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Alon Kahana, Phillip E Kish, Alfonso Saera-Vila; Differential expression analysis for early gene activation patterns in regenerating lateral rectus muscle.. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
Using an adult zebrafish model of EOM injury in which residual myocytes undergo cellular reprogramming to regenerate a functional muscle, a transcriptome analysis of early genetic response to injury was performed in order to identify key regulators.
Lateral rectus (LR) RNA was isolated by laser-microdissection before injury and at 9 and 18 hours post injury (hpi) and purified by sequential Trizol and column purification with DNAse treatment. Following ribosomal RNA depletion and linear amplification, an Illumina Hi-Seq was used for sequencing. FastCQ was used to assess raw data for each sample, and Tuxedo Suite (Tophat, Bowtie and Cufflinks/CuffDiff) was used for alignment, differential expression analysis, and diagnostics. NCBI GRCz10.fa and .gft were used as reference sequence and annotation, respectively. Transcripts were identified as differentially expressed (DE) based on three criteria: test status=“OK”, FDR<0.05 and fold change ≥1.5. Transcripts were annotated using NCBI Entrez GeneIDs and Gene Ontology (GO) terms using NCBI and DAVID to identify significantly enriched functional categories.
Early changes in gene expression pattern were observed at both time points. More than 4300 genes were DE at both 9 & 18 hpi. Of these, 1,250 (9 hpi) and 1,199 genes (18 hpi) were differentially expressed only at 9 & 18 hpi, respectively, indicating DE patterns of very-early versus early-late genes. GO annotation showed several patterns which included transcription factor activity, sequence-specific DNA binding, transcription and epigenetic regulation, and negative regulation of apoptosis, to name a few. In addition to the large number of genes identified as being differentially expressed, we have tentatively identified DE of 106 long-noncoding RNAs (lncRNA) @ 9 hpi and 109 lncRNA @ 18 hpi.
The large number of genes differentially expressed (DE) at such early time points in regeneration suggests that there is extensive reprogramming of the genome and cellular machinery rapidly following injury. These genes are likely early regulators of gene pathways that are activated in a controlled manner both sequentially and in parallel. Understanding the regenerative response of adult zebrafish EOMs at a molecular genetic level will facilitate development of regenerative strategies to strabismic disorders affecting visual function.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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