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Christopher Sifuentes, Jung-Woong Kim, Hyun-Jin Yang, Matthew Brooks, Anand Swaroop, Pamela Raymond, RC; Transcriptional regulation of the Müller glia stem cell response to acute light injury. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5486.
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© ARVO (1962-2015); The Authors (2016-present)
Zebrafish Müller glial cells act as adult stem cells, able to regenerate lost retinal neurons following a variety of insults. Analysis of transcriptional changes in zebrafish Müller glia after retinal injury may provide insights into regulation of stem cell properties. Past transcriptome analyses of zebrafish Müller glia were limited by the technology available, thus gene datasets are incomplete. The goal of this study is to provide a comprehensive analysis of early transcriptional changes in zebrafish Müller glia in response to acute loss of photoreceptors.
Müller glia were isolated by FACS from Tg(gfap:GFP)mi2002 transgenic zebrafish controls and at 8 and 16 hours after acute light lesion. The post-lesion time-points were chosen to examine the pre-mitotic response of Müller glia to light-induced photoreceptor death. RNA was isolated from sorted Müller glia, reverse transcribed into cDNA, and used to create TruSeq libraries that were sequenced on Illumina GAIIx. ChIP-seq libraries are being sequenced on Illumina HiSeq. Differential expression analysis was performed using Bowtie2, eXpress, edgeR, and limma.
Analysis of the RNA-seq dataset revealed high quality of the sequencing data. The previously reported genes whose expression changes in injury-induced zebrafish Müller glia (tgif1, hspd1, hspe1, socs3, stat3, and neurod) exhibit expected differential expression. Our analyses demonstrate additional transcriptional changes that provide evidence of specific regulation of genes implicated in retinal regeneration. Pathway and network analyses and experimental validation of the RNA-seq data are underway. We are also performing ChIP-seq to determine how epigenetic marks influence gene expression changes in the injury induced Müller glial stem cells by correlating sites of enrichment of specific histone markers with gene expression changes.
This comprehensive RNA-seq dataset will identify additional genes and pathways not described in previous gene profiling experiments. A better understanding of the transcriptional changes that trigger the regenerative response of zebrafish Müller glia, will allow a more thorough comparison with injury responses in mammalian Müller glia. We hope to discover alternative regulatory mechanisms that lead to differential outcomes following retinal injury in fish and mammalian retinas.
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