Fig 1: Ecrg4 over-expression inhibits injury-induced proliferation of subependymal progenitor cells. A: Transduction of ependyma with adenovirus vector containing the green fluorescent protein gene (AdGFP). If AdGFP was injected i.c.v. such that target cells were labeled prior to the penetrating injury, ependymal cells throughout the ventricles were transduced as indicated by the GFP staining of ependyma (green). Scale bar = 100 μm. B: Cell proliferation after penetrating injury after AdGFP. To monitor cell growth after injury, animals that had been injected with AdGFP, received daily injections of BrdU i.p. as described in the text and the incorporation of BrdU into DNA was evaluated by immunohistochemistry. Brdu: red, DAPI: blue, scale bar = 20 μm. C: Cell proliferation after penetrating injury and AdEcrg4. If instead of AdGFP as above, the target ependymal cells were transduced by i.c.v. injection of adenovirus containing Ecrg4 (AdEcrg4), there was a decrease in BrdU-labeled cells in the subependymal zone. Scale bar = 20 μm. D: Quantification of cell proliferation after penetrating injury after AdGFP or AdEcrg4. When apparent differences in proliferating cell number were quantified, a significant difference in proliferating cell number was observed (p < 0.01). Error bars represent mean ± standard deviation (n = 4).
Fig 2: Augurin immunoreactivity and Ecrg4 gene expression decreases in the CP following CNS injury. A and B: Augurin immunoreactivity decreased in rat choroid plexus after penetrating CNS injury. A: Augurin immunoreactivity in the brains of control rats was, as anticipated, readily detectable in the cytoplasm of CPe cells. In certain areas, this staining appeared apical. B: There was an apparent decrease in augurin at 1 day post injury (d.p.i.). Augurin: red, DAPI: blue, scale bar = 20 μm. C and D: Ecrg4 gene expression decreased in rat choroid plexus after penetrating CNS injury. C: Ecrg4 gene expression in the brains of control rats was, as anticipated, readily detectable by in situ hybridization in the cytoplasm of CP epithelial cells. D: As shown in Panel D however, there was an apparent decrease in Ecrg4 gene expression staining in the CP-containing sections processed from animals 24 h post injury (d.p.i.). Scale bar = 20 μm.
Fig 3: ECRG4 gene knockdown causes enlarged hindbrain ventricles and a hyper proliferative response in the developing zebrafish. A: Development of Zebrafish after morpholino injection 48 hours post fertilization. In control (A1) and Ecrg4 (A2) morpholino treatments, the head, eyes, heart, yolk and hind ventricle (arrows) were photographed for analysis. The anti-sense Ecrg4a morpholino caused readily detectable hindbrain ventriculomegaly resembling a hydrocephalus-edema-like response in the CNS. The ventricles can be isolated from images for analyses (n = 8). B: Quantification of developmental effects of morpholino injection. Hindbrain ventricle size was quantified by pixel area of microscope images from n = 8 control (MO ctl), n = 8 Ecrg4a MO-treated embryos or (n = 8) Ecrg4a MO co-injected with the Zebrafish mRNA ortholog A to neutralize specific inhibition (error bars: mean ± standard deviation). C-E: Cell growth in Zebrafish 24 hours after control or Ecrg4a morpholino injection 24 hours post fertilization. Proliferating cells in control MO (top panels) from sagittal (C1), dorsal (D1) or after cross sections at dashed line (E1) can be compared to similar sections of Ecrg4a MO (bottom panel) injected animals by staining for phosphorylated Histone 3 (H3P, green), a marker of cell growth. H3P-positive proliferating cells are compared to that of cytoplasmic glial fibrillary acidic protein (red). The arrow (D2) highlights space corresponding to enlarged ventricles in MO-Ecrg4 treated embryos. The H3P positive cells are on the ventricle surface and there are more H3P cells and thicker GFAP staining in Ecrg4a MO. Scale bar is 100 μm F Quantification of cell growth in Zebrafish after control or Ecrg4a morpholino injection. The amount of H3P-postive cell staining was determined as described in the text and compared between treatment groups (n = 7, mean ± SEM). There were significantly more labeled cells in the Ecrg4a morpholino group. Scale bar is 100 μm.
Fig 4: ECRG4 homologies and its predicted processing: A: ECRG4 encodes conserved, potentially secreted proteins. Homologies between species of Ecrg4 encoded proteins are evident after the primary sequence alignment of the proteins encoded by mouse, rat, human, chimpanzee, dog, cow, chicken and zebrafish Ecrg4(a) gene were collated from Pubmed (nucleotide) and compared. To aid in functional comparisons, the sequences were labeled red for small amino acids, blue for acidic and magenta for basic amino acids, green for hydroxyl-, sulfhydryl, aminated or glycine amino acids. Actual homologies are presented in table 1. Consensus processing sites for (1) removal of the signal peptide, (2) processing by furin-like hormone substrates and (3) thrombin is highlighted by a line over the sequences. B: Candidate proteins generated from Ecrg4 gene expression. Eight potential protein products are predicted by algorithms to be generated from the single gene: intact Ecrg4(1-148); its leader sequence, augurin, argilin and ecilin and their ∆16C-terminal cleaved homologs: C∆16-augurin, C∆16-argilin and the ∆16 peptide itself.
Fig 5: The choroid plexus and ependyma are major sites of Ecrg4 expression. A: Ecrg4 gene expression in the mouse embryo. In situ hybridization of mouse embryo day E14.5 were obtained from the GenePaint consortium [51]. Significantly more data is available at: http://www.genepaint.org/cgi-bin/mgrqcgi94?APPNAME=genepaint&PRGNAME=analysis_viewer&ARGUMENTS=-AQ64622747128404,-AEG,-A804,-Asetstart,-A5 B: Ecrg4 gene expression in the developing mouse brain. Higher magnification analysis of the CNS localization (from samples shown in A), shows the in situ hybridization signal localizing primarily to choroid plexus and ependyma. C: Ecrg4 gene expression in the adult mouse brain. In situ hybridization of adult mouse brain were obtained from open source data generated by the Allen Brain Atlas [21]. Significantly more data throughout the brain are available for analyses at: http://mouse.brain-map.org/brain/1500015O10Rik/70429477/thumbnails.html?ispopup=1. D: Ecrg4 gene expression in the mouse choroid plexus and ependyma. A higher magnification analysis of the CNS localization (from samples shown in Panel C), show that the in situ hybridization signal localizes primarily to choroid plexus epithelial cells and to a lesser extent ventricular ependymal cells. E: Ecrg4 gene expression in mouse central canal ependymal/epithelial cells in the spinal cord. In situ hybridization maps of adult mouse spinal cord were selected from open source data generated by the Allen Brain Atlas Consortium [21]. Significantly more data on the distribution of Ecrg4 (Riken150001O15) gene expression throughout the mouse spinal cord, including other cell types, is available at http://mousespinal.brain-map.org/imageseries/show.html?id=100028769. F: Quantitative gene expression by RT-qPCR. After extracting tissues from donor adult mice (n = 12), highest Ecrg4 expression was found in the dissected choroid plexus. Data was calculated using the ∆ΔCt method and normalized to the values obtained in testes. Similar findings were obtained in a survey of male rats (not shown) and low gene expression was detectable in all tissues. Error bars represent mean ± standard deviation, n = 5.
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