The Experts below are selected from a list of 249 Experts worldwide ranked by ideXlab platform
Sue A. Menko - One of the best experts on this subject based on the ideXlab platform.
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Lens Differentiation is characterized by stage specific changes in chromatin accessibility correlating with Differentiation state specific gene expression
Developmental Biology, 2019Co-Authors: Joshua Disatham, Sue A. Menko, Daniel Chauss, Rifah Gheyas, Lisa A Brennan, David Blanco, Lauren Daley, Marc KantorowAbstract:Abstract Changes in chromatin accessibility regulate the expression of multiple genes by controlling transcription factor access to key gene regulatory sequences. Here, we sought to establish a potential function for altered chromatin accessibility in control of key gene expression events during Lens cell Differentiation by establishing genome-wide chromatin accessibility maps specific for four distinct stages of Lens cell Differentiation and correlating specific changes in chromatin accessibility with genome-wide changes in gene expression. ATAC sequencing was employed to generate chromatin accessibility profiles that were correlated with the expression profiles of over 10,000 Lens genes obtained by high-throughput RNA sequencing at the same stages of Lens cell Differentiation. Approximately 90,000 regions of the Lens genome exhibited distinct changes in chromatin accessibility at one or more stages of Lens Differentiation. Over 1000 genes exhibited high Pearson correlation coefficients (r > 0.7) between altered expression levels at specific stages of Lens cell Differentiation and changes in chromatin accessibility in potential promoter (−7.5kbp/+2.5kbp of the transcriptional start site) and/or other potential cis-regulatory regions ( ±10 kb of the gene body). Analysis of these regions identified consensus binding sequences for multiple transcription factors including members of the TEAD, FOX, and NFAT families of transcription factors as well as HIF1a, RBPJ and IRF1. Functional mapping of genes with high correlations between altered chromatin accessibility and Differentiation state-specific gene expression changes identified multiple families of proteins whose expression could be regulated through changes in chromatin accessibility including those governing Lens structure (BFSP1,BFSP2), gene expression (Pax-6, Sox 2), translation (TDRD7), cell-cell communication (GJA1), autophagy (FYCO1), signal transduction (SMAD3, EPHA2), and Lens transparency (CRYBB1, CRYBA4). These data provide a novel relationship between altered chromatin accessibility and Lens Differentiation and they identify a wide-variety of Lens genes and functions that could be regulated through altered chromatin accessibility. The data also point to a large number of potential DNA regulatory sequences and transcription factors whose functional analysis is likely to provide insight into novel regulatory mechanisms governing the Lens Differentiation program.
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α6 integrin transactivates insulin like growth factor receptor 1 igf 1r to regulate caspase 3 mediated Lens epithelial cell Differentiation initiation
Journal of Biological Chemistry, 2014Co-Authors: Subhasree Basu, Liping Zhang, Suren Rajakaruna, Adele De Arcangelis, Elisabeth Georgeslabouesse, Sue A. MenkoAbstract:The canonical mitochondrial death pathway was first discovered for its role in signaling apoptosis. It has since been found to have a requisite function in Differentiation initiation in many cell types including the Lens through low level activation of the caspase-3 protease. The ability of this pathway to function as a molecular switch in Lens Differentiation depends on the concurrent induction of survival molecules in the Bcl-2 and IAP families, induced downstream of an IGF-1R/NFκB coordinate survival signal, to regulate caspase-3 activity. Here we investigated whether α6 integrin signals upstream to this IGF-1R-mediated survival-linked Differentiation signal. Our findings show that IGF-1R is recruited to and activated specifically in α6 integrin receptor signaling complexes in the Lens equatorial region, where Lens epithelial cells initiate their Differentiation program. In studies with both α6 integrin knock-out mice Lenses and primary Lens cell cultures following α6 integrin siRNA knockdown, we show that IGF-1R activation is dependent on α6 integrin and that this transactivation requires Src kinase activity. In addition, without α6 integrin, activation and expression of NFκB was diminished, and expression of Bcl-2 and IAP family members were down-regulated, resulting in high levels of caspase-3 activation. As a result, a number of hallmarks of Lens Differentiation failed to be induced; including nuclear translocation of Prox1 in the Differentiation initiation zone and apoptosis was promoted. We conclude that α6 integrin is an essential upstream regulator of the IGF-1R survival pathway that regulates the activity level of caspase-3 for it to signal Differentiation initiation of Lens epithelial cells.
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phosphatidylinositol 3 kinase is necessary for Lens fiber cell Differentiation and survival
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Gregory F Weber, Sue A. MenkoAbstract:PURPOSE. To determine the mechanisms of action of phosphatidylinositol 3-kinase (PI3K) in Lens cell Differentiation and survival. METHODS. Primary quail Lens cell cultures were treated at different stages of Differentiation with the PI3K inhibitor LY294002, and expression of survival proteins and Differentiation markers were determined by immunoblot analysis. The connection between PI3K regulation of Lens Differentiation and actin cytoskeleton reorganization was examined by fluorescent-phalloidin staining and Rac activity assay. Survival in the absence of PI3K signaling was examined by TUNEL and DAPI staining. Phosphorylation of the PI3K effector glycogen synthase kinase-3 (GSK3) in the absence of PI3K signaling was induced with lithium chloride. RESULTS. Exposure to LY294002 blocked Lens epithelial cell Differentiation initiation. This result was linked to attenuation of Rac activity and inhibition of actin filament reorganization from stress fibers to cortical fibers, which has been shown to signal Lens Differentiation initiation. The survival of Lens epithelial cells in the absence of PI3K signaling correlated with induction of numerous survival factors, including Bcl-2. In contrast, inhibition of PI3K signaling in differentiating Lens fiber cells induced apoptosis by blocking inactivation of GSK3, showing that PI3K/GSK3 signaling has a protective role in the late stages of Differentiation as nuclei and organelles are lost. CONCLUSIONS. PI3K signaling regulates Lens cell Differentiation initiation through its ability to signal reorganization of the actin cytoskeleton from stress fibers to cortical fibers. In differentiating Lens fiber cells, PI3K has a protective function, signaling survival through inactivation of its downstream effector GSK3.
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Lens epithelial cell Differentiation
Experimental Eye Research, 2002Co-Authors: Sue A. MenkoAbstract:The Differentiation of Lens epithelial cells is an elaborate process that requires the coordination of many different cellular signaling pathways (Fig. 2). The process begins as cells in the anterior Lens epithelium, which are committed to the Lens lineage but are not differentiated, receive signals to enter the cell cycle. These signals require input from both integrins and growth factors and are mediated by Src family kinases and the ERK signaling pathway. The Lens epithelial cells proliferate in the anterior aspects of the equatorial epithelium and before they begin to express characteristics specific to Lens fiber cells, they withdraw from the cell cycle in the transition zone. This proliferation/Differentiation decision is a crucial step for these cells and common to the regulation of many cell Differentiation pathways. In the Lens this transition requires the suppression of Src family kinase activity and the coordinated actions of molecules such as the CDK inhibitors p27 and p57, and Rb proteins. The ability of the Lens epithelial cells to transition from proliferating to post-mitotic cells is correlated with their establishment of stable N-cadherin junctions. The initiation of the Lens Differentiation program requires the coordinated action of both integrins and growth factor receptors and their activation of downstream signaling pathways including the ERK MAP kinase pathway. Completion of the Differentiation program of the Lens fiber cell then involves molecules that regulate cell elongation, membrane stabilization, cell-cell communication and the establishment of cell transparency. While many of the aspects of Lens epithelial cell Differentiation are now understood, much more remains to be elucidated. Progress will be greatest if investigators expand their focus to include the notion that many different signaling pathways will be found to be involved in regulating the various incremental steps that lead a Lens epithelial cell to become a fiber cell. Most important to this success will be the realization that creation of this exquisite tissue, the Lens, is only possible through the cooperation of many different signaling effectors.
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n cadherin function is required for Differentiation dependent cytoskeletal reorganization in Lens cells in vitro
Experimental Cell Research, 2000Co-Authors: Celeste M Ferreiracornwell, Robert W Veneziale, Gerald B Grunwald, Sue A. MenkoAbstract:Abstract Members of the cadherin family of cell adhesion molecules participate in calcium-dependent cell-cell adhesions that are necessary for the cell sorting events that regulate early developmental processes. Although individual cadherin molecules have been shown to participate in tissue histogenesis, the regulation of function of these receptors in cell Differentiation has been more difficult to identify. We have determined that N-cadherin linkage to the cytoskeleton is correlated with Lens cell Differentiation in vivo. Through the use of a chick embryo Lens culture system that mimics Differentiation in vivo, we have determined that N-cadherin linkage to the cytoskeleton is altered and Lens Differentiation is blocked by function-blocking antibodies to N-cadherin. In the presence of the N-cadherin function-blocking antibody, NCD-2, both N-cadherin and filamentous actin are prevented from organizing at the cortical membranes. This correlates with an inhibition of Lens morphogenesis and Differentiation. These results are paralleled by changes in the expression of the molecular components of the cadherin-catenin complex and their linkage to the actin cytoskeleton. In the presence of NCD-2, expression of N-cadherin, α-catenin, and β-catenin is inhibited and their association with the cytoskeleton blocked. Overall cadherin expression, however, remains unchanged as demonstrated by studies with a pan-cadherin antibody. This is accompanied by an increase in expression of the cadherin cytoskeletal protein plakoglobin. Although the cells have tried to compensate for the loss of N-cadherin by up-regulation of another cadherin(s) and plakoglobin, this is unable to compensate for N-cadherin function. The data strongly suggest that N-cadherin and its associated cytoskeleton play an important role in the Differentiation process that leads to the formation of the crystalline Lens.
Ales Cvekl - One of the best experts on this subject based on the ideXlab platform.
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profiling of chromatin accessibility and identification of general cis regulatory mechanisms that control two ocular Lens Differentiation pathways
Epigenetics & Chromatin, 2019Co-Authors: Yilin Zhao, Deyou Zheng, Ales CveklAbstract:Promoters and enhancers are cis-regulatory DNA sequences that control specificity and quantity of transcription. Both are rich on clusters of cis-acting sites that interact with sequence-specific DNA-binding transcription factors (TFs). At the level of chromatin, these regions display increased nuclease sensitivity, reduced nucleosome density, including nucleosome-free regions, and specific combinations of posttranslational modifications of core histone proteins. Together, “open” and “closed” chromatins represent transcriptionally active and repressed states of individual genes, respectively. Cellular Differentiation is marked by changes in local chromatin structure. Lens morphogenesis, regulated by TF Pax6, includes Differentiation of epithelial precursor cells into Lens fibers in parallel with Differentiation of epithelial precursors into the mature Lens epithelium. Using ATAC-seq, we investigated dynamics of chromatin changes during mouse Lens fibers and epithelium Differentiation. Tissue-specific features of these processes are demonstrated via comparative studies of embryonic stem cells, forebrain, and liver chromatins. Unbiased analysis reveals cis-regulatory logic of Lens Differentiation through known (e.g., AP-1, Ets, Hsf4, Maf, and Pax6 sites) and novel (e.g., CTCF, Tead, and NF1) motifs. Twenty-six DNA-binding TFs, recognizing these cis-motifs, are markedly up-regulated in differentiating Lens fibers. As specific examples, our ATAC-seq data uncovered both the regulatory regions and TF binding motifs in Foxe3, Prox1, and Mip loci that are consistent with previous, though incomplete, experimental data. A cross-examination of Pax6 binding with ATAC-seq data demonstrated that Pax6 bound to both open (H3K27ac and P300-enriched) and closed chromatin domains in Lens and forebrain. Our study has generated the first Lens chromatin accessibility maps that support a general model of stage-specific chromatin changes associated with transcriptional activities of batteries of genes required for Lens fiber cell formation. Analysis of active (or open) promoters and enhancers reveals important cis-DNA motifs that establish the molecular foundation for temporally and spatially regulated gene expression in Lens. Together, our data and models open new avenues for the field to conduct mechanistic studies of transcriptional control regions, reconstruction of gene regulatory networks that govern Lens morphogenesis, and identification of cataract-causing mutations in noncoding sequences.
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transcriptional burst fraction and size dynamics during Lens fiber cell Differentiation and detailed insights into the denucleation process
Journal of Biological Chemistry, 2018Co-Authors: Saima Limi, Adrien Senecal, Robert A Coleman, Melissa Lopezjones, Christina Polumbo, Robert H Singer, Arthur I Skoultchi, Ales CveklAbstract:Genes are transcribed in irregular pulses of activity termed transcriptional bursts. Cellular Differentiation requires coordinated gene expression; however, it is unknown whether the burst fraction (i.e. the number of active phases of transcription) or size/intensity (the number of RNA molecules produced within a burst) changes during cell Differentiation. In the ocular Lens, the positions of Lens fiber cells correlate precisely with their Differentiation status, and the most advanced cells degrade their nuclei. Here, we examined the transcriptional parameters of the β-actin and Lens Differentiation–specific α-, β-, and γ-crystallin genes by RNA fluorescent in situ hybridization (FISH) in the Lenses of embryonic day (E) E12.5, E14.5, and E16.5 mouse embryos and newborns. We found that cellular Differentiation dramatically alters the burst fraction in synchronized waves across the Lens fiber cell compartment with less dramatic changes in burst intensity. Surprisingly, we observed nascent transcription of multiple genes in nuclei just before nuclear destruction. Nuclear condensation was accompanied by transfer of nuclear proteins, including histone and nonhistone proteins, to the cytoplasm. Although Lens-specific deletion of the chromatin remodeler SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (Smarca5/Snf2h) interfered with denucleation, persisting nuclei remained transcriptionally competent and exhibited changes in both burst intensity and fraction depending on the gene examined. Our results uncover the mechanisms of nascent transcriptional control during Differentiation and chromatin remodeling, confirm the burst fraction as the major factor adjusting gene expression levels, and reveal transcriptional competence of fiber cell nuclei even as they approach disintegration.
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identification and characterization of fgf2 dependent mrna microrna networks during Lens fiber cell Differentiation
G3: Genes Genomes Genetics, 2013Co-Authors: Louise Wolf, Karen Gueta, Tiphaine Chevallier, Nikhil R Podduturi, Ivan Conte, Peggy S Zelenka, Ruth Asherypadan, Jiri Zavadil, Ales CveklAbstract:MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, Differentiation, and survival. In Lens, both these systems control Lens fiber cell Differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating Lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) Lens. To dissect the miRNA-dependent pathways during Lens Differentiation, we used a rat (Rattus norvegicus) Lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in Lens Differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3′-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in Lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate Lens Differentiation.
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Lens Differentiation from Embryonic Stem (ES) and Induced Pluripotent Stem (iPS) Cells
Stem Cell Biology and Regenerative Medicine in Ophthalmology, 2012Co-Authors: Ales Cvekl, Ying Yang, Yang JingAbstract:The formation of Lens progenitor cells and differentiated Lens tissue in cell culture conditions presents a number of experimental challenges, even though Lens lineage formation and Lens fiber cell Differentiation are among the best characterized model systems at both genetic and molecular levels. Lens Differentiation from ES cells in vitro appears to be a feasible goal. This chapter describes the significance of using ES and iPS cells for better understanding of embryonic Lens development and formation of congenital cataracts. A discussion of how iPS cells can help studies of age-related cataract is also included. The chapter summarizes the current data on lentoid body formation from human and primate ES cells, and the molecular basis of directed Differentiation of human ES cells into Lens progenitor cells and lentoid bodies. Finally, current gaps in Lens research and future directions to address these problems are discussed.
Kunio Yasuda - One of the best experts on this subject based on the ideXlab platform.
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Lens Differentiation and crystallin regulation: a chick model.
The International journal of developmental biology, 2020Co-Authors: Hasan M Reza, Kunio YasudaAbstract:The vertebrate Lens is a transparent polarized tissue that acts as the gateway for vision. The chick Lens is an excellent model for studying tissue organogenesis, since it is both accessible and easily manipulated during embryonic stages. The chick Lens consists of two morphologically discrete compartments, the epithelium and the fiber-cell mass. Evidence indicates that the early phases of Lens development involve several sequential events, including tissue interactions, cell proliferation and Differentiation. The morphological change during Lens development is associated with the concurrent and distinct functions of numerous transcription factors. Diffusible molecules from the complementary neural tissue play vital roles during the entire process of Lens development. Lens tissue is characterized by the ample production of crystallins, Lens specific proteins which provide structural integrity and functional properties to the Lens. Thus, the study of crystallin regulation should provide insight into the development of a functional Lens during embryogenesis. This process has been shown to involve a complex and evolutionary conserved pathway supported by different regulatory proteins.
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Transcription factors involved in Lens development from the preplacodal ectoderm
Developmental Biology, 2012Co-Authors: Hajime Ogino, Haruki Ochi, Hasan Mahmud Reza, Kunio YasudaAbstract:article i nfo Lens development is a stepwise process accompanied by the sequential activation of transcription factors. Transcription factor genes can be classified into three groups according to their functions: the first group comprises preplacodal genes, which are implicated in the formation of the preplacodal ectoderm that serves as a common primordium for cranial sensory tissues, including the Lens. The second group comprises Lens- specification genes, which establish the Lens-field within the preplacodal ectoderm. The third group com- prises Lens-Differentiation genes, which promote Lens morphogenesis after the optic vesicle makes contact with the presumptive Lens ectoderm. Analyses of the regulatory interactions between these genes have pro- vided an overview of Lens development, highlighting crucial roles for positive cross-regulation in fate speci- fication and for feed-forward regulation in the execution of terminal Differentiation. This overview also sheds light upon the mechanisms of how preplacodal gene activities lead to the activation of genes involved in Lens- specification.
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the stability of the Lens specific maf protein is regulated by fibroblast growth factor fgf erk signaling in Lens fiber Differentiation
Journal of Biological Chemistry, 2003Co-Authors: Haruki Ochi, Hajime Ogino, Yuji Kageyama, Kunio YasudaAbstract:Abstract Fibroblast growth factor (FGF) signaling is necessary for both proliferation and Differentiation of Lens cells. However, the molecular mechanisms by which FGFs exert their effects on the Lens remain poorly understood. In this study, we show that FGF-2 repressed the expression of Lens-specific genes at the proliferative phase in primary cultured Lens cells. Using transfected cells, we also found that the activity of L-Maf, a Lens Differentiation factor, is repressed by FGF/ERK signaling. L-Maf is shown to be phosphorylated by ERK, and introduction of mutations into the ERK target sites on L-Maf promotes its stabilization. The stable L-Maf mutant protein promotes the Differentiation of Lens cells from neural retina cells. Taken together, these results indicate that FGF/ERK signaling negatively regulates the function of L-Maf in proliferative Lens cells and that stabilization of the L-Maf protein is important for Lens fiber Differentiation.
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Characterization of the chicken L‐Maf, MafB and c‐Maf in crystallin gene regulation and Lens Differentiation
Genes to Cells, 2002Co-Authors: Tomonori Yoshida, Kunio YasudaAbstract:Background: Members of the Maf family, including L-Maf, MafB and c-Maf, are ‘basic region/leucine zipper’ (bZIP) transcription factors. Maf proteins contain a highly conserved acidic transactivation domain (AD), and a bZIP region that mediates DNA-binding activity. The hinge region between AD and bZIP varies considerably in length between different proteins. Recent studies reveal that L-Maf, c-Maf and MafB play key roles in vertebrate Lens development. Results: We investigated the transactivation activity of individual factors in culture cells to analyse their specific functions. In transient transfection assays with a reporter gene containing Maf responsive elements, MafB and c-Maf activated higher levels of the reporter gene than L-Maf. However, L-Maf transactivated the αA-crystallin promoter as effectively as MafB and c-Maf, and induced the expression of the endogenous δ-crystallin gene more efficiently than the other two proteins. Domain-swapping experiments reveal that the bZIP region of MafB takes part in strong transcriptional activity, while the acidic and hinge regions (AH) of c-Maf collectively serve as a strong transactivation domain. The AH region of L-Maf (but not c-Maf) conferred transactivation activity to induce δ-crystallin gene expression. Conclusions: These results suggest that despite their similar DNA binding properties, L-Maf, MafB and c-Maf regulate different sets of target genes by complex interactions with multiple factors that recognize cis-elements in promoters. The AH region of L-Maf has a distinct role in inducing endogenous δ-crystallin gene.
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Sequential activation of transcription factors in Lens induction
Development Growth & Differentiation, 2000Co-Authors: Hajime Ogino, Kunio YasudaAbstract:Since the pioneering work of the early 1900s, the Lens has been used as a model system for the study of tissue development in vertebrates. A number of embryological transplantation experiments designed to elucidate the role of tissue interactions in the formation of the Lens have led to the proposal of a stepwise determination model. This model has recently been refined through the identification of certain transcription factor genes, which exhibit distinct expression patterns and functional properties in the Lens cell lineage. Otx2, Pax6, and Lens1 are induced by the adjacent anterior neural plate and expressed in predifferentiated Lens ectoderm. Contact between the optic vesicle and Lens ectoderm promotes expression of mafs, Soxs, and Prox1, which are responsible for the initiation of Lens Differentiation programs including crystallin expression, cell elongation, and cell cycle arrest. Further analysis of the expression and functional characteristics of these transcription factors will allow greater detail when describing the orchestration of genetic programs, which control tissue development from induction to maturation.
Frank J. Lovicu - One of the best experts on this subject based on the ideXlab platform.
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prox1 and fibroblast growth factor receptors form a novel regulatory loop controlling Lens fiber Differentiation and gene expression
Development, 2016Co-Authors: Dylan S Audette, Frank J. Lovicu, Deepti Anand, Tammy So, Troy Rubenstein, Salil A Lachke, Melinda K DuncanAbstract:Lens epithelial cells differentiate into Lens fibers (LFs) in response to a fibroblast growth factor (FGF) gradient. This cell fate decision requires the transcription factor Prox1, which has been hypothesized to promote cell cycle exit in differentiating LF cells. However, we find that conditional deletion of Prox1 from mouse Lenses results in a failure in LF Differentiation despite maintenance of normal cell cycle exit. Instead, RNA-seq demonstrated that Prox1 functions as a global regulator of LF cell gene expression. Intriguingly, Prox1 also controls the expression of fibroblast growth factor receptors (FGFRs) and can bind to their promoters, correlating with decreased downstream signaling through MAPK and AKT in Prox1 mutant Lenses. Further, culturing rat Lens explants in FGF increased their expression of Prox1, and this was attenuated by the addition of inhibitors of MAPK. Together, these results describe a novel feedback loop required for Lens Differentiation and morphogenesis, whereby Prox1 and FGFR signaling interact to mediate LF Differentiation in response to FGF.
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development and use of the Lens epithelial explant system to study Lens Differentiation and cataractogenesis
Progress in Retinal and Eye Research, 2010Co-Authors: Judith A Westmays, Guiseppe Pino, Frank J. LovicuAbstract:Abstract Over the last two decades much progress has been made in identifying and characterizing many of the molecules involved in understanding normal Lens biology and its pathology. Much of this has been made possible through the establishment and use of the Lens epithelial explant system. This simplistic tissue culture model, comprised of a sheet of Lens epithelium on its native substratum, has been used effectively to study many cellular processes, including Lens epithelial cell proliferation, fiber cell Differentiation, cell apoptosis as well as epithelial-to-mesenchymal transformation of cells. In doing so, a number of key growth factors and cytokines, including members of the FGF, Wnt and TGFβ family have been shown to play essential roles in many of these cellular events. This has led to further studies exploring the signaling pathways downstream of these molecules in the Lens, paving the way for the development of a number of in situ models (primarily transgenic mouse lines) to further explore in more detail the nature of these molecular and cellular interactions. To reciprocate, the Lens epithelial explant system is increasingly being used to further characterize the nature of many complex phenotypes and pathologies observed in these in situ models, allowing us to selectively isolate and examine the direct impact of an individual molecule on a specific cellular response in Lens cells. There is no question that the Lens epithelial explant system has served as a powerful tool to further our understanding of Lens biology and pathology, and there is no doubt that it will continue to serve in such a capacity, as new developments are realized and putative treatments for aberrant Lens cell behavior are to be trialed.
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Expression of FGF-1 and FGF-2 mRNA during Lens morphogenesis, Differentiation and growth
Current Eye Research, 1997Co-Authors: Frank J. Lovicu, Robbert U. De Iongh, John W. McavoyAbstract:Purpose. There is now considerable evidence that FGF is involved in Lens Differentiation and growth throughout life. The aim of this study was to determine potential sites of FGF production in and near the Lens during morphogenesis, Differentiation and growth. Methods. The distribution of FGF-1 and FGF-2 mRNAs was analysed in embryonic, weanling and adult rat eyes by in situ hybridization. Results. During Lens morphogenesis, there was distinct expression of FGF-1, but not FGF-2, in the Lens placode and retinal disc cells. Subsequently, both forms of FGF showed similar expression patterns. During Lens Differentiation, distinct expression of FGFs was associated with elongating primary fiber cells. From embryonic day 20 onwards, Lenses showed strongest expression of FGF mRNAs in the transitional zone, where epithelial cells differentiate into fibers, with weaker expression in the anterior epithelium. Messenger RNAs for both FGFs were also localised in ocular tissues near the Lens and bordering the ocular med...
Fu Shang - One of the best experts on this subject based on the ideXlab platform.
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ubiquitin proteasome pathway function is required for Lens cell proliferation and Differentiation
Investigative Ophthalmology & Visual Science, 2006Co-Authors: Fu Shang, Lyudmila Urim, Minlei Zhang, Allen TaylorAbstract:PURPOSE. The ubiquitin proteasome pathway is involved in the regulation of many cellular processes, such as cell cycle control, signal transduction, transcription, and removal of obsolete proteins. The objective of this work was to investigate roles for this proteolytic pathway in controlling the Differentiation of Lens epithelial cells into Lens fibers. METHODS. bFGF-induced cell proliferation was monitored in rat Lens epithelial explants by bromodeoxyuridine (BrdU) incorporation. Indicators of Lens Differentiation included expression of crystallins, Lens major intrinsic protein 26 (MIP26), CP49, and fiLensin and morphologic changes such as cell multilayering and elongation or loss of nuclei. Clasto-lactacystin-β-lactone, the proteasome-specific inhibitor, was used to study the role of the proteasome in controlling the proliferation and Differentiation processes. RESULTS. Explants treated with bFGF initially underwent enhanced proliferation, as indicated by BrdU incorporation and multilayering of the epithelial cells. By 4 days of bFGF treatment, most cells withdrew from the cell cycle, as indicated by diminished BrdU incorporation. After 7 days of treatment with bFGF, Lens epithelial explants displayed characteristics of Lens fibers, including higher ratios of crystallins to other cytoplasmic proteins and expression of large quantities of MIP26, CP49, and fiLensin. Adding the proteasome inhibitor to the medium simultaneously with bFGF (day 0) or at day 4 prohibited or delayed bFGF-induced cell proliferation and Differentiation. This was indicated by reduced BrdU incorporation and decreased expression of β- and γ-crystallins, MIP26, CP49, and fiLensin. Proteasome inhibition also significantly decreased the number of layers and the sizes of differentiating fibers. CONCLUSIONS. These data show that proteasome activity is required not only for Lens cell proliferation but also required for the transition from the epithelial phenotype to the fiber phenotype.
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subcellular redistribution of components of the ubiquitin proteasome pathway during Lens Differentiation and maturation
Investigative Ophthalmology & Visual Science, 2005Co-Authors: Henrique Girao, Paulo Pereira, Allen Taylor, Fu ShangAbstract:PURPOSE. To determine the subcellular distribution of components of the ubiquitin-proteasome pathway (UPP) in Lens epithelium and differentiating fibers and to evaluate potential roles of the UPP in eliminating nuclei and other organelles during maturation of Lens fibers. METHODS. Adult bovine Lens cryosections were stained for immunofluorescence and analyzed by confocal microscopy. The specificities of the antibodies used in this study were determined by Western blot. RESULTS Cryosections of bovine Lenses show that E1 and Ubc1 were present in both the cytoplasm and the nucleus in epithelial cells, whereas Ubc3 and ubiquitin conjugates were mostly confined to the nucleus, and Ubc4/5 was preferentially localized in clusters in the vicinity of the nuclear membrane. The 19S and 20S proteasome complexes were preferentially localized in the cytoplasm. When the epithelial cells differentiated into fiber cells at the transition zone, all components of the UPP were primarily present in the nucleus, with the exception of Ubc4/5, which was associated with the nuclear membrane. CONCLUSIONS The results show that during Lens fiber Differentiation and maturation, components of the UPP are redistributed at subcellular levels. Subcellular localization of an enzyme indicates where the reaction takes place. The primary nuclear localization of the UPP components in the differentiating fibers supports the hypothesis that the UPP may play a role in elimination of nuclei and other organelles during Differentiation and maturation of Lens fibers.
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Subcellular Redistribution of Components of the Ubiquitin–Proteasome Pathway during Lens Differentiation and Maturation
Investigative Ophthalmology & Visual Science, 2005Co-Authors: Henrique Girao, Paulo Pereira, Allen Taylor, Fu ShangAbstract:PURPOSE. To determine the subcellular distribution of components of the ubiquitin-proteasome pathway (UPP) in Lens epithelium and differentiating fibers and to evaluate potential roles of the UPP in eliminating nuclei and other organelles during maturation of Lens fibers. METHODS. Adult bovine Lens cryosections were stained for immunofluorescence and analyzed by confocal microscopy. The specificities of the antibodies used in this study were determined by Western blot. RESULTS Cryosections of bovine Lenses show that E1 and Ubc1 were present in both the cytoplasm and the nucleus in epithelial cells, whereas Ubc3 and ubiquitin conjugates were mostly confined to the nucleus, and Ubc4/5 was preferentially localized in clusters in the vicinity of the nuclear membrane. The 19S and 20S proteasome complexes were preferentially localized in the cytoplasm. When the epithelial cells differentiated into fiber cells at the transition zone, all components of the UPP were primarily present in the nucleus, with the exception of Ubc4/5, which was associated with the nuclear membrane. CONCLUSIONS The results show that during Lens fiber Differentiation and maturation, components of the UPP are redistributed at subcellular levels. Subcellular localization of an enzyme indicates where the reaction takes place. The primary nuclear localization of the UPP components in the differentiating fibers supports the hypothesis that the UPP may play a role in elimination of nuclei and other organelles during Differentiation and maturation of Lens fibers.