverse transcription (RT)-quantitative PCR (qPCR). Results: Here, we present the first application of RNA-Seq to understand the transcriptional changes underlying the differentiation of epithelial Cells into Fiber Cells in the newborn mouse lens. In total, 6,022 protein-coding genes exhibited differential expression between lens epithelial Cells and lens Fiber Cells. To our knowledge, this is the first study identify ing the expression of 254 long intergenic non-coding RNAs (lincRNAs) in the lens, of which 86 lincRNAs displayed differential expression between the two cell types. We found that RNA-Seq identified more differentially expressed genes and correlated with RT-qPCR quantification better than previously published microarray data. Gene Ontology analysis showed that genes upregulated in the epithelial Cells were enriched for extracellular matrix production, cell division, migration, protein kinase activity, growth factor binding, and calcium ion binding. Genes upregulated in the Fiber Cells were enriched for proteosome complexes, unfolded protein responses, phosphatase activity, and ubiquitin binding. Differentially expressed genes involved in several important signaling pathways, lens structural components, organelle loss, and denucleation were also highlighted to provide insights into lens development and lens Fiber differentiation. Conclusions: RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of protein-coding and non-coding transcripts from lens epithelial Cells and lens Fiber Cells. This information provides a valuable resource for studying lens development, nuclear degradation, and organelle loss during Fiber differentiation, and associated diseases.

  • nuclear removal during terminal lens Fiber cell differentiation requires cdk1 activity appropriating mitosis related nuclear disassembly
    Development, 2014
    Co-Authors: Blake R Chaffee, Masaki Nakahara, Shigekazu Nagata, Michael L. Robinson, Fu Shang, Min Lee Chang, Tracy M Clement, E M Eddy, Brad D Wagner, Allen Taylor
    Abstract:

    Lens epithelial Cells and early lens Fiber Cells contain the typical complement of intracellular organelles. However, as lens Fiber Cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens Fiber Cells requires the nuclease DNase IIβ (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic Cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic Cells lack CDK1 and cyclins, lens Fiber Cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial Cells, with the remaining Cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during Fiber cell differentiation.

  • Lens Fiber Cell Differentiation
    Encyclopedia of the Eye, 2010
    Co-Authors: Michael L. Robinson
    Abstract:

    The Fiber cell is the definitive lens cell type. Although only one of the two cell types comprising the lens, the Fiber Cells make up the vast majority of the lens volume; and specialized adaptations of lens Fiber Cells give the lens its characteristic spheroid shape, transparency, and its high refractive index. In this article, the differentiation of lens Fiber Cells from their precursor cell types is discussed with an emphasis on the important adaptations that characterize each stage of lens Fiber cell development. Although all vertebrates undergo similar lens development, emphasis in the article is on mammalian lens development, with exceptions noted.

  • signaling through fgf receptor 2 is required for lens cell survival and for withdrawal from the cell cycle during lens Fiber cell differentiation
    Developmental Dynamics, 2005
    Co-Authors: C M Garcia, Michael L. Robinson, Kai Yu, Haotian Zhao, Ruth Asherypadan, David M Ornitz, David C Beebe
    Abstract:

    Fibroblast growth factors (FGFs) play important roles in many aspects of development, including lens development. The lens is derived from the surface ectoderm and consists of an anterior layer of epithelial Cells and elongated, terminally differentiated Fiber Cells that form the bulk of the tissue. FGF signaling has been implicated in lens induction, proliferation, and differentiation. To address the role of FGFs in lens development, we inactivated FGF receptor-2 (Fgfr2) using a Cre transgene that is expressed in all prospective lens Cells from embryonic day 9.0. Inactivation of Fgfr2 shows that signaling through this receptor is not required for lens induction or for the proliferation of lens epithelial Cells. However, Fgfr2 signaling is needed to drive lens Fiber Cells out of the cell cycle during their terminal differentiation. It also contributes to the normal elongation of primary lens Fiber Cells and to the survival of lens epithelial Cells. Developmental Dynamics 233:516 –527, 2005. © 2005 Wiley-Liss, Inc.

  • ectopic pax6 expression disturbs lens Fiber cell differentiation
    Investigative Ophthalmology & Visual Science, 2004
    Co-Authors: Melinda K. Duncan, Michael L. Robinson, Jennifer R. Taube, Larry L. David, Lixing W Reneker
    Abstract:

    PURPOSE. Pax6 is a transcription factor necessary for the specification and subsequent formation of the ocular lens. It is expressed in all lens Cells at early stages of development. After lens formation, Pax6 expression is maintained in the lens epithelium, whereas its level abruptly decreases in differentiated Fiber Cells. This study is to test the hypothesis that normal Fiber cell differentiation would be perturbed by sustained Pax6 expression. METHODS. Transgenic mice expressing the canonical form of mouse Pax6 were created under the control of a modified mouse aA-crystallin promoter. The phenotypic changes in the transgenic lens were analyzed by light and electron microscopy. The effect of ectopic Pax6 expression on the lens Fiber Cells was investigated by in situ hybridization, immunohistochemical staining, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and two-dimensional (2-D) gel electrophoresis. RESULTS. Transgenic mice from seven different lines all had cataracts with severity that correlated with the transgene expression level in lens Fiber Cells. In severely affected lines, a lumen was present between the apical surfaces of the epithelial and Fiber Cells, suggesting that secondary Fiber cell elongation is incomplete. Electron microscopy analysis showed that the ball-and-socket interdigitations between neighboring Fiber Cells were underdeveloped or attenuated in the transgenic lens. Most interesting, elevated levels of Pax6 in Fiber Cells reduced the protein levels of transcription factor cMaf, which is known to be essential in Fiber cell differentiation. Furthermore, the total amount of lens proteins was 60% less than normal in the Pax6 transgenic lens. Among the crystallins examined, the relative ratio of intact βB1-crystallin protein to total lens protein was significantly reduced. Real-time reverse transcriptase PCR showed that the ratio of βB1-crystallin transcript levels to total mRNA levels were reduced by 87%. CONCLUSIONS. The data demonstrate that high levels of Pax6 expression disrupt normal Fiber cell differentiation and maturation.

Xiaohua Gong - One of the best experts on this subject based on the ideXlab platform.

  • Diverse Roles of Eph/ephrin Signaling in the Mouse Lens
    PLOS ONE, 2011
    Co-Authors: Catherine Cheng, Xiaohua Gong
    Abstract:

    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.

  • Diverse roles of Eph/ephrin signaling in the mouse lens.
    PloS one, 2011
    Co-Authors: Catherine Cheng, Xiaohua Gong
    Abstract:

    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.

  • Absence of α3 (Cx46) and α8 (Cx50) connexins leads to cataracts by affecting lens inner Fiber Cells
    Experimental eye research., 2006
    Co-Authors: Chun Hong Xia, Catherine Cheng, Irene Dunia, Qingling Huang, Debra Cheung, Lucio Benedetti, Joseph Horwitz, Xiaohua Gong
    Abstract:

    Lens development and transparency have been hypothesized to depend on intercellular gap junction channels, consisting of α3 (Cx46) and α8 (Cx50) connexin subunits, to transport metabolites, secondary messages and ions between lens Cells. To evaluate this hypothesis, we have generated α3(−/−) α8(−/−) double knockout mice and characterized their lens phenotypes. Without gap junctions between lens Fiber Cells, α3(−/−) α8(−/−) lenses displayed severe cataracts resulting from cell swelling and degeneration of inner Fibers while normal peripheral Fiber Cells continued to form throughout life. Neither an increase of degraded crystallins nor an increase of water-insoluble crystallins was found in α3(−/−) α8(−/−) lenses. However, a substantial reduction of γ-crystallin proteins, but not α- and β-crystallins, was detected. These results suggest that gap junction communication is important for maintaining lens homeostasis of inner Fiber Cells and that a loss of gap junctions leads to cataract formation as well as reductions of γ-crystallin proteins and transcripts.

  • Structural and immunocytochemical alterations in eye lens Fiber Cells from Cx46 and Cx50 knockout mice
    European journal of cell biology, 2006
    Co-Authors: Irene Dunia, Xiaohua Gong, Christian Cibert, Chun Hong Xia, Michel Recouvreur, Essy Levy, Nalin M. Kumar, Hans Bloemendal, E. Lucio Benedetti
    Abstract:

    In the current study we describe the changes of overall organization of lens Fiber Cells in connexin 46 (Cx46) and connexin 50 (Cx50) knockout mice. Morphometric analyses and the application of immunocytochemical techniques revealed that in Cx46 knockout lens (Cx46 -/-), where Cx50 is expressed alone, the postnatal differentiation of secondary Fiber Cells proceeds faster and is characterized by an increased number of smaller Fiber Cells. Conversely, in Cx50 knockout mice (Cx50 -/-), the lenticular mass is considerably reduced and characterized by a small number of Fiber Cells added during the postnatal period. The process of terminal differentiation was impaired and generated larger Fiber Cells still possessing cytoplasmic organelles. Freeze-fracture and fracture labeling revealed that the junctional assembly, packing organization and topographic interactions between connexons and MP26 differed when Cx46 and Cx50 were co-assembled in the wild-type or expressed separately in the two distinct knockout phenotypes. Filipin cytochemistry provided indirect evidence that Cx46 and Cx50 expressed alone are recruited into different lipid environments. Our results represent the structural proof that interaction of connexins and MP26 contributes to the overall organization of the Fiber Cells.

  • diverse gap junctions modulate distinct mechanisms for Fiber cell formation during lens development and cataractogenesis
    Development, 2006
    Co-Authors: Debra Cheung, Catherine Cheng, Woo-kuen Lo, Eddie Wang, Xin Du, Bruce Beutler, Xiaohua Gong
    Abstract:

    Different mutations of α3 connexin (Cx46 or Gja8 ) andα 8 connexin (Cx50 or Gja8 ), subunits of lens gap junction channels, cause a variety of cataracts via unknown mechanisms. We identified a dominant cataractous mouse line ( L1 ), caused by a missense α8 connexin mutation that resulted in the expression of α8-S50P mutant proteins. Histology studies showed that primary lens Fiber Cells failed to fully elongate in heterozygous α8 S50P/+ embryonic lenses, but not in homozygous α8 S50P/S50P , α8 -/- andα 3 -/- α8 -/- mutant embryonic lenses. We hypothesized that α8-S50P mutant subunits interacted with wild-typeα 3 or α8, or with both subunits to affect Fiber cell formation. We found that the combination of mutant α8-S50P and wild-type α8 subunits specifically inhibited the elongation of primary Fiber Cells, while the combination of α8-S50P and wild-type α3 subunits disrupted the formation of secondary Fiber Cells. Thus, this work provides the first in vivo evidence that distinct mechanisms, modulated by diverse gap junctions, control the formation of primary and secondary Fiber Cells during lens development. This explains why and how different connexin mutations lead to a variety of cataracts. The principle of this explanation can also be applied to mutations of other connexin isoforms that cause different diseases in other organs.

Melinda K. Duncan - One of the best experts on this subject based on the ideXlab platform.

  • Beta-1 integrin is important for the structural maintenance and homeostasis of differentiating Fiber Cells.
    The international journal of biochemistry & cell biology, 2014
    Co-Authors: David A. Scheiblin, Richard T. Mathias, Junyuan Gao, Jeffrey L. Caplan, Vladimir N. Simirskii, Kirk J. Czymmek, Melinda K. Duncan
    Abstract:

    Abstract β1-Integrin is a heterodimeric transmembrane protein that has roles in both cell–extra-cellular matrix and cell–cell interactions. Conditional deletion of β1-integrin from all lens Cells during embryonic development results in profound lens defects, however, it is less clear whether this reflects functions in the lens epithelium alone or whether this protein plays a role in lens Fibers. Thus, a conditional approach was used to delete β1-integrin solely from the lens Fiber Cells. This deletion resulted in two distinct phenotypes with some lenses exhibiting cataracts while others were clear, albeit with refractive defects. Analysis of “clear” conditional knockout lenses revealed that they had profound defects in Fiber cell morphology associated with the loss of the F-actin network. Physiological measurements found that the lens Fiber Cells had a twofold increase in gap junctional coupling, perhaps due to differential localization of connexins 46 and 50, as well as increased water permeability. This would presumably facilitate transport of ions and nutrients through the lens, and may partially explain how lenses with profound structural abnormalities can maintain transparency. In summary, β1-integrin plays a role in maintaining the cellular morphology and homeostasis of the lens Fiber Cells.

  • ectopic pax6 expression disturbs lens Fiber cell differentiation
    Investigative Ophthalmology & Visual Science, 2004
    Co-Authors: Melinda K. Duncan, Michael L. Robinson, Jennifer R. Taube, Larry L. David, Lixing W Reneker
    Abstract:

    PURPOSE. Pax6 is a transcription factor necessary for the specification and subsequent formation of the ocular lens. It is expressed in all lens Cells at early stages of development. After lens formation, Pax6 expression is maintained in the lens epithelium, whereas its level abruptly decreases in differentiated Fiber Cells. This study is to test the hypothesis that normal Fiber cell differentiation would be perturbed by sustained Pax6 expression. METHODS. Transgenic mice expressing the canonical form of mouse Pax6 were created under the control of a modified mouse aA-crystallin promoter. The phenotypic changes in the transgenic lens were analyzed by light and electron microscopy. The effect of ectopic Pax6 expression on the lens Fiber Cells was investigated by in situ hybridization, immunohistochemical staining, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and two-dimensional (2-D) gel electrophoresis. RESULTS. Transgenic mice from seven different lines all had cataracts with severity that correlated with the transgene expression level in lens Fiber Cells. In severely affected lines, a lumen was present between the apical surfaces of the epithelial and Fiber Cells, suggesting that secondary Fiber cell elongation is incomplete. Electron microscopy analysis showed that the ball-and-socket interdigitations between neighboring Fiber Cells were underdeveloped or attenuated in the transgenic lens. Most interesting, elevated levels of Pax6 in Fiber Cells reduced the protein levels of transcription factor cMaf, which is known to be essential in Fiber cell differentiation. Furthermore, the total amount of lens proteins was 60% less than normal in the Pax6 transgenic lens. Among the crystallins examined, the relative ratio of intact βB1-crystallin protein to total lens protein was significantly reduced. Real-time reverse transcriptase PCR showed that the ratio of βB1-crystallin transcript levels to total mRNA levels were reduced by 87%. CONCLUSIONS. The data demonstrate that high levels of Pax6 expression disrupt normal Fiber cell differentiation and maturation.

  • Ectopic Pax6 expression disturbs lens Fiber cell differentiation.
    Investigative ophthalmology & visual science, 2004
    Co-Authors: Melinda K. Duncan, Michael L. Robinson, Jennifer R. Taube, Larry L. David, Leike Xie, Wenwu Cui, Lixing W Reneker
    Abstract:

    PURPOSE Pax6 is a transcription factor necessary for the specification and subsequent formation of the ocular lens. It is expressed in all lens Cells at early stages of development. After lens formation, Pax6 expression is maintained in the lens epithelium, whereas its level abruptly decreases in differentiated Fiber Cells. This study is to test the hypothesis that normal Fiber cell differentiation would be perturbed by sustained Pax6 expression. METHODS Transgenic mice expressing the canonical form of mouse Pax6 were created under the control of a modified mouse alphaA-crystallin promoter. The phenotypic changes in the transgenic lens were analyzed by light and electron microscopy. The effect of ectopic Pax6 expression on the lens Fiber Cells was investigated by in situ hybridization, immunohistochemical staining, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and two-dimensional (2-D) gel electrophoresis. RESULTS Transgenic mice from seven different lines all had cataracts with severity that correlated with the transgene expression level in lens Fiber Cells. In severely affected lines, a lumen was present between the apical surfaces of the epithelial and Fiber Cells, suggesting that secondary Fiber cell elongation is incomplete. Electron microscopy analysis showed that the ball-and-socket interdigitations between neighboring Fiber Cells were underdeveloped or attenuated in the transgenic lens. Most interesting, elevated levels of Pax6 in Fiber Cells reduced the protein levels of transcription factor cMaf, which is known to be essential in Fiber cell differentiation. Furthermore, the total amount of lens proteins was 60% less than normal in the Pax6 transgenic lens. Among the crystallins examined, the relative ratio of intact betaB1-crystallin protein to total lens protein was significantly reduced. Real-time reverse transcriptase PCR showed that the ratio of betaB1-crystallin transcript levels to total mRNA levels were reduced by 87%. CONCLUSIONS The data demonstrate that high levels of Pax6 expression disrupt normal Fiber cell differentiation and maturation.

  • General Utility of the Chicken βB1-Crystallin Promoter to Drive Protein Expression in Lens Fiber Cells of Transgenic Mice
    Transgenic Research, 2002
    Co-Authors: Jennifer R. Taube, Peggy S. Zelenka, Yoji Ueda, Larry L. David, Melinda K. Duncan
    Abstract:

    Transgenic mouse technology has been very valuable for the study of lens Fiber Cells since they can not be propagated in cell culture. The targeting of transgenes to the lens has traditionally been done with the αA-crystallin promoter. However, while lens-specific, transgenic lines made with the αA-crystallin promoter express the transgene at levels 100–300-fold lower than endogenous αA-crystallin. Here we propose an alternative, the chicken βB1-crystallin promoter (−432/+30). Transgenic mice made with this promoter have successfully expressed CAT, d/n m-calpain, Wee1, and βB2-crystallin mRNA at levels comparable to the endogenous βB1-crystallin gene and no eye abnormalities such as cataracts, have resulted. All of the transgenic lines made with the chicken βB1-crystallin promoter have expressed the transgene in the lens Fiber Cells, and the best lines express at levels close to endogenous βB1-crystallin. While RNA expression is very high, only moderate protein expression has been achieved, implying that the high protein expression of the crystallins is partially controlled at the level of translation. Thus, the chicken βB1-crystallin promoter directs high level RNA expression to lens Fiber Cells, which may be especially useful for the expression of ribozyme and anti-sense RNAs in addition to ectopic proteins.