Transcriptome Profiling of Developing Murine Lens Through RNA Sequencing.

The ocular lens is an excellent model for investigating both intricate details of development and pathophysiological mechanism of disease. The ocular lens is a transparent biconvex structure that helps focus light on the retina. The maintenance of lens transparency is essential for the normal visual function, and lack thereof leads to visual impairment by development of lens opacities. The lens arises from the head ectoderm that thickens to form the lens placode.1,2 The lens placode invaginates together with the optic vesicle to form the lens pit and the optic cup, respectively.1,2 Subsequently, the lens pit separates from the ectoderm to form the lens vesicle that gives rise to anterior and posterior single layers of cells.1,2 Cells of the former layer differentiate into the epithelium while cells forming the posterior layer differentiate into primary fiber cells forming the lens nucleus.1,2 The lens grows rapidly during late embryonic and early postnatal stages due to proliferation of epithelial cells that elongate and differentiate into secondary fiber cells continuously adding to the fiber mass.1,2 Cataract, clouding of the lens, is the leading cause of blindness worldwide and accounts for one-third of the total cases of blindness in children.3,4 A significant fraction of cataractogenesis is familial with a total of 35 genes associated with congenital cataracts.5,6 Nonetheless, these genes constitute a small fraction of the total genetic load, and the total number of genes associated with this debilitating disorder is presumed to be much higher. Previously, serial analysis of gene expression (SAGE) and RNA microarrays have been used to investigate transcriptional activities of the mouse and human ocular lens.7–9 However, both of these techniques have inherent limitations including the extremely short length of the SAGE tags that may be shared by multiple genes, and the inbuilt limitation of microarrays that can detect only those transcripts included on the array itself. Next-generation sequencing (NGS) technology provides an efficient and cost-effective platform to investigate the transcriptional landscape accurately and with much greater sensitivity, which is essential to reliably detect and quantitate rare but physiologically relevant coding as well as noncoding RNA molecules. Recently, two studies reported to use NGS to investigate the mouse lens transcriptome.10,11 However, neither study examined the lens transcriptome through different developmental stages nor was microRNA (miRNA) investigated in the lens transcriptome. miRNAs are important regulatory molecules that act as post transcriptional regulators and according to some estimates each miRNA can regulate the expression of 100 to 200 target genes.12 Severe microphthalmia with no visible lens and a poorly stratified corneal epithelium is observed in Dicer conditional knockout mice.13 Wolf and colleagues14 confirmed the indispensable role of miRNA-based regulation in differentiation and development of the lens tissue. This notion was further strengthened with the identification of the role of Pax6 in the regulation of miR-204, where the latter acts as a negative modulator of gene expression during lens development in vertebrates.15 In this study, we employed NGS to create mouse lens expression profiles for both mRNA and miRNA at six developmental stages. Deep sequencing of mRNA libraries generated 187.56 million unique reads that identified expression of 14,465 genes in the mouse lens transcriptome. Likewise, sequencing of the small RNA libraries generated 154.22 million unique reads that retrieved 379 miRNAs expressed at different developmental stages.