Project description:Total RNA was isolated from three separate populations of human lens epithelial cells and three matching populations of lens cortical fiber cells. All samples were analyzed on separate microarrays.
Project description:The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that require a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected over 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, over 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic, autophagy, and mitophagy transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells. Differentiation-state transcriptional analysis of embryonic chicken lenses was performed following microdissection of 100 embryonic day 13 (E13) chicken lenses into four distinct regions that represent a continuum of lens cell differentiation states: lens central epithelium (EC), equatorial epithelium (EQ), cortical fibers (FP), and central fibers (FC). Further analysis of the transcriptional content of biologically replicate samples was performed by Illumina directional mRNA sequencing and resulting reads mapped by TopHat and assembled with Cufflinks.
Project description:The eye lens is composed of fiber cells, which differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. To understand the molecular mechanism of the lens cell differentiation, we compared the gene expression profiles between epithelial and cortical fiber cells of young mouse lens using a microarray analysis. Keywords: cell-type comparison
Project description:Total RNA was isolated from three separate populations of human lens epithelial cells and three matching populations of lens cortical fiber cells. All samples were analyzed on separate microarrays. Keywords: repeat sample
Project description:The mature eye lens contains a surface layer of epithelial cells called the lens epithelium that require a functional mitochondrial population to maintain the homeostasis and transparency of the entire lens. The lens epithelium overlies a core of terminally differentiated fiber cells that must degrade their mitochondria to achieve lens transparency. These distinct mitochondrial populations make the lens a useful model system to identify those genes that regulate the balance between mitochondrial homeostasis and elimination. Here we used an RNA sequencing and bioinformatics approach to identify the transcript levels of all genes expressed by distinct regions of the lens epithelium and maturing fiber cells of the embryonic Gallus gallus (chicken) lens. Our analysis detected over 15,000 unique transcripts expressed by the embryonic chicken lens. Of these, over 3000 transcripts exhibited significant differences in expression between lens epithelial cells and fiber cells. Multiple transcripts coding for separate mitochondrial homeostatic and degradation mechanisms were identified to exhibit preferred patterns of expression in lens epithelial cells that require mitochondria relative to lens fiber cells that require mitochondrial elimination. These included differences in the expression levels of metabolic, autophagy, and mitophagy transcripts between lens epithelial cells and lens fiber cells. These data provide a comprehensive window into all genes transcribed by the lens and those mitochondrial regulatory and degradation pathways that function to maintain mitochondrial populations in the lens epithelium and to eliminate mitochondria in maturing lens fiber cells.
Project description:This study investigates the response of human lens epithelial cells to mechanical injury. Human geriatric lenses obtained from cadaver eyes from donated to an eye bank for research were subject to in-vitro capsulotomy mimicking the injury sustained during cataract surgery. The anterior capsule was dissected using a curvilinear capsulorhexis technique, and central lens epithelial cells attached to the patch of anterior capsule (Rhexis) were immediately stabilized in RNAlater. The fiber cells were then removed, and the cortical fibers were immediately stabilized in RNA later. The remaining equatorial lens epithelial cells attached to the capsular bag from one eye were stabilized in RNA later immediately while the equatorial lens epithelial cells from the other eye were cultured for 24 hours then stabilized in RNAlater.
Project description:Epithelial cells and differentiated fiber cells represent distinct compartments in the ocular lens. While previous studies have revealed proteins that are preferentially expressed in epithelial vs. fiber cells, a comprehensive proteomics library comparing the molecular composition of epithelial vs. fiber cells is essential for understanding lens formation, function, disease and regenerative potential, and for efficient differentiation of pluripotent stem cells for modeling of lens development and pathology in vitro. To compare protein composition between the lens epithelium and fibers, we employed tandem mass spectrometry (2DLC/ MS) analysis of micro-dissected mouse P0.5 lenses. Functional classifications of the top 525 identified proteins into gene ontology categories by molecular process and subcellular localization, were adapted for lens. Expression levels of both epithelial and fiber proteomes were compared with their temporal and spatial mRNA levels using E14.5, E16.5, E18.5, and P0.5 RNA-Seq data sets. During this developmental time window, multiple complex biosynthetic and catabolic processes generate the molecular and structural foundation for lens transparency. As expected, crystallins showed a high correlation between their mRNA and protein levels. Comprehensive data analysis confirmed and/or predicted roles for transcription factors (TFs), RNA-binding proteins, translational apparatus including ribosomal heterogeneity and initiation factors, microtubules, cytoskeletal and membrane proteins in lens formation and maturation. Our data highlighted many proteins with unknown function in the lens that were preferentially enriched in epithelium or fibers, setting the stage for future studies to further dissect the roles of these proteins in fiber cell differentiation vs. epithelial cell maintenance. In conclusion, the present proteomic datasets established reference mouse lens epithelium and fiber cell proteomes, provided quantitative analyses of protein and RNA-Seq data, and probed the major proteome remodeling required to form the mature lens fiber cells.
Project description:Methylation at cytosines (mCG) is a well-known regulator of gene expression but its requirements for cellular differentiation have yet to be fully elucidated. A well-studied cellular differentiation model system is the eye lens, consisting of a single anterior layer of epithelial cells that migrate laterally and differentiate into a core of fiber cells. Here, we explore the genome-wide relationships between mCG methylation, chromatin accessibility and gene expression during differentiation of eye lens epithelial cells into fiber cells. Whole genome bisulfite sequencing identified 7621 genomic loci exhibiting significant differences in mCG levels between lens epithelial and fiber cells. Changes in mCG levels were inversely correlated with the differentiation state-specific expression of 1285 genes preferentially expressed in either lens fiber or lens epithelial cells (Pearson correlation r = -0.37, p < 1x10-42). mCG levels were inversely correlated with chromatin accessibility determined by Assay for transposase-accessible sequencing (ATAC-seq) (Pearson correlation r = -0.86, p < 1x10-300). Many of the genes exhibiting altered regions of DNA methylation, chromatin accessibility and gene expression levels in fiber cells relative to epithelial cells are associated with lens fiber cell structure, homeostasis and transparency. These include lens crystallins (CRYBA4, CRYBB1, CRYGN, CRYBB2), lens beaded filament proteins (BFSP1, BFSP2), transcription factors (HSF4, SOX2, HIF1A), and Notch signaling pathway members (NOTCH1, NOTCH2, HEY1, HES5). Analysis of regions exhibiting cell-type specific alterations in DNA methylation revealed an overrepresentation of consensus sequences of multiple transcription factors known to play key roles in lens cell differentiation including HIF1A, SOX2, and the MAF family of transcription factors. Collectively, these results link DNA methylation with control of chromatin accessibility and gene expression changes required for eye lens differentiation. The results also point to a role for DNA methylation in the regulation of transcription factors previously identified to be important for lens cell differentiation.
Project description:Methylation at cytosines (mCG) is a well-known regulator of gene expression but its requirements for cellular differentiation have yet to be fully elucidated. A well-studied cellular differentiation model system is the eye lens, consisting of a single anterior layer of epithelial cells that migrate laterally and differentiate into a core of fiber cells. Here, we explore the genome-wide relationships between mCG methylation, chromatin accessibility and gene expression during differentiation of eye lens epithelial cells into fiber cells. Whole genome bisulfite sequencing identified 7621 genomic loci exhibiting significant differences in mCG levels between lens epithelial and fiber cells. Changes in mCG levels were inversely correlated with the differentiation state-specific expression of 1285 genes preferentially expressed in either lens fiber or lens epithelial cells (Pearson correlation r = -0.37, p < 1x10-42). mCG levels were inversely correlated with chromatin accessibility determined by Assay for transposase-accessible sequencing (ATAC-seq) (Pearson correlation r = -0.86, p < 1x10-300). Many of the genes exhibiting altered regions of DNA methylation, chromatin accessibility and gene expression levels in fiber cells relative to epithelial cells are associated with lens fiber cell structure, homeostasis and transparency. These include lens crystallins (CRYBA4, CRYBB1, CRYGN, CRYBB2), lens beaded filament proteins (BFSP1, BFSP2), transcription factors (HSF4, SOX2, HIF1A), and Notch signaling pathway members (NOTCH1, NOTCH2, HEY1, HES5). Analysis of regions exhibiting cell-type specific alterations in DNA methylation revealed an overrepresentation of consensus sequences of multiple transcription factors known to play key roles in lens cell differentiation including HIF1A, SOX2, and the MAF family of transcription factors. Collectively, these results link DNA methylation with control of chromatin accessibility and gene expression changes required for eye lens differentiation. The results also point to a role for DNA methylation in the regulation of transcription factors previously identified to be important for lens cell differentiation.
Project description:Lens epithelial explants consist of lens epithelial cells (P8 FVB/N mice) grown in vitro on their native basement membrane, the lens capsule. For decades, biologists have used lens epithelial explants to study lens fiber cell differentiation. However, the global change in the accessibility of the chromatin and transcriptome during the process of explanting and culture is unknown. Therefore, P8 FVB/N lens epithelial explants cultured in either unsupplemented media or media containing 50% bovine vitreous humor for one or five days were collected. Chromatin and RNA was collected for ATAC-sequencing and RNA-sequencing respectively. Differentially accessible regions and differentially expressed genes were identified for each condition to provide a genome wide view of chromatin architecture and gene expression during fiber cell differentiation in vitro. Vitreous humor generally increased chromatin accessibility in promoter regions of genes associated with fiber differentiation and immune response, and this was associated with increased transcript levels for these genes. In contrast, vitreous had relatively little effect on the accessibility of most of the genes highly expressed in the lens epithelium despite dramatic reductions in the transcript levels of these genes.