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Comparative transcriptome analysis of epithelial and fiber cells in newborn mouse lenses with RNA sequencing.

Hoang TV, Kumar PK, Sutharzan S, Tsonis PA, Liang C, Robinson ML - Mol. Vis. (2014)

Bottom Line: 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).We found that RNA-Seq identified more differentially expressed genes and correlated with RT-qPCR quantification better than previously published microarray data.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Miami University, Oxford, OH.

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 comprehensively 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 identifying 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.

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Top 30 most significant differentially expressed protein-coding genes between epithelial and fiber cells ranked by adjusted p value. A: Genes with upregulated expression in epithelial cells. B: Genes with upregulated expression in fiber cells. In the heatmap, white represents low levels of expression while red represents high levels. Sample abbreviations: E1, E2, and E3: epithelial replicates; F1, F2, and F3: fiber replicates.
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f2: Top 30 most significant differentially expressed protein-coding genes between epithelial and fiber cells ranked by adjusted p value. A: Genes with upregulated expression in epithelial cells. B: Genes with upregulated expression in fiber cells. In the heatmap, white represents low levels of expression while red represents high levels. Sample abbreviations: E1, E2, and E3: epithelial replicates; F1, F2, and F3: fiber replicates.

Mentions: Genes differentially expressed (DEGs) in the epithelial cell and fiber cell fractions were first filtered based on whether they were upregulated in the epithelial cells or fiber cells. Genes were then ranked by the lowest adjusted p value rather than the greatest fold change to avoid large fold-change differences that failed to achieve sufficient significance as a result of low expression levels. The most significantly differentially expressed genes, ranked by adjusted p value, in the epithelial cells included arylsulfatase family member I (Arsi), immunoglobulin superfamily containing leucine-rich repeat protein (Islr), sulfatase-1 (Sulf1), folate receptor-1 (Folr1), B-cell translocation gene-1 (Btg1), nepronectin (Npnt), vascular endothelial growth factor receptor-1 (Flt1), and cyclin dependent kinase-1 (Cdk1; Figure 2A). The most significantly DEGs in the fiber cells tended to be those that encode lens structural proteins and membrane proteins, including several different crystallins, filensin (Bfsp1), gap junction protein epsilon-1 (Gje1), aquaporin0 (Aqp0 also known as Mip), tropomodulin1 (Tmod1), CD24 molecule (Cd24a), solute carrier family 24 (Slc24a4), and lens fiber membrane intrinsic protein (Lim2; Figure 2B). The structure and transparency of the lens require a high concentration (about 350 mg/ml) of crystallins, which represent the largest class of lens structural proteins. Mutations in several different crystallin genes result in cataract in mice and humans [43]. Len cell membranes contain high concentrations of the lens-specific water channel, Aqp0, as well as gap junctions and ion channels, which cooperate to allow the lens to transport nutrients and waste products despite the lack of vasculature in the mature lens [44].


Comparative transcriptome analysis of epithelial and fiber cells in newborn mouse lenses with RNA sequencing.

Hoang TV, Kumar PK, Sutharzan S, Tsonis PA, Liang C, Robinson ML - Mol. Vis. (2014)

Top 30 most significant differentially expressed protein-coding genes between epithelial and fiber cells ranked by adjusted p value. A: Genes with upregulated expression in epithelial cells. B: Genes with upregulated expression in fiber cells. In the heatmap, white represents low levels of expression while red represents high levels. Sample abbreviations: E1, E2, and E3: epithelial replicates; F1, F2, and F3: fiber replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4225139&req=5

f2: Top 30 most significant differentially expressed protein-coding genes between epithelial and fiber cells ranked by adjusted p value. A: Genes with upregulated expression in epithelial cells. B: Genes with upregulated expression in fiber cells. In the heatmap, white represents low levels of expression while red represents high levels. Sample abbreviations: E1, E2, and E3: epithelial replicates; F1, F2, and F3: fiber replicates.
Mentions: Genes differentially expressed (DEGs) in the epithelial cell and fiber cell fractions were first filtered based on whether they were upregulated in the epithelial cells or fiber cells. Genes were then ranked by the lowest adjusted p value rather than the greatest fold change to avoid large fold-change differences that failed to achieve sufficient significance as a result of low expression levels. The most significantly differentially expressed genes, ranked by adjusted p value, in the epithelial cells included arylsulfatase family member I (Arsi), immunoglobulin superfamily containing leucine-rich repeat protein (Islr), sulfatase-1 (Sulf1), folate receptor-1 (Folr1), B-cell translocation gene-1 (Btg1), nepronectin (Npnt), vascular endothelial growth factor receptor-1 (Flt1), and cyclin dependent kinase-1 (Cdk1; Figure 2A). The most significantly DEGs in the fiber cells tended to be those that encode lens structural proteins and membrane proteins, including several different crystallins, filensin (Bfsp1), gap junction protein epsilon-1 (Gje1), aquaporin0 (Aqp0 also known as Mip), tropomodulin1 (Tmod1), CD24 molecule (Cd24a), solute carrier family 24 (Slc24a4), and lens fiber membrane intrinsic protein (Lim2; Figure 2B). The structure and transparency of the lens require a high concentration (about 350 mg/ml) of crystallins, which represent the largest class of lens structural proteins. Mutations in several different crystallin genes result in cataract in mice and humans [43]. Len cell membranes contain high concentrations of the lens-specific water channel, Aqp0, as well as gap junctions and ion channels, which cooperate to allow the lens to transport nutrients and waste products despite the lack of vasculature in the mature lens [44].

Bottom Line: 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).We found that RNA-Seq identified more differentially expressed genes and correlated with RT-qPCR quantification better than previously published microarray data.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Miami University, Oxford, OH.

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 comprehensively 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 identifying 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.

Show MeSH
Related in: MedlinePlus