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Ke'ale W Louie, Alfonso Saera-Vila, Estelle F Chiari, Phillip E Kish, Alon Kahana; Regulation of extraocular muscle cell identity and dedifferentiation by epigenetic regulators of chromatin structure. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4572. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Extraocular muscle (EOM) function is required for proper binocular vision. Disorders that affect EOM function are common causes of visual morbidity, yet therapeutic approaches to EOM dysfunction are mostly limited to strabismus surgery, with varying success. Using a zebrafish model of EOM injury and repair, we characterized the role of epigenetic regulators in EOM regenerative response following severe injury. The long-term goal is to develop targeted therapy that would reduce morbidity and improve response to treatment for vision-threatening EOM disorders.
Injury to adult zebrafish EOMs was a unilateral excision of ~0.75mm segment (50% myectomy) of the lateral rectus muscle (LR). Transcriptome analysis of the injured LR was performed to identify important genetic regulators of regeneration. The transcriptome was analyzed via deep sequencing for genes known to affect chromatin structure. Using antisense morpholino oligonucleotides (MO), the role of these epigenetic regulators in EOM regeneration was assessed. MOs were microinjected and electroporated into the LR muscle of adult zebrafish 2 hours before myectomy. Cell proliferation was assessed at different time points using 5-ethynyl-2’-deoxyuridine (EdU) labeling, along with histologic characterization and a regeneration assay.
Members of the Polycomb Repressor Complex 2 (PRC2) were identified as early responders to EOM injury. MOs were designed to target ezh2 and suz12a, both of which were induced after injury. Following MO downregulation of ezh2 or suz12a, myoblast cell proliferation was significantly reduced: ezh2-MO = 27% reduction at 24hpi (p<0.001) and 35% at 48hpi (p<0.01); suz12a-MO = 28% reduction at 24hpi (p<0.05) and 29% at 48hpi (p<0.05). Histologic and molecular characterizations of muscle response to PRC2 downregulation are ongoing.
Knockdown of PRC2 members ezh2 and suz12a significantly reduced cell proliferation at 24 and 48hpi, revealing an important role for histone trimethylation-induced chromatin remodeling in the response of adult zebrafish EOMs to severe injury. Epigenetic regulators of EOM myocytes and myoblasts may represent new targets for diagnostic tools and therapeutic interventions. Additional studies are warranted.
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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