Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome Profiling of Developing Murine Lens through RNA Sequencing

ABSTRACT: Purpose: Transcriptome is the entire repertoire of all transcripts present in a cell at any particular time. We undertook next-generation whole transcriptome sequencing approach to gain insight of the transcriptional landscape of the developing mouse lens. Methods: We ascertained mice lenses at six developmental time points including two embryonic (E15 and E18) and four postnatal stages (P0, P3, P6, and P9). The ocular tissue at each time point was maintained as two distinct pools serving as biological replicates for each developmental stage. The mRNA and small RNA libraries were paired-end sequenced on Illumina HiSeq 2000 and subsequently analyzed using bioinformatics tools. Results: Mapping of mRNA and small RNA libraries generated 187.56 and 154.22 million paired-end reads, respectively. We detected a total of 14,465 genes in the mouse ocular lens. Of these, 46 genes exhibited 40-fold differential expression compared to transcriptional levels at E15. Likewise, small RNA profiling identified 379 microRNAs (miRNAs) expressed in mouse lens. Of these, 49 miRNAs manifested an 8-fold or higher differential expression when compared, as above to the microRNA expression at E15. Conclusion: We report the first comprehensive profile of developing murine lens transcriptome including both mRNA and miRNA through next-generation RNA sequencing. A complete repository of the lens transcriptome of six developmental time points will be monumental in elucidating processes essential for development of the ocular lens and maintenance its transparency. Whole transcrtiome and microRNA profilling of mouse lens using 2 embryonic (E15 and E18) and 4 postnatal stages (P0, P3, P6 and P9) in duplicates through high-throughput sequening using Illumina HiSeq2000.

ORGANISM(S): Mus musculus

SUBMITTER: S. Amer Riazuddin

PROVIDER: E-GEOD-69221 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptome Profiling of Developing Murine Lens Through RNA Sequencing.

Khan Shahid Y SY Hackett Sean F SF Lee Mei-Chong W MC Pourmand Nader N Talbot C Conover CC Riazuddin S Amer SA

Investigative ophthalmology & visual science 20150701 8

<h4>Purpose</h4>Transcriptome is the entire repertoire of transcripts present in a cell at any particular time. We undertook a next-generation whole transcriptome sequencing approach to gain insight into the transcriptional landscape of the developing mouse lens.<h4>Methods</h4>We ascertained mouse lenses at six developmental time points including two embryonic (E15 and E18) and four postnatal stages (P0, P3, P6, and P9). The ocular tissue at each time point was maintained as two distinct pools ...[more]

PMID: 26225632

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Project description:We report the application of RNA-sequencing technology for the high-throughput profiling of mammalian lens gene expression at embryonic day 15.5. The lens has a particularly biased transcriptome, with the top 50 genes encoding approximately 90% of the protein content. Using RNA-Seq, we have shown that there are over 7,700 genes being expressed in the lens at embryonic day 15.5. As expected, the crystallins and many structural genes were amoung the most highly expressed; however, numerous genes expressed at lower transcript abundance were also identified. This study provides a framework for the application of RNA-Seq technology towards characterization of the mammalian lens transcriptome during development. Using high-throughput RNA-sequencing, gene expression in the mammalian lens was compared between an inbred C56Bl/6<har> strain and a mix background strain at E15.5. This analysis identifies almost 2,000 genes being differentially expressed between the inbred and mixed background lenses, ranging from 6.5 fold upregulated to 5.2 fold downregulated in the mixed background compared to the inbred strain. This list does not include unknown/predicted genes or pseudogenes which are known to change between strains. Further, it appears that approximately 98% of these genes are altered at levels less than 2.5 fold. This study therefore provides a fold change threshold cutoff (2.5 fold) to use in the analysis of differentially expressed lens genes at E15.5 using RNA-Seq technology as it takes into account genetic variation due to background strain differences. SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes as highlighted by the pleiotropic defects observed in Mowat-Wilson Syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases where it functionally links TGFb signaling to the loss of epithelial preferred gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes involved in EMT/cancer. Furthermore, 34% of the upregulated genes in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes downregulated in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts. RNA-Seq of inbred background wild type lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and inbred wild type (C57Bl/6<har>) lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and Sip1 conditional knockout lenses at E15.5

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.

Dataset Information

Transcriptome Profiling of Developing Murine Lens through RNA Sequencing

Publications

Transcriptome Profiling of Developing Murine Lens Through RNA Sequencing.

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