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RNA-seq Analysis of Msx2fl/fl(Msx2 WT) and Le-Cre+/- Msx2fl/fl(Msx2 CKO) Lens Transcriptomes

ABSTRACT: RNA-seq Analysis of Msx2fl/fl(Msx2 WT) and Le-Cre+/- Msx2fl/fl(Msx2 CKO) Lens Transcriptomes

PROVIDER: PRJNA472869 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7212480_1.fastq.gz	Fastqsanger.gz
	SRR7212480_2.fastq.gz	Fastqsanger.gz
	SRR7212481_1.fastq.gz	Fastqsanger.gz
	SRR7212481_2.fastq.gz	Fastqsanger.gz

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RNA-seq Analysis of Msx2fl/fl(Msx2 WT) and Le-Cre+/- Msx2fl/fl(Msx2 CKO) Lens Transcriptomes

Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived lens transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Lens RNA was extracted from Msx2 WT and Msx2 CKO mice at postnatal day 60 (P60) using RNAeasy™ Animal RNA Isolation Kit. The freshly frozen RNA solution was prepared for RNA sequencing on the same day. The RNA-seq was carried out using Illumina HiSeq sequencing (Novogene Bioinformatics Institute, Beijing, China). The insert size of the library was tested by Agilent 2100 Bioanalyzer to ensure library quality before Illumina HiSeq sequencing. The company used STAR software to compare RNA-seq sequencing data, HTSeq to analyze the quantitative of gene levels, RSEM to analyze transcript levels quantitatively, ClusterProfiler software to analyze differential gene enrichment, rMATS software to analyze alternative splicing, GATK software to analyze mutation sites and SnpEff software to analyze annotation of the variation sites. After mutation site detection and annotation, statistical analysis was done by Ts/Tv and Hom/Het, respectively. Results: RNA-sequencing results showed that 1,911 differentially expressed genes (DEGs) were detected. Among them, the expression of 1,586 genes were upregulated and the rest downregulated.

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Crim1 regulates integrin signaling in murine lens development

Project description:The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1-glcr11, which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1-null and Crim1-cko, we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active β1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development.

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Gene expression at E9.5 after deletion of type I BMP receptors in the mouse lens placode

Project description:The type I BMP receptors, Bmpr1a and Acvr1 were conditionally deleted from the mouse lens placode with Le-Cre. The heads of wild type and knockout E9.5 mouse embryos were laser micodissected to isolate the lens ectoderm from three embryos of each genotype. Total RNA was purified and reverse transcribed and amplified using a NuGEN Ovation Pico WTA system kit. cDNA was biotinylated and hybridized to Illumina Mouse6 V1.1 bead arrays.

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