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.