Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Comparison of lens gene expression between the control and DicerCN mouse embryos at E13.5

ABSTRACT: In order to investigate the mechanism for the progressive lens degeneration caused by targeted deletion of Dicer, we compared expression profiles of protein-coding genes in wild type and DicerCN lenses at E13.5, at a time before gross morphological changes had occurred. We identified distinct classes of differentially expressed genes in the conditional knockout lenses. Intact lenses were dissected from five control and five DicerCN mice at E13.5 for RNA extraction and hybridization on Affymetrix microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Yan Li

PROVIDER: E-GEOD-16209 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Targeted deletion of Dicer disrupts lens morphogenesis, corneal epithelium stratification, and whole eye development.

Li Yan Y Piatigorsky Joram J

Developmental dynamics : an official publication of the American Association of Anatomists 20090901 9

Dicer, a ribonuclease essential for miRNA processing, is expressed abundantly in developing mouse cornea and lens. We studied the roles of Dicer and miRNAs in eye development by conditionally deleting the Dicer gene in the mouse lens and corneal epithelium. Adult Dicer conditional null (DicerCN) mice had severe microphthalmia with no discernible lens and a poorly stratified corneal epithelium. Targeted deletion of Dicer effectively inhibited miRNA processing in the developing lens at 12.5 day of ...[more]

PMID: 19681134

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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:Microarray expression profiling has become a valuable tool in the evaluation of the genetic consequences of metabolic disease. Although 3â??-biased gene expression microarray platforms were the first generation to have widespread availability, newer platforms are gradually emerging that have more up-to-date content and/or higher cost efficiency. Deciphering the relative strengths and weaknesses of these various platforms for metabolic pathway level analyses can be daunting. We sought to determine the practical strengths and weaknesses of four leading commercially-available expression array platforms relative to biologic investigations, as well as assess the feasibility of cross-platform data integration for purposes of biochemical pathway analyses. METHODS: Liver RNA from B6.Alb/cre,Pdss2loxP/loxP mice having primary Coenzyme Q deficiency was extracted either at baseline or following treatment with an antioxidant/antihyperlipidemic agent, probucol. Target RNA samples were prepared and hybridized to Affymetrix 430 2.0, Affymetrix Gene 1.0 ST, Affymetrix Exon 1.0 ST, and Illumina Mouse WG-6 expression arrays. Probes on all platforms were re-mapped to coding sequences in the current version of the mouse genome. Data processing and statistical analysis were performed by R/Bioconductor functions, and pathway analyses were carried out by KEGG Atlas and GSEA. RESULTS: Expression measurements were generally consistent across platforms. However, intensive probe-level comparison suggested that differences in probe locations were a major source of inter-platform variance. In addition, genes expressed at low or intermediate levels had lower inter-platform reproducibility than highly expressed genes. All platforms showed similar patterns of differential expression between sample groups, with â??steroid biosynthesisâ?? consistently identified as the most down-regulated metabolic pathway by probucol treatment. CONCLUSIONS: This work offers a timely guide for metabolic disease investigators to enable informed end-user decisions regarding choice of expression microarray platform best-suited to specific research project goals. Successful cross-platform integration of biochemical pathway expression data is also demonstrated, especially for well-annotated and highly-expressed genes. However, integration of gene-level expression data is limited by individual platform probe design and the expression level of target genes. Cross-platform analyses of biochemical pathway data will require additional data processing and novel computational bioinformatics tools to address unique statistical challenges. 4 samples are analyzed on each of 4 different Mouse Microarray Expression platforms (Affymetrix Mo430 2.0, Affymetrix Gene, Affymetrix Exon 1.0 ST, and Illumina Whole Genome 6).

Dataset Information

Comparison of lens gene expression between the control and DicerCN mouse embryos at E13.5

Publications

Targeted deletion of Dicer disrupts lens morphogenesis, corneal epithelium stratification, and whole eye development.

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