Martinez-De Luna RI , Ku RY , Aruck AM , Santiago F , Viczian AS , San Mauro D , Zuber ME .

R01 EY015748 NEI NIH HHS , R01 EY017964 NEI NIH HHS

	Fig. 1. GFAP-like immunolabeling in the normal and injured X. laevis retina. Retinal sections of wild-type (A-D, I-L) and XOPNTR transgenic (E-H) tadpoles were co-stained with GFAP pAb and XAP2 mAb (A, B, E, F, I, J) or GFAP mAb and GαT1 pAb (C, D, G, H, K, L). Green fluorescent secondary antibodies were used to visualize GFAP antibody staining, while red fluorescent secondary antibodies detect XAP2 (unknown epitope) and GαT1 (Transducin) antibodies, which stain rod photoreceptors. Wild-type controls and XOPNTR transgenic tadpoles were treated for 17 days with either DMSO-alone (A, C, E, G) or Mtz (B, D, F, H) to ablate rod photoreceptors. Retinal sections of wild-type tadpoles were stained three days following retinal axotomy. The left, unoperated eyes (I and K) are compared to the right, operated eyes (J and L) of the same animals. Hoescht (blue) stains nuclei of the ONL, INL and GCL, which are the outer nuclear, inner nuclear, and ganglion cell layers (asterisks in I-L), respectively. Scale bar=25 µm.
	Fig. 2. Sequences most similar to GFAP in Xenopus. (A) GFAP consensus used to distinguish candidate GFAP sequences from other intermediate filament proteins. Conserved regions (labeled a–e) are present in all GFAP orthologs identified and separated by regions of varying length (Xn). The gfap exon in which each region is coded is shown. The numbering system below the consensus is for human GFAP Isoform 1 (NP_002046). All residues shown are invariant in the GFAP orthologs aligned ( Fig. S1). The residues (*) at positions 347 (Q) and 357 (L) are unique to GFAP. Consensus region b is coded for in two exons (4 and 5). Vertical line (|) demarcates sequence coding for exons 4 and 5. (B) Cladogram of GFAP orthologs, Xenopus IFPs and untitled Xenopus proteins with similarity to GFAP. Midpoint rooted RAxML Polar Tree based on MAFFT alignment of GFAP orthologs, selected known X. laevis and X. tropicalis IFPs and the 17 untitled X. laevis and X. tropicalis sequences showing greatest similarity to GFAP ( Katoh and Standley, 2013). Untitled X. laevis (Xla) and X. tropicalis (Xt) sequences are shown in red. Green branches show location of the GFAP clade. Groupings of IFP types are shown. Maximum likelihood bootstrap (above) and Bayesian inference posterior probabilities (below) for each branch are included ( Felsenstein, 1981 and Huelsenbeck et al., 2001). Accession numbers for each sequence in the tree can be found in Supplementary Table S2.
	Fig. 3. Syntenic analyses of gfap genomic regions. The genomic regions surrounding gfap genes in select vertebrates are illustrated. For clarity, homologous genes have been similarly colored. Non-coding RNAs and pseudogenes were not included. Although human FAM187a is not annotated on the most recent reference assembly (GRCh38), it was included on previous assemblies and its location is included here. Chromosome number as well as the size of the regions depicted in the schematic are shown in parenthesis. The reverse complement of sequences were used so the gfap gene appears 5′ (left) to 3′ (right) in all species. The location of hypothesized deletions, duplications and inversions are also illustrated. The sequence source and locations used to build these syntenic schematics can be found in Table S4.
	Fig. 4. Specificity of intermediate filament protein antibodies. Western blots were used to determine the specificity of GFAP pAb (A), GFAP mAb (B), Prph pAb 1 (C), Prph pAb 2 (D), Vim mAb (E), and R5 mAb (F). Extracts were prepared from embryos injected with mRNA coding for the indicated myc-tagged IFP. Blots were probed with the indicated combination of primary antibodies. Green fluorescent secondary antibodies were used to detect the IFP and myc primary antibodies, respectively (A-E). Red fluorescent signals were pseudocolored to make visualization easier. Merged images indicate myc-IFP proteins detected by both antibodies (A–E, yellow). Enhanced chemiluminescence was used to test the specificity of the R5 mAb and myc pAb (F). One-sixth the volume of extract from myc-MmGFAP expressing embryos in lane 2 was used in lane 3. Immunohistochemistry was use to compare the staining pattern of the GFAP pAb (G-G′′′) and GFAP mAb (H-H′′′) in sections of stage 35/36 X. laevis embryos. G′/H′, G′/H′ and G′′′/H′′′ show magnified views of the brain, notochord and retina, respectively. Arrow and arrowheads, indicate the location of skin epidermis and ocular motorneuron projections dorsal and ventral to the optic cup, respectively. Scale bar, 50 µm.
	Fig. 5. Expression patterns of vim, des, prph, ina, and nes in the pre-metamorphic tadpole nervous system. In situ hybridization on retinal, brain and spinal cord sections for vim (A–C), des (D–F), prph (G-I), ina (J–L), and nes (M–O). Sections were obtained from pre-metamorphic stage 50 tadpoles. Location of the lens (L), outer (O), inner (I) and ganglion (G) cell layers are indicated. Scale bars, 100 µm in A, D, G, J and M; all others are 50 µm.
	Fig. 6. Retinal expression of intermediate filament proteins after rod photoreceptor ablation. vim (A and B), prph (E and F), and ina (I and J) in situ hybridization in retinal sections from wild-type (A, E, and I) and XOPNTR (B, F, and J) tadpoles treated with Mtz for 7 days. Dashed white lines indicate the boundary between the RPE (dark pigment) and neural retina. White asterisks in B and F indicates expression of vim (B) and prph (F) adjacent to the RPE in the subretinal space. Immunolabeling of retinal sections with anti-Vim mAb (C and D), Prph pAb 1 (G and H), and R5 mAb (K and L) in Mtz-treated wild-type (C, G, and K) or XOPNTR (D, H, and L) retinas. Retinal sections were co-stained with DAPI to visualize nuclei. Scale bars, 50 µm.
	Fig. 7. Retinal expression of intermediate filament proteins after retinal ganglion cell axotomy. vim (A and B), prph (E and F), and ina (I and J) in situ hybridization on retinal sections from control unoperated (A, E, and I) and operated (retinal axotomy) (B, F, and J) eyes. Vim mAb (C and D), Prph pAb 1 (G and H), and R5 mAb (K and L) immunolabeling of retinal sections from control (C, G, and K) and operated (D, H, and L) eyes. Rod photoreceptors were labeled with either GαT1 (Transducin) pAb (C and D) or XAP-2 mAb (G, H, K, and L). Sections were counterstained with DAPI to visualize nuclei. Scale bars, 50 µm.
	Fig. 8. Schematic representation of the evolutionary relatedness of species in the survey for gfap and vim genes. Orders for amphibian species are indicated. Asterisk illustrates last common ancestor shared by Anura and Caudata. + and − indicate presence or absence of indicated gene in the PCR surveys, respectively. Photo credits: B. bufo, R. bivittatum (DSM), C. lusitanica (Benny Trapp: https://commons.wikimedia.org/wiki/User:Benny_Trapp).
	Fig. S15. Vim expression in the subretinal space after rod ablation. Wild-type (A) and XOPNTR (B) stage 50 tadpoles were treated for seven days with Mtz. Retinal sections were stained by in situ hybridization for vim expression and then bleached with hydrogen peroxide to clear RPE pigment. White dashed lines in both panels mark the boundary between RPE and photoreceptors. White asterisk in B labels vim expression in the subretinal space in response to rod ablation. Scale bar, 50 µm.
	Antibody gnat Ab1 (guanine nucleotide binding protein (G protein), alpha transducing activity polypeptide 1), in sectioned eye of Xenopus laevis, at NF stage 55.
	ina (internexin neuronal intermediate filament protein alpha) gene expression in Xenopus laevistdpole, seen in eye (J), brain (L) and spinal cord (K) sections , at NF stage 50.

Abascal, ProtTest: selection of best-fit models of protein evolution. 2005, Pubmed

Abascal, ProtTest: selection of best-fit models of protein evolution. 2005, Pubmed
Araki, Regeneration of the amphibian retina: role of tissue interaction and related signaling molecules on RPE transdifferentiation. 2007, Pubmed , Xenbase
Barbosa-Sabanero, Lens and retina regeneration: new perspectives from model organisms. 2012, Pubmed
Bignami, The radial glia of Müller in the rat retina and their response to injury. An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein. 1979, Pubmed
Bisbee, Albumin phylogeny for clawed frogs (Xenopus). 1977, Pubmed , Xenbase
Bordeaux, Antibody validation. 2010, Pubmed
Bringmann, Müller cells in the healthy and diseased retina. 2006, Pubmed
Bringmann, Cellular signaling and factors involved in Müller cell gliosis: neuroprotective and detrimental effects. 2009, Pubmed
Bringmann, Müller glial cells in retinal disease. 2012, Pubmed
Castresana, Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. 2000, Pubmed
Cerdà, Zebrafish vimentin: molecular characterization, assembly properties and developmental expression. 1998, Pubmed
Chain, Multiple mechanisms promote the retained expression of gene duplicates in the tetraploid frog Xenopus laevis. 2006, Pubmed , Xenbase
Chernoivanenko, Mitochondrial membrane potential is regulated by vimentin intermediate filaments. 2015, Pubmed
Chernyatina, Intermediate filament structure: the bottom-up approach. 2015, Pubmed
Chiba, The retinal pigment epithelium: an important player of retinal disorders and regeneration. 2014, Pubmed , Xenbase
Choi, Cone degeneration following rod ablation in a reversible model of retinal degeneration. 2011, Pubmed , Xenbase
Darriba, ProtTest 3: fast selection of best-fit models of protein evolution. 2011, Pubmed
Davidson, Intermediate filament complement of the normal and gliotic canine retina. 1990, Pubmed
Debus, Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides. 1983, Pubmed
Dent, A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. 1989, Pubmed , Xenbase
Dräger, Antibodies against filamentous components in discrete cell types of the mouse retina. 1984, Pubmed , Xenbase
Edgar, MUSCLE: a multiple sequence alignment method with reduced time and space complexity. 2004, Pubmed
Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput. 2004, Pubmed
Erickson, Glial fibrillary acidic protein increases in Müller cells after retinal detachment. 1987, Pubmed
Evans, A mitochondrial DNA phylogeny of African clawed frogs: phylogeography and implications for polyploid evolution. 2004, Pubmed , Xenbase
Felsenstein, Evolutionary trees from DNA sequences: a maximum likelihood approach. 1981, Pubmed
Filoni, Retina and lens regeneration in anuran amphibians. 2009, Pubmed , Xenbase
Fischer, Transitin, a nestin-related intermediate filament, is expressed by neural progenitors and can be induced in Müller glia in the chicken retina. 2005, Pubmed
Fuchs, Intermediate filaments: structure, dynamics, function, and disease. 1994, Pubmed
Gervasi, Xenopus laevis peripherin (XIF3) is expressed in radial glia and proliferating neural epithelial cells as well as in neurons. 2000, Pubmed , Xenbase
Gervasi, Increased expression of multiple neurofilament mRNAs during regeneration of vertebrate central nervous system axons. 2003, Pubmed , Xenbase
Giménez Y Ribotta, Comparative anatomy of the cerebellar cortex in mice lacking vimentin, GFAP, and both vimentin and GFAP. 2000, Pubmed
Glasgow, Plasticin, a novel type III neurofilament protein from goldfish retina: increased expression during optic nerve regeneration. 1992, Pubmed
Godsave, The appearance and distribution of intermediate filament proteins during differentiation of the central nervous system, skin and notochord of Xenopus laevis. 1986, Pubmed , Xenbase
Goldman, The function of intermediate filaments in cell shape and cytoskeletal integrity. 1996, Pubmed
Gray, Genenames.org: the HGNC resources in 2015. 2015, Pubmed
Grosche, Retinal light damage vs. normal aging of rats: altered morphology, intermediate filament expression, and nuclear organization of Müller (glial) cells. 1997, Pubmed
Guo, Probing the stochastic, motor-driven properties of the cytoplasm using force spectrum microscopy. 2014, Pubmed
Hatzfeld, A synthetic peptide representing the consensus sequence motif at the carboxy-terminal end of the rod domain inhibits intermediate filament assembly and disassembles preformed filaments. 1992, Pubmed
Helfand, A role for intermediate filaments in determining and maintaining the shape of nerve cells. 2003, Pubmed
Hellsten, Accelerated gene evolution and subfunctionalization in the pseudotetraploid frog Xenopus laevis. 2007, Pubmed , Xenbase
Hemmati-Brivanlou, A protein expressed in the growth cones of embryonic vertebrate neurons defines a new class of intermediate filament protein. 1992, Pubmed , Xenbase
Herrmann, Expression of intermediate filament proteins during development of Xenopus laevis. II. Identification and molecular characterization of desmin. 1989, Pubmed , Xenbase
Herrmann, Expression of intermediate filament proteins during development of Xenopus laevis. I. cDNA clones encoding different forms of vimentin. 1989, Pubmed , Xenbase
Huelsenbeck, Bayesian inference of phylogeny and its impact on evolutionary biology. 2001, Pubmed
Ivaska, Novel functions of vimentin in cell adhesion, migration, and signaling. 2007, Pubmed
Jones, The rapid generation of mutation data matrices from protein sequences. 1992, Pubmed
Katoh, MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. 2002, Pubmed
Katoh, Recent developments in the MAFFT multiple sequence alignment program. 2008, Pubmed
Katoh, MAFFT multiple sequence alignment software version 7: improvements in performance and usability. 2013, Pubmed
Kinouchi, Robust neural integration from retinal transplants in mice deficient in GFAP and vimentin. 2003, Pubmed
Klymkowsky, Polar asymmetry in the organization of the cortical cytokeratin system of Xenopus laevis oocytes and embryos. 1987, Pubmed , Xenbase
Kohno, Induction of nestin, Ki-67, and cyclin D1 expression in Müller cells after laser injury in adult rat retina. 2006, Pubmed
Kornreich, Order and disorder in intermediate filament proteins. 2015, Pubmed
Lenkowski, Müller glia: Stem cells for generation and regeneration of retinal neurons in teleost fish. 2014, Pubmed
Lewis, Up-regulation of glial fibrillary acidic protein in response to retinal injury: its potential role in glial remodeling and a comparison to vimentin expression. 2003, Pubmed
Lewis, Changes in the expression of specific Müller cell proteins during long-term retinal detachment. 1989, Pubmed
Liedtke, GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination. 1996, Pubmed
Lowery, Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function. 2015, Pubmed
Lu, Reactive glial cells: increased stiffness correlates with increased intermediate filament expression. 2011, Pubmed
Luna, Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin. 2010, Pubmed
Lundkvist, Under stress, the absence of intermediate filaments from Müller cells in the retina has structural and functional consequences. 2004, Pubmed
MacDonald, Müller glia provide essential tensile strength to the developing retina. 2015, Pubmed
Martinez-De Luna, Maturin is a novel protein required for differentiation during primary neurogenesis. 2013, Pubmed , Xenbase
Martinez-De Luna, Putting regeneration into regenerative medicine. 2014, Pubmed
Martinez-De Luna, The Retinal Homeobox (Rx) gene is necessary for retinal regeneration. 2011, Pubmed , Xenbase
Matsuda, Dkk1-dependent inhibition of Wnt signaling activates Hesx1 expression through its 5' enhancer and directs forebrain precursor development. 2014, Pubmed
Matveeva, Vimentin is involved in regulation of mitochondrial motility and membrane potential by Rac1. 2015, Pubmed
McCall, Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology. 1996, Pubmed
McLenachan, Transgenic mice expressing the Peripherin-EGFP genomic reporter display intrinsic peripheral nervous system fluorescence. 2008, Pubmed
Menet, Inactivation of the glial fibrillary acidic protein gene, but not that of vimentin, improves neuronal survival and neurite growth by modifying adhesion molecule expression. 2001, Pubmed
Menet, Axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes. 2003, Pubmed
Messenger, The appearance of neural and glial cell markers during early development of the nervous system in the amphibian embryo. 1989, Pubmed , Xenbase
Mitashov, Retinal regeneration in amphibians. 1997, Pubmed
Nakazawa, Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin. 2007, Pubmed
Okada, Müller cells in detached human retina express glial fibrillary acidic protein and vimentin. 1990, Pubmed
Pekny, Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally. 1995, Pubmed
Raymond, Molecular characterization of retinal stem cells and their niches in adult zebrafish. 2006, Pubmed
Reeves, Heterogeneity in the substitution process of amino acid sites of proteins coded for by mitochondrial DNA. 1992, Pubmed , Xenbase
Ronquist, MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. 2012, Pubmed
Sakaguchi, Growth cone interactions with a glial cell line from embryonic Xenopus retina. 1989, Pubmed , Xenbase
San Mauro, Molecular systematics: A synthesis of the common methods and the state of knowledge. 2010, Pubmed
San Mauro, A multilocus timescale for the origin of extant amphibians. 2010, Pubmed
Saper, A guide to the perplexed on the specificity of antibodies. 2009, Pubmed
Sethi, Glial remodeling and neural plasticity in human retinal detachment with proliferative vitreoretinopathy. 2005, Pubmed
Sharpe, Developmental expression of a neurofilament-M and two vimentin-like genes in Xenopus laevis. 1988, Pubmed , Xenbase
Sievers, Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. 2011, Pubmed
Stamatakis, RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. 2006, Pubmed
Strelkov, Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly. 2002, Pubmed
Szaro, Immunocytochemical identification of non-neuronal intermediate filament proteins in the developing Xenopus laevis nervous system. 1988, Pubmed , Xenbase
Szeverenyi, The Human Intermediate Filament Database: comprehensive information on a gene family involved in many human diseases. 2008, Pubmed
Talavera, Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. 2007, Pubmed
Thompson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. 1994, Pubmed
Verardo, Abnormal reactivity of muller cells after retinal detachment in mice deficient in GFAP and vimentin. 2008, Pubmed
Vergara, Retinal regeneration in the Xenopus laevis tadpole: a new model system. 2009, Pubmed , Xenbase
Viczian, XOtx5b and XOtx2 regulate photoreceptor and bipolar fates in the Xenopus retina. 2003, Pubmed , Xenbase
Viczian, Expression of Xenopus laevis Lhx2 during eye development and evidence for divergent expression among vertebrates. 2006, Pubmed , Xenbase
Viczian, A simple behavioral assay for testing visual function in Xenopus laevis. 2014, Pubmed , Xenbase
Wang, Axonal and nonneuronal cell responses to spinal cord injury in mice lacking glial fibrillary acidic protein. 1997, Pubmed
Wang, Disease gene candidates revealed by expression profiling of retinal ganglion cell development. 2007, Pubmed
Wiens, Global patterns of diversification and species richness in amphibians. 2007, Pubmed
Wilhelmsson, Absence of glial fibrillary acidic protein and vimentin prevents hypertrophy of astrocytic processes and improves post-traumatic regeneration. 2004, Pubmed
Wong, Efficient retina formation requires suppression of both Activin and BMP signaling pathways in pluripotent cells. 2015, Pubmed , Xenbase
Xue, Nestin expression in Müller glial cells in postnatal rat retina and its upregulation following optic nerve transection. 2006, Pubmed
Xue, Nestin, a new marker, expressed in Müller cells following retinal injury. 2010, Pubmed
Yang, Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. 1994, Pubmed
Zhao, Xefiltin, a Xenopus laevis neuronal intermediate filament protein, is expressed in actively growing optic axons during development and regeneration. 1997, Pubmed , Xenbase
Zhao, Xefiltin, a new low molecular weight neuronal intermediate filament protein of Xenopus laevis, shares sequence features with goldfish gefiltin and mammalian alpha-internexin and differs in expression from XNIF and NF-L. 1997, Pubmed , Xenbase