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Reyna Isabel Martinez-De Luna, Ray Yueh Ku, Alexandria M. Aruck, Francesca N. Santiago, Diego San Mauro, Michael Ezra Zuber; Müller Glia reactivity follows retinal injury despite the absence of the Glial Fibrillary Acidic Protein gene in Xenopus. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© 2017 Association for Research in Vision and Ophthalmology.
Glial fibrillary acidic protein (GFAP) is a class III intermediate filament protein (IFP) that is up-regulated in Müller glia in response to injury or disease, and is thought to play a role in the extensive structural changes observed during Müller cell hypertrophy and glial scar formation. We previously demonstrated that Xenopus laevis Müller glia become reactive and hypertrophy following rod loss in an inducible (XOPNTR) model of retinal degeneration. The purpose of this study was to determine if GFAP is required for Müller cell reactivity in X. laevis.
Blastn, blastp, and tblastn searches of NCBI and Ensembl databases were used in an attempt to identify a Xenopus GFAP ortholog. MAFFT was used to align IFP sequences, identify conserved regions, and generate phylogenetic trees. Retinal injuries included photoreceptor ablation by metronidazole (Mtz) treatment of XOPNTR transgenic tadpoles and retinal ganglion cell (RGC) axotomy. Antibody specificity and changes in IFP expression were determined by western blot, immunohistochemistry and in situ hybridization of retinal sections. Degenerate PCR was used to detect gfap genes in amphibian and other vertebrate species.
In spite of the morphological changes and GFAP-like immunoreactivity in Müller cells following retinal injury, we discovered that Xenopus lack a gene for gfap. Commonly used GFAP antibodies were not specific and also detect the class III IFPs Vim, Prph, Des, and Ina; suggested that one or more of these proteins was up-regulated in Müller cells following retinal injury in Xenopus. Consistent with this observation, we found that Vim and Prph were significantly induced following both rod photoreceptor ablation and RGC axotomy. Analyses of the X. tropicalis and X. laevis genomes indicated either a small deletion or incomplete inversion event resulted in deletion of the gfap gene during evolution. A PCR-based survey of representative species from all three extant amphibian orders (Anura, Caudata and Gymnophiona) suggests deletion of the gfap locus occurred in the ancestor of all Anura after its divergence from the Caudata ancestor ~290 million years ago.
Our results demonstrate that extensive changes in Müller cell morphology following retinal injury do not require GFAP in X. laevis. Instead, Müller cell changes may require other class III IFPs, potentially including Vim and Prph.
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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