XB-ART-51990Dev Biol January 1, 2017; 426 (2): 219-235.
Müller glia reactivity follows retinal injury despite the absence of the glial fibrillary acidic protein gene in Xenopus.
Intermediate filament proteins are structural components of the cellular cytoskeleton with cell-type specific expression and function. Glial fibrillary acidic protein (GFAP) is a type III intermediate filament protein and is up-regulated in glia of the nervous system in response to injury and during neurodegenerative diseases. In the retina, GFAP levels are dramatically increased in Müller glia and are thought to play a role in the extensive structural changes resulting in Müller cell hypertrophy and glial scar formation. In spite of similar changes to the morphology of Xenopus Müller cells following injury, we found that Xenopus lack a gfap gene. Other type III intermediate filament proteins were, however, significantly induced following rod photoreceptor ablation and retinal ganglion cell axotomy. The recently available X. tropicalis and X. laevis genomes indicate a small deletion most likely resulted in the loss of the gfap gene during anuran evolution. Lastly, a survey of representative species from all three extant amphibian orders including the Anura (frogs, toads), Caudata (salamanders, newts), and Gymnophiona (caecilians) suggests that deletion of the gfap locus occurred in the ancestor of all Anura after its divergence from the Caudata ancestor around 290 million years ago. Our results demonstrate that extensive changes in Müller cell morphology following retinal injury do not require GFAP in Xenopus, and other type III intermediate filament proteins may be involved in the gliotic response.
PubMed ID: 26996101
PMC ID: PMC5026855
Article link: Dev Biol
Genes referenced: des.1 gnat1 ina nes prph rpe vim vim.2
GO keywords: intermediate filament
Antibodies: GFAP Ab2 GFAP Ab3 Gnat1 Ab1 Muller Glia Ab1 Myc Ab12 Myc Ab6 Photoreceptors Ab1 Vim Ab1
Article Images: [+] show captions
|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.|
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