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  • 94
    Millipore rabbit anti tbr 2 antibody
    Effects of cold challenge or CL 316,243 treatment for 1 week or 4 weeks on the <t>TBR-2</t> and Ki-67 positive cells in the dentate gyrus. Note that the TBR-2 and Ki-67 positive cells are abundant in the Cold1W and Cold4W groups when compared with that in the CON Cold1W and CON Cold 4W groups, respectively (A,C,E,G) . However, the CON CL1W, CL1W, CON CL4W, and CL4W groups show similar populations of TBR-2 and Ki-67 positive cells in the dentate gyrus (A,C,E,G) . GCL, granule cell layer; MoL, molecular layer; PoL, polymorphic layer. Quantitative analysis of TBR-2 and Ki-67 positive cells per section in the CON Cold1W, Cold1W, CON Cold4W, Cold4W, CON CL1W, CL1W, CON CL4W, and CL4W groups (B,D,F,H) ( n = 5 per group); ∗ indicates a significant difference when compared with the CON group ( p
    Rabbit Anti Tbr 2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti tbr 2
    Impaired adult hippocampal neurogenesis in Sox21 −/− mice. A , Experimental scheme for long-term BrdU labeling analysis used to detect slowly dividing cells and neurons. B , C , To analyze type 1 cells, marker+/BrdU+/fiber+ cells (arrows) in wild-type and Sox21 −/− mice were quantified 4 weeks after the last BrdU administration. The graphs show the numbers of FABP7+/BrdU+/fiber+ cells ( B ) and GFAP+/BrdU+/fiber+ cells ( C ) in the DG. D , E , To analyze newly generated mature and immature neurons, the numbers of BrdU+/NeuN+ ( D ) and BrdU+/DCX+ ( E ) cells were quantified 4 weeks after the last BrdU administration. F , Experimental scheme for the short-term BrdU labeling analysis used to detect rapidly dividing cells. G , H , The number of BrdU+/Ascl1+ type 2a cells ( G , arrows) increased, while the number of <t>BrdU+/Tbr2+</t> type 2b cells ( H , arrows) decreased, in Sox21 −/− mice. The top planes are through the x–z axes, and the right planes are through the y-z axes. Data represent the mean ± SE. ** p
    Anti Tbr 2, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore tbr 2
    Immunohistological images of a <t>Tbr2</t> eGFP pseudo-lineage tracing study. Tbr2 expressing IPs were marked by EGFP through crossing the Fln cKO-NPC mice with the Tg(Eomes-EGFP) line. Due to the perdurance of EGFP, many newly generated daughter neurons from Tbr2+ IPs also express EGFP. In a Tg( Eomes -EGFP) :: Flna cKO Emx1Cre+ ;Flnb -/- brain at P0, PH was identified by periventricular NeuN or MAP2 immunosignals (red); the co-presence of EGFP+ cells (green) in PH is shown, demonstrating that neurons in PH were generated directly by mislocalized Tbr2+ IPs. Nuclei DNA was stained with Hoechst 33,342 and shown in blue in all fluorescence images. DOI: http://dx.doi.org/10.7554/eLife.17823.009
    Tbr 2, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Merck KGaA rabbit anti tbr 2
    Immunohistological images of a <t>Tbr2</t> eGFP pseudo-lineage tracing study. Tbr2 expressing IPs were marked by EGFP through crossing the Fln cKO-NPC mice with the Tg(Eomes-EGFP) line. Due to the perdurance of EGFP, many newly generated daughter neurons from Tbr2+ IPs also express EGFP. In a Tg( Eomes -EGFP) :: Flna cKO Emx1Cre+ ;Flnb -/- brain at P0, PH was identified by periventricular NeuN or MAP2 immunosignals (red); the co-presence of EGFP+ cells (green) in PH is shown, demonstrating that neurons in PH were generated directly by mislocalized Tbr2+ IPs. Nuclei DNA was stained with Hoechst 33,342 and shown in blue in all fluorescence images. DOI: http://dx.doi.org/10.7554/eLife.17823.009
    Rabbit Anti Tbr 2, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biorbyt tbr2 antibody
    Immunohistological images of a <t>Tbr2</t> eGFP pseudo-lineage tracing study. Tbr2 expressing IPs were marked by EGFP through crossing the Fln cKO-NPC mice with the Tg(Eomes-EGFP) line. Due to the perdurance of EGFP, many newly generated daughter neurons from Tbr2+ IPs also express EGFP. In a Tg( Eomes -EGFP) :: Flna cKO Emx1Cre+ ;Flnb -/- brain at P0, PH was identified by periventricular NeuN or MAP2 immunosignals (red); the co-presence of EGFP+ cells (green) in PH is shown, demonstrating that neurons in PH were generated directly by mislocalized Tbr2+ IPs. Nuclei DNA was stained with Hoechst 33,342 and shown in blue in all fluorescence images. DOI: http://dx.doi.org/10.7554/eLife.17823.009
    Tbr2 Antibody, supplied by Biorbyt, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Millipore anti tbr 2
    Incorrectly positioned <t>Tbr2</t> + cells in the tish −/− neocortex are mitotic progenitor cells. Confocal images were taken of coronal sections of embryonic neocortex at E17. In E, J , and O , compressed stacks are presented. Vertical and horizontal
    Anti Tbr 2, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher tbr2 conjugated to phycoerythrin tbr2 pe
    Treatment with poly(I ⋅ C) during gestation negatively regulates the transition of PAX6 + radial glia (RG) cells to <t>TBR2</t> + PAX6 + intermediate progenitor cells (IPC) in a TLR3-dependent manner. WT C57BL/6 or TLR3 −/− C57BL/6 pregnant mice were treated with either PBS or poly(I ⋅ C) for three consecutive days (GD15 to GD17) and injected with BrdU on GD16. Cerebral cortical cells were isolated from embryos on GD18. Total viable cells were analyzed for the expression of TBR2 and PAX6 by flow cytometry. (a) Representative histograms showing TBR2 + PAX6 + positive IPC in WT and TLR3 −/− cerebral cortical cells isolated from embryos ( n = 3) obtained from mice treated with PBS or poly(I ⋅ C). The numbers above the brackets indicate the percentages of TBR2 + PAX6 + IPC. (b) Representative histograms showing PAX6 + RG in WT and TLR3 −/− cerebral cortical cells isolated from embryos obtained from PBS- and poly(I ⋅ C)-treated mice. The numbers above the brackets indicate the percentages of PAX6 + RG cells. Gating for the PAX6- or TBR2-positive cells was based on the staining observed with the isotype control. (c) Representative histograms showing the mean fluorescence intensity of TBR2 and PAX6 expression. The mean fluorescence intensity (MFI) is shown as a percentage of the maximum expression. MFI values for the different groups are indicated as follows: PBS-treated group (red line), poly(I ⋅ C)-treated group (blue line), and isotype control group (gray line). The MFI for PBS-treated mice was 1,158.7 ± 57.4, and the MFI for poly(I ⋅ C)-treated mice was 809.3 ± 28.4. (d) Representative histograms showing the MFI of PAX6 expression. Experiments were performed with cerebral cortical cells isolated from embryos ( n = 3); data from one representative cortex are shown. MFI values for the different groups are indicated as follows: PBS-treated group (red line), poly(I ⋅ C)-treated group (blue line), and isotype control group (gray line). The MFI for PBS-treated mice was 13,784.0 ± 1,527.3, and the MFI for poly(I ⋅ C)-treated mice was 16,842.3 ± 799.1. (e) Percentages of TBR2 + PAX6 + IPC and PAX6 + RG in WT and TLR3 −/− cerebral cortical cells isolated from embryos obtained from PBS- and poly(I ⋅ C)-treated mice. Results are expressed as percentages of total viable cells. The values for PBS-treated mice (white bars) and poly(I ⋅ C)-treated mice (hatched bars) are shown. Values that are significantly different ( P
    Tbr2 Conjugated To Phycoerythrin Tbr2 Pe, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tbr2  (Abcam)
    93
    Abcam tbr2
    Schematic showing fetal telencephalon development and Imp1 expression. ( A ) Anatomy during fetal telencephalon development (D: dorsal, V:ventral, L:left, and R:right). At E10.5, two signalling centers, floor plate (FP) and roof plate (RP) exist in the ventral and dorsal telencephalon. At E12.5 and later, dorsal telencephalon can be divided into dorsomedial telencephalon (DMT, light blue) and dorsolateral telencephalon (DLT, pink). Choloid plexus (CP) and cortical hem (CH) constitute the dorsal end of telencephalon. ( B ) Regions where Imp1 was expressed are indicated in green. At E10.5, Imp1 was expressed throughout the forebrain, except in the floor plate and roof plate. At E12.5, Imp1 was expressed typically in undifferentiated cells in the dorsal telencephalon but not in ventral telencephalon or in basal neurons. At E14.5 and later, Imp1 expression was gradually confined to the apical region of the dorsomedial telencephalon. ( C and D ) Schematics show a close up of the boxed regions in panel B to illustrate the cellular layers during cortical development and the cells that expressed Imp1 in those layers. At E10.5, undifferentiated Pax6+ neural stem cells (blue ovals in D ) dominate all layers of the developing telencephalon and all cells express Imp1 (green in C ). At E12.5, neural stem cells start to differentiate into <t>Tbr2+</t> intermediate neural progenitors (yellow ovals in D ) and Tuj1+ neurons (red ovals in D ) that form the preplate (PP in panel C ). At E14.5 and E18.5, newly formed neurons constitute the marginal zone (MZ), cortical plate (CP) and subplate (SP) on the basal side of the developing cortex. The intermediate zone (IZ) is a cell sparse region that lies between the SP and VZ. Imp1 is expressed in undifferentiated cells at these stages (green ovals in C ). DOI: http://dx.doi.org/10.7554/eLife.00924.005
    Tbr2, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher tbr2 alexa488
    Schematic showing fetal telencephalon development and Imp1 expression. ( A ) Anatomy during fetal telencephalon development (D: dorsal, V:ventral, L:left, and R:right). At E10.5, two signalling centers, floor plate (FP) and roof plate (RP) exist in the ventral and dorsal telencephalon. At E12.5 and later, dorsal telencephalon can be divided into dorsomedial telencephalon (DMT, light blue) and dorsolateral telencephalon (DLT, pink). Choloid plexus (CP) and cortical hem (CH) constitute the dorsal end of telencephalon. ( B ) Regions where Imp1 was expressed are indicated in green. At E10.5, Imp1 was expressed throughout the forebrain, except in the floor plate and roof plate. At E12.5, Imp1 was expressed typically in undifferentiated cells in the dorsal telencephalon but not in ventral telencephalon or in basal neurons. At E14.5 and later, Imp1 expression was gradually confined to the apical region of the dorsomedial telencephalon. ( C and D ) Schematics show a close up of the boxed regions in panel B to illustrate the cellular layers during cortical development and the cells that expressed Imp1 in those layers. At E10.5, undifferentiated Pax6+ neural stem cells (blue ovals in D ) dominate all layers of the developing telencephalon and all cells express Imp1 (green in C ). At E12.5, neural stem cells start to differentiate into <t>Tbr2+</t> intermediate neural progenitors (yellow ovals in D ) and Tuj1+ neurons (red ovals in D ) that form the preplate (PP in panel C ). At E14.5 and E18.5, newly formed neurons constitute the marginal zone (MZ), cortical plate (CP) and subplate (SP) on the basal side of the developing cortex. The intermediate zone (IZ) is a cell sparse region that lies between the SP and VZ. Imp1 is expressed in undifferentiated cells at these stages (green ovals in C ). DOI: http://dx.doi.org/10.7554/eLife.00924.005
    Tbr2 Alexa488, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam tbr2 blocking peptide
    Loss of basal progenitors in NfiB −/− brains during late coticogenesis. Immunohistochemistry and confocal microscopy of coronal brain sections were used to compare the number and distribution of <t>TBR2-expressing</t> cells in NfiB +/+ and NfiB
    Tbr2 Blocking Peptide, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti tbr2 antibody
    The distribution of Pax6-, pVim- and <t>Tbr2-positive</t> cells in the cerebral cortex of developing TD ferrets. GFP and FGF8 were expressed in the ferret cerebral cortex at E33 using in utero electroporation, and the brain was prepared at P6. Coronal sections were stained with Hoechst 33342 (blue) plus either anti-Pax6 antibody, anti-phosphorylated vimentin (pVim) antibody or anti-Tbr2 antibody (red). ( a – c ) The cerebral cortex containing the transfected GFP-positive area (green) is shown. Note that Pax6-positive cells (arrows), pVim-positive cells and Tbr2-positive cells were markedly increased in TD ferrets. ( d ) Pax6-positive cells in the OSVZ. Confocal images in the white boxes in ( a ) are shown. ( e ) Pax6-positive cells in the CP. Confocal images in the white boxes in ( a ) are shown. Note that Pax6-positive cells were markedly increased both in the OSVZ and in the CP. ( f – h ) The cerebral cortex was divided into 10 regions along the radial axis from the ventricular surface (1) to the pial surface (10). The numbers of Pax6-, pVim- and Tbr2-positive cells in each region were counted and were divided by the numbers of Hoechst 33342-positive cells in the same region. The percentages of positive cells are shown. Bars represent mean ± SD. *p
    Anti Tbr2 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    R&D Systems tbr2
    Neural progenitor subtypes in the developing tree shrew neocortex. (A–F) Triple-immunofluorescence for phospho-vimentin (pVim, white), Pax6 (red), and <t>Tbr2</t> (green) and DAPI staining (blue) on 30 μm-cryosections of E37 tree shrew neocortex. The merge images show combined immunofluorescence for pVim, Pax6, and Tbr2. Open arrowheads, cell bodies; solid arrowheads, pVim+ processes. Scale bars, 20 μm. VZ, ventricular zone; SVZ, subventricular zone. (G–I) Quantification of the distinct progenitor cell types at M-phase, identified by their location of mitosis and morphology at M-phase, in the E37 tree shrew neocortex, expressed as the percentage of total pVim+ cells. Data are the sum of two brains. The total number of pVim+ mitoses quantified was 642. (J–M) Quantification of the distinct progenitor cell types at M-phase, identified by pVim staining, that are Pax6+/Tbr2– (red), Pax6+/Tbr2+ (red/green), Pax6–/Tbr2+ (green), or Pax6–/Tbr2– (white) in the E37 tree shrew neocortex. Data are the sum of two brains. The total number of pVim+ mitoses analyzed was as in (G–I) . (G–M) AP, apical progenitor; BP, basal progenitor; bIP, basal intermediate progenitor; bRG, basal radial glia, bTG; basal tangential glia; -P, process.
    Tbr2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher tbr2
    Raldh2cKO does not affect cortical NPCs. (A-D′) Immunolabellings on brain sections from E14.5 control and Raldh2cKO animals for Pax6 (A-B′) or <t>Tbr2</t> (C-D′) at rostral (A-D) and more caudal levels (A′-D′) used for quantification. (G-J′) Immunolabellings on brain sections from E16.5 control and Raldh2cKO animals for Pax6 (G-H′) or Tbr2 (I-J′) at rostral (G-J) and medial levels (G′-J′) used for quantification. (E) Quantification of Pax6-positive cells at E14.5; rostrally: 477.13±13.10 for control and 456±10.48 for Raldh2cKO; caudally: 412.73±6.14 for control and 408.2±3.49 for Raldh2cKO. (F) Quantification of Tbr2-positive cells at E14.5; rostrally: 398.66±9.23 for control and 390.47±11.37 for Raldh2cKO; caudally: 339.47±9.89 for control and 320.13±12.49 for Raldh2cKO. (K) Quantification of Pax6-positive cells at E16.5; rostrally: 374.06±21.71 for control and 357.26±22.71 for Raldh2cKO; caudally: 319±16.42 for control and 306.99±15.28 for Raldh2cKO. (L) Quantification of Tbr2-positive cells at E16.5; rostrally: 353.2±20.93 for control and 341.2±20.23 for Raldh2cKO; caudally: 298.46±16.96 for control and 276.66±13.27 for Raldh2cKO. Data presented as mean±s.e.m.; n =5 brains; ns, not significant by two-tailed Student's t -test. Scale bars: 50 μm.
    Tbr2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA tbr2
    Radmis expression during embryonic CNS development. ( A – C ) E10.5 neural tube of the metencephalon immunostained for radmis ( A , green ) and counterstained with propidium iodide (PI) ( B , red ), showing uniformly distributed radmis in neuroepithelial cells throughout the neural tube. Note the significantly lower immunoreactivity of radmis in the connective tissue outside the neural tube. ( D – F ) Higher magnification view of the ventricular region of an E10.5 neural tube double-stained for radmis ( D , green ) and nestin ( E , red ). Radmis is expressed in the mitotic spindles ( arrowheads ) and nestin-positive radial fibers ( arrows ) of neuroepithelial cells. ( G – J ) E12.5 forebrain immunostained for radmis ( green ) and PI stained ( red ). ( H ) Cerebral neocortex at E12.5. ( I ) Magnified view of the ventricular surface of (H) showing accumulation of radmis in the mitotic spindles ( arrowhead , prometaphase; arrows , metaphase; double arrowhead , anaphase) of dividing NSPCs located at the ventricular surface, and their radial fibers ( double arrows ). ( J ) A dividing NSC at metaphase showing robust distribution of radmis in the mitotic spindle and its extending radial fiber ( double arrows ). ( K – L ) E15.5 neocortex immunostained for radmis ( green ) and <t>Tbr2</t> ( red ). ( L , L ’) Higher magnification of the boxed area in K. Arrows depicted the radmis immunoreactivity ( L ) in mitotic spindles of Tbr2 ( L ’)-positive dividing cells within the SVZ. Chromosome staining ( blue ) indicated that these two dividing cells were in anaphase. Scale bars: A – C , 250 μm; D – E , 20 μm; G , 200 μm; H , 31 μm; I , 12 μm; J , 3 μm; K , 250 μm; L , 5 μm. lv , lateral ventricle; mge , medial ganglionic eminence; lge , lateral ganglionic eminence.
    Tbr2, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Mutant Mouse Resource & Research Center tbr2
    Deregulation of progenitor pools was revealed by cell sorting in RP58 mutant cortices. ( a ) FACS graph showing separation of E14.5 cortical cells depending on their expression of CD133 (Prominin1) and <t>EGFP,</t> the latter driven from the <t>tbr2</t> regulatory region in tbr2: EGFP mice. The subpopulations of cells include the following: Group-A: CD133 + /EGFP − -containing RGCs in the VZ; Group-B: double positives, containing INPs in the SVZ; and Group-C: CD133 − /EGFP + , containing differentiating/differentiated cortical neurons. ( b ) Characterization of the three cell populations described in panel a by expression of key molecular markers of RGCs ( blbp , glast , pax6 ), INPs ( tbr2 ) and neurons ( tbr1 , MAP2 ). ( c ) Quantification of the pool size of A and B cells in control and RP58 KO cortices. A small increase in A cells and a slight decrease in B cells were observed in the mutant cortices at E14.5. The asterisks denote significant changes ( P
    Tbr2, supplied by Mutant Mouse Resource & Research Center, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Novus Biologicals tbr2
    Unfolded protein response pathway in hCS exposed to low oxygen. ( a ) Dendrogram for the Weighted gene co-expression network analysis (WGCNA), which identified genes with similar expression profiles and grouped them into modules (represented by colors). ( b ) Eigengene expression profiles (‘average’ expression profile of all module genes) for the top WGCNA modules associated with exposure of hCS to low O 2 for 48 hours. The blue module (upper) contains genes strongly enriched for annotation in biological pathways related to the hypoxia response (HIF-1α transcription factor network), while the turquoise module (lower) is enriched for genes involved in the unfolded protein response (UPR) pathway. ( c ) Quantification of the density of <t>TBR2</t> + cells in hCS after 48 h exposure to
    Tbr2, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam rabbit anti tbr2 mab
    In vitro-prepared CP-like neurons changed subtype marker expression upon exposure to microglia. a Immunostaining for d4Venus (Gadd45g; green), <t>Tbr2</t> (red), MAP2 (cyan), and DAPI (blue) in E14 Gadd45g-d4Venus Tg mouse cerebral walls. The broken lines indicate apical and basal surfaces. Scale bar, 50 µm. b The protocol for the in vitro preparation of CP-like neurons, which were obtained from Gadd45g-d4Venus + cells isolated from the E14 Gadd45g-d4Venus cortex by FACS, after culture for 2 days. c A time-lapse series showing the reduction in Gadd45g-dVenus expression during culture. Scale bar, 20 µm. d CX3CR1-GFP + microglia harvested from E15 CX3CR1-GFP pallial walls by FACS were added to the CP-like neuronal cultures. e Representative immunostaining (shown in pseudo color) for βIII-tubulin (cyan), Tbr1/Satb2/Cux1 (red), and Ctip2 (green). The arrowhead indicates microglia. Scale bar, 30 µm. f The proportion of the βIII-tubulin + cells that expressed Tbr1, Ctip2, Satb2, or Cux1 in cocultures with or without microglia (two-sided Mann–Whitney U test; n = 10 independent experiments; P = 0.0021 for Tbr1, P = 7.6 × 10 −5 for Ctip2, P = 7.6 × 10 −5 for Satb2, P = 1.5 × 10 −4 for Cux1). g An experimental schematic of the coculture of youngest (0 day, Gadd45g-d4Venus + ) neurons and CX3CR1-GFP + microglia. h Representative immunostaining (shown in pseudo color) for βIII-tubulin (cyan), Tbr1/Satb2/Cux1 (red), and Ctip2 (green). The arrowhead indicates microglia. Scale bar, 30 µm. i The proportion of βIII-tubulin + cells that expressed Tbr1, Ctip2, Satb2, or Cux1 in cocultures with or without microglia (two-sided Mann–Whitney U test; n = 10 independent cultures; P = 0.529 for Tbr1, P = 0.289 for Ctip2, P = 0.631 for Satb2, P = 0.912 for Cux1). Data are presented as the mean values ± S.D. Source data are provided as a Source Data File .
    Rabbit Anti Tbr2 Mab, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    cell signaling technology inc tbr2
    ABHD4 is not required for proliferation, differentiation and lamination in the developing neocortex. a,b, Phospho-histone H3 (PHH3)-immunostaining visualizes cell proliferation during the M phase of the cell cycle at the ventricular border. c, Quantification of PHH3-positive cell density (two-sided Student’s unpaired t-test, P = 0.688; n = 26 sections from n = 3 animals per wild-type (+/+) mice, n = 30 sections from n = 3 animals per Abhd4- knockout (-/-) mice). d,e, High density of T-box brain protein 2 <t>(TBR2)-positive</t> intermediate progenitor cells in the subventricular zone. f, Quantification of TBR2-positive cell density (two-sided Student’s unpaired t-test, P = 0.194; n = 18 sections from n = 3 animals per wild-type (+/+) mice, n = 15 sections from n = 3 animals per Abhd4- knockout (-/-) mice). g,h , Distribution of TBR1-positive postmitotic neurons in the cortical plate. i, Quantification of TBR1-positive cell density (two-sided Mann-Whitney U test, P = 0.074; n = 17 sections from n = 3 animals per wild-type (+/+) mice, n = 16 sections from n = 3 animals per Abhd4- knockout (-/-) mice). MZ: marginal zone, CP: cortical plate, IZ: intermediate zone, SVZ: subventricular zone, VZ: ventricular zone. Graphs show raw data and mean ± 2 x standard error ( c,f ), or raw data and median ± interquartile range ( i ).
    Tbr2, supplied by cell signaling technology inc, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of cold challenge or CL 316,243 treatment for 1 week or 4 weeks on the TBR-2 and Ki-67 positive cells in the dentate gyrus. Note that the TBR-2 and Ki-67 positive cells are abundant in the Cold1W and Cold4W groups when compared with that in the CON Cold1W and CON Cold 4W groups, respectively (A,C,E,G) . However, the CON CL1W, CL1W, CON CL4W, and CL4W groups show similar populations of TBR-2 and Ki-67 positive cells in the dentate gyrus (A,C,E,G) . GCL, granule cell layer; MoL, molecular layer; PoL, polymorphic layer. Quantitative analysis of TBR-2 and Ki-67 positive cells per section in the CON Cold1W, Cold1W, CON Cold4W, Cold4W, CON CL1W, CL1W, CON CL4W, and CL4W groups (B,D,F,H) ( n = 5 per group); ∗ indicates a significant difference when compared with the CON group ( p

    Journal: Frontiers in Neuroscience

    Article Title: Adult Hippocampal Neurogenesis Can Be Enhanced by Cold Challenge Independently From Beigeing Effects

    doi: 10.3389/fnins.2019.00092

    Figure Lengend Snippet: Effects of cold challenge or CL 316,243 treatment for 1 week or 4 weeks on the TBR-2 and Ki-67 positive cells in the dentate gyrus. Note that the TBR-2 and Ki-67 positive cells are abundant in the Cold1W and Cold4W groups when compared with that in the CON Cold1W and CON Cold 4W groups, respectively (A,C,E,G) . However, the CON CL1W, CL1W, CON CL4W, and CL4W groups show similar populations of TBR-2 and Ki-67 positive cells in the dentate gyrus (A,C,E,G) . GCL, granule cell layer; MoL, molecular layer; PoL, polymorphic layer. Quantitative analysis of TBR-2 and Ki-67 positive cells per section in the CON Cold1W, Cold1W, CON Cold4W, Cold4W, CON CL1W, CL1W, CON CL4W, and CL4W groups (B,D,F,H) ( n = 5 per group); ∗ indicates a significant difference when compared with the CON group ( p

    Article Snippet: For TBR-2 immunofluorescence, sections were incubated with a rabbit anti-TBR-2 antibody (1:200; Millipore, Temecula, CA, United States) for 2 h at 25°C, followed by overnight incubation at 4°C.

    Techniques:

    Impaired adult hippocampal neurogenesis in Sox21 −/− mice. A , Experimental scheme for long-term BrdU labeling analysis used to detect slowly dividing cells and neurons. B , C , To analyze type 1 cells, marker+/BrdU+/fiber+ cells (arrows) in wild-type and Sox21 −/− mice were quantified 4 weeks after the last BrdU administration. The graphs show the numbers of FABP7+/BrdU+/fiber+ cells ( B ) and GFAP+/BrdU+/fiber+ cells ( C ) in the DG. D , E , To analyze newly generated mature and immature neurons, the numbers of BrdU+/NeuN+ ( D ) and BrdU+/DCX+ ( E ) cells were quantified 4 weeks after the last BrdU administration. F , Experimental scheme for the short-term BrdU labeling analysis used to detect rapidly dividing cells. G , H , The number of BrdU+/Ascl1+ type 2a cells ( G , arrows) increased, while the number of BrdU+/Tbr2+ type 2b cells ( H , arrows) decreased, in Sox21 −/− mice. The top planes are through the x–z axes, and the right planes are through the y-z axes. Data represent the mean ± SE. ** p

    Journal: The Journal of Neuroscience

    Article Title: Sox21 Promotes Hippocampal Adult Neurogenesis via the Transcriptional Repression of the Hes5 Gene

    doi: 10.1523/JNEUROSCI.5803-11.2012

    Figure Lengend Snippet: Impaired adult hippocampal neurogenesis in Sox21 −/− mice. A , Experimental scheme for long-term BrdU labeling analysis used to detect slowly dividing cells and neurons. B , C , To analyze type 1 cells, marker+/BrdU+/fiber+ cells (arrows) in wild-type and Sox21 −/− mice were quantified 4 weeks after the last BrdU administration. The graphs show the numbers of FABP7+/BrdU+/fiber+ cells ( B ) and GFAP+/BrdU+/fiber+ cells ( C ) in the DG. D , E , To analyze newly generated mature and immature neurons, the numbers of BrdU+/NeuN+ ( D ) and BrdU+/DCX+ ( E ) cells were quantified 4 weeks after the last BrdU administration. F , Experimental scheme for the short-term BrdU labeling analysis used to detect rapidly dividing cells. G , H , The number of BrdU+/Ascl1+ type 2a cells ( G , arrows) increased, while the number of BrdU+/Tbr2+ type 2b cells ( H , arrows) decreased, in Sox21 −/− mice. The top planes are through the x–z axes, and the right planes are through the y-z axes. Data represent the mean ± SE. ** p

    Article Snippet: The following primary antibodies were used in this study: anti-βIII-tubulin (rabbit IgG; Covance, PRB-435P; 1:500), anti-glial fibrillary acidic protein (GFAP; rabbit IgG; DAKO, Z0334; 1:500), anti-GFAP (mouse IgG; Sigma-Aldrich, G3893; 1:200), anti-fatty acid binding protein 7 (FABP7; rabbit IgG; Millipore, AB9558; 1:250), anti-Sox21 (goat IgG; R & D Systems, AF3538; 1:100), anti-Sox2 (rabbit IgG; Millipore, AB5603; 1:200), anti-Sox1/(2)/3 (rabbit IgG; gift from Dr. H. Kondoh, Osaka University, Osaka, Japan; 1:5000) , anti-Nestin (mouse IgG; BD Biosciences/PharMingen, 556309; 1:200), anti-doublecortin (DCX; goat IgG; Santa Cruz Biotechnology, sc-8066; 1:100), anti- DCX (rabbit IgG; Abcam, ab18723; 1:100), anti-polysialylated neuronal cell adhesion molecule (PSA-NCAM; mouse IgMκ; gift from Dr. T. Seki, Tokyo Medical University, Tokyo, Japan; 1:200), anti-NeuN (mouse IgG; Millipore, MAB377; 1:100), anti-S100β (mouse IgG; Sigma-Aldrich, S2532; 1:200), anti-glutathione S -transferase π (mouse IgG; BD Biosciences/PharMingen, 610719; 1:200), anti-Brn2 (Santa Cruz Biotechnology, sc-6029; 1:100), anti-Tbr1 (rabbit IgG; gift from Dr. R. F. Hevner, University of Washington, Seattle, WA; 1:10,000), anti-Ascl1 (mouse IgG; BD Biosciences/PharMingen, 556604; 1:100), anti-Tbr2 (rabbit IgG; Abcam, ab23345; 1:200), anti-Ki67 (rabbit IgG; Abcam, ab15580; 1:3000), anti-GFP (rabbit IgG, Medical and Biological Laboratories, 598, 1:500; goat IgG, Rockland, 600-101-215, 1:500), anti-BrdU (rat IgG; Abcam, ab6326; 1:100), anti-lacZ (rabbit IgG, Cappel, 55976, 1:500; goat IgG, Cappel, 56028, 1:500), and normal IgG (rabbit IgG, Millipore, DAM1421465; goat IgG, Santa Cruz Biotechnology, sc-2028).

    Techniques: Mouse Assay, Labeling, Marker, Generated

    Immunohistological images of a Tbr2 eGFP pseudo-lineage tracing study. Tbr2 expressing IPs were marked by EGFP through crossing the Fln cKO-NPC mice with the Tg(Eomes-EGFP) line. Due to the perdurance of EGFP, many newly generated daughter neurons from Tbr2+ IPs also express EGFP. In a Tg( Eomes -EGFP) :: Flna cKO Emx1Cre+ ;Flnb -/- brain at P0, PH was identified by periventricular NeuN or MAP2 immunosignals (red); the co-presence of EGFP+ cells (green) in PH is shown, demonstrating that neurons in PH were generated directly by mislocalized Tbr2+ IPs. Nuclei DNA was stained with Hoechst 33,342 and shown in blue in all fluorescence images. DOI: http://dx.doi.org/10.7554/eLife.17823.009

    Journal: eLife

    Article Title: Upregulation of neurovascular communication through filamin abrogation promotes ectopic periventricular neurogenesis

    doi: 10.7554/eLife.17823

    Figure Lengend Snippet: Immunohistological images of a Tbr2 eGFP pseudo-lineage tracing study. Tbr2 expressing IPs were marked by EGFP through crossing the Fln cKO-NPC mice with the Tg(Eomes-EGFP) line. Due to the perdurance of EGFP, many newly generated daughter neurons from Tbr2+ IPs also express EGFP. In a Tg( Eomes -EGFP) :: Flna cKO Emx1Cre+ ;Flnb -/- brain at P0, PH was identified by periventricular NeuN or MAP2 immunosignals (red); the co-presence of EGFP+ cells (green) in PH is shown, demonstrating that neurons in PH were generated directly by mislocalized Tbr2+ IPs. Nuclei DNA was stained with Hoechst 33,342 and shown in blue in all fluorescence images. DOI: http://dx.doi.org/10.7554/eLife.17823.009

    Article Snippet: 5) The statement that Tbr2-GFP analysis demonstrates that PH neurons are daughters of Tbr2+ IPs is not correct.

    Techniques: Expressing, Mouse Assay, Generated, Staining, Fluorescence

    Effective Flna protein abrogation from NPCs lineages and lack of cortical structural impairment in all Flna-Flnb compound mutant brains at E12.5. ( A ) Filamin A (green) and Pax6 (red) double immunohistological stained brain sections from a control and a Flna flox Emx1Cre+ ;Flnb -/- embryo at E12.5, indicating the absence of filamin A in NPC-derived neural tissue. Note the high filamin A on blood vessels. ( B ) Double immunostaining of cortical sections from a Flna flox Emx1Cre+ ;Flnb -/- and control embryos show normal number and distribution of both S-phase and M-phase NPCs at E12.5. S phase was identified by BrdU pulse labeling and immunostaining (red); M phase was identified by immunostaining with an antibody to phospho-Histone 3(PH3, green). ( C ) Double immunostaining with antibodies to panCadherin (green) and neuron marker Tuj1(red) showing normal RG adhesion, cortical histogenesis and neurogenesis in both Flna k/y ;Flnb +/- and Flna flox Emx1Cre+ ;Flnb -/- embryos at E12.5. ( D ) Double immunohistological staining with AJ marker β-Catenin (red) and IP marker Tbr2 (green), showing undisturbed NPC adhesion as well as normal production and localization of IPs in both Flna k/y ;Flnb +/- and Flna flox Emx1Cre+ ;Flnb -/- embryos at E12.5. No Flna k/y ;Flnb -/- embryos were recovered at E12.5 due to early embryonic lethality. Nuclei DNA was stained with Hoechst 33,342 and shown in blue in all fluorescence images. DOI: http://dx.doi.org/10.7554/eLife.17823.007

    Journal: eLife

    Article Title: Upregulation of neurovascular communication through filamin abrogation promotes ectopic periventricular neurogenesis

    doi: 10.7554/eLife.17823

    Figure Lengend Snippet: Effective Flna protein abrogation from NPCs lineages and lack of cortical structural impairment in all Flna-Flnb compound mutant brains at E12.5. ( A ) Filamin A (green) and Pax6 (red) double immunohistological stained brain sections from a control and a Flna flox Emx1Cre+ ;Flnb -/- embryo at E12.5, indicating the absence of filamin A in NPC-derived neural tissue. Note the high filamin A on blood vessels. ( B ) Double immunostaining of cortical sections from a Flna flox Emx1Cre+ ;Flnb -/- and control embryos show normal number and distribution of both S-phase and M-phase NPCs at E12.5. S phase was identified by BrdU pulse labeling and immunostaining (red); M phase was identified by immunostaining with an antibody to phospho-Histone 3(PH3, green). ( C ) Double immunostaining with antibodies to panCadherin (green) and neuron marker Tuj1(red) showing normal RG adhesion, cortical histogenesis and neurogenesis in both Flna k/y ;Flnb +/- and Flna flox Emx1Cre+ ;Flnb -/- embryos at E12.5. ( D ) Double immunohistological staining with AJ marker β-Catenin (red) and IP marker Tbr2 (green), showing undisturbed NPC adhesion as well as normal production and localization of IPs in both Flna k/y ;Flnb +/- and Flna flox Emx1Cre+ ;Flnb -/- embryos at E12.5. No Flna k/y ;Flnb -/- embryos were recovered at E12.5 due to early embryonic lethality. Nuclei DNA was stained with Hoechst 33,342 and shown in blue in all fluorescence images. DOI: http://dx.doi.org/10.7554/eLife.17823.007

    Article Snippet: 5) The statement that Tbr2-GFP analysis demonstrates that PH neurons are daughters of Tbr2+ IPs is not correct.

    Techniques: Mutagenesis, Staining, Derivative Assay, Double Immunostaining, Labeling, Immunostaining, Marker, Fluorescence

    Ectopic division of IPs and presence of periventricular neurons without neocortical impairment or apoptosis in Fln cKO-NPC and Flna null ( Flna Ky ) mutants. ( A–C ) Double immunohistological analysis with antibodies to N-Cadherin (green) and BrdU (red) on cortical sections of Flna flox Emx1Cre+ ;Flnb -/- (PH) and their control (Ctrl) littermates at E13.5, E15.5, and E16.5. Embryos were fixed 30 min after BrdU pulse labeling. Arrows indicate mislocalized S-phase nuclei adjacent to the ventricular surface where N-Cadherin signals were reduced. High magnification images of E16.5 also show the concurrence of ectopic S-phase nuclei with local loss of RG polarity and adhesion in the mutant. ( D ) Double immunohistological analysis with antibodies to Tbr1 (green) and Tuj1 (red) to reveal newborn neurons in a Flna flox Emx1Cre+ ;Flnb -/- (PH) and its control (Ctrl) littermate at E16.5. Note the normal cortical distribution of Tbr1 and Tuj1 despite their periventricular appearance (indicated by arrows). ( E ) Double immunohistological analysis with antibodies to Pax6 (normally expressed by RGs, red) and M-phase marker (PH3, green) on control (Ctrl) and Flna flox Emx1Cre+ ;Flnb -/- (PH) cortical sections at E14.5, showing reduced Pax6+ cells and their mitosis in affected regions. ( F , G ) Double immunohistological analysis with antibodies to Pax6 (red) and BrdU (green), showing the association of PH formation with loss of Pax6+ RGs (arrows) at E14.5 and E15.5, respectively. Embryos were fixed 30 min after BrdU pulse labeling. ( H ) Double immunohistological analysis of Flna flox Emx1Cre+ ;Flnb -/- (PH) embryos at E14.5 with antibodies to Ki67 (green) and Tuj1 or Pax6 (red, as indicated on the image), showing a well-organized cortical plate despite ectopic periventricular neurogenesis. The figure also demonstrates the presence of Ki67+ cells in regions where RGs lost Pax6 expression. ( I , J ) Double immunohistological analysis with antibodies to Tbr2 (green) and βCatenin (red), or Tbr2(green) and BrdU (red) on cortical sections of a wild type and a Flna null (Ky) embryo at E15.5, showing the phenocopy of Fln cKO-NPC and Flna null mutants. Embryos were fixed 30 min after BrdU pulse labeling. Arrows indicate ectopic Tbr2 IPs that incorporate BrdU at the ventricular surface and their correlation with loss of AJs in the mutant RGs. ( K ) Double immunohistological analysis with antibodies to cleaved caspase 3 (CC3, red) and Na-K ATPase (green) on a cortical section of a Flna flox Emx1Cre+ ;Flnb -/- embryo at E14.5, showing the absence of apoptosis. Nuclei DNA was stained with Hoechst 33,342 and shown in blue in all fluorescence images. DOI: http://dx.doi.org/10.7554/eLife.17823.008

    Journal: eLife

    Article Title: Upregulation of neurovascular communication through filamin abrogation promotes ectopic periventricular neurogenesis

    doi: 10.7554/eLife.17823

    Figure Lengend Snippet: Ectopic division of IPs and presence of periventricular neurons without neocortical impairment or apoptosis in Fln cKO-NPC and Flna null ( Flna Ky ) mutants. ( A–C ) Double immunohistological analysis with antibodies to N-Cadherin (green) and BrdU (red) on cortical sections of Flna flox Emx1Cre+ ;Flnb -/- (PH) and their control (Ctrl) littermates at E13.5, E15.5, and E16.5. Embryos were fixed 30 min after BrdU pulse labeling. Arrows indicate mislocalized S-phase nuclei adjacent to the ventricular surface where N-Cadherin signals were reduced. High magnification images of E16.5 also show the concurrence of ectopic S-phase nuclei with local loss of RG polarity and adhesion in the mutant. ( D ) Double immunohistological analysis with antibodies to Tbr1 (green) and Tuj1 (red) to reveal newborn neurons in a Flna flox Emx1Cre+ ;Flnb -/- (PH) and its control (Ctrl) littermate at E16.5. Note the normal cortical distribution of Tbr1 and Tuj1 despite their periventricular appearance (indicated by arrows). ( E ) Double immunohistological analysis with antibodies to Pax6 (normally expressed by RGs, red) and M-phase marker (PH3, green) on control (Ctrl) and Flna flox Emx1Cre+ ;Flnb -/- (PH) cortical sections at E14.5, showing reduced Pax6+ cells and their mitosis in affected regions. ( F , G ) Double immunohistological analysis with antibodies to Pax6 (red) and BrdU (green), showing the association of PH formation with loss of Pax6+ RGs (arrows) at E14.5 and E15.5, respectively. Embryos were fixed 30 min after BrdU pulse labeling. ( H ) Double immunohistological analysis of Flna flox Emx1Cre+ ;Flnb -/- (PH) embryos at E14.5 with antibodies to Ki67 (green) and Tuj1 or Pax6 (red, as indicated on the image), showing a well-organized cortical plate despite ectopic periventricular neurogenesis. The figure also demonstrates the presence of Ki67+ cells in regions where RGs lost Pax6 expression. ( I , J ) Double immunohistological analysis with antibodies to Tbr2 (green) and βCatenin (red), or Tbr2(green) and BrdU (red) on cortical sections of a wild type and a Flna null (Ky) embryo at E15.5, showing the phenocopy of Fln cKO-NPC and Flna null mutants. Embryos were fixed 30 min after BrdU pulse labeling. Arrows indicate ectopic Tbr2 IPs that incorporate BrdU at the ventricular surface and their correlation with loss of AJs in the mutant RGs. ( K ) Double immunohistological analysis with antibodies to cleaved caspase 3 (CC3, red) and Na-K ATPase (green) on a cortical section of a Flna flox Emx1Cre+ ;Flnb -/- embryo at E14.5, showing the absence of apoptosis. Nuclei DNA was stained with Hoechst 33,342 and shown in blue in all fluorescence images. DOI: http://dx.doi.org/10.7554/eLife.17823.008

    Article Snippet: 5) The statement that Tbr2-GFP analysis demonstrates that PH neurons are daughters of Tbr2+ IPs is not correct.

    Techniques: Labeling, Mutagenesis, Marker, Expressing, Staining, Fluorescence

    Aberrant cerebral angiogenesis with PH-genesis was observed in Fln cKO-NPC but not Fln cKO-EC mutants. ( A ) VE-Cadherin (red) and Tbr2 (green) double immunostained cortical sections from a control and its Fln cKO-NPC (PH) littermate at E13.5. Note the spatial correlation of enlarged ectopic blood vessels with mislocalized IPs at the ventricular surface. ( B ) Double immunostained cortical sections from a Fln cKO-NPC (PH) and its control littermate at E14.5 with biotinylated IB4 (green) and Tbr2, or BrdU (red) antibodies, showing the intermingling of aberrant blood vessels with apicalized IPs. Embryos were pulse labeled by BrdU. The dotted lines mark the ventricular surface. ( C ) Triple immunostained cortical sections from a control and its Fln cKO-NPC (PH) littermate at E15.5 with IB4 (blue) and antibodies to beta-Catenin (red) and Tbr2 (green). ( D ) A double immunostained cortical section from an E15.5 Fln cKO-NPC (PH) embryo with antibodies to PDGFβ (red, indicates vascular pericytes) and DCX (green, indicates new neurons). ( E ) Brain images of a control and a Flna flox Nes8Cre+ ;Flnb -/- (PH) littermate at postnatal day 12, showing the bilateral hemorrhage of the PH brain. ( F ) Double stained E14.5 cortical sections with biotinylated IB4 (red) and the anti-Flna antibody (green), showing effective abrogation of Flna protein in ECs or neural tissues by Tie2 Cre and Emx1 Cre , respectively. ( G ) Quantification of vascular density in both Fln cKO-NPC (Nes8 Cre)and Fln cKO-EC cortices. Data are presented as Mean + SD from a minimum of 3 independent litters. *p

    Journal: eLife

    Article Title: Upregulation of neurovascular communication through filamin abrogation promotes ectopic periventricular neurogenesis

    doi: 10.7554/eLife.17823

    Figure Lengend Snippet: Aberrant cerebral angiogenesis with PH-genesis was observed in Fln cKO-NPC but not Fln cKO-EC mutants. ( A ) VE-Cadherin (red) and Tbr2 (green) double immunostained cortical sections from a control and its Fln cKO-NPC (PH) littermate at E13.5. Note the spatial correlation of enlarged ectopic blood vessels with mislocalized IPs at the ventricular surface. ( B ) Double immunostained cortical sections from a Fln cKO-NPC (PH) and its control littermate at E14.5 with biotinylated IB4 (green) and Tbr2, or BrdU (red) antibodies, showing the intermingling of aberrant blood vessels with apicalized IPs. Embryos were pulse labeled by BrdU. The dotted lines mark the ventricular surface. ( C ) Triple immunostained cortical sections from a control and its Fln cKO-NPC (PH) littermate at E15.5 with IB4 (blue) and antibodies to beta-Catenin (red) and Tbr2 (green). ( D ) A double immunostained cortical section from an E15.5 Fln cKO-NPC (PH) embryo with antibodies to PDGFβ (red, indicates vascular pericytes) and DCX (green, indicates new neurons). ( E ) Brain images of a control and a Flna flox Nes8Cre+ ;Flnb -/- (PH) littermate at postnatal day 12, showing the bilateral hemorrhage of the PH brain. ( F ) Double stained E14.5 cortical sections with biotinylated IB4 (red) and the anti-Flna antibody (green), showing effective abrogation of Flna protein in ECs or neural tissues by Tie2 Cre and Emx1 Cre , respectively. ( G ) Quantification of vascular density in both Fln cKO-NPC (Nes8 Cre)and Fln cKO-EC cortices. Data are presented as Mean + SD from a minimum of 3 independent litters. *p

    Article Snippet: 5) The statement that Tbr2-GFP analysis demonstrates that PH neurons are daughters of Tbr2+ IPs is not correct.

    Techniques: Labeling, Staining

    Incorrectly positioned Tbr2 + cells in the tish −/− neocortex are mitotic progenitor cells. Confocal images were taken of coronal sections of embryonic neocortex at E17. In E, J , and O , compressed stacks are presented. Vertical and horizontal

    Journal: Neuroscience

    Article Title: Disturbances in the positioning, proliferation, and apoptosis of neural progenitors contribute to subcortical band heterotopia formation

    doi: 10.1016/j.neuroscience.2010.12.003

    Figure Lengend Snippet: Incorrectly positioned Tbr2 + cells in the tish −/− neocortex are mitotic progenitor cells. Confocal images were taken of coronal sections of embryonic neocortex at E17. In E, J , and O , compressed stacks are presented. Vertical and horizontal

    Article Snippet: The following primary antibodies were used: anti-Pax6 (rabbit, 1:300, Covance), anti-Tbr2 (rabbit, 1:300, Millipore), anti-phosphorylated vimentin 4A4 clone (mouse, 1:500, Assay Designs), anti-BrdU (mouse, 1:10, BD Biosciences), anti-BrdU (rat, 1:40, Abcam), anti-βIII tubulin (mouse, 1:100, Sigma-Aldritch), anti-cleaved caspase 3 (rabbit, 1:200, Cell Signaling Technology), anti-aPKC-λ (mouse, 1:100, BD Transduction Labs), anti-PAR3 (rabbit, 1:100, Millipore), anti-GFP (rabbit, 1:500, Invitrogen), anti-vimentin (mouse, 1:40, Sigma).

    Techniques:

    Pax6 + and Tbr2+ cells are incorrectly positioned in the tish +/− and tish −/− neocortex. Light microscopic images were taken of coronal sections of embryonic neocortex during timepoints corresponding to early, middle, at late cortical

    Journal: Neuroscience

    Article Title: Disturbances in the positioning, proliferation, and apoptosis of neural progenitors contribute to subcortical band heterotopia formation

    doi: 10.1016/j.neuroscience.2010.12.003

    Figure Lengend Snippet: Pax6 + and Tbr2+ cells are incorrectly positioned in the tish +/− and tish −/− neocortex. Light microscopic images were taken of coronal sections of embryonic neocortex during timepoints corresponding to early, middle, at late cortical

    Article Snippet: The following primary antibodies were used: anti-Pax6 (rabbit, 1:300, Covance), anti-Tbr2 (rabbit, 1:300, Millipore), anti-phosphorylated vimentin 4A4 clone (mouse, 1:500, Assay Designs), anti-BrdU (mouse, 1:10, BD Biosciences), anti-BrdU (rat, 1:40, Abcam), anti-βIII tubulin (mouse, 1:100, Sigma-Aldritch), anti-cleaved caspase 3 (rabbit, 1:200, Cell Signaling Technology), anti-aPKC-λ (mouse, 1:100, BD Transduction Labs), anti-PAR3 (rabbit, 1:100, Millipore), anti-GFP (rabbit, 1:500, Invitrogen), anti-vimentin (mouse, 1:40, Sigma).

    Techniques:

    Treatment with poly(I ⋅ C) during gestation negatively regulates the transition of PAX6 + radial glia (RG) cells to TBR2 + PAX6 + intermediate progenitor cells (IPC) in a TLR3-dependent manner. WT C57BL/6 or TLR3 −/− C57BL/6 pregnant mice were treated with either PBS or poly(I ⋅ C) for three consecutive days (GD15 to GD17) and injected with BrdU on GD16. Cerebral cortical cells were isolated from embryos on GD18. Total viable cells were analyzed for the expression of TBR2 and PAX6 by flow cytometry. (a) Representative histograms showing TBR2 + PAX6 + positive IPC in WT and TLR3 −/− cerebral cortical cells isolated from embryos ( n = 3) obtained from mice treated with PBS or poly(I ⋅ C). The numbers above the brackets indicate the percentages of TBR2 + PAX6 + IPC. (b) Representative histograms showing PAX6 + RG in WT and TLR3 −/− cerebral cortical cells isolated from embryos obtained from PBS- and poly(I ⋅ C)-treated mice. The numbers above the brackets indicate the percentages of PAX6 + RG cells. Gating for the PAX6- or TBR2-positive cells was based on the staining observed with the isotype control. (c) Representative histograms showing the mean fluorescence intensity of TBR2 and PAX6 expression. The mean fluorescence intensity (MFI) is shown as a percentage of the maximum expression. MFI values for the different groups are indicated as follows: PBS-treated group (red line), poly(I ⋅ C)-treated group (blue line), and isotype control group (gray line). The MFI for PBS-treated mice was 1,158.7 ± 57.4, and the MFI for poly(I ⋅ C)-treated mice was 809.3 ± 28.4. (d) Representative histograms showing the MFI of PAX6 expression. Experiments were performed with cerebral cortical cells isolated from embryos ( n = 3); data from one representative cortex are shown. MFI values for the different groups are indicated as follows: PBS-treated group (red line), poly(I ⋅ C)-treated group (blue line), and isotype control group (gray line). The MFI for PBS-treated mice was 13,784.0 ± 1,527.3, and the MFI for poly(I ⋅ C)-treated mice was 16,842.3 ± 799.1. (e) Percentages of TBR2 + PAX6 + IPC and PAX6 + RG in WT and TLR3 −/− cerebral cortical cells isolated from embryos obtained from PBS- and poly(I ⋅ C)-treated mice. Results are expressed as percentages of total viable cells. The values for PBS-treated mice (white bars) and poly(I ⋅ C)-treated mice (hatched bars) are shown. Values that are significantly different ( P

    Journal: mBio

    Article Title: Induction of Toll-Like Receptor 3-Mediated Immunity during Gestation Inhibits Cortical Neurogenesis and Causes Behavioral Disturbances

    doi: 10.1128/mBio.00176-10

    Figure Lengend Snippet: Treatment with poly(I ⋅ C) during gestation negatively regulates the transition of PAX6 + radial glia (RG) cells to TBR2 + PAX6 + intermediate progenitor cells (IPC) in a TLR3-dependent manner. WT C57BL/6 or TLR3 −/− C57BL/6 pregnant mice were treated with either PBS or poly(I ⋅ C) for three consecutive days (GD15 to GD17) and injected with BrdU on GD16. Cerebral cortical cells were isolated from embryos on GD18. Total viable cells were analyzed for the expression of TBR2 and PAX6 by flow cytometry. (a) Representative histograms showing TBR2 + PAX6 + positive IPC in WT and TLR3 −/− cerebral cortical cells isolated from embryos ( n = 3) obtained from mice treated with PBS or poly(I ⋅ C). The numbers above the brackets indicate the percentages of TBR2 + PAX6 + IPC. (b) Representative histograms showing PAX6 + RG in WT and TLR3 −/− cerebral cortical cells isolated from embryos obtained from PBS- and poly(I ⋅ C)-treated mice. The numbers above the brackets indicate the percentages of PAX6 + RG cells. Gating for the PAX6- or TBR2-positive cells was based on the staining observed with the isotype control. (c) Representative histograms showing the mean fluorescence intensity of TBR2 and PAX6 expression. The mean fluorescence intensity (MFI) is shown as a percentage of the maximum expression. MFI values for the different groups are indicated as follows: PBS-treated group (red line), poly(I ⋅ C)-treated group (blue line), and isotype control group (gray line). The MFI for PBS-treated mice was 1,158.7 ± 57.4, and the MFI for poly(I ⋅ C)-treated mice was 809.3 ± 28.4. (d) Representative histograms showing the MFI of PAX6 expression. Experiments were performed with cerebral cortical cells isolated from embryos ( n = 3); data from one representative cortex are shown. MFI values for the different groups are indicated as follows: PBS-treated group (red line), poly(I ⋅ C)-treated group (blue line), and isotype control group (gray line). The MFI for PBS-treated mice was 13,784.0 ± 1,527.3, and the MFI for poly(I ⋅ C)-treated mice was 16,842.3 ± 799.1. (e) Percentages of TBR2 + PAX6 + IPC and PAX6 + RG in WT and TLR3 −/− cerebral cortical cells isolated from embryos obtained from PBS- and poly(I ⋅ C)-treated mice. Results are expressed as percentages of total viable cells. The values for PBS-treated mice (white bars) and poly(I ⋅ C)-treated mice (hatched bars) are shown. Values that are significantly different ( P

    Article Snippet: The primary antibodies used included BrdU conjugated to fluorescein isothiocyanate (BrdU-FITC) (clone BU1/75 ICR1 with cross-reactivity to CldU; Abcam, Cambridge, MA), BrdU (clone B44 with cross-reactivity to IdU; Becton Dickinson, San Jose, CA), PAX6 (developed by Atsushi Kawakami and obtained from Development Studies Hybridoma Bank, University of Iowa, Iowa City, IA), TBR2 and TBR2 conjugated to phycoerythrin (TBR2-PE) (clone 21Mgs8; eBioscience, San Diego, CA), TLR3 (Sigma-Aldrich, St. Louis, MO), TLR3 (eBioscience, San Diego, CA), and CUX1 (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Mouse Assay, Injection, Isolation, Expressing, Flow Cytometry, Cytometry, Staining, Fluorescence

    Schematic showing fetal telencephalon development and Imp1 expression. ( A ) Anatomy during fetal telencephalon development (D: dorsal, V:ventral, L:left, and R:right). At E10.5, two signalling centers, floor plate (FP) and roof plate (RP) exist in the ventral and dorsal telencephalon. At E12.5 and later, dorsal telencephalon can be divided into dorsomedial telencephalon (DMT, light blue) and dorsolateral telencephalon (DLT, pink). Choloid plexus (CP) and cortical hem (CH) constitute the dorsal end of telencephalon. ( B ) Regions where Imp1 was expressed are indicated in green. At E10.5, Imp1 was expressed throughout the forebrain, except in the floor plate and roof plate. At E12.5, Imp1 was expressed typically in undifferentiated cells in the dorsal telencephalon but not in ventral telencephalon or in basal neurons. At E14.5 and later, Imp1 expression was gradually confined to the apical region of the dorsomedial telencephalon. ( C and D ) Schematics show a close up of the boxed regions in panel B to illustrate the cellular layers during cortical development and the cells that expressed Imp1 in those layers. At E10.5, undifferentiated Pax6+ neural stem cells (blue ovals in D ) dominate all layers of the developing telencephalon and all cells express Imp1 (green in C ). At E12.5, neural stem cells start to differentiate into Tbr2+ intermediate neural progenitors (yellow ovals in D ) and Tuj1+ neurons (red ovals in D ) that form the preplate (PP in panel C ). At E14.5 and E18.5, newly formed neurons constitute the marginal zone (MZ), cortical plate (CP) and subplate (SP) on the basal side of the developing cortex. The intermediate zone (IZ) is a cell sparse region that lies between the SP and VZ. Imp1 is expressed in undifferentiated cells at these stages (green ovals in C ). DOI: http://dx.doi.org/10.7554/eLife.00924.005

    Journal: eLife

    Article Title: A network of heterochronic genes including Imp1 regulates temporal changes in stem cell properties

    doi: 10.7554/eLife.00924

    Figure Lengend Snippet: Schematic showing fetal telencephalon development and Imp1 expression. ( A ) Anatomy during fetal telencephalon development (D: dorsal, V:ventral, L:left, and R:right). At E10.5, two signalling centers, floor plate (FP) and roof plate (RP) exist in the ventral and dorsal telencephalon. At E12.5 and later, dorsal telencephalon can be divided into dorsomedial telencephalon (DMT, light blue) and dorsolateral telencephalon (DLT, pink). Choloid plexus (CP) and cortical hem (CH) constitute the dorsal end of telencephalon. ( B ) Regions where Imp1 was expressed are indicated in green. At E10.5, Imp1 was expressed throughout the forebrain, except in the floor plate and roof plate. At E12.5, Imp1 was expressed typically in undifferentiated cells in the dorsal telencephalon but not in ventral telencephalon or in basal neurons. At E14.5 and later, Imp1 expression was gradually confined to the apical region of the dorsomedial telencephalon. ( C and D ) Schematics show a close up of the boxed regions in panel B to illustrate the cellular layers during cortical development and the cells that expressed Imp1 in those layers. At E10.5, undifferentiated Pax6+ neural stem cells (blue ovals in D ) dominate all layers of the developing telencephalon and all cells express Imp1 (green in C ). At E12.5, neural stem cells start to differentiate into Tbr2+ intermediate neural progenitors (yellow ovals in D ) and Tuj1+ neurons (red ovals in D ) that form the preplate (PP in panel C ). At E14.5 and E18.5, newly formed neurons constitute the marginal zone (MZ), cortical plate (CP) and subplate (SP) on the basal side of the developing cortex. The intermediate zone (IZ) is a cell sparse region that lies between the SP and VZ. Imp1 is expressed in undifferentiated cells at these stages (green ovals in C ). DOI: http://dx.doi.org/10.7554/eLife.00924.005

    Article Snippet: Cells infected with the Imp1 shRNA were significantly less likely to express Pax6 or Ki67 and significantly more likely to express Tbr2 or Tuj1 ( ; ).

    Techniques: Expressing

    BrdU+/Ki67+ cells were Pax6+ while BrdU+/Ki67- cells were Tbr2+ or Tuj1+ in E14.5 dorsomedial telencephalon. Brains were dissected from E14.5 wild-type or Imp1 β-geo/β-geo mice after a 24 hr pulse of BrdU. Coronal sections were immunostained with antibodies against BrdU, Ki67, and Pax6 ( A ), Tbr2 ( B ), or Tuj1( C ). Most BrdU+Ki67+ cells (yellow cells in Ki67/BrdU images) expressed Pax6 and most BrdU+Ki67- cells (red cells in Ki67/BrdU images) were either Tbr2+ or Tuj1+. DOI: http://dx.doi.org/10.7554/eLife.00924.015

    Journal: eLife

    Article Title: A network of heterochronic genes including Imp1 regulates temporal changes in stem cell properties

    doi: 10.7554/eLife.00924

    Figure Lengend Snippet: BrdU+/Ki67+ cells were Pax6+ while BrdU+/Ki67- cells were Tbr2+ or Tuj1+ in E14.5 dorsomedial telencephalon. Brains were dissected from E14.5 wild-type or Imp1 β-geo/β-geo mice after a 24 hr pulse of BrdU. Coronal sections were immunostained with antibodies against BrdU, Ki67, and Pax6 ( A ), Tbr2 ( B ), or Tuj1( C ). Most BrdU+Ki67+ cells (yellow cells in Ki67/BrdU images) expressed Pax6 and most BrdU+Ki67- cells (red cells in Ki67/BrdU images) were either Tbr2+ or Tuj1+. DOI: http://dx.doi.org/10.7554/eLife.00924.015

    Article Snippet: Cells infected with the Imp1 shRNA were significantly less likely to express Pax6 or Ki67 and significantly more likely to express Tbr2 or Tuj1 ( ; ).

    Techniques: Mouse Assay

    Loss of basal progenitors in NfiB −/− brains during late coticogenesis. Immunohistochemistry and confocal microscopy of coronal brain sections were used to compare the number and distribution of TBR2-expressing cells in NfiB +/+ and NfiB

    Journal: The Journal of comparative neurology

    Article Title: Nuclear Factor One B regulates neural stem cell differentiation and axonal projection of corticofugal neurons

    doi: 10.1002/cne.23373

    Figure Lengend Snippet: Loss of basal progenitors in NfiB −/− brains during late coticogenesis. Immunohistochemistry and confocal microscopy of coronal brain sections were used to compare the number and distribution of TBR2-expressing cells in NfiB +/+ and NfiB

    Article Snippet: However, no band was detected when using the TBR2 antibody on human mesoderm whole cell lysate incubated with TBR2 blocking peptide (Abcam, #ab25698), or on EL4 cells expressing an empty vector or V5 tagged vector, thus validating antibody specificity (manufacturer’s results).

    Techniques: Immunohistochemistry, Confocal Microscopy, Expressing

    The distribution of Pax6-, pVim- and Tbr2-positive cells in the cerebral cortex of developing TD ferrets. GFP and FGF8 were expressed in the ferret cerebral cortex at E33 using in utero electroporation, and the brain was prepared at P6. Coronal sections were stained with Hoechst 33342 (blue) plus either anti-Pax6 antibody, anti-phosphorylated vimentin (pVim) antibody or anti-Tbr2 antibody (red). ( a – c ) The cerebral cortex containing the transfected GFP-positive area (green) is shown. Note that Pax6-positive cells (arrows), pVim-positive cells and Tbr2-positive cells were markedly increased in TD ferrets. ( d ) Pax6-positive cells in the OSVZ. Confocal images in the white boxes in ( a ) are shown. ( e ) Pax6-positive cells in the CP. Confocal images in the white boxes in ( a ) are shown. Note that Pax6-positive cells were markedly increased both in the OSVZ and in the CP. ( f – h ) The cerebral cortex was divided into 10 regions along the radial axis from the ventricular surface (1) to the pial surface (10). The numbers of Pax6-, pVim- and Tbr2-positive cells in each region were counted and were divided by the numbers of Hoechst 33342-positive cells in the same region. The percentages of positive cells are shown. Bars represent mean ± SD. *p

    Journal: Scientific Reports

    Article Title: Pathophysiological analyses of cortical malformation using gyrencephalic mammals

    doi: 10.1038/srep15370

    Figure Lengend Snippet: The distribution of Pax6-, pVim- and Tbr2-positive cells in the cerebral cortex of developing TD ferrets. GFP and FGF8 were expressed in the ferret cerebral cortex at E33 using in utero electroporation, and the brain was prepared at P6. Coronal sections were stained with Hoechst 33342 (blue) plus either anti-Pax6 antibody, anti-phosphorylated vimentin (pVim) antibody or anti-Tbr2 antibody (red). ( a – c ) The cerebral cortex containing the transfected GFP-positive area (green) is shown. Note that Pax6-positive cells (arrows), pVim-positive cells and Tbr2-positive cells were markedly increased in TD ferrets. ( d ) Pax6-positive cells in the OSVZ. Confocal images in the white boxes in ( a ) are shown. ( e ) Pax6-positive cells in the CP. Confocal images in the white boxes in ( a ) are shown. Note that Pax6-positive cells were markedly increased both in the OSVZ and in the CP. ( f – h ) The cerebral cortex was divided into 10 regions along the radial axis from the ventricular surface (1) to the pial surface (10). The numbers of Pax6-, pVim- and Tbr2-positive cells in each region were counted and were divided by the numbers of Hoechst 33342-positive cells in the same region. The percentages of positive cells are shown. Bars represent mean ± SD. *p

    Article Snippet: The sections were permeabilized with 0.5% Triton X-100/PBS and incubated overnight with primary antibodies, which included anti-Ki-67 antibody (Leica), anti-phospho-histone H3 (pH3) antibody (Upstate Biotechnology), anti-pH3 antibody (Millipore), biotin-conjugated anti-pH3 antibody (Millipore), anti-Olig2 antibody (Immuno-Biological Laboratories, Japan), anti-Pax6 antibody (Abcam), anti-Pax6 antibody (Covance), anti-phosphorylated vimentin (pVim) antibody (Medical & Biological Laboratories, Japan), anti-Tbr2 antibody (Abcam), anti-NeuN antibody (Chemicon), anti-GFAP antibody (Sigma-Aldrich), anti-APC antibody (Calbiochem), anti-Brn2 antibody (Santa Cruz Biotechnology), anti-Ctip2 antibody (Abcam) and anti-FOXP2 antibody (Abcam).

    Techniques: In Utero, Electroporation, Staining, Transfection

    Neural progenitor subtypes in the developing tree shrew neocortex. (A–F) Triple-immunofluorescence for phospho-vimentin (pVim, white), Pax6 (red), and Tbr2 (green) and DAPI staining (blue) on 30 μm-cryosections of E37 tree shrew neocortex. The merge images show combined immunofluorescence for pVim, Pax6, and Tbr2. Open arrowheads, cell bodies; solid arrowheads, pVim+ processes. Scale bars, 20 μm. VZ, ventricular zone; SVZ, subventricular zone. (G–I) Quantification of the distinct progenitor cell types at M-phase, identified by their location of mitosis and morphology at M-phase, in the E37 tree shrew neocortex, expressed as the percentage of total pVim+ cells. Data are the sum of two brains. The total number of pVim+ mitoses quantified was 642. (J–M) Quantification of the distinct progenitor cell types at M-phase, identified by pVim staining, that are Pax6+/Tbr2– (red), Pax6+/Tbr2+ (red/green), Pax6–/Tbr2+ (green), or Pax6–/Tbr2– (white) in the E37 tree shrew neocortex. Data are the sum of two brains. The total number of pVim+ mitoses analyzed was as in (G–I) . (G–M) AP, apical progenitor; BP, basal progenitor; bIP, basal intermediate progenitor; bRG, basal radial glia, bTG; basal tangential glia; -P, process.

    Journal: Frontiers in Neuroanatomy

    Article Title: Neural Progenitors in the Developing Neocortex of the Northern Tree Shrew (Tupaia belangeri) Show a Closer Relationship to Gyrencephalic Primates Than to Lissencephalic Rodents

    doi: 10.3389/fnana.2018.00029

    Figure Lengend Snippet: Neural progenitor subtypes in the developing tree shrew neocortex. (A–F) Triple-immunofluorescence for phospho-vimentin (pVim, white), Pax6 (red), and Tbr2 (green) and DAPI staining (blue) on 30 μm-cryosections of E37 tree shrew neocortex. The merge images show combined immunofluorescence for pVim, Pax6, and Tbr2. Open arrowheads, cell bodies; solid arrowheads, pVim+ processes. Scale bars, 20 μm. VZ, ventricular zone; SVZ, subventricular zone. (G–I) Quantification of the distinct progenitor cell types at M-phase, identified by their location of mitosis and morphology at M-phase, in the E37 tree shrew neocortex, expressed as the percentage of total pVim+ cells. Data are the sum of two brains. The total number of pVim+ mitoses quantified was 642. (J–M) Quantification of the distinct progenitor cell types at M-phase, identified by pVim staining, that are Pax6+/Tbr2– (red), Pax6+/Tbr2+ (red/green), Pax6–/Tbr2+ (green), or Pax6–/Tbr2– (white) in the E37 tree shrew neocortex. Data are the sum of two brains. The total number of pVim+ mitoses analyzed was as in (G–I) . (G–M) AP, apical progenitor; BP, basal progenitor; bIP, basal intermediate progenitor; bRG, basal radial glia, bTG; basal tangential glia; -P, process.

    Article Snippet: The oSVZ corresponded to the diffuse outer band of Tbr2+ cells, which was localized to the Tau1-striated zone (Supplementary Figure ).

    Techniques: Immunofluorescence, Staining

    Pax6 and Tbr2 expression in the germinal zones of the developing tree shrew neocortex. (A–D) Double-Immunofluorescence for Pax6 (red) and Tbr2 (green) and DAPI staining (blue) on 30 μm-cryosections of E32–P1 tree shrew neocortex. The top margin of the image corresponds to the transition zone SVZ/intermediate zone. Scale bars, 50 μm. VZ, ventricular zone; SVZ, subventricular zone; iSVZ, inner SVZ; oSVZ, outer SVZ. (E,F) Quantification of Pax6+/Tbr2–, Pax6+/Tbr2+ and Pax6–/Tbr2+ NPCs in the VZ (E) and SVZ (F) of the E32–P1 tree shrew neocortex, expressed as number of cells per 200 μm ventricular surface. Color legend is shown in (F) . (G) Quantification of Pax6+ (Pax6+/Tbr2–, Pax6+/Tbr2+) NPCs (G) and Tbr2+ (Pax6–/Tbr2+, Pax6+/Tbr2+) NPCs (H) in the VZ and SVZ of the E32–P1 tree shrew neocortex, expressed as percentage of the sum of VZ and SVZ. Color legend is shown in (H) . (I,J) Quantification of Pax6+ (Pax6+/Tbr2–, Pax6+/Tbr2+) NPCs (I) and Tbr2+ (Pax6–/Tbr2+, Pax6+/Tbr2+) NPCs (J) in the iSVZ and oSVZ of the E32–P1 tree shrew neocortex, expressed as percentage of the sum of iSVZ and oSVZ. Color legend is shown in (J) . (E–J) Cortical wall corresponding to a total ventricular surface of 4.12–12.081 mm was analyzed. Data represent mean ± SD and are from two brains each.

    Journal: Frontiers in Neuroanatomy

    Article Title: Neural Progenitors in the Developing Neocortex of the Northern Tree Shrew (Tupaia belangeri) Show a Closer Relationship to Gyrencephalic Primates Than to Lissencephalic Rodents

    doi: 10.3389/fnana.2018.00029

    Figure Lengend Snippet: Pax6 and Tbr2 expression in the germinal zones of the developing tree shrew neocortex. (A–D) Double-Immunofluorescence for Pax6 (red) and Tbr2 (green) and DAPI staining (blue) on 30 μm-cryosections of E32–P1 tree shrew neocortex. The top margin of the image corresponds to the transition zone SVZ/intermediate zone. Scale bars, 50 μm. VZ, ventricular zone; SVZ, subventricular zone; iSVZ, inner SVZ; oSVZ, outer SVZ. (E,F) Quantification of Pax6+/Tbr2–, Pax6+/Tbr2+ and Pax6–/Tbr2+ NPCs in the VZ (E) and SVZ (F) of the E32–P1 tree shrew neocortex, expressed as number of cells per 200 μm ventricular surface. Color legend is shown in (F) . (G) Quantification of Pax6+ (Pax6+/Tbr2–, Pax6+/Tbr2+) NPCs (G) and Tbr2+ (Pax6–/Tbr2+, Pax6+/Tbr2+) NPCs (H) in the VZ and SVZ of the E32–P1 tree shrew neocortex, expressed as percentage of the sum of VZ and SVZ. Color legend is shown in (H) . (I,J) Quantification of Pax6+ (Pax6+/Tbr2–, Pax6+/Tbr2+) NPCs (I) and Tbr2+ (Pax6–/Tbr2+, Pax6+/Tbr2+) NPCs (J) in the iSVZ and oSVZ of the E32–P1 tree shrew neocortex, expressed as percentage of the sum of iSVZ and oSVZ. Color legend is shown in (J) . (E–J) Cortical wall corresponding to a total ventricular surface of 4.12–12.081 mm was analyzed. Data represent mean ± SD and are from two brains each.

    Article Snippet: The oSVZ corresponded to the diffuse outer band of Tbr2+ cells, which was localized to the Tau1-striated zone (Supplementary Figure ).

    Techniques: Expressing, Immunofluorescence, Staining

    Raldh2cKO does not affect cortical NPCs. (A-D′) Immunolabellings on brain sections from E14.5 control and Raldh2cKO animals for Pax6 (A-B′) or Tbr2 (C-D′) at rostral (A-D) and more caudal levels (A′-D′) used for quantification. (G-J′) Immunolabellings on brain sections from E16.5 control and Raldh2cKO animals for Pax6 (G-H′) or Tbr2 (I-J′) at rostral (G-J) and medial levels (G′-J′) used for quantification. (E) Quantification of Pax6-positive cells at E14.5; rostrally: 477.13±13.10 for control and 456±10.48 for Raldh2cKO; caudally: 412.73±6.14 for control and 408.2±3.49 for Raldh2cKO. (F) Quantification of Tbr2-positive cells at E14.5; rostrally: 398.66±9.23 for control and 390.47±11.37 for Raldh2cKO; caudally: 339.47±9.89 for control and 320.13±12.49 for Raldh2cKO. (K) Quantification of Pax6-positive cells at E16.5; rostrally: 374.06±21.71 for control and 357.26±22.71 for Raldh2cKO; caudally: 319±16.42 for control and 306.99±15.28 for Raldh2cKO. (L) Quantification of Tbr2-positive cells at E16.5; rostrally: 353.2±20.93 for control and 341.2±20.23 for Raldh2cKO; caudally: 298.46±16.96 for control and 276.66±13.27 for Raldh2cKO. Data presented as mean±s.e.m.; n =5 brains; ns, not significant by two-tailed Student's t -test. Scale bars: 50 μm.

    Journal: Biology Open

    Article Title: Meningeal retinoic acid contributes to neocortical lamination and radial migration during mouse brain development

    doi: 10.1242/bio.021063

    Figure Lengend Snippet: Raldh2cKO does not affect cortical NPCs. (A-D′) Immunolabellings on brain sections from E14.5 control and Raldh2cKO animals for Pax6 (A-B′) or Tbr2 (C-D′) at rostral (A-D) and more caudal levels (A′-D′) used for quantification. (G-J′) Immunolabellings on brain sections from E16.5 control and Raldh2cKO animals for Pax6 (G-H′) or Tbr2 (I-J′) at rostral (G-J) and medial levels (G′-J′) used for quantification. (E) Quantification of Pax6-positive cells at E14.5; rostrally: 477.13±13.10 for control and 456±10.48 for Raldh2cKO; caudally: 412.73±6.14 for control and 408.2±3.49 for Raldh2cKO. (F) Quantification of Tbr2-positive cells at E14.5; rostrally: 398.66±9.23 for control and 390.47±11.37 for Raldh2cKO; caudally: 339.47±9.89 for control and 320.13±12.49 for Raldh2cKO. (K) Quantification of Pax6-positive cells at E16.5; rostrally: 374.06±21.71 for control and 357.26±22.71 for Raldh2cKO; caudally: 319±16.42 for control and 306.99±15.28 for Raldh2cKO. (L) Quantification of Tbr2-positive cells at E16.5; rostrally: 353.2±20.93 for control and 341.2±20.23 for Raldh2cKO; caudally: 298.46±16.96 for control and 276.66±13.27 for Raldh2cKO. Data presented as mean±s.e.m.; n =5 brains; ns, not significant by two-tailed Student's t -test. Scale bars: 50 μm.

    Article Snippet: After antigen unmasking in citrate buffer (0.01 M, pH 6) during 15 min in a microwave oven, slides were blocked with 5% donkey serum, 0.1% Triton X-100 in phosphate-buffered saline (PBS) and incubated overnight with the following primary antibodies: bromodeoxyuridine (BrdU) (1:500, AbD Serotec #OBT0030G), Ki67 (1:300, Novocastra #NCL-KI67P), phospho-histone H3 (1:500, Upstate #05-806); Pax6 (1:300, Covance #PRB-278P); Tbr2 (1:300, eBioscience #14-4875); βIII-tubulin/Tuj1 (1:200, Covance #MMS-435P-100); Tbr1 (1:100, Abcam #ab31940); Ctip2 (1:500, Abcam #ab18465); Cux1 (1:100, Santa Cruz Biotechnologies #sc13024); cleaved caspase-3 (1:200, R & D Systems #NB100-56113); Brn2 (1:1000 Santa Cruz Biotechnologies #sc6029X), RORβ (1:500, Santa Cruz Biotechnologies #sc21354); Calretinin (1:2000, Swant #7699/4); Raldh2 (1:75, Santa Cruz Biotechnologies #sc22591); nestin (1:100; Developmental Studies Hybridoma Bank #rat-401); laminin (1:500, Sigma #L9393).

    Techniques: Two Tailed Test

    Lack of RA impairs cell migration of early - born cortical neurons. Brain sections from E17.5 mice electroporated at E13.5 with a GFP reporter construct were analysed for GFP (C-D′,G-H′), and by immunolabelling for Tbr2 and Ctip2 (A-B′,E-F′). Single-color immunolabelling for the two markers help to define the upper layers (UL) and deeper layers (DL) of the cortical plate and the intermediate zone (IZ). (I,J) Histograms depict the percentage of GFP-positive cells per zone (UL, DL and IZ) of the rostral (I) and caudal (J) levels of developing cortex. (K-L′,O) Quantification of GFP-positive cells (green) expressing Tbr1 (red) in the cortical plate of control and Raldh2cKO animals. (M-N′,P) Quantification of GFP-positive cells (green) expressing Ctip2 (red) in the cortical plate of control and Raldh2cKO animals. The histograms (O,P) show the percentage of double-labelled cells over GFP+ cells. Data presented as mean±s.e.m.; n =5 brains; * P

    Journal: Biology Open

    Article Title: Meningeal retinoic acid contributes to neocortical lamination and radial migration during mouse brain development

    doi: 10.1242/bio.021063

    Figure Lengend Snippet: Lack of RA impairs cell migration of early - born cortical neurons. Brain sections from E17.5 mice electroporated at E13.5 with a GFP reporter construct were analysed for GFP (C-D′,G-H′), and by immunolabelling for Tbr2 and Ctip2 (A-B′,E-F′). Single-color immunolabelling for the two markers help to define the upper layers (UL) and deeper layers (DL) of the cortical plate and the intermediate zone (IZ). (I,J) Histograms depict the percentage of GFP-positive cells per zone (UL, DL and IZ) of the rostral (I) and caudal (J) levels of developing cortex. (K-L′,O) Quantification of GFP-positive cells (green) expressing Tbr1 (red) in the cortical plate of control and Raldh2cKO animals. (M-N′,P) Quantification of GFP-positive cells (green) expressing Ctip2 (red) in the cortical plate of control and Raldh2cKO animals. The histograms (O,P) show the percentage of double-labelled cells over GFP+ cells. Data presented as mean±s.e.m.; n =5 brains; * P

    Article Snippet: After antigen unmasking in citrate buffer (0.01 M, pH 6) during 15 min in a microwave oven, slides were blocked with 5% donkey serum, 0.1% Triton X-100 in phosphate-buffered saline (PBS) and incubated overnight with the following primary antibodies: bromodeoxyuridine (BrdU) (1:500, AbD Serotec #OBT0030G), Ki67 (1:300, Novocastra #NCL-KI67P), phospho-histone H3 (1:500, Upstate #05-806); Pax6 (1:300, Covance #PRB-278P); Tbr2 (1:300, eBioscience #14-4875); βIII-tubulin/Tuj1 (1:200, Covance #MMS-435P-100); Tbr1 (1:100, Abcam #ab31940); Ctip2 (1:500, Abcam #ab18465); Cux1 (1:100, Santa Cruz Biotechnologies #sc13024); cleaved caspase-3 (1:200, R & D Systems #NB100-56113); Brn2 (1:1000 Santa Cruz Biotechnologies #sc6029X), RORβ (1:500, Santa Cruz Biotechnologies #sc21354); Calretinin (1:2000, Swant #7699/4); Raldh2 (1:75, Santa Cruz Biotechnologies #sc22591); nestin (1:100; Developmental Studies Hybridoma Bank #rat-401); laminin (1:500, Sigma #L9393).

    Techniques: Migration, Mouse Assay, Construct, Expressing

    Aberrant morphology of newborn migrating neurons in Raldh2cKO brains. In vivo electroporation of the GFP reporter was performed on E14.5 cortices, which were harvested at E17.5. (A,B,E,F) Immunolabellings on electroporated control and Raldh2cKO animals for Tbr2 and Ctip2 (red) to define the upper layers (UL), the deeper layers (DL) and the intermediate zone (IZ). (C,D,G,H) GFP signal in electroporated control and Raldh2cKO animals. (C′,D′,G′,H′) Higher magnification views of the areas boxed in C,D,G,H representing the counted areas. More electroporated cells in the IZ exhibit a multipolar morphology in Raldh2cKO mutants (white arrows). (I,J) Histograms showing the distribution of bipolar and multipolar cells in the IZ at rostral (I) and caudal (J) levels. Quantifications. Bipolar cells rostrally: 87.64±1.41% for control and 71.14±1.5% for Raldh2cKO; caudally : 84.17±1.15% for control and 69.70±2.43% for Raldh2cKO. Multipolar cells rostrally: 12.35±1.4% for control and 28.85±1.5% for Raldh2cKO; caudally: 15.82±1.15% for control and 30.29±2.43% for Raldh2cKO. Data presented as mean±s.e.m.; n =5 brains; ** P

    Journal: Biology Open

    Article Title: Meningeal retinoic acid contributes to neocortical lamination and radial migration during mouse brain development

    doi: 10.1242/bio.021063

    Figure Lengend Snippet: Aberrant morphology of newborn migrating neurons in Raldh2cKO brains. In vivo electroporation of the GFP reporter was performed on E14.5 cortices, which were harvested at E17.5. (A,B,E,F) Immunolabellings on electroporated control and Raldh2cKO animals for Tbr2 and Ctip2 (red) to define the upper layers (UL), the deeper layers (DL) and the intermediate zone (IZ). (C,D,G,H) GFP signal in electroporated control and Raldh2cKO animals. (C′,D′,G′,H′) Higher magnification views of the areas boxed in C,D,G,H representing the counted areas. More electroporated cells in the IZ exhibit a multipolar morphology in Raldh2cKO mutants (white arrows). (I,J) Histograms showing the distribution of bipolar and multipolar cells in the IZ at rostral (I) and caudal (J) levels. Quantifications. Bipolar cells rostrally: 87.64±1.41% for control and 71.14±1.5% for Raldh2cKO; caudally : 84.17±1.15% for control and 69.70±2.43% for Raldh2cKO. Multipolar cells rostrally: 12.35±1.4% for control and 28.85±1.5% for Raldh2cKO; caudally: 15.82±1.15% for control and 30.29±2.43% for Raldh2cKO. Data presented as mean±s.e.m.; n =5 brains; ** P

    Article Snippet: After antigen unmasking in citrate buffer (0.01 M, pH 6) during 15 min in a microwave oven, slides were blocked with 5% donkey serum, 0.1% Triton X-100 in phosphate-buffered saline (PBS) and incubated overnight with the following primary antibodies: bromodeoxyuridine (BrdU) (1:500, AbD Serotec #OBT0030G), Ki67 (1:300, Novocastra #NCL-KI67P), phospho-histone H3 (1:500, Upstate #05-806); Pax6 (1:300, Covance #PRB-278P); Tbr2 (1:300, eBioscience #14-4875); βIII-tubulin/Tuj1 (1:200, Covance #MMS-435P-100); Tbr1 (1:100, Abcam #ab31940); Ctip2 (1:500, Abcam #ab18465); Cux1 (1:100, Santa Cruz Biotechnologies #sc13024); cleaved caspase-3 (1:200, R & D Systems #NB100-56113); Brn2 (1:1000 Santa Cruz Biotechnologies #sc6029X), RORβ (1:500, Santa Cruz Biotechnologies #sc21354); Calretinin (1:2000, Swant #7699/4); Raldh2 (1:75, Santa Cruz Biotechnologies #sc22591); nestin (1:100; Developmental Studies Hybridoma Bank #rat-401); laminin (1:500, Sigma #L9393).

    Techniques: In Vivo, Electroporation

    Lack of RA perturbs cell migration of late - born cortical neurons. Brain sections from E17.5 embryos electroporated at E14.5 with the GFP reporter were analysed by immunolabelling for Tbr2 and Ctip2 (A-B′,E-F′) and for GFP-expressing cells (C-D′,G-H′), at rostral (A-D′) and caudal levels (E-H′) of the cortex. (I,J) Histograms show the percentage of GFP-positive cells in control and Raldh2cKO animals per upper layer (UL), deeper layer (DL) and intermediate zone (IZ) of the developing cortex. Data presented as mean±s.e.m.; n =5 brains; ** P

    Journal: Biology Open

    Article Title: Meningeal retinoic acid contributes to neocortical lamination and radial migration during mouse brain development

    doi: 10.1242/bio.021063

    Figure Lengend Snippet: Lack of RA perturbs cell migration of late - born cortical neurons. Brain sections from E17.5 embryos electroporated at E14.5 with the GFP reporter were analysed by immunolabelling for Tbr2 and Ctip2 (A-B′,E-F′) and for GFP-expressing cells (C-D′,G-H′), at rostral (A-D′) and caudal levels (E-H′) of the cortex. (I,J) Histograms show the percentage of GFP-positive cells in control and Raldh2cKO animals per upper layer (UL), deeper layer (DL) and intermediate zone (IZ) of the developing cortex. Data presented as mean±s.e.m.; n =5 brains; ** P

    Article Snippet: After antigen unmasking in citrate buffer (0.01 M, pH 6) during 15 min in a microwave oven, slides were blocked with 5% donkey serum, 0.1% Triton X-100 in phosphate-buffered saline (PBS) and incubated overnight with the following primary antibodies: bromodeoxyuridine (BrdU) (1:500, AbD Serotec #OBT0030G), Ki67 (1:300, Novocastra #NCL-KI67P), phospho-histone H3 (1:500, Upstate #05-806); Pax6 (1:300, Covance #PRB-278P); Tbr2 (1:300, eBioscience #14-4875); βIII-tubulin/Tuj1 (1:200, Covance #MMS-435P-100); Tbr1 (1:100, Abcam #ab31940); Ctip2 (1:500, Abcam #ab18465); Cux1 (1:100, Santa Cruz Biotechnologies #sc13024); cleaved caspase-3 (1:200, R & D Systems #NB100-56113); Brn2 (1:1000 Santa Cruz Biotechnologies #sc6029X), RORβ (1:500, Santa Cruz Biotechnologies #sc21354); Calretinin (1:2000, Swant #7699/4); Raldh2 (1:75, Santa Cruz Biotechnologies #sc22591); nestin (1:100; Developmental Studies Hybridoma Bank #rat-401); laminin (1:500, Sigma #L9393).

    Techniques: Migration, Expressing

    Radmis expression during embryonic CNS development. ( A – C ) E10.5 neural tube of the metencephalon immunostained for radmis ( A , green ) and counterstained with propidium iodide (PI) ( B , red ), showing uniformly distributed radmis in neuroepithelial cells throughout the neural tube. Note the significantly lower immunoreactivity of radmis in the connective tissue outside the neural tube. ( D – F ) Higher magnification view of the ventricular region of an E10.5 neural tube double-stained for radmis ( D , green ) and nestin ( E , red ). Radmis is expressed in the mitotic spindles ( arrowheads ) and nestin-positive radial fibers ( arrows ) of neuroepithelial cells. ( G – J ) E12.5 forebrain immunostained for radmis ( green ) and PI stained ( red ). ( H ) Cerebral neocortex at E12.5. ( I ) Magnified view of the ventricular surface of (H) showing accumulation of radmis in the mitotic spindles ( arrowhead , prometaphase; arrows , metaphase; double arrowhead , anaphase) of dividing NSPCs located at the ventricular surface, and their radial fibers ( double arrows ). ( J ) A dividing NSC at metaphase showing robust distribution of radmis in the mitotic spindle and its extending radial fiber ( double arrows ). ( K – L ) E15.5 neocortex immunostained for radmis ( green ) and Tbr2 ( red ). ( L , L ’) Higher magnification of the boxed area in K. Arrows depicted the radmis immunoreactivity ( L ) in mitotic spindles of Tbr2 ( L ’)-positive dividing cells within the SVZ. Chromosome staining ( blue ) indicated that these two dividing cells were in anaphase. Scale bars: A – C , 250 μm; D – E , 20 μm; G , 200 μm; H , 31 μm; I , 12 μm; J , 3 μm; K , 250 μm; L , 5 μm. lv , lateral ventricle; mge , medial ganglionic eminence; lge , lateral ganglionic eminence.

    Journal: PLoS ONE

    Article Title: Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

    doi: 10.1371/journal.pone.0079895

    Figure Lengend Snippet: Radmis expression during embryonic CNS development. ( A – C ) E10.5 neural tube of the metencephalon immunostained for radmis ( A , green ) and counterstained with propidium iodide (PI) ( B , red ), showing uniformly distributed radmis in neuroepithelial cells throughout the neural tube. Note the significantly lower immunoreactivity of radmis in the connective tissue outside the neural tube. ( D – F ) Higher magnification view of the ventricular region of an E10.5 neural tube double-stained for radmis ( D , green ) and nestin ( E , red ). Radmis is expressed in the mitotic spindles ( arrowheads ) and nestin-positive radial fibers ( arrows ) of neuroepithelial cells. ( G – J ) E12.5 forebrain immunostained for radmis ( green ) and PI stained ( red ). ( H ) Cerebral neocortex at E12.5. ( I ) Magnified view of the ventricular surface of (H) showing accumulation of radmis in the mitotic spindles ( arrowhead , prometaphase; arrows , metaphase; double arrowhead , anaphase) of dividing NSPCs located at the ventricular surface, and their radial fibers ( double arrows ). ( J ) A dividing NSC at metaphase showing robust distribution of radmis in the mitotic spindle and its extending radial fiber ( double arrows ). ( K – L ) E15.5 neocortex immunostained for radmis ( green ) and Tbr2 ( red ). ( L , L ’) Higher magnification of the boxed area in K. Arrows depicted the radmis immunoreactivity ( L ) in mitotic spindles of Tbr2 ( L ’)-positive dividing cells within the SVZ. Chromosome staining ( blue ) indicated that these two dividing cells were in anaphase. Scale bars: A – C , 250 μm; D – E , 20 μm; G , 200 μm; H , 31 μm; I , 12 μm; J , 3 μm; K , 250 μm; L , 5 μm. lv , lateral ventricle; mge , medial ganglionic eminence; lge , lateral ganglionic eminence.

    Article Snippet: EGFP (rabbit polyclonal, Life Technologies, 1:1000), EGFP (chick IgY, AvesLab, Oregon, 1:1000), Tbr2 (chick IgY, Merck Millipore, 1:1000), Pax6 (rabbit polyclonal, MBL, Nagoya, Japan, 1:1000), anti-bromodeoxyuridine (BrdU) (sheep polyclonal, Abcam, 1:1500), Ki-67 (rabbit monoclonal clone SP6, Lab Vision, CA, 1:1000).

    Techniques: Expressing, Staining

    Sustained expression of radmis results in decreased cell proliferation and increased cell-cycle exit of NSPCs. Control EGFP or EGFP-radmis-mut constructs were electroporated into E14.5 embryonic brains that were analyzed after 72 h (E17.5). ( A , B ) Confocal images of E17.5 brain sections electroporated with control EGFP ( A ) or EGFP-radmis-mut ( B ). Sections through the neocortex were stained for EGFP ( green ), Tbr2 ( red ), phosphoH3 ( light blue ), and DNA ( blue ). Hatched areas in panels B and B’ show the cell distributions of cells electroporated with EGFP-radmis-mut. By E17.5, the Tbr2-positive SVZ layer became much thinner upon electroporation of EGFP-radmis-mut (compare with the thickness of the neighboring Tbr2-positive SVZ outside the hatched areas in panel B”). ( C , D ) Magnified views of VZ/SVZ regions in A and B, respectively. Virtually none of the cells overexpressing EGFP-radmis-mut are Tbr2-positive. ( E ) Confocal image of electroporated brain immunostained for EGFP-radmis-mut ( green ), Pax6 ( red ), and phosphoH3 ( blue ). Compared with the control, the majority of cells overexpressing EGFP-radmis-mut no longer express Tbr2, and most of them translocated to the IZL. Arrow indicates persistent expression of radmis-mut in Pax6-positive radial glial cells in the VZ. ( F – I ) Prolonged expression of radmis-mut results in reduced cell proliferation, and increased cell-cycle exit and cell death. E14.5 brains were electroporated with control EGFP ( F ) or EGFP-radmis-mut ( G ) constructs. BrdU was consecutively administered into dams after 24 h post-electroporation (E15.5–E17.5), followed by analysis of brains at 72 h (E17.5). Left column shows merged confocal images of sections stained for EGFP ( green ), BrdU ( red ), and Ki67 ( blue ). Middle and right columns show BrdU-, and Ki67-labeled cells, respectively. Arrowheads indicate EGFP, BrdU, and Ki67 triple-positive cells, and arrows indicate EGFP- and BrdU-positive, but Ki67-negative, cells. ( H , I ) Apoptotic cells ( arrows ) were detected by TUNEL staining in control ( H ) or radmis-mut electroporated brains ( I , I ’). EGFP ( green ) and TUNEL-positive cells ( red ). ( J ) Quantification of 72 h BrdU labeling index. Data are presented as mean ± SEM (%). Student’s t -tests (n = 5 embryos); *p

    Journal: PLoS ONE

    Article Title: Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

    doi: 10.1371/journal.pone.0079895

    Figure Lengend Snippet: Sustained expression of radmis results in decreased cell proliferation and increased cell-cycle exit of NSPCs. Control EGFP or EGFP-radmis-mut constructs were electroporated into E14.5 embryonic brains that were analyzed after 72 h (E17.5). ( A , B ) Confocal images of E17.5 brain sections electroporated with control EGFP ( A ) or EGFP-radmis-mut ( B ). Sections through the neocortex were stained for EGFP ( green ), Tbr2 ( red ), phosphoH3 ( light blue ), and DNA ( blue ). Hatched areas in panels B and B’ show the cell distributions of cells electroporated with EGFP-radmis-mut. By E17.5, the Tbr2-positive SVZ layer became much thinner upon electroporation of EGFP-radmis-mut (compare with the thickness of the neighboring Tbr2-positive SVZ outside the hatched areas in panel B”). ( C , D ) Magnified views of VZ/SVZ regions in A and B, respectively. Virtually none of the cells overexpressing EGFP-radmis-mut are Tbr2-positive. ( E ) Confocal image of electroporated brain immunostained for EGFP-radmis-mut ( green ), Pax6 ( red ), and phosphoH3 ( blue ). Compared with the control, the majority of cells overexpressing EGFP-radmis-mut no longer express Tbr2, and most of them translocated to the IZL. Arrow indicates persistent expression of radmis-mut in Pax6-positive radial glial cells in the VZ. ( F – I ) Prolonged expression of radmis-mut results in reduced cell proliferation, and increased cell-cycle exit and cell death. E14.5 brains were electroporated with control EGFP ( F ) or EGFP-radmis-mut ( G ) constructs. BrdU was consecutively administered into dams after 24 h post-electroporation (E15.5–E17.5), followed by analysis of brains at 72 h (E17.5). Left column shows merged confocal images of sections stained for EGFP ( green ), BrdU ( red ), and Ki67 ( blue ). Middle and right columns show BrdU-, and Ki67-labeled cells, respectively. Arrowheads indicate EGFP, BrdU, and Ki67 triple-positive cells, and arrows indicate EGFP- and BrdU-positive, but Ki67-negative, cells. ( H , I ) Apoptotic cells ( arrows ) were detected by TUNEL staining in control ( H ) or radmis-mut electroporated brains ( I , I ’). EGFP ( green ) and TUNEL-positive cells ( red ). ( J ) Quantification of 72 h BrdU labeling index. Data are presented as mean ± SEM (%). Student’s t -tests (n = 5 embryos); *p

    Article Snippet: EGFP (rabbit polyclonal, Life Technologies, 1:1000), EGFP (chick IgY, AvesLab, Oregon, 1:1000), Tbr2 (chick IgY, Merck Millipore, 1:1000), Pax6 (rabbit polyclonal, MBL, Nagoya, Japan, 1:1000), anti-bromodeoxyuridine (BrdU) (sheep polyclonal, Abcam, 1:1500), Ki-67 (rabbit monoclonal clone SP6, Lab Vision, CA, 1:1000).

    Techniques: Expressing, Construct, Staining, Electroporation, Labeling, TUNEL Assay

    In vivo overexpression of radmis increases the rate of mitotic cells in VZ/SVZ. In utero electroporation of EGFP-radmis, EGFP-radmis KEN mut (radmis-mut), or control EGFP was performed at E14.5, followed by analysis at 24 h post-electroporation (E15.5). ( A – C ) Distribution of cells electroporated with EGFP-radmis ( B ), or control EGFP ( A ) in E15.5 neocortex. Sections were stained for EGFP ( green ) and phosphoH3 ( red ). Arrows indicate EGFP and phosphoH3 double-positive cells in the VZ/SVZ. The ventricular surface is at the bottom, and the pial surface is at the top. ( C ) Magnified view of the ventricular surface of an EGFP-radmis electroporated brain. ( D – G ) Distribution of cells electroporated with control EGFP ( D , F ) or EGFP-radmis-mut ( E , G ). ( D , E ) Double immunostaining for EGFP ( green ) and phosphoH3 ( red ). Hatched areas in panels E and E’ denote the distribution of cells electroporated with EGFP-radmis-mut. NSPCs overexpressing radmis-mut remain mostly in the SVZ/VZ, and many of them express phosphoH3. ( F , G ) Confocal images of electroporated sections stained for EGFP and Tbr2 ( red ). Many cells expressing control EGFP are translocated to the upper IZ toward the pial surface, whereas cells expressing EGFP-radmis-mut remain in the SVZ/VZ and most of them show immunoreactivity for Tbr2. ( H , I ) Representative magnified images of radial glia cells that overexpressed EGFP-radmis-mut. EGFP-radmis-mut expression is frequently found in radial fibers ( arrow in H) and mitotic spindles ( arrowhead ) of radial glial cells, and induces the formation of a monopolar mitotic spindle during mitosis (hatched circle in I) in the VZ. EGFP-radmis-mut ( green ), phosphoH3 ( red ), and DNA ( blue ). ( J ) Quantification of electroporated NSPCs that are positive for phosphoH3 or Tbr2. The ratio of phosphoH3-positive M phase cells, or Tbr2-positive cells, to total EGFP-positive cells in the neocortex was calculated for each electroporation construct, and is presented as the mean ± SEM (%) (group, number of embryo analyzed; EGFP, n = 15; EGFP-Rad, n = 4; EGFP-Rad mut, n = 12). Student’s t -tests; *

    Journal: PLoS ONE

    Article Title: Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors

    doi: 10.1371/journal.pone.0079895

    Figure Lengend Snippet: In vivo overexpression of radmis increases the rate of mitotic cells in VZ/SVZ. In utero electroporation of EGFP-radmis, EGFP-radmis KEN mut (radmis-mut), or control EGFP was performed at E14.5, followed by analysis at 24 h post-electroporation (E15.5). ( A – C ) Distribution of cells electroporated with EGFP-radmis ( B ), or control EGFP ( A ) in E15.5 neocortex. Sections were stained for EGFP ( green ) and phosphoH3 ( red ). Arrows indicate EGFP and phosphoH3 double-positive cells in the VZ/SVZ. The ventricular surface is at the bottom, and the pial surface is at the top. ( C ) Magnified view of the ventricular surface of an EGFP-radmis electroporated brain. ( D – G ) Distribution of cells electroporated with control EGFP ( D , F ) or EGFP-radmis-mut ( E , G ). ( D , E ) Double immunostaining for EGFP ( green ) and phosphoH3 ( red ). Hatched areas in panels E and E’ denote the distribution of cells electroporated with EGFP-radmis-mut. NSPCs overexpressing radmis-mut remain mostly in the SVZ/VZ, and many of them express phosphoH3. ( F , G ) Confocal images of electroporated sections stained for EGFP and Tbr2 ( red ). Many cells expressing control EGFP are translocated to the upper IZ toward the pial surface, whereas cells expressing EGFP-radmis-mut remain in the SVZ/VZ and most of them show immunoreactivity for Tbr2. ( H , I ) Representative magnified images of radial glia cells that overexpressed EGFP-radmis-mut. EGFP-radmis-mut expression is frequently found in radial fibers ( arrow in H) and mitotic spindles ( arrowhead ) of radial glial cells, and induces the formation of a monopolar mitotic spindle during mitosis (hatched circle in I) in the VZ. EGFP-radmis-mut ( green ), phosphoH3 ( red ), and DNA ( blue ). ( J ) Quantification of electroporated NSPCs that are positive for phosphoH3 or Tbr2. The ratio of phosphoH3-positive M phase cells, or Tbr2-positive cells, to total EGFP-positive cells in the neocortex was calculated for each electroporation construct, and is presented as the mean ± SEM (%) (group, number of embryo analyzed; EGFP, n = 15; EGFP-Rad, n = 4; EGFP-Rad mut, n = 12). Student’s t -tests; *

    Article Snippet: EGFP (rabbit polyclonal, Life Technologies, 1:1000), EGFP (chick IgY, AvesLab, Oregon, 1:1000), Tbr2 (chick IgY, Merck Millipore, 1:1000), Pax6 (rabbit polyclonal, MBL, Nagoya, Japan, 1:1000), anti-bromodeoxyuridine (BrdU) (sheep polyclonal, Abcam, 1:1500), Ki-67 (rabbit monoclonal clone SP6, Lab Vision, CA, 1:1000).

    Techniques: In Vivo, Over Expression, In Utero, Electroporation, Staining, Double Immunostaining, Expressing, Construct

    Deregulation of progenitor pools was revealed by cell sorting in RP58 mutant cortices. ( a ) FACS graph showing separation of E14.5 cortical cells depending on their expression of CD133 (Prominin1) and EGFP, the latter driven from the tbr2 regulatory region in tbr2: EGFP mice. The subpopulations of cells include the following: Group-A: CD133 + /EGFP − -containing RGCs in the VZ; Group-B: double positives, containing INPs in the SVZ; and Group-C: CD133 − /EGFP + , containing differentiating/differentiated cortical neurons. ( b ) Characterization of the three cell populations described in panel a by expression of key molecular markers of RGCs ( blbp , glast , pax6 ), INPs ( tbr2 ) and neurons ( tbr1 , MAP2 ). ( c ) Quantification of the pool size of A and B cells in control and RP58 KO cortices. A small increase in A cells and a slight decrease in B cells were observed in the mutant cortices at E14.5. The asterisks denote significant changes ( P

    Journal: Cell Death and Differentiation

    Article Title: RP58/ZNF238 directly modulates proneurogenic gene levels and is required for neuronal differentiation and brain expansion

    doi: 10.1038/cdd.2011.144

    Figure Lengend Snippet: Deregulation of progenitor pools was revealed by cell sorting in RP58 mutant cortices. ( a ) FACS graph showing separation of E14.5 cortical cells depending on their expression of CD133 (Prominin1) and EGFP, the latter driven from the tbr2 regulatory region in tbr2: EGFP mice. The subpopulations of cells include the following: Group-A: CD133 + /EGFP − -containing RGCs in the VZ; Group-B: double positives, containing INPs in the SVZ; and Group-C: CD133 − /EGFP + , containing differentiating/differentiated cortical neurons. ( b ) Characterization of the three cell populations described in panel a by expression of key molecular markers of RGCs ( blbp , glast , pax6 ), INPs ( tbr2 ) and neurons ( tbr1 , MAP2 ). ( c ) Quantification of the pool size of A and B cells in control and RP58 KO cortices. A small increase in A cells and a slight decrease in B cells were observed in the mutant cortices at E14.5. The asterisks denote significant changes ( P

    Article Snippet: RP58 +/− mice were crossed with transgenic mice expressing EGFP under tbr2 regulatory sequences. tbr2::EGFP mice (GENSAT project) were obtained from the MMRRC (Mutant Mouse Regional Resource Center).

    Techniques: FACS, Mutagenesis, Expressing, Mouse Assay

    Unfolded protein response pathway in hCS exposed to low oxygen. ( a ) Dendrogram for the Weighted gene co-expression network analysis (WGCNA), which identified genes with similar expression profiles and grouped them into modules (represented by colors). ( b ) Eigengene expression profiles (‘average’ expression profile of all module genes) for the top WGCNA modules associated with exposure of hCS to low O 2 for 48 hours. The blue module (upper) contains genes strongly enriched for annotation in biological pathways related to the hypoxia response (HIF-1α transcription factor network), while the turquoise module (lower) is enriched for genes involved in the unfolded protein response (UPR) pathway. ( c ) Quantification of the density of TBR2 + cells in hCS after 48 h exposure to

    Journal: Nature medicine

    Article Title: Human 3D Cellular Model of Hypoxic Brain Injury of Prematurity

    doi: 10.1038/s41591-019-0436-0

    Figure Lengend Snippet: Unfolded protein response pathway in hCS exposed to low oxygen. ( a ) Dendrogram for the Weighted gene co-expression network analysis (WGCNA), which identified genes with similar expression profiles and grouped them into modules (represented by colors). ( b ) Eigengene expression profiles (‘average’ expression profile of all module genes) for the top WGCNA modules associated with exposure of hCS to low O 2 for 48 hours. The blue module (upper) contains genes strongly enriched for annotation in biological pathways related to the hypoxia response (HIF-1α transcription factor network), while the turquoise module (lower) is enriched for genes involved in the unfolded protein response (UPR) pathway. ( c ) Quantification of the density of TBR2 + cells in hCS after 48 h exposure to

    Article Snippet: The following primary antibodies were used for immunohistochemistry: PAX6 (rabbit, 1:300, Biolegend, PRB-278P), TBR2 (sheep, 1:100, Novus Biologicals, AF6166).

    Techniques: Expressing

    Proportion of TBR2 + cells in hCS exposed to low oxygen. ( a , b ) Representative images of proliferative areas in hCS maintained in 21% O 2 (upper) or exposed to

    Journal: Nature medicine

    Article Title: Human 3D Cellular Model of Hypoxic Brain Injury of Prematurity

    doi: 10.1038/s41591-019-0436-0

    Figure Lengend Snippet: Proportion of TBR2 + cells in hCS exposed to low oxygen. ( a , b ) Representative images of proliferative areas in hCS maintained in 21% O 2 (upper) or exposed to

    Article Snippet: The following primary antibodies were used for immunohistochemistry: PAX6 (rabbit, 1:300, Biolegend, PRB-278P), TBR2 (sheep, 1:100, Novus Biologicals, AF6166).

    Techniques:

    In vitro-prepared CP-like neurons changed subtype marker expression upon exposure to microglia. a Immunostaining for d4Venus (Gadd45g; green), Tbr2 (red), MAP2 (cyan), and DAPI (blue) in E14 Gadd45g-d4Venus Tg mouse cerebral walls. The broken lines indicate apical and basal surfaces. Scale bar, 50 µm. b The protocol for the in vitro preparation of CP-like neurons, which were obtained from Gadd45g-d4Venus + cells isolated from the E14 Gadd45g-d4Venus cortex by FACS, after culture for 2 days. c A time-lapse series showing the reduction in Gadd45g-dVenus expression during culture. Scale bar, 20 µm. d CX3CR1-GFP + microglia harvested from E15 CX3CR1-GFP pallial walls by FACS were added to the CP-like neuronal cultures. e Representative immunostaining (shown in pseudo color) for βIII-tubulin (cyan), Tbr1/Satb2/Cux1 (red), and Ctip2 (green). The arrowhead indicates microglia. Scale bar, 30 µm. f The proportion of the βIII-tubulin + cells that expressed Tbr1, Ctip2, Satb2, or Cux1 in cocultures with or without microglia (two-sided Mann–Whitney U test; n = 10 independent experiments; P = 0.0021 for Tbr1, P = 7.6 × 10 −5 for Ctip2, P = 7.6 × 10 −5 for Satb2, P = 1.5 × 10 −4 for Cux1). g An experimental schematic of the coculture of youngest (0 day, Gadd45g-d4Venus + ) neurons and CX3CR1-GFP + microglia. h Representative immunostaining (shown in pseudo color) for βIII-tubulin (cyan), Tbr1/Satb2/Cux1 (red), and Ctip2 (green). The arrowhead indicates microglia. Scale bar, 30 µm. i The proportion of βIII-tubulin + cells that expressed Tbr1, Ctip2, Satb2, or Cux1 in cocultures with or without microglia (two-sided Mann–Whitney U test; n = 10 independent cultures; P = 0.529 for Tbr1, P = 0.289 for Ctip2, P = 0.631 for Satb2, P = 0.912 for Cux1). Data are presented as the mean values ± S.D. Source data are provided as a Source Data File .

    Journal: Nature Communications

    Article Title: Transient microglial absence assists postmigratory cortical neurons in proper differentiation

    doi: 10.1038/s41467-020-15409-3

    Figure Lengend Snippet: In vitro-prepared CP-like neurons changed subtype marker expression upon exposure to microglia. a Immunostaining for d4Venus (Gadd45g; green), Tbr2 (red), MAP2 (cyan), and DAPI (blue) in E14 Gadd45g-d4Venus Tg mouse cerebral walls. The broken lines indicate apical and basal surfaces. Scale bar, 50 µm. b The protocol for the in vitro preparation of CP-like neurons, which were obtained from Gadd45g-d4Venus + cells isolated from the E14 Gadd45g-d4Venus cortex by FACS, after culture for 2 days. c A time-lapse series showing the reduction in Gadd45g-dVenus expression during culture. Scale bar, 20 µm. d CX3CR1-GFP + microglia harvested from E15 CX3CR1-GFP pallial walls by FACS were added to the CP-like neuronal cultures. e Representative immunostaining (shown in pseudo color) for βIII-tubulin (cyan), Tbr1/Satb2/Cux1 (red), and Ctip2 (green). The arrowhead indicates microglia. Scale bar, 30 µm. f The proportion of the βIII-tubulin + cells that expressed Tbr1, Ctip2, Satb2, or Cux1 in cocultures with or without microglia (two-sided Mann–Whitney U test; n = 10 independent experiments; P = 0.0021 for Tbr1, P = 7.6 × 10 −5 for Ctip2, P = 7.6 × 10 −5 for Satb2, P = 1.5 × 10 −4 for Cux1). g An experimental schematic of the coculture of youngest (0 day, Gadd45g-d4Venus + ) neurons and CX3CR1-GFP + microglia. h Representative immunostaining (shown in pseudo color) for βIII-tubulin (cyan), Tbr1/Satb2/Cux1 (red), and Ctip2 (green). The arrowhead indicates microglia. Scale bar, 30 µm. i The proportion of βIII-tubulin + cells that expressed Tbr1, Ctip2, Satb2, or Cux1 in cocultures with or without microglia (two-sided Mann–Whitney U test; n = 10 independent cultures; P = 0.529 for Tbr1, P = 0.289 for Ctip2, P = 0.631 for Satb2, P = 0.912 for Cux1). Data are presented as the mean values ± S.D. Source data are provided as a Source Data File .

    Article Snippet: Sections or cells were treated with the following primary antibodies: mouse anti-βIII-tubulin mAb (1:1000, Cat#MMS-435P, Covance, Princeton, NJ, USA); rabbit anti-βIII-tubulin pAb (1:1000, Cat#802001, BioLegend, San Diego, CA, USA); rabbit anti-Cl-Casp-3 pAb (1:500, Cat#9661s, Cell Signaling Technology Inc., Beverly, MA, USA); goat anti-CD206 pAb (1:200, Cat#AF2535, R & D systems); rat anti-Ctip2 mAb (1:1000, Cat#ab18465, Abcam, Cambridge, UK); rabbit anti-Cux1 pAb (1:200, Cat#sc-13024, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); chicken anti-GFP pAb (1:1000, Cat#GFP-1020, Aves Labs, Tigard, OR, USA); rat anti-GFP mAb (1:500, Cat#GF090R, Nacalai Tesque, Kyoto, Japan); rabbit anti-Iba1 pAb (1:2000, Cat#019–19741, FUJIFILM Wako Pure Chemical Corp.); mouse anti-MAP2 mAb (1:5000, Cat#M1406, Sigma-Aldrich); rabbit anti-P2RY12 pAb (1:500, Cat#AS–55043A, AnaSpec, San Jose, CA, USA); rabbit anti-RFP pAb (1:1000, Cat#PM005, MBL);mouse anti-Satb2 mAb (1:400, Cat#ab51502, Abcam), rabbit anti-Tbr1 pAb (1:500, Cat#ab31940, Abcam), and rabbit anti-Tbr2 mAb (1:500, Cat#ab183991, Abcam).

    Techniques: In Vitro, Marker, Expressing, Immunostaining, Isolation, FACS, MANN-WHITNEY

    Inward attraction from the SVZ/VZ contributes to microglial disappearance from the midembryonic CP. a Typical migratory behavior of CX3CR1-GFP + microglia (arrowhead) that were initially in the inner (VZ/SVZ/IZ) region. Broken line, apical surface. b The trajectories of the microglia in the inner region. A comparison of microglial translocation patterns ( c two-sided Pearson’s chi-squared test; P = 2.2 × 10 −16 ) and the migratory distance toward the meninges ( d two-sided Mann–Whitney U test; n = 30 outer cells and 33 inner cells, P = 3.1 × 10 −8 ). e Immunostaining for CX3CR1 (microglia), Tbr2 (intermediate progenitors and young neurons positioned in the SVZ), and MAP2 (neurons). Broken line, apical surface. f The trajectories of the microglia were categorized into two groups: cells 0–100 µm deep relative to the apical surface and cells 100–250 µm deep relative to the apical surface. A comparison of microglial translocation patterns ( g two-sided Pearson’s chi-squared test; P 0–100 µm = 3.3 × 10 −13 , P 100–250 µm = 2.2 × 10 −16 ) and the migratory distance toward the meninges ( h two-sided Steel–Dwass test; n = 30, 13, and 20 cells [left to right]; P 0–100 µm = 0.004, P 100–250 µm = 1.5 × 10 −6 ). i FACS analysis for pallial cells collected from E14 CX3CR1-GFP Tg mice. j A schematic showing the coculturing of CX3CR1-GFP + microglia with the SVZ/VZ and CP explants. k A bright-field image of microglia (white square space) adjacently cocultured with the explants. l Representative images of the monitoring of microglial accumulation in the SVZ/VZ (pink) and CP (yellow) explants. m The density of microglia in each explant (two-sided Mann–Whitney U test; n = 4 individual experiments; P = 0.029 for 0.5 day, P = 0.029 for 1 day, and P = 0.029 for 3 days). Data are presented as the mean values ± S.D. Scale bar, 50 µm. Source data are provided as a Source Data File .

    Journal: Nature Communications

    Article Title: Transient microglial absence assists postmigratory cortical neurons in proper differentiation

    doi: 10.1038/s41467-020-15409-3

    Figure Lengend Snippet: Inward attraction from the SVZ/VZ contributes to microglial disappearance from the midembryonic CP. a Typical migratory behavior of CX3CR1-GFP + microglia (arrowhead) that were initially in the inner (VZ/SVZ/IZ) region. Broken line, apical surface. b The trajectories of the microglia in the inner region. A comparison of microglial translocation patterns ( c two-sided Pearson’s chi-squared test; P = 2.2 × 10 −16 ) and the migratory distance toward the meninges ( d two-sided Mann–Whitney U test; n = 30 outer cells and 33 inner cells, P = 3.1 × 10 −8 ). e Immunostaining for CX3CR1 (microglia), Tbr2 (intermediate progenitors and young neurons positioned in the SVZ), and MAP2 (neurons). Broken line, apical surface. f The trajectories of the microglia were categorized into two groups: cells 0–100 µm deep relative to the apical surface and cells 100–250 µm deep relative to the apical surface. A comparison of microglial translocation patterns ( g two-sided Pearson’s chi-squared test; P 0–100 µm = 3.3 × 10 −13 , P 100–250 µm = 2.2 × 10 −16 ) and the migratory distance toward the meninges ( h two-sided Steel–Dwass test; n = 30, 13, and 20 cells [left to right]; P 0–100 µm = 0.004, P 100–250 µm = 1.5 × 10 −6 ). i FACS analysis for pallial cells collected from E14 CX3CR1-GFP Tg mice. j A schematic showing the coculturing of CX3CR1-GFP + microglia with the SVZ/VZ and CP explants. k A bright-field image of microglia (white square space) adjacently cocultured with the explants. l Representative images of the monitoring of microglial accumulation in the SVZ/VZ (pink) and CP (yellow) explants. m The density of microglia in each explant (two-sided Mann–Whitney U test; n = 4 individual experiments; P = 0.029 for 0.5 day, P = 0.029 for 1 day, and P = 0.029 for 3 days). Data are presented as the mean values ± S.D. Scale bar, 50 µm. Source data are provided as a Source Data File .

    Article Snippet: Sections or cells were treated with the following primary antibodies: mouse anti-βIII-tubulin mAb (1:1000, Cat#MMS-435P, Covance, Princeton, NJ, USA); rabbit anti-βIII-tubulin pAb (1:1000, Cat#802001, BioLegend, San Diego, CA, USA); rabbit anti-Cl-Casp-3 pAb (1:500, Cat#9661s, Cell Signaling Technology Inc., Beverly, MA, USA); goat anti-CD206 pAb (1:200, Cat#AF2535, R & D systems); rat anti-Ctip2 mAb (1:1000, Cat#ab18465, Abcam, Cambridge, UK); rabbit anti-Cux1 pAb (1:200, Cat#sc-13024, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); chicken anti-GFP pAb (1:1000, Cat#GFP-1020, Aves Labs, Tigard, OR, USA); rat anti-GFP mAb (1:500, Cat#GF090R, Nacalai Tesque, Kyoto, Japan); rabbit anti-Iba1 pAb (1:2000, Cat#019–19741, FUJIFILM Wako Pure Chemical Corp.); mouse anti-MAP2 mAb (1:5000, Cat#M1406, Sigma-Aldrich); rabbit anti-P2RY12 pAb (1:500, Cat#AS–55043A, AnaSpec, San Jose, CA, USA); rabbit anti-RFP pAb (1:1000, Cat#PM005, MBL);mouse anti-Satb2 mAb (1:400, Cat#ab51502, Abcam), rabbit anti-Tbr1 pAb (1:500, Cat#ab31940, Abcam), and rabbit anti-Tbr2 mAb (1:500, Cat#ab183991, Abcam).

    Techniques: Translocation Assay, MANN-WHITNEY, Immunostaining, FACS, Mouse Assay

    The CXCL12/CXCR4 system plays a pivotal role in the bidirectional exit of microglia from the CP and their proper positioning in the cerebral wall. a In situ hybridization for Cxcl12 mRNA expression (left) and immunostaining for Tbr2/MAP2/DAPI (right) in the E14 cerebral wall. Scale bar, 100 µm. b Immunostaining for P2RY12/CD206/DAPI in E15 cerebral walls. Arrowheads, microglia in the MZ. Scale bar, 100 µm. The density of P2RY12 + microglia in the MZ ( c P = 6.7 × 10 −5 ) and in the meninges ( d P = 0.286) and of CD206 + macrophages ( e P = 0.198) (two-sided Mann–Whitney U test; n = 12 sections from four mice). f Immunostaining for Iba1/DAPI (left) and MAP2/Tbr2/DAPI (right) in E15 cerebral walls. Scale bar, 100 µm. The percentage of Iba1 + cells in each bin (the inner part of the cerebral wall than the CP was equally divided into three parts) ( g P = 9.2 × 10 −10 [bin 1], P = 6.1 × 10 −5 [bin 2], P = 3.4 × 10 −10 [bin 3], P = 0.045 [bin CP]) and the density of pallial Iba1 + cells ( h P = 0.0019) (two-sided Mann–Whitney U test; n = 30 sections from six mice). Microglial translocation patterns in the CP ( i two-sided Pearson’s chi-squared test; P = 7.2 × 10 −7 ) and the migratory distance toward the meninges ( j two-sided Mann–Whitney U test; n = 30 cells; P = 1.5 × 10 −4 ). Microglial translocation patterns in the IZ ( k two-sided Pearson’s chi-squared test; P = 1.7 × 10 −9 ) and the migratory distance toward the apical surface ( l two-sided Mann–Whitney U test; n = 20 WT cells, 17 Cxcr4 −/− cells; P = 0.017). Data are presented as the mean values ± S.D. Source data are provided as a Source Data File .

    Journal: Nature Communications

    Article Title: Transient microglial absence assists postmigratory cortical neurons in proper differentiation

    doi: 10.1038/s41467-020-15409-3

    Figure Lengend Snippet: The CXCL12/CXCR4 system plays a pivotal role in the bidirectional exit of microglia from the CP and their proper positioning in the cerebral wall. a In situ hybridization for Cxcl12 mRNA expression (left) and immunostaining for Tbr2/MAP2/DAPI (right) in the E14 cerebral wall. Scale bar, 100 µm. b Immunostaining for P2RY12/CD206/DAPI in E15 cerebral walls. Arrowheads, microglia in the MZ. Scale bar, 100 µm. The density of P2RY12 + microglia in the MZ ( c P = 6.7 × 10 −5 ) and in the meninges ( d P = 0.286) and of CD206 + macrophages ( e P = 0.198) (two-sided Mann–Whitney U test; n = 12 sections from four mice). f Immunostaining for Iba1/DAPI (left) and MAP2/Tbr2/DAPI (right) in E15 cerebral walls. Scale bar, 100 µm. The percentage of Iba1 + cells in each bin (the inner part of the cerebral wall than the CP was equally divided into three parts) ( g P = 9.2 × 10 −10 [bin 1], P = 6.1 × 10 −5 [bin 2], P = 3.4 × 10 −10 [bin 3], P = 0.045 [bin CP]) and the density of pallial Iba1 + cells ( h P = 0.0019) (two-sided Mann–Whitney U test; n = 30 sections from six mice). Microglial translocation patterns in the CP ( i two-sided Pearson’s chi-squared test; P = 7.2 × 10 −7 ) and the migratory distance toward the meninges ( j two-sided Mann–Whitney U test; n = 30 cells; P = 1.5 × 10 −4 ). Microglial translocation patterns in the IZ ( k two-sided Pearson’s chi-squared test; P = 1.7 × 10 −9 ) and the migratory distance toward the apical surface ( l two-sided Mann–Whitney U test; n = 20 WT cells, 17 Cxcr4 −/− cells; P = 0.017). Data are presented as the mean values ± S.D. Source data are provided as a Source Data File .

    Article Snippet: Sections or cells were treated with the following primary antibodies: mouse anti-βIII-tubulin mAb (1:1000, Cat#MMS-435P, Covance, Princeton, NJ, USA); rabbit anti-βIII-tubulin pAb (1:1000, Cat#802001, BioLegend, San Diego, CA, USA); rabbit anti-Cl-Casp-3 pAb (1:500, Cat#9661s, Cell Signaling Technology Inc., Beverly, MA, USA); goat anti-CD206 pAb (1:200, Cat#AF2535, R & D systems); rat anti-Ctip2 mAb (1:1000, Cat#ab18465, Abcam, Cambridge, UK); rabbit anti-Cux1 pAb (1:200, Cat#sc-13024, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); chicken anti-GFP pAb (1:1000, Cat#GFP-1020, Aves Labs, Tigard, OR, USA); rat anti-GFP mAb (1:500, Cat#GF090R, Nacalai Tesque, Kyoto, Japan); rabbit anti-Iba1 pAb (1:2000, Cat#019–19741, FUJIFILM Wako Pure Chemical Corp.); mouse anti-MAP2 mAb (1:5000, Cat#M1406, Sigma-Aldrich); rabbit anti-P2RY12 pAb (1:500, Cat#AS–55043A, AnaSpec, San Jose, CA, USA); rabbit anti-RFP pAb (1:1000, Cat#PM005, MBL);mouse anti-Satb2 mAb (1:400, Cat#ab51502, Abcam), rabbit anti-Tbr1 pAb (1:500, Cat#ab31940, Abcam), and rabbit anti-Tbr2 mAb (1:500, Cat#ab183991, Abcam).

    Techniques: In Situ Hybridization, Expressing, Immunostaining, MANN-WHITNEY, Mouse Assay, Translocation Assay

    ABHD4 is not required for proliferation, differentiation and lamination in the developing neocortex. a,b, Phospho-histone H3 (PHH3)-immunostaining visualizes cell proliferation during the M phase of the cell cycle at the ventricular border. c, Quantification of PHH3-positive cell density (two-sided Student’s unpaired t-test, P = 0.688; n = 26 sections from n = 3 animals per wild-type (+/+) mice, n = 30 sections from n = 3 animals per Abhd4- knockout (-/-) mice). d,e, High density of T-box brain protein 2 (TBR2)-positive intermediate progenitor cells in the subventricular zone. f, Quantification of TBR2-positive cell density (two-sided Student’s unpaired t-test, P = 0.194; n = 18 sections from n = 3 animals per wild-type (+/+) mice, n = 15 sections from n = 3 animals per Abhd4- knockout (-/-) mice). g,h , Distribution of TBR1-positive postmitotic neurons in the cortical plate. i, Quantification of TBR1-positive cell density (two-sided Mann-Whitney U test, P = 0.074; n = 17 sections from n = 3 animals per wild-type (+/+) mice, n = 16 sections from n = 3 animals per Abhd4- knockout (-/-) mice). MZ: marginal zone, CP: cortical plate, IZ: intermediate zone, SVZ: subventricular zone, VZ: ventricular zone. Graphs show raw data and mean ± 2 x standard error ( c,f ), or raw data and median ± interquartile range ( i ).

    Journal: bioRxiv

    Article Title: ABHD4-dependent developmental anoikis protects the prenatal brain from pathological insults

    doi: 10.1101/2019.12.17.879551

    Figure Lengend Snippet: ABHD4 is not required for proliferation, differentiation and lamination in the developing neocortex. a,b, Phospho-histone H3 (PHH3)-immunostaining visualizes cell proliferation during the M phase of the cell cycle at the ventricular border. c, Quantification of PHH3-positive cell density (two-sided Student’s unpaired t-test, P = 0.688; n = 26 sections from n = 3 animals per wild-type (+/+) mice, n = 30 sections from n = 3 animals per Abhd4- knockout (-/-) mice). d,e, High density of T-box brain protein 2 (TBR2)-positive intermediate progenitor cells in the subventricular zone. f, Quantification of TBR2-positive cell density (two-sided Student’s unpaired t-test, P = 0.194; n = 18 sections from n = 3 animals per wild-type (+/+) mice, n = 15 sections from n = 3 animals per Abhd4- knockout (-/-) mice). g,h , Distribution of TBR1-positive postmitotic neurons in the cortical plate. i, Quantification of TBR1-positive cell density (two-sided Mann-Whitney U test, P = 0.074; n = 17 sections from n = 3 animals per wild-type (+/+) mice, n = 16 sections from n = 3 animals per Abhd4- knockout (-/-) mice). MZ: marginal zone, CP: cortical plate, IZ: intermediate zone, SVZ: subventricular zone, VZ: ventricular zone. Graphs show raw data and mean ± 2 x standard error ( c,f ), or raw data and median ± interquartile range ( i ).

    Article Snippet: The following primary antibodies were used: rabbit antibody to phospho-histone H3 (PHH3, 1:500, Millipore), rabbit antibody to the transcription factor T-box, brain, 1 (TBR1, 1:500, Abcam), rabbit antibody to T-box, brain, 2 (TBR2, 1:500, Abcam), rabbit antibody to laminin subunit alpha 1 (LAMA1, 1:500, Sigma).

    Techniques: Immunostaining, Mouse Assay, Knock-Out, MANN-WHITNEY

    Abhd4 mRNA is expressed by radial glia progenitor cells in the germinative niches. a-h, Abhd4 mRNA is present exclusively in the ventricular zone along the lateral ( b,g ) and third ventricles ( c,h ) at both E16.5 ( a-d ) and P1 ( f-h ) wild-type (+/+) mice. The specificity of the Abhd4 riboprobe is validated in Abhd4-knockout (-/-) animals ( e ). CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. i-l, High-power confocal imaging outlines the plasma membrane of ChR2-GFP -electroporated cells and delimits multi-color RNAscope analysis into single cells within the heterogeneous and densely packed cell layer of the ventricular zone. Abhd4 mRNA typically colocalizes with the radial glia progenitor cell marker Slc1a3 mRNA (encoding GLAST1 protein) ( i ), whereas other cells are often devoid of both markers ( k ), or either express Eomes (encoding TBR2 protein), a marker for committed daughter cells ( j ), or Abhd4 alone ( l ). m, Correlation analysis of Abhd4 mRNA levels with Glast1 or Tbr2 mRNA levels in single cells (Spearman’s rank correlation, Abhd4/Glast1: R = 0.4814, P

    Journal: bioRxiv

    Article Title: ABHD4-dependent developmental anoikis protects the prenatal brain from pathological insults

    doi: 10.1101/2019.12.17.879551

    Figure Lengend Snippet: Abhd4 mRNA is expressed by radial glia progenitor cells in the germinative niches. a-h, Abhd4 mRNA is present exclusively in the ventricular zone along the lateral ( b,g ) and third ventricles ( c,h ) at both E16.5 ( a-d ) and P1 ( f-h ) wild-type (+/+) mice. The specificity of the Abhd4 riboprobe is validated in Abhd4-knockout (-/-) animals ( e ). CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. i-l, High-power confocal imaging outlines the plasma membrane of ChR2-GFP -electroporated cells and delimits multi-color RNAscope analysis into single cells within the heterogeneous and densely packed cell layer of the ventricular zone. Abhd4 mRNA typically colocalizes with the radial glia progenitor cell marker Slc1a3 mRNA (encoding GLAST1 protein) ( i ), whereas other cells are often devoid of both markers ( k ), or either express Eomes (encoding TBR2 protein), a marker for committed daughter cells ( j ), or Abhd4 alone ( l ). m, Correlation analysis of Abhd4 mRNA levels with Glast1 or Tbr2 mRNA levels in single cells (Spearman’s rank correlation, Abhd4/Glast1: R = 0.4814, P

    Article Snippet: The following primary antibodies were used: rabbit antibody to phospho-histone H3 (PHH3, 1:500, Millipore), rabbit antibody to the transcription factor T-box, brain, 1 (TBR1, 1:500, Abcam), rabbit antibody to T-box, brain, 2 (TBR2, 1:500, Abcam), rabbit antibody to laminin subunit alpha 1 (LAMA1, 1:500, Sigma).

    Techniques: Mouse Assay, Knock-Out, Imaging, Marker