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RNA-Seq transcriptome analysis of whole newborn lenses from FVB/N, Le-Cre and P0-3.9GFPCre mice

ABSTRACT: Here we report RNA-Seq data from RNA isolated from newborn lenses from FVB/N control mice as well as from newborn lenses of two different transgenic mouse lines (Le-Cre and P0-3.9GFPCre). Sequence reads of 51 bp were obtained from an illumine HiSeq 2000 system and mapped to the C57BL/6 reference genome (assembly GRCm38 (mm10)) using GSNAP software. Adapters and poor-quality regions were trimmed using Trimmomatic-0.36 software. Gene and isoform abundance was quantified using RSEM-1.3.0 software. Differential expression analysis was completed using DESeq2-1.10.1 software. For differential expression we used a cutoff value of equal to or greater than 1.5-fold change with an adjusted p value ≤ 0.05. The transcriptomes of both Le-Cre and P0-3.9GFPCre lenses closely matched the FVB/N control lenses. However, Le-Cre lenses exhibited deregulation of 15 murine genes, several of which are associated with apoptosis. In contrast, P0-3.9GFPCre lenses only deregulated two murine genes.

ORGANISM(S): Mus musculus

PROVIDER: GSE125108 | GEO | 2019/02/26

REPOSITORIES: GEO

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Project description:Glutathione (GSH) is a critical endogenous antioxidant that protects against intracellular oxidative stress. As such, pathological alterations in GSH levels are linked to a myriad of diseases including cancer, neurodegeneration and cataract. The rate limiting step in GSH biosynthesis is catalyzed by the glutamate cysteine ligase catalytic subunit (GCLC). The high expression of GCLC in the lens supports the synthesis of millimolar concentrations of GSH in this tissue. Herein, we describe the morphological consequences of deleting (knocking out) Gclc from surface ectoderm-derived ocular tissues (using the Le-Cre transgene; Gclc KO) which includes an overt microphthalmia phenotype and severely disrupted formation of multiple ocular structures (i.e., cornea, iris, lens, retina). Controlling for the Le-Cre transgene revealed that the deletion of Gclc significantly exacerbated the microphthalmia phenotype in Le-Cre hemizygous mice and resulted in dysregulated gene expression that was unique to only the lenses of KO mice. We further characterized the impaired lens development by conducting an RNA-seq experiment on KO and Gclc control (CON) mouse lens at the day of birth. RNA-sequencing revealed significant differences between Gclc knockout (KO) and Gclc control (CON) lenses, including down-regulation of crystallins and lens fiber cell identity genes, and up-regulation of lens epithelial cell identity genes. In addition, genes related to the immune system (e.g., immune system process, inflammatory response, neutrophil chemotaxis) were upregulated, and genes related to eye/lens development were downregulated. TRANSFAC analysis of differentially expressed genes (DEGs) in the lens of Gclc KO mice implicated PAX6 as a key upstream regulator of Gclc KO sensitive genes. This was further supported by a strong positive correlation between the transcriptomes of the lenses of Gclc KO and Pax6 KO mice. Strikingly, the dysregulation of PAX6-regulated genes in Gclc KO mice was observed despite no change in the ocular localization of PAX6 or decrease in the expression of PAX6 in the lens. In vitro experiments demonstrated that suppression of intracellular GSH concentrations resulted in impairment of PAX6 transactivation activity. Taken together, the present results elucidate a novel mechanism wherein intracellular GSH concentrations may modulate PAX6 activity.

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RNA-Seq transcriptome analysis of whole newborn lenses from FVB/N, Le-Cre and P0-3.9GFPCre mice

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