Fig 1: Proliferating cell nuclear antigen (PCNA) is located primarily in the PPa4/PPp1 transition region. (A–J) Transverse paraffin-sections of 5 µm of POA. (A–E) Vim+ cells (green) are localized to the dorsal region of the POA, in contrast, PCNA+ cells (red, arrow (B,C,E)) are found in the ventral of the POA. Vim+ labeling ((D’,D’’’), green) does not co-localize with anti-PCNA labeling ((D’’,D’’’), red). (F–H) Many Sox2+ cells (green) were observed in PPa1-3 and PPp1 (J) with few PCNA+ cells (red, arrows). (I) Sox2+ cells (green) and PCNA+ (red) in the ventral PPa4 region of the POA (boxed area) where the signals were co-localized in some cells ((I’–I’’’), arrowheads). All sections were labeled with DAPI (blue). Scale bar: 30 μm.
Fig 2: Summary of the distribution of neural progenitors in the POA. (A) Distribution of neural progenitors lining the DiV in the hypothalamus of mammals according to [17]. α2 tanycytes with proliferative capacity express: Vim, Nestin [34], Sox2, and GFAP, while the non-proliferative tanycytes do not express GFAP. Ependymal cells are distributed in the dorsal region. (B) In zebrafish, we described a low-proliferative cell Vim+, Sox2+, and Zrf1+, and high proliferative Sox2+ cells, previously reported to express nestin [42]. Cytoplasmic Sox2/Fezf2:GFP cells were observed in the region previously shown to express cytoplasmic Sox2 [29]. (C) Cytokeratin cells are express in the ventral region of the POA in cytokeratin [43] positive ependymal-like cells similar to those seen in mammals.
Fig 3: Cells lining the diencephalic ventricle (DiV) express neural progenitor markers. (A–J) Expression of Vim, Zrf1, and Sox2 in transverse paraffin-sections of 10 μm of the POA. (A–E) Cells immuno-positive for anti-Vim (red) and anti-Zrf1 (green). (A,B) Vim+/Zrf1+ positive cells are distributed dorsomedially in the wall of the third ventricle (boxed area), and signals are co-localized ((A’,B’), arrows). (C) Fewer Vim+/Zrf1+ cells are observed in PPa3 (boxed area) with limited co-localization ((C’), arrow). ((A–C), asterisks) Vim+/Zrf1+ cells are located in the ventrolateral region with long processes that extend towards the DiV (arrowheads). (D) Vim-/Zrf1- cells with fusiform nuclei are found in at ventral sur-face of POA ((D’), asterisk). (E) Vim+ and Zrf1+ label in dorsomedial region (boxed area), is co-localized ((E’), arrow). (F–J) Cells immune-positive for anti-Vim and anti-Sox2. (F–H) Vim+ cells also label with anti-Sox2 (red) (boxed area), and Sox2+ cells are observed in dorsal-ventral regions of the ventricle wall. (F’–H’) Higher magnification views of box areas in F-H: Vim+/Sox2+ co-localization in cells (arrows). (I) Cells with fusiform nuclei are Sox2+ and are located in the ventral wall ((I’), arrow). (J) Vim+ and Sox2+ boxed area, co-localize in ventricle wall ((J’), arrow). Diencephalic ventricle (DiV); magnocellular nucleus (PM); optic nerve (ON); anterior part of the parvocellular preoptic nucleus (PPa); posterior part parvocellular nucleus (PPp); suprachiasmatic nucleus (SC). All sections labeled with DAPI (blue). Scale bar: 30 μm.
Fig 4: Phenotypes of ifnlr1 zebrafish morphants. (A,B) Effectiveness of ifnlr1 knockdown was confirmed by RT-PCR. (A) The zebrafish ifnlr1 gene was targeted by specific MO antisense to prevent the appropriate splicing of exon 4 (E4I4-MO). Primers 3F and 5R detected the presence of WT (non-mutant) transcripts or those in which exon 4 had been skipped. The diagram below is a schematic depiction of the exon 4-skipped transcript in the E4I4-MO-injected embryos compared with control embryos. (B) RT-PCR of ifnlr1 transcript from control-MO and E4I4-MO MO-injected embryos at 4 dpf demonstrates skipping of exon 4. Injection of 4 ng of ifnlr1 MO altered the splicing between exon 4 and intron 4, as revealed by a shift in PCR bands between control (249 bp) and ifnlr1 MO-injected embryos (67 bp). (C–G) At late stages, knockdown ifnlr1 caused non-inflation of the SB (F, blue arrow) and expansion of the gut lumen area (F, red dashed lines). The bar graph in Panel G shows the percentage of embryos with development defects at 7 dpf. (H–N) ifnlr1 knockdown induces potent neuromasts loss in zebrafish. Control embryos and embryos injected with 4 ng ifnlr1-e4i4-MO were stained with the mitochondrial potentiometric dye DASPEI at 7 dpf. Neuromasts stereotypically located on the lateral line were stained as green dots (H,J: white arrow). Control MO-injected zebrafish had normal numbers of neuromasts. In contrast, significantly decreased hair cell staining (I,K: asterisk) and head neuromast staining (L: white arrow; M: asterisk) were observed in ifnlr1 morphants. Fluorescent DASPEI images were inverted for particle analysis. The fluorescence particle signals of neuromasts were quantified using morphometric analysis. (O) Hair cells, supporting cells and proliferated cells per neuromast were counted and analysed in 6dpf Tg (Brn3c:mGFP). Transgenic zebrafish larvae using four markers GFP (hair cells, green), SOX2 (supporting cells, white), BrdU (proliferated cells, red) and DAPI (nucleus, blue). Four dpf embryos were exposed to BrdU for 48 hours and visualised the cell proliferation. There were fewer hair cells, supporting cells and proliferated cells in morphants than WT. (N,P–R) Quantification of the average number of neuromasts (N), hair cells (P), support cells (Q) and proliferated cells (R) shows significantly decrease in ifnlr1 morphants. Error bars, SEM; ***P<0.0001 (n=10; Student’s t-test). BrdU, bromodeoxyuridine; DASPEI, 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide; dpf, days postfertilisation; E, ear; H, heart; L, liver; MO, Morpholino; SB, swim bladder; WT, wild type.
Fig 5: Glioblastoma and HGA stem-like glioma-initiating cells stained positive for SOX-2 and Nestin. Exemplary immunofluorescence staining of SOX-2 (red), Nestin (green), and DAPI (blue) in pre-treatment MGMT+ (a), post-5-days/-23 days DMSO treatment HGA (b), and monolayer cell cultures and 6 weeks of TMZ treatment MGMT+ neurosphere culture (c). The scale bars represent 100 µm each. Abbreviations: HGA, astrocytoma, isocitrate dehydrogenase mutant, CNS WHO grade 4; DAPI, 4′,6-Diamidin-2-phenylindol; MGMT+, glioblastoma cell line with >50% O6-methylguanine-DNA-methyltransferase promoter methylation; and DMSO, dimethyl sulfoxide.
Supplier Page from Abcam for Anti-SOX2 antibody