anti-otx2 Search Results


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  • 91
    Thermo Fisher anti otx2 antiserum
    A, An EMSA was performed using the consensus <t>OTX2</t> binding sequence incubated with in vitro transcribed and translated empty vector, WT or N233S (MUT) OTX2 protein. Lanes 2–4 and 5–7 demonstrate increased intensity of binding with the addition of increasing quantities of in vitro translated OTX2 (2, 4, or 8 μl). The OTX2 complex is denoted by an arrowhead . B, Probes containing consensus, BDI, BDII, and BDIII sites were incubated with in vitro translated WT or MUT OTX2. Binding of both WT and MUT OTX2 was observed, as shown in lanes 1 and 2 (CON), 3 and 4 (BDI), and 7 and 8 (BDIII), whereas no binding was seen in lanes 5 and 6 (BDII) of the EMSA (denoted by arrowhead ). Lane 9 is empty vector incubated with consensus sequence. C, Consensus, BDI, and BDIII probes were incubated with WT or MUT OTX2 protein, followed by addition of a polyclonal OTX2 antibody resulting in “supershifted” (SS) protein-DNA complexes by EMSA (denoted by the double arrowhead ).
    Anti Otx2 Antiserum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore anti otx2
    Inactivation of Lhx2 with Hes1 CreERT2 at E15.5 results in the overproduction of rods. A–H , Sections from control and Hes1 CreERT2 /+ ;Lhx2 f /− eyes stained with antibodies against Pou4f (RGC precursors, A , B ), <t>Otx2</t> (photoreceptor precursors, C , D ), Nr2e3 (rod precursors, E , F ), and Rxrγ (cone precursors, G , H ). I , The number of RGC precursors is unchanged, while the number of photoreceptor precursors in the NBL is increased due to the selective overproduction of rods (* p = 0.0007 and * p = 0.0004, respectively; n = 3 mice for each genotype; n > 200 marker+ cells for each genotype). J–O , Immunostaining for Bhlhb5 ( J , K ), Calretinin ( L , M ), and Gaba ( N , O ) show that amacrine cell precursors are slightly increased. P–S , Immunostaining for Vsx2 ( P , Q ) and Vsx1 ( R , S ) show that bipolar cells are decreased. T–W , At P9, Lhx2, predominantly expressed in Müller glia, is largely absent in the Hes1 CreERT2 /+ ;Lhx2 f /− retina ( T , U ). Müller glia are still present, but disorganized, as indicated by Sox9 ( V , W ). X–AA , Immunostaining for Gfap ( X , Y ) and Pcna ( Z , AA ) show that the Lhx2 -inactivated Müller glia are reactive and proliferative. Time points above each part indicate the time of inactivation and analysis, respectively. Scale bars:100 μm. INL, inner nuclear layer.
    Anti Otx2, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore orthodenticle homeobox 2 otx2
    Tau deficiency reduced the expression of transcription factor <t>OTX2</t> in mDANs of the VTA. (A) Western blot showed the expression level of Lmx1b, Pitx3, and OTX2 in the midbrain homogenates of tau +/+ , tau +/ − , and tau − / − mice at 18 months of age. (B) Densitometry analyses depicted the Lmx1b, Pitx3, and OTX2 expression level, normalized with β-actin ( n = 5 per genotype, P > 0.05). (C) OTX2 (green) and TH (red) co-staining in the VTA coronal sections of tau +/+ , tau +/ − , and tau − / − mice at 18 months of age. Scale bar = 20 lm. (D) Densitometry analyses of (C) showed OTX2 signals intensity in TH-positive neurons and TH-negative neurons ( n = 5 per genotype). All data were presented as mean ± SEM, * P
    Orthodenticle Homeobox 2 Otx2, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti otx2
    Expression of Otx1 and <t>Otx2</t> in adult mouse brain. ( A ) Analysis of Otx1 and Otx2 expression by quantitative RT-PCR on extracts from lateral geniculate nucleus (LGN), visual cortex (V Cx), superior colliculus (S Col) and cerebellum (Cb). The fold-difference in expression is calculated relative to Otx1 in LGN. The Otx2 locus is silent in visual cortex. ( B ) Non-cell autonomous Otx2 is found in visual cortex. Immunostaining in wild type mice reveals Otx1/2 cells in layers IV and V of visual cortex, including cells with perineuronal nets (stained by WFA lectin) enriched in layer IV. Staining for Otx2 persists in Otx1 null mice (Otx1 KO). Scale bar, 50 µm.
    Anti Otx2, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti otx2 antibody
    Immunohistochemistry reveals that both predicted activin receptors (Acvr2a and Acvr2b) are expressed in photoreceptors. To validate the cytokine receptor prediction by receptoR, sections of post-natal day 4 mouse retina (without RPE) were immunostained for activin type 2 receptors and well-known photoreceptor/retina markers. (A) Co-localization of rhodopsin (green) and Acvr2a (magenta) indicates the expression of this activin receptor in photoreceptor cells. (B) In contrast, Acvr2b (green) shows weaker, less specific staining throughout the retina, similar to the expression of <t>Otx2</t> (magenta). Sections are 20 μm thick and were counterstained with DAPI (gray).
    Anti Otx2 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Neuromics anti otx2
    Abnormal neuronal migration and distribution of neurons in ak/ak mesencephalon. ( A-M ) The migrational profile of E11-labeled neuronal progenitors was analyzed in WT (A-C,G) and ak/ak mutant (D-F,H-M) mice at E17. White asterisks indicate areas free of BrdU-labeled cells in WT mesencephalon (A-C,G) and blue asterisks indicate abnormal cell clusters in the red nucleus of the ak/ak mutant (D-F,H). (G,H) Higher magnifications of C and F displaying failed perpendicular migration (arrowheads) in the ak/ak mutant. (I-M) Stalled cells in the ak/ak mutant are double positive for <t>BrdU/Otx2</t> (arrowheads, I), BrdU/Lmx1b (J,K) and BrdU/Foxa2 (L,M). The boxed regions in J and L are magnified in K and M, respectively, and white arrows indicate double-positive cells. ( N ) Quantification of E11 BrdU-labeled cells distributed in the red nucleus (both hemispheres) of WT and ak/ak mutant (mean density of BrdU + cells ± s.d.). A significant increase in BrdU + cells was observed in the ak/ak mutant. * P
    Anti Otx2, supplied by Neuromics, used in various techniques. Bioz Stars score: 89/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    R&D Systems otx2
    DAPT induces retinal progenitor differentiation and inhibits proliferation. hESCs were differentiated toward retinal neuronal fates. (A) Brightfield and immunostaining of control and DAPT-treated cells after 5 days. A decrease in the number of rosettes, LHX2 and PAX6 expression, along with decreased PH3 staining of mitotic cells was observed in DAPT-treated cells. (B) DAPT treatment resulted in increased TUJ1- and HuC/D-expressing retinal cells. (C) Immunostaining reveals retinal neurons expressing photoreceptor markers, recoverin, and <t>OTX2</t> ( arrows ). (D) RT-PCR analysis demonstrated decreased PAX6 and increased BRN3B and RCVRN expression in DAPT-treated cells compared to DMSO controls. Mean ± SEM, * P
    Otx2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 269 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology otx2
    Delayed PV+ cell maturation and visual cortical plasticity in <t>Otx2</t> +/AA mice. ( a ) A GAG-binding motif is found between the N-terminal (Nter) and the homeodomain of Otx2. The ‘RK’ doublet is mutated to ‘AA’ in the resulting AA mutant. ( b ) Representative images of WFA (staining for PNN) and Otx2 co-labeling in primary visual cortex (V1) layer IV (L4) at P30 (scale bar: 100 µm), comparing +/+ (WT) and +/AA (heterozygous Otx2 +/AA ). ( c-e ) Quantification of Otx2 immunostaining intensity (arbitrary unit, a.u.) in V1 L4 WFA+ cells from P20 to P100 ( c , N=3-5 mice per group), total number of Otx2+ cells at P60 ( d , N=9-12 mice per group) and percentage of Otx2+ cells not stained with WFA at P60 ( e , N=9-12 mice per group). ( f, g ) WFA staining intensity (a.u.) per pixel ( f ) and PV staining intensity (a.u.) per cell ( g ) in V1 L4, quantified from P14 to P200 (N=3-10 mice per group). Shaded area indicates WT ocular dominance critical period. ( h ) Visual acuity measurements of Otx2 +/AA at P30, P100 and P200, with or without short-term (4-day) monocular deprivation (MD; N=4-5 mice per group). (All values: mean ± SEM; t-test; *p
    Otx2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Merck KGaA anti otx2
    Delayed PV+ cell maturation and visual cortical plasticity in <t>Otx2</t> +/AA mice. ( a ) A GAG-binding motif is found between the N-terminal (Nter) and the homeodomain of Otx2. The ‘RK’ doublet is mutated to ‘AA’ in the resulting AA mutant. ( b ) Representative images of WFA (staining for PNN) and Otx2 co-labeling in primary visual cortex (V1) layer IV (L4) at P30 (scale bar: 100 µm), comparing +/+ (WT) and +/AA (heterozygous Otx2 +/AA ). ( c-e ) Quantification of Otx2 immunostaining intensity (arbitrary unit, a.u.) in V1 L4 WFA+ cells from P20 to P100 ( c , N=3-5 mice per group), total number of Otx2+ cells at P60 ( d , N=9-12 mice per group) and percentage of Otx2+ cells not stained with WFA at P60 ( e , N=9-12 mice per group). ( f, g ) WFA staining intensity (a.u.) per pixel ( f ) and PV staining intensity (a.u.) per cell ( g ) in V1 L4, quantified from P14 to P200 (N=3-10 mice per group). Shaded area indicates WT ocular dominance critical period. ( h ) Visual acuity measurements of Otx2 +/AA at P30, P100 and P200, with or without short-term (4-day) monocular deprivation (MD; N=4-5 mice per group). (All values: mean ± SEM; t-test; *p
    Anti Otx2, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex otx2
    Clinical and genetic heterogeneity among nine subtypes of PFA ependymoma. Nine subtypes of PFA ependymoma displaying variability in: (A) age at diagnosis (years), (B) gender ratio, (C) ratio of pathological grade, (D) progression-free survival (PFS), and (E) overall survival (OS). (F) Elevated <t>OTX2</t> expression in PFA-2c ependymomas demonstrated by gene expression profiling (2x Affymetrix u133 v2 array probes) and immunohistochemistry (G). Immunonegative PFA ependymoma of another subtype (H). Varying frequencies across subtypes of the four commonest chromosome arm copy number alterations found in PFA ependymomas; 1q gain (I), 6q loss (J), 10q loss (K), 22q loss (L). Scale bars (G/H) = 100μm .
    Otx2, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Developmental Studies Hybridoma Bank otx2
    Generation of highly-pure population of MNPs from hPSCs (A) Schematics showing the time course and small molecule cocktail for hPSC differentiation into MNPs. (B) Representative images of SOX1 + NEPs after 6 days of culture in CHIR+SB+DMH vs. SB+DMH condition. The regional identity ( <t>OTX2</t> + vs. HOXA3 +) were stained. Scale bars: 50μm. Quantification of SOX1 + NEP percentage and number is shown on right ( > 500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (C) Representative images of pure MNPs at Day12 under different conditions, which express OLIG2 (green) but not NKX2.2 (red). Scale bars: 50μm. Quantification of OLIG2 +, NKX2.2 + and OLIG2 +/ NKX2.2 + cells is shown on right ( > 500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (D) The efficiency of OLIG2 + MNP differentiation from multiple hPSC lines ( > 500 cells from random fields were manually counted in each cell line). The bar graph shows the mean±s.d. (n=3 in each cell line).
    Otx2, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc anti otx2
    Methylation of the <t>OTX2</t> promoter is sufficient to suppress OTX2 in medulloblastoma. (A) Representative methylation patterns of medulloblastoma samples, with samples organized by row and each CpG location organized in columns. Leftmost column indicates OTX2 expression status (red, OTX2-expressing; green, OTX2-nonexpressing), letters to the right of this column indicate OTX2 copy number status (N, normal; G, gained; HL, heterozygous loss), if known [5] , [19] , [32] , [33] . (B) OTX2 mRNA level, as determined by RT-qPCR, in tumors exhibiting either a methylated or unmethylated OTX2 promoter. ND, not detected. (C) Semi-quantitative RT-PCR of OTX2-nonexpressing cells treated with the indicated concentrations of 5′-aza-2′-deoxycytidine for 5 days. D283 medulloblastoma cells serve as a positive control for OTX2 detection.
    Anti Otx2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher otx2
    Primitive neuroectodermal specification of human TiPSCs through EB formation. A : Phase contrast images of an established Tenon’s-derived induced pluripotent stem cell (TiPSC) line at passage P 33, displaying typical undifferentiated human embryonic stem cell (hESC)-like colony morphology. Scale bar = 100 μm. B – D : Confocal immunofluorescent images of undifferentiated TiPSC clones, stained positive for pluripotency associated markers Oct-4, Nanog, and Sox2 but negative for panneural markers Pax6, <t>Otx2,</t> and Nestin. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars = 50 μm. E : Quantitative real-time PCR analysis confirmed pluripotent genes Oct3/4 and Nanog markedly decreased after 6 days of suspension culture (* p
    Otx2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    A, An EMSA was performed using the consensus OTX2 binding sequence incubated with in vitro transcribed and translated empty vector, WT or N233S (MUT) OTX2 protein. Lanes 2–4 and 5–7 demonstrate increased intensity of binding with the addition of increasing quantities of in vitro translated OTX2 (2, 4, or 8 μl). The OTX2 complex is denoted by an arrowhead . B, Probes containing consensus, BDI, BDII, and BDIII sites were incubated with in vitro translated WT or MUT OTX2. Binding of both WT and MUT OTX2 was observed, as shown in lanes 1 and 2 (CON), 3 and 4 (BDI), and 7 and 8 (BDIII), whereas no binding was seen in lanes 5 and 6 (BDII) of the EMSA (denoted by arrowhead ). Lane 9 is empty vector incubated with consensus sequence. C, Consensus, BDI, and BDIII probes were incubated with WT or MUT OTX2 protein, followed by addition of a polyclonal OTX2 antibody resulting in “supershifted” (SS) protein-DNA complexes by EMSA (denoted by the double arrowhead ).

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: A Novel Dominant Negative Mutation of OTX2 Associated with Combined Pituitary Hormone Deficiency

    doi: 10.1210/jc.2008-1189

    Figure Lengend Snippet: A, An EMSA was performed using the consensus OTX2 binding sequence incubated with in vitro transcribed and translated empty vector, WT or N233S (MUT) OTX2 protein. Lanes 2–4 and 5–7 demonstrate increased intensity of binding with the addition of increasing quantities of in vitro translated OTX2 (2, 4, or 8 μl). The OTX2 complex is denoted by an arrowhead . B, Probes containing consensus, BDI, BDII, and BDIII sites were incubated with in vitro translated WT or MUT OTX2. Binding of both WT and MUT OTX2 was observed, as shown in lanes 1 and 2 (CON), 3 and 4 (BDI), and 7 and 8 (BDIII), whereas no binding was seen in lanes 5 and 6 (BDII) of the EMSA (denoted by arrowhead ). Lane 9 is empty vector incubated with consensus sequence. C, Consensus, BDI, and BDIII probes were incubated with WT or MUT OTX2 protein, followed by addition of a polyclonal OTX2 antibody resulting in “supershifted” (SS) protein-DNA complexes by EMSA (denoted by the double arrowhead ).

    Article Snippet: Anti-OTX2 antiserum was generated in rabbits immunized with the peptide SCPAATPRKQRRERT (residues 37–51) (Invitrogen).

    Techniques: Binding Assay, Sequencing, Incubation, In Vitro, Plasmid Preparation

    Schematic illustration of OTX2 (297aa) showing the N-terminal domain (1–47aa), homeodomain (HD) (47–102aa), poly-glutamine stretch (PolyQ) (102–109aa), SIWSPA region (158–163aa), and repeated OTX-tail motif (Otx1 TF) (263–275aa and 281–293aa). A heterozygous mutation, A698G, was visualized by electropherogram and results in a change from an asparagine to a serine residue (codon 233). This mutation is located in the previously described OTX1 transcription factor region and was found in two unrelated patients of nonconsanguineous mating. ex, Exon.

    Journal: The Journal of Clinical Endocrinology and Metabolism

    Article Title: A Novel Dominant Negative Mutation of OTX2 Associated with Combined Pituitary Hormone Deficiency

    doi: 10.1210/jc.2008-1189

    Figure Lengend Snippet: Schematic illustration of OTX2 (297aa) showing the N-terminal domain (1–47aa), homeodomain (HD) (47–102aa), poly-glutamine stretch (PolyQ) (102–109aa), SIWSPA region (158–163aa), and repeated OTX-tail motif (Otx1 TF) (263–275aa and 281–293aa). A heterozygous mutation, A698G, was visualized by electropherogram and results in a change from an asparagine to a serine residue (codon 233). This mutation is located in the previously described OTX1 transcription factor region and was found in two unrelated patients of nonconsanguineous mating. ex, Exon.

    Article Snippet: Anti-OTX2 antiserum was generated in rabbits immunized with the peptide SCPAATPRKQRRERT (residues 37–51) (Invitrogen).

    Techniques: Mutagenesis

    OTX2 is associated with higher levels of activity when paired with NEUROD1 and arranged in clusters A. OTX2 bound distal sites show a wide range of H3K27ac levels. Heatmaps depicting OTX2 ChIP-seq (purple) in cell lines and H3K27ac ChIP-seq (green) in cell lines and tumors. Sites where OTX2 is arranged as clusters of at least three peaks are marked. Rows are ordered by the average H3K27ac levels in cell lines and tumors and show a 10 kb region centered on each OTX2 binding site. Two categories of sites are shown: consistently Active sites (top; n=7053) and consistently Inactive sites (bottom; n=5751). B. One example of each category of OTX2 binding sites in D341 cell line: Active site (top) Inactive site (bottom). OTX2 ChIP-seq signals are shown in purple and H3K27ac ChIP-seq signals are shown in green. C. OTX2 enhancer activity is linked to higher gene expression levels in medulloblastoma primary tumors and D341 medulloblastoma cells. FPKM levels of the closest genes in each category of OTX2 peak: Active (green) and Inactive (yellow). *** Indicates p value

    Journal: Cancer discovery

    Article Title: OTX2 activity at distal regulatory elements shapes the chromatin landscape of Group 3 medulloblastoma

    doi: 10.1158/2159-8290.CD-16-0844

    Figure Lengend Snippet: OTX2 is associated with higher levels of activity when paired with NEUROD1 and arranged in clusters A. OTX2 bound distal sites show a wide range of H3K27ac levels. Heatmaps depicting OTX2 ChIP-seq (purple) in cell lines and H3K27ac ChIP-seq (green) in cell lines and tumors. Sites where OTX2 is arranged as clusters of at least three peaks are marked. Rows are ordered by the average H3K27ac levels in cell lines and tumors and show a 10 kb region centered on each OTX2 binding site. Two categories of sites are shown: consistently Active sites (top; n=7053) and consistently Inactive sites (bottom; n=5751). B. One example of each category of OTX2 binding sites in D341 cell line: Active site (top) Inactive site (bottom). OTX2 ChIP-seq signals are shown in purple and H3K27ac ChIP-seq signals are shown in green. C. OTX2 enhancer activity is linked to higher gene expression levels in medulloblastoma primary tumors and D341 medulloblastoma cells. FPKM levels of the closest genes in each category of OTX2 peak: Active (green) and Inactive (yellow). *** Indicates p value

    Article Snippet: Cells were permeabilized 10 minutes at RT in 1X PBS containing 0.5 % Triton X-100 then blocked for 30 minutes at RT, stained with OTX2 antibody for 2 hours at RT and with Alexa-Fluor 546-conjugated secondary antibody (Life Technologies) for 1h at RT.

    Techniques: Activity Assay, Chromatin Immunoprecipitation, Binding Assay, Expressing

    The majority of active enhancers shared by primary Group 3 medulloblastomas are bound by the transcription factor OTX2 A. Identification of a set of 9621 shared active enhancers in primary Group 3 medulloblastoma tumors. Heatmaps depict H3K27ac (green) and H3K4me1 (blue) ChIP-seq signals in 5 frozen primary medulloblastoma tumors. Similar chromatin signals are found in two Group 3 cell lines (D341 and D283). Each row shows a 10 kb region centered on the active enhancer coordinates, ranked by average H3K27ac signals. B. Two examples of the set of active enhancers shared by Group 3 medulloblastoma tumors. H3K27ac ChIP-seq signals are shown in green and consistently active regions are marked in light gray. C. Genes associated with the Group 3 active enhancer set are expressed at higher levels in primary tumors and in the D341 cell line. Boxplot of RNA-seq FPKM expression values for genes closest to Group 3 active enhancers (red) compared to other loci (blue). *** Indicates p value

    Journal: Cancer discovery

    Article Title: OTX2 activity at distal regulatory elements shapes the chromatin landscape of Group 3 medulloblastoma

    doi: 10.1158/2159-8290.CD-16-0844

    Figure Lengend Snippet: The majority of active enhancers shared by primary Group 3 medulloblastomas are bound by the transcription factor OTX2 A. Identification of a set of 9621 shared active enhancers in primary Group 3 medulloblastoma tumors. Heatmaps depict H3K27ac (green) and H3K4me1 (blue) ChIP-seq signals in 5 frozen primary medulloblastoma tumors. Similar chromatin signals are found in two Group 3 cell lines (D341 and D283). Each row shows a 10 kb region centered on the active enhancer coordinates, ranked by average H3K27ac signals. B. Two examples of the set of active enhancers shared by Group 3 medulloblastoma tumors. H3K27ac ChIP-seq signals are shown in green and consistently active regions are marked in light gray. C. Genes associated with the Group 3 active enhancer set are expressed at higher levels in primary tumors and in the D341 cell line. Boxplot of RNA-seq FPKM expression values for genes closest to Group 3 active enhancers (red) compared to other loci (blue). *** Indicates p value

    Article Snippet: Cells were permeabilized 10 minutes at RT in 1X PBS containing 0.5 % Triton X-100 then blocked for 30 minutes at RT, stained with OTX2 antibody for 2 hours at RT and with Alexa-Fluor 546-conjugated secondary antibody (Life Technologies) for 1h at RT.

    Techniques: Chromatin Immunoprecipitation, RNA Sequencing Assay, Expressing

    OTX2 maintains enhancer activity in Group 3 medulloblastoma and can operate as a pioneer factor in primary cells A. OTX2 is required to maintain marks of enhancer activity in D341 medulloblastoma cells. Top: A heatmap shows changes in H3K27ac ChIP-seq at distal sites with decreased OTX2 binding following knockdown with lentiviral shRNA (19513 sites are shown). Bottom: Immunoblotting for OTX2 in the two D341 knockdown experiments shows decreases in OTX2 protein levels. B. Depletion of OTX2 has a strong effect on chromatin states of Active OTX2 sites compared to Inactive sites. Histograms show changes in H3K27ac at sites with decreased OTX2 after infection of D341 medulloblastoma cells with shRNA lentiviruses. Top: H3K27ac decreases in Active sites after OTX2 depletion (n=2649). Bottom: Inactive sites are mostly unaffected by OTX2 depletion (n=2240). The red line indicates no variation; blue bars correspond to more than two-fold decrease and red bars to more than two-fold increase. C. Examples of ChIP-seq tracks of H3K27ac on Active and Inactive OTX2 distal sites following OTX2 knockdown in D341 medulloblastoma cells. OTX2 is shown in purple and H3K27ac in green. Regions of interest are shown in light gray. D. Depletion of NEUROD1 affects chromatin states at OTX2 bound enhancers. Left: NEUROD1 immunoblotting in D341 knockdown experiments. Right: Histogram plot showing H3K27ac ChIP-seq changes at OTX2 distal sites with decreased NEUROD1 after infection of D341 medulloblastoma cells with lentiviral shRNA (3377 sites are shown). The red line indicates no variation; blue bars correspond to more than two-fold decrease and red bars to more than two-fold increase. E. Expression of ectopic OTX2 in primary mesenchymal stem cells (MSCs). Top: OTX2 protein is detected in MSCs after lentiviral infection. Bottom: Immunofluorescence demonstrates the absence of OTX2 signals in control cells and the presence of nuclear signals after introduction of ectopic OTX2 in MSCs (72 hours post-infection). F. OTX2 creates medulloblastoma enhancers de novo in MSCs. Heatmaps depict OTX2 (purple), H3K4me1 (blue) and H3K27ac (green) ChIP-seq signals as well as ATAC-seq (black) on 12286 de novo enhancers 72 hours after OTX2 expression in MSCs. Each row shows a 10 kb region centered on OTX2 binding sites in medulloblastoma. G. Examples of OTX2 induced de novo enhancer creation in MSCs. OTX2 (purple), H3K4me1 (blue) and H3K27ac (green) ChIP-seq as well as ATAC-seq (black). Regions of interest are shown in light gray. H. OTX2 interacts with chromatin modifying complexes associated with enhancer chromatin states. Co-immunoprecipitation experiments were performed in D341 nuclear extracts and show interactions with components involved in chromatin opening (SMARCA4, SMARCA2, SMARCC1), and deposition of the enhancer marks H3K4me1 (UTX, ASH2) and H3K27ac (p300).

    Journal: Cancer discovery

    Article Title: OTX2 activity at distal regulatory elements shapes the chromatin landscape of Group 3 medulloblastoma

    doi: 10.1158/2159-8290.CD-16-0844

    Figure Lengend Snippet: OTX2 maintains enhancer activity in Group 3 medulloblastoma and can operate as a pioneer factor in primary cells A. OTX2 is required to maintain marks of enhancer activity in D341 medulloblastoma cells. Top: A heatmap shows changes in H3K27ac ChIP-seq at distal sites with decreased OTX2 binding following knockdown with lentiviral shRNA (19513 sites are shown). Bottom: Immunoblotting for OTX2 in the two D341 knockdown experiments shows decreases in OTX2 protein levels. B. Depletion of OTX2 has a strong effect on chromatin states of Active OTX2 sites compared to Inactive sites. Histograms show changes in H3K27ac at sites with decreased OTX2 after infection of D341 medulloblastoma cells with shRNA lentiviruses. Top: H3K27ac decreases in Active sites after OTX2 depletion (n=2649). Bottom: Inactive sites are mostly unaffected by OTX2 depletion (n=2240). The red line indicates no variation; blue bars correspond to more than two-fold decrease and red bars to more than two-fold increase. C. Examples of ChIP-seq tracks of H3K27ac on Active and Inactive OTX2 distal sites following OTX2 knockdown in D341 medulloblastoma cells. OTX2 is shown in purple and H3K27ac in green. Regions of interest are shown in light gray. D. Depletion of NEUROD1 affects chromatin states at OTX2 bound enhancers. Left: NEUROD1 immunoblotting in D341 knockdown experiments. Right: Histogram plot showing H3K27ac ChIP-seq changes at OTX2 distal sites with decreased NEUROD1 after infection of D341 medulloblastoma cells with lentiviral shRNA (3377 sites are shown). The red line indicates no variation; blue bars correspond to more than two-fold decrease and red bars to more than two-fold increase. E. Expression of ectopic OTX2 in primary mesenchymal stem cells (MSCs). Top: OTX2 protein is detected in MSCs after lentiviral infection. Bottom: Immunofluorescence demonstrates the absence of OTX2 signals in control cells and the presence of nuclear signals after introduction of ectopic OTX2 in MSCs (72 hours post-infection). F. OTX2 creates medulloblastoma enhancers de novo in MSCs. Heatmaps depict OTX2 (purple), H3K4me1 (blue) and H3K27ac (green) ChIP-seq signals as well as ATAC-seq (black) on 12286 de novo enhancers 72 hours after OTX2 expression in MSCs. Each row shows a 10 kb region centered on OTX2 binding sites in medulloblastoma. G. Examples of OTX2 induced de novo enhancer creation in MSCs. OTX2 (purple), H3K4me1 (blue) and H3K27ac (green) ChIP-seq as well as ATAC-seq (black). Regions of interest are shown in light gray. H. OTX2 interacts with chromatin modifying complexes associated with enhancer chromatin states. Co-immunoprecipitation experiments were performed in D341 nuclear extracts and show interactions with components involved in chromatin opening (SMARCA4, SMARCA2, SMARCC1), and deposition of the enhancer marks H3K4me1 (UTX, ASH2) and H3K27ac (p300).

    Article Snippet: Cells were permeabilized 10 minutes at RT in 1X PBS containing 0.5 % Triton X-100 then blocked for 30 minutes at RT, stained with OTX2 antibody for 2 hours at RT and with Alexa-Fluor 546-conjugated secondary antibody (Life Technologies) for 1h at RT.

    Techniques: Activity Assay, Chromatin Immunoprecipitation, Binding Assay, shRNA, Infection, Expressing, Immunofluorescence, Immunoprecipitation

    Inactivation of Lhx2 with Hes1 CreERT2 at E15.5 results in the overproduction of rods. A–H , Sections from control and Hes1 CreERT2 /+ ;Lhx2 f /− eyes stained with antibodies against Pou4f (RGC precursors, A , B ), Otx2 (photoreceptor precursors, C , D ), Nr2e3 (rod precursors, E , F ), and Rxrγ (cone precursors, G , H ). I , The number of RGC precursors is unchanged, while the number of photoreceptor precursors in the NBL is increased due to the selective overproduction of rods (* p = 0.0007 and * p = 0.0004, respectively; n = 3 mice for each genotype; n > 200 marker+ cells for each genotype). J–O , Immunostaining for Bhlhb5 ( J , K ), Calretinin ( L , M ), and Gaba ( N , O ) show that amacrine cell precursors are slightly increased. P–S , Immunostaining for Vsx2 ( P , Q ) and Vsx1 ( R , S ) show that bipolar cells are decreased. T–W , At P9, Lhx2, predominantly expressed in Müller glia, is largely absent in the Hes1 CreERT2 /+ ;Lhx2 f /− retina ( T , U ). Müller glia are still present, but disorganized, as indicated by Sox9 ( V , W ). X–AA , Immunostaining for Gfap ( X , Y ) and Pcna ( Z , AA ) show that the Lhx2 -inactivated Müller glia are reactive and proliferative. Time points above each part indicate the time of inactivation and analysis, respectively. Scale bars:100 μm. INL, inner nuclear layer.

    Journal: The Journal of Neuroscience

    Article Title: Lhx2 Balances Progenitor Maintenance with Neurogenic Output and Promotes Competence State Progression in the Developing Retina

    doi: 10.1523/JNEUROSCI.1494-13.2013

    Figure Lengend Snippet: Inactivation of Lhx2 with Hes1 CreERT2 at E15.5 results in the overproduction of rods. A–H , Sections from control and Hes1 CreERT2 /+ ;Lhx2 f /− eyes stained with antibodies against Pou4f (RGC precursors, A , B ), Otx2 (photoreceptor precursors, C , D ), Nr2e3 (rod precursors, E , F ), and Rxrγ (cone precursors, G , H ). I , The number of RGC precursors is unchanged, while the number of photoreceptor precursors in the NBL is increased due to the selective overproduction of rods (* p = 0.0007 and * p = 0.0004, respectively; n = 3 mice for each genotype; n > 200 marker+ cells for each genotype). J–O , Immunostaining for Bhlhb5 ( J , K ), Calretinin ( L , M ), and Gaba ( N , O ) show that amacrine cell precursors are slightly increased. P–S , Immunostaining for Vsx2 ( P , Q ) and Vsx1 ( R , S ) show that bipolar cells are decreased. T–W , At P9, Lhx2, predominantly expressed in Müller glia, is largely absent in the Hes1 CreERT2 /+ ;Lhx2 f /− retina ( T , U ). Müller glia are still present, but disorganized, as indicated by Sox9 ( V , W ). X–AA , Immunostaining for Gfap ( X , Y ) and Pcna ( Z , AA ) show that the Lhx2 -inactivated Müller glia are reactive and proliferative. Time points above each part indicate the time of inactivation and analysis, respectively. Scale bars:100 μm. INL, inner nuclear layer.

    Article Snippet: Primary antibodies used were as follows: anti-LHX2 (Edwin Monuki, University of California, Irvine; 1:50), anti-LHX2 (Santa Cruz Biotechnology; 1:1000), anti-Calretinin (Millipore Bioscience Research Reagents; 1:1000), anti-P27 (BD Bioscience; 1:100), anti-CCND1 (Lab Vision; 1:400), anti-PCNA (DAKO; 1:500), anti-BRN3 (Santa Cruz Biotechnology; 1:50), anti-SOX2 (Abcam; 1:400), anti-AQP4 (Santa Cruz Biotechnology; 1:300), anti-RXRγ (Santa Cruz Biotechnology; 1:200), anti-NR2E3 (Anand Swaroop, National Eye Institute; 1:100), anti-BHLHB5 (Santa Cruz Biotechnology; 1:1000), anti-GABA (Sigma; 1:1000), anti-VSX2 (Exα Biologicals; 1:300), anti-VSX1 ( ; 1:500), anti-SOX9 (Millipore Bioscience Research Reagents; 1:400), anti-CRALBP (John Saari, University of Washington; 1:1000), anti-GFAP (Lipshaw; 1:1000), anti-βIII-Tubulin (Covance; 1:1000), anti-PTF1A (Helena Edlund, Umea University; 1:800), anti-PTF1A (Jane Johnson, University of Texas Southwestern; 1:8000), and anti-OTX2 (Millipore Bioscience Research Reagents; 1:15,000).

    Techniques: Staining, Mouse Assay, Marker, Immunostaining

    Inactivation of Lhx2 at E12.5 does not result in overproduction of other early born cell types. A–L , Sections from control and Hes1 CreERT2 /+ ;Lhx2 f /− eyes stained with antibodies against Lhx2, Pou4f (RGC precursors, C , D ), Ptf1a (horizontal and amacrine cell precursors, E , F ), Sox2 (amacrine cell precursors, G , H ), Bhlhb5 (amacrine cell precursors, I , J ), and Otx2 (photoreceptor precursors, K , L ) show that, similar to inactivation at E10.5, RGC precursors are selectively overproduced while other early born cell types are unchanged or decreased in number. M–T , The progeny of Lhx2 -inactivated RPCs are more likely to adopt the RGC fate and subsequently express the precursor marker Pou4f (arrowheads, O–T ). Tomato expression marks the recombined population in both control and Hes1 CreERT2 /+ ;Lhx2 f /− eyes ( O , Q , and S show the boxed area in M ; P , R , and T show the boxed area in N ). U , The percentage of the recombined population expressing Pou4f+ is significantly increased while the percentages expressing Ptf1a+ and Otx2+ are decreased (* p = 0.0429, * p = 0.0021, and p = 0.066, respectively; n = 3 mice for each genotype; n > 1000 Tomato+ cells for each experiment). V–Y , Lhx2 inactivation proceeds with similar kinetics whether initiated at E10.5 ( V , W ) or E12.5 ( X , Y ). In each case, only a few Lhx2+ cells remain after 2 d. Error bars indicate SEM. Scale bars: 100 μm.

    Journal: The Journal of Neuroscience

    Article Title: Lhx2 Balances Progenitor Maintenance with Neurogenic Output and Promotes Competence State Progression in the Developing Retina

    doi: 10.1523/JNEUROSCI.1494-13.2013

    Figure Lengend Snippet: Inactivation of Lhx2 at E12.5 does not result in overproduction of other early born cell types. A–L , Sections from control and Hes1 CreERT2 /+ ;Lhx2 f /− eyes stained with antibodies against Lhx2, Pou4f (RGC precursors, C , D ), Ptf1a (horizontal and amacrine cell precursors, E , F ), Sox2 (amacrine cell precursors, G , H ), Bhlhb5 (amacrine cell precursors, I , J ), and Otx2 (photoreceptor precursors, K , L ) show that, similar to inactivation at E10.5, RGC precursors are selectively overproduced while other early born cell types are unchanged or decreased in number. M–T , The progeny of Lhx2 -inactivated RPCs are more likely to adopt the RGC fate and subsequently express the precursor marker Pou4f (arrowheads, O–T ). Tomato expression marks the recombined population in both control and Hes1 CreERT2 /+ ;Lhx2 f /− eyes ( O , Q , and S show the boxed area in M ; P , R , and T show the boxed area in N ). U , The percentage of the recombined population expressing Pou4f+ is significantly increased while the percentages expressing Ptf1a+ and Otx2+ are decreased (* p = 0.0429, * p = 0.0021, and p = 0.066, respectively; n = 3 mice for each genotype; n > 1000 Tomato+ cells for each experiment). V–Y , Lhx2 inactivation proceeds with similar kinetics whether initiated at E10.5 ( V , W ) or E12.5 ( X , Y ). In each case, only a few Lhx2+ cells remain after 2 d. Error bars indicate SEM. Scale bars: 100 μm.

    Article Snippet: Primary antibodies used were as follows: anti-LHX2 (Edwin Monuki, University of California, Irvine; 1:50), anti-LHX2 (Santa Cruz Biotechnology; 1:1000), anti-Calretinin (Millipore Bioscience Research Reagents; 1:1000), anti-P27 (BD Bioscience; 1:100), anti-CCND1 (Lab Vision; 1:400), anti-PCNA (DAKO; 1:500), anti-BRN3 (Santa Cruz Biotechnology; 1:50), anti-SOX2 (Abcam; 1:400), anti-AQP4 (Santa Cruz Biotechnology; 1:300), anti-RXRγ (Santa Cruz Biotechnology; 1:200), anti-NR2E3 (Anand Swaroop, National Eye Institute; 1:100), anti-BHLHB5 (Santa Cruz Biotechnology; 1:1000), anti-GABA (Sigma; 1:1000), anti-VSX2 (Exα Biologicals; 1:300), anti-VSX1 ( ; 1:500), anti-SOX9 (Millipore Bioscience Research Reagents; 1:400), anti-CRALBP (John Saari, University of Washington; 1:1000), anti-GFAP (Lipshaw; 1:1000), anti-βIII-Tubulin (Covance; 1:1000), anti-PTF1A (Helena Edlund, Umea University; 1:800), anti-PTF1A (Jane Johnson, University of Texas Southwestern; 1:8000), and anti-OTX2 (Millipore Bioscience Research Reagents; 1:15,000).

    Techniques: Staining, Marker, Expressing, Mouse Assay

    Tau deficiency reduced the expression of transcription factor OTX2 in mDANs of the VTA. (A) Western blot showed the expression level of Lmx1b, Pitx3, and OTX2 in the midbrain homogenates of tau +/+ , tau +/ − , and tau − / − mice at 18 months of age. (B) Densitometry analyses depicted the Lmx1b, Pitx3, and OTX2 expression level, normalized with β-actin ( n = 5 per genotype, P > 0.05). (C) OTX2 (green) and TH (red) co-staining in the VTA coronal sections of tau +/+ , tau +/ − , and tau − / − mice at 18 months of age. Scale bar = 20 lm. (D) Densitometry analyses of (C) showed OTX2 signals intensity in TH-positive neurons and TH-negative neurons ( n = 5 per genotype). All data were presented as mean ± SEM, * P

    Journal: Neuroscience

    Article Title: Tau Deficiency Down-Regulated Transcription Factor Orthodenticle Homeobox 2 Expression in the Dopaminergic Neurons in Ventral Tegmental Area and Caused No Obvious Motor Deficits in Mice

    doi: 10.1016/j.neuroscience.2018.01.002

    Figure Lengend Snippet: Tau deficiency reduced the expression of transcription factor OTX2 in mDANs of the VTA. (A) Western blot showed the expression level of Lmx1b, Pitx3, and OTX2 in the midbrain homogenates of tau +/+ , tau +/ − , and tau − / − mice at 18 months of age. (B) Densitometry analyses depicted the Lmx1b, Pitx3, and OTX2 expression level, normalized with β-actin ( n = 5 per genotype, P > 0.05). (C) OTX2 (green) and TH (red) co-staining in the VTA coronal sections of tau +/+ , tau +/ − , and tau − / − mice at 18 months of age. Scale bar = 20 lm. (D) Densitometry analyses of (C) showed OTX2 signals intensity in TH-positive neurons and TH-negative neurons ( n = 5 per genotype). All data were presented as mean ± SEM, * P

    Article Snippet: The membranes were transferred to polyvinylidene fluoride (PVDF) (Millipore) and then they were blocked with 5% not-fat milk for 1 h. The membranes were then incubated with the following primary antibodies: Pitx3 (1:1000, abcam), TH (1:500, Santa Cruz), OTX2 (1:1000, Sigma), Lmx1b (1:500, Sigma), DAT (1:1000, Millipore), Girk2 (1:1000, Alomone labs), and β-actin (1:5000; Sigma).

    Techniques: Expressing, Western Blot, Mouse Assay, Staining

    Tau deficiency influenced the expression level of OTX2 in some brain regions. (A) OTX2 staining (green) in the superficial subdivision of SC, deep subdivision of SC and visual cortex of 18-month-old tau +/+ , tau +/ − and tau − / − mice. Scale bar = 20 lm. (B) Densitometry analyses of A, showed OTX2 signals intensity in the neurons from superficial subdivision of SC, deep subdivision of SC and visual cortex ( n = 5 per genotype). All data were presented as mean ± SEM, * P

    Journal: Neuroscience

    Article Title: Tau Deficiency Down-Regulated Transcription Factor Orthodenticle Homeobox 2 Expression in the Dopaminergic Neurons in Ventral Tegmental Area and Caused No Obvious Motor Deficits in Mice

    doi: 10.1016/j.neuroscience.2018.01.002

    Figure Lengend Snippet: Tau deficiency influenced the expression level of OTX2 in some brain regions. (A) OTX2 staining (green) in the superficial subdivision of SC, deep subdivision of SC and visual cortex of 18-month-old tau +/+ , tau +/ − and tau − / − mice. Scale bar = 20 lm. (B) Densitometry analyses of A, showed OTX2 signals intensity in the neurons from superficial subdivision of SC, deep subdivision of SC and visual cortex ( n = 5 per genotype). All data were presented as mean ± SEM, * P

    Article Snippet: The membranes were transferred to polyvinylidene fluoride (PVDF) (Millipore) and then they were blocked with 5% not-fat milk for 1 h. The membranes were then incubated with the following primary antibodies: Pitx3 (1:1000, abcam), TH (1:500, Santa Cruz), OTX2 (1:1000, Sigma), Lmx1b (1:500, Sigma), DAT (1:1000, Millipore), Girk2 (1:1000, Alomone labs), and β-actin (1:5000; Sigma).

    Techniques: Expressing, Staining, Mouse Assay

    Expression of Otx1 and Otx2 in adult mouse brain. ( A ) Analysis of Otx1 and Otx2 expression by quantitative RT-PCR on extracts from lateral geniculate nucleus (LGN), visual cortex (V Cx), superior colliculus (S Col) and cerebellum (Cb). The fold-difference in expression is calculated relative to Otx1 in LGN. The Otx2 locus is silent in visual cortex. ( B ) Non-cell autonomous Otx2 is found in visual cortex. Immunostaining in wild type mice reveals Otx1/2 cells in layers IV and V of visual cortex, including cells with perineuronal nets (stained by WFA lectin) enriched in layer IV. Staining for Otx2 persists in Otx1 null mice (Otx1 KO). Scale bar, 50 µm.

    Journal: F1000Research

    Article Title: Immunoprecipitation and mass spectrometry identify non-cell autonomous Otx2 homeoprotein in the granular and supragranular layers of mouse visual cortex

    doi: 10.12688/f1000research.4869.1

    Figure Lengend Snippet: Expression of Otx1 and Otx2 in adult mouse brain. ( A ) Analysis of Otx1 and Otx2 expression by quantitative RT-PCR on extracts from lateral geniculate nucleus (LGN), visual cortex (V Cx), superior colliculus (S Col) and cerebellum (Cb). The fold-difference in expression is calculated relative to Otx1 in LGN. The Otx2 locus is silent in visual cortex. ( B ) Non-cell autonomous Otx2 is found in visual cortex. Immunostaining in wild type mice reveals Otx1/2 cells in layers IV and V of visual cortex, including cells with perineuronal nets (stained by WFA lectin) enriched in layer IV. Staining for Otx2 persists in Otx1 null mice (Otx1 KO). Scale bar, 50 µm.

    Article Snippet: Membranes were incubated overnight at 4°C with anti-Otx2 (rabbit polyclonal, 1/1,000, Abcam ab21990) and then with HRP-coupled anti-rabbit IgG (1/2,000, GE Healthcare NA934) 1 h at RT.

    Techniques: Expressing, Quantitative RT-PCR, Immunostaining, Mouse Assay, Staining

    Otx2 protein in the granular and supragranular layers of adult mouse visual cortex. ( A ) Immunoblots for Otx1/2 of immunoprecipitation (IP) experiments on extracts from visual cortex and choroid plexus. ( B ) Diagram of finely dissected region for extracts containing granular (IV) and supragranular (I-III) layers of visual cortex. ( C ) Immunoblot for Otx1/2 of samples from IP using cross-linked magnetic beads and finely dissected extracts. Choroid plexus (Ch Pl) extract was used to control for Otx2 migration velocity. ( D ) The peptides (red, bold) matching Otx2 protein identified by high-resolution mass spectrometry. Only the homeodomain sequence of Otx2 (amino acids 38–97) is shown. The amino acid differing in Otx1 is highlighted in green, while amino acids that differ in Crx are highlighted in green and in yellow.

    Journal: F1000Research

    Article Title: Immunoprecipitation and mass spectrometry identify non-cell autonomous Otx2 homeoprotein in the granular and supragranular layers of mouse visual cortex

    doi: 10.12688/f1000research.4869.1

    Figure Lengend Snippet: Otx2 protein in the granular and supragranular layers of adult mouse visual cortex. ( A ) Immunoblots for Otx1/2 of immunoprecipitation (IP) experiments on extracts from visual cortex and choroid plexus. ( B ) Diagram of finely dissected region for extracts containing granular (IV) and supragranular (I-III) layers of visual cortex. ( C ) Immunoblot for Otx1/2 of samples from IP using cross-linked magnetic beads and finely dissected extracts. Choroid plexus (Ch Pl) extract was used to control for Otx2 migration velocity. ( D ) The peptides (red, bold) matching Otx2 protein identified by high-resolution mass spectrometry. Only the homeodomain sequence of Otx2 (amino acids 38–97) is shown. The amino acid differing in Otx1 is highlighted in green, while amino acids that differ in Crx are highlighted in green and in yellow.

    Article Snippet: Membranes were incubated overnight at 4°C with anti-Otx2 (rabbit polyclonal, 1/1,000, Abcam ab21990) and then with HRP-coupled anti-rabbit IgG (1/2,000, GE Healthcare NA934) 1 h at RT.

    Techniques: Western Blot, Immunoprecipitation, Magnetic Beads, Migration, Mass Spectrometry, Sequencing

    OTX2 knockdown significantly inhibits cell proliferation and self-renewal in both trans-hENs and D283 MB cells. (A) Western blot validation of OTX2 knockdown in three independent siRNA sequences relative to scramble siRNA. β-actin serves as a loading control. (B) Representative images of D283 MB cells in adherent culture following 4 days knockdown of OTX2. Scale bars: 400 µm. (C-F) Quantification of total cell number (C,E) and cell viability (D,F) in D283 (C,D) and trans-hENs (E,F) following knockdown of OTX2 over 4 days. (G-N) Self-renewal capacity following OTX2 knockdown in D283 (G-J) and trans-hENs (K-N). In D283, self-renewal capacity was almost completely inhibited following passage to secondary spheres (G-H), and this was evident in representative brightfield images of secondary spheres in (J). (K-M) Significant decreases in neurosphere-forming capacity were also evident in trans-hENs (K,L) following OTX2 knockdown and similar to D283, self-renewal capacity was significantly inhibited (N). For both cell lines, cell viability was not significantly decreased (H,M). Error bars: s.e.m. * P

    Journal: Disease Models & Mechanisms

    Article Title: OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

    doi: 10.1242/dmm.020594

    Figure Lengend Snippet: OTX2 knockdown significantly inhibits cell proliferation and self-renewal in both trans-hENs and D283 MB cells. (A) Western blot validation of OTX2 knockdown in three independent siRNA sequences relative to scramble siRNA. β-actin serves as a loading control. (B) Representative images of D283 MB cells in adherent culture following 4 days knockdown of OTX2. Scale bars: 400 µm. (C-F) Quantification of total cell number (C,E) and cell viability (D,F) in D283 (C,D) and trans-hENs (E,F) following knockdown of OTX2 over 4 days. (G-N) Self-renewal capacity following OTX2 knockdown in D283 (G-J) and trans-hENs (K-N). In D283, self-renewal capacity was almost completely inhibited following passage to secondary spheres (G-H), and this was evident in representative brightfield images of secondary spheres in (J). (K-M) Significant decreases in neurosphere-forming capacity were also evident in trans-hENs (K,L) following OTX2 knockdown and similar to D283, self-renewal capacity was significantly inhibited (N). For both cell lines, cell viability was not significantly decreased (H,M). Error bars: s.e.m. * P

    Article Snippet: Blots were incubated in primary rabbit anti-OTX2 (1:500 dilution; Abcam, ab21990) or rabbit anti-SOX2 antibody (1:500 dilution; Cell Signaling Technologies, #3579) for 2 h at room temperature. β-actin (Sigma) was used as a loading control.

    Techniques: Western Blot

    Overexpression of SOX2 in OTX2+ Daoy and hEN cells rescues self-renewal and migration deficits. (A) pReceiver-Lv-105 vector construct used for stable overexpession of SOX2 in hEN and Daoy SHH MB cells in the presence or absence of OTX2 overexpression. (B,C) Western blots depicting overexpression of SOX2 in Daoy SHH MB (B) and hEN cells (C) in the presence or absence of OTX2 overexpression. (D-K) SOX2 overexpression in OTX2+ cells results in a significant recovery in self-renewal for both Daoy (D-G) and hEN (H-K). (D,F) Representative images of spheres over subsequent passage following SOX2 overexpression in OTX2+ Daoy. (H,J) Representative images of spheres over subsequent passage following SOX2 overexpression in OTX2+ hENs. Scale bars: 400 µm. (L-O) SOX2 overexpression in OTX2+ cells rescues cell migration deficits in both Daoy (L,M) and hEN (N,O). Error bars: s.e.m. * P

    Journal: Disease Models & Mechanisms

    Article Title: OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

    doi: 10.1242/dmm.020594

    Figure Lengend Snippet: Overexpression of SOX2 in OTX2+ Daoy and hEN cells rescues self-renewal and migration deficits. (A) pReceiver-Lv-105 vector construct used for stable overexpession of SOX2 in hEN and Daoy SHH MB cells in the presence or absence of OTX2 overexpression. (B,C) Western blots depicting overexpression of SOX2 in Daoy SHH MB (B) and hEN cells (C) in the presence or absence of OTX2 overexpression. (D-K) SOX2 overexpression in OTX2+ cells results in a significant recovery in self-renewal for both Daoy (D-G) and hEN (H-K). (D,F) Representative images of spheres over subsequent passage following SOX2 overexpression in OTX2+ Daoy. (H,J) Representative images of spheres over subsequent passage following SOX2 overexpression in OTX2+ hENs. Scale bars: 400 µm. (L-O) SOX2 overexpression in OTX2+ cells rescues cell migration deficits in both Daoy (L,M) and hEN (N,O). Error bars: s.e.m. * P

    Article Snippet: Blots were incubated in primary rabbit anti-OTX2 (1:500 dilution; Abcam, ab21990) or rabbit anti-SOX2 antibody (1:500 dilution; Cell Signaling Technologies, #3579) for 2 h at room temperature. β-actin (Sigma) was used as a loading control.

    Techniques: Over Expression, Migration, Plasmid Preparation, Construct, Western Blot

    Overexpression of OTX2 in Daoy and hEN cells decreases cell growth. (A,B) Endogenous protein levels of OTX2 in hEN versus trans-hEN cultured in neural precursor conditions for 7 days (A) and Daoy (SHH MB variant) versus D283 (Group 4 MB variant; B). β-actin serves as a loading control. (C) LentiORF cassette used for stable overexpression of OTX2 in hEN and Daoy cells. (D,E) Schematic representation of stable Blasticidin selection for OTX2-Daoy and RFP-Daoy cells (D) and sorting procedure used to distinguish between infected (GFP+) and non-infected (GFP−) hEN cells as well as RFP+ control hENs (E). (F,G) Blasticidin-selected Daoy populations (F) and sorted hEN cell populations (G) used for cell proliferation, self-renewal and migration assays in vitro . Scale bars: 200 µm. (H,I) Validation of stable OTX2 overexpression in Daoy (H) and hEN (I) by qPCR and western blot. β-actin serves as a loading control. Error bars: s.e.m. ** P

    Journal: Disease Models & Mechanisms

    Article Title: OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

    doi: 10.1242/dmm.020594

    Figure Lengend Snippet: Overexpression of OTX2 in Daoy and hEN cells decreases cell growth. (A,B) Endogenous protein levels of OTX2 in hEN versus trans-hEN cultured in neural precursor conditions for 7 days (A) and Daoy (SHH MB variant) versus D283 (Group 4 MB variant; B). β-actin serves as a loading control. (C) LentiORF cassette used for stable overexpression of OTX2 in hEN and Daoy cells. (D,E) Schematic representation of stable Blasticidin selection for OTX2-Daoy and RFP-Daoy cells (D) and sorting procedure used to distinguish between infected (GFP+) and non-infected (GFP−) hEN cells as well as RFP+ control hENs (E). (F,G) Blasticidin-selected Daoy populations (F) and sorted hEN cell populations (G) used for cell proliferation, self-renewal and migration assays in vitro . Scale bars: 200 µm. (H,I) Validation of stable OTX2 overexpression in Daoy (H) and hEN (I) by qPCR and western blot. β-actin serves as a loading control. Error bars: s.e.m. ** P

    Article Snippet: Blots were incubated in primary rabbit anti-OTX2 (1:500 dilution; Abcam, ab21990) or rabbit anti-SOX2 antibody (1:500 dilution; Cell Signaling Technologies, #3579) for 2 h at room temperature. β-actin (Sigma) was used as a loading control.

    Techniques: Over Expression, Cell Culture, Variant Assay, Selection, Infection, Migration, In Vitro, Real-time Polymerase Chain Reaction, Western Blot

    OTX2 overexpression results in dysregulation of transcriptome networks associated with cell proliferation and cell motility. Top dysregulated cellular networks consisting of transcripts associated with cell proliferation (A) and cell migration (B). Shaded green areas denote transcripts that are significantly downregulated and red areas denote significantly upregulated transcripts in OTX2 hENs versus hENs. Note that the vast majority of transcripts are downregulated in OTX2 hENs. n =3 biological replicates or independent infections.

    Journal: Disease Models & Mechanisms

    Article Title: OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

    doi: 10.1242/dmm.020594

    Figure Lengend Snippet: OTX2 overexpression results in dysregulation of transcriptome networks associated with cell proliferation and cell motility. Top dysregulated cellular networks consisting of transcripts associated with cell proliferation (A) and cell migration (B). Shaded green areas denote transcripts that are significantly downregulated and red areas denote significantly upregulated transcripts in OTX2 hENs versus hENs. Note that the vast majority of transcripts are downregulated in OTX2 hENs. n =3 biological replicates or independent infections.

    Article Snippet: Blots were incubated in primary rabbit anti-OTX2 (1:500 dilution; Abcam, ab21990) or rabbit anti-SOX2 antibody (1:500 dilution; Cell Signaling Technologies, #3579) for 2 h at room temperature. β-actin (Sigma) was used as a loading control.

    Techniques: Over Expression, Migration

    Overexpression of OTX2 significantly decreases self-renewal and migration in vitro and tumor growth in vivo. (A,B) Representative images of spheres over subsequent passage following OTX2 overexpression in Daoy (A) and hEN cells (B). (C,D) OTX2 overexpression decreases self-renewal capacity in both Daoy (C) and hEN spheres (D). Error bars: s.e.m. * P

    Journal: Disease Models & Mechanisms

    Article Title: OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

    doi: 10.1242/dmm.020594

    Figure Lengend Snippet: Overexpression of OTX2 significantly decreases self-renewal and migration in vitro and tumor growth in vivo. (A,B) Representative images of spheres over subsequent passage following OTX2 overexpression in Daoy (A) and hEN cells (B). (C,D) OTX2 overexpression decreases self-renewal capacity in both Daoy (C) and hEN spheres (D). Error bars: s.e.m. * P

    Article Snippet: Blots were incubated in primary rabbit anti-OTX2 (1:500 dilution; Abcam, ab21990) or rabbit anti-SOX2 antibody (1:500 dilution; Cell Signaling Technologies, #3579) for 2 h at room temperature. β-actin (Sigma) was used as a loading control.

    Techniques: Over Expression, Migration, In Vitro, In Vivo

    OTX2 overexpression significantly decreases levels of hESC genes. (A) Results from global gene expression analysis demonstrating overall downregulation of hESC pluripotency genes in OTX2-overexpressing hENs. Graph represents n =3 biological replicate pairs of OTX2 hEN and hENs. All transcripts are significantly different. (B) Upregulation of representative miRNA targets of LIN28A following OTX2 overexpression in hENs. n =3 independent biological replicates. (C,D) Validation of hESC transcript downregulation in hENs (C) and Daoy (D) following OTX2 overexpression by qPCR. Error bars: s.e.m. * P

    Journal: Disease Models & Mechanisms

    Article Title: OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

    doi: 10.1242/dmm.020594

    Figure Lengend Snippet: OTX2 overexpression significantly decreases levels of hESC genes. (A) Results from global gene expression analysis demonstrating overall downregulation of hESC pluripotency genes in OTX2-overexpressing hENs. Graph represents n =3 biological replicate pairs of OTX2 hEN and hENs. All transcripts are significantly different. (B) Upregulation of representative miRNA targets of LIN28A following OTX2 overexpression in hENs. n =3 independent biological replicates. (C,D) Validation of hESC transcript downregulation in hENs (C) and Daoy (D) following OTX2 overexpression by qPCR. Error bars: s.e.m. * P

    Article Snippet: Blots were incubated in primary rabbit anti-OTX2 (1:500 dilution; Abcam, ab21990) or rabbit anti-SOX2 antibody (1:500 dilution; Cell Signaling Technologies, #3579) for 2 h at room temperature. β-actin (Sigma) was used as a loading control.

    Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction

    Knockdown of OTX2 decreases tumor growth in vivo , and working models depicting divergent roles of OTX2 and relationship to cell state. (A-D) Tumorigenic capacity of D283 OTX2 knockdown (KD; A,B) and trans-hEN OTX2 knockdown (C,D) relative to respective scramble control cells in NOD SCID mice. Scale bars: 200 µm. (E,F) The relationship between hESC genes and normal versus aberrant embryonic neural development and how OTX2 expression is predicted to regulate this process in hEN and SHH MB cells. Following OTX2 overexpression, pluripotent genes are downregulated, and our study provides evidence for a relationship between OTX2 and SOX2. (G) The oncogenic role of OTX2 in trans-hENs or Group 3 and 4 MB cells.

    Journal: Disease Models & Mechanisms

    Article Title: OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells

    doi: 10.1242/dmm.020594

    Figure Lengend Snippet: Knockdown of OTX2 decreases tumor growth in vivo , and working models depicting divergent roles of OTX2 and relationship to cell state. (A-D) Tumorigenic capacity of D283 OTX2 knockdown (KD; A,B) and trans-hEN OTX2 knockdown (C,D) relative to respective scramble control cells in NOD SCID mice. Scale bars: 200 µm. (E,F) The relationship between hESC genes and normal versus aberrant embryonic neural development and how OTX2 expression is predicted to regulate this process in hEN and SHH MB cells. Following OTX2 overexpression, pluripotent genes are downregulated, and our study provides evidence for a relationship between OTX2 and SOX2. (G) The oncogenic role of OTX2 in trans-hENs or Group 3 and 4 MB cells.

    Article Snippet: Blots were incubated in primary rabbit anti-OTX2 (1:500 dilution; Abcam, ab21990) or rabbit anti-SOX2 antibody (1:500 dilution; Cell Signaling Technologies, #3579) for 2 h at room temperature. β-actin (Sigma) was used as a loading control.

    Techniques: In Vivo, Mouse Assay, Expressing, Over Expression

    EGFP Expression in Newly Born Post-mitotic RXRγ-Positive Cones of E15.5 Retinas (A–CII) Transversal sections of E15.5 retinas from Chrnb4- EGFP mice. (A–AII) OTX2 detection is shown in EGFP-positive photoreceptors at the apical retinal side. (B–BII) Double RXRγ- EGFP-positive cones localized at both the apical and basal sides of the retina. (C–CII) EGFP co-expression with PAX6-positive cells adjacent to the basal retinal side is shown. (D) Percentage of double EGFP- RXRγ-positive cells was counted over the EGFP-positive cells detected in the whole, apical, and basal retina. (E) Percentage of RXRγ + -EGFP + was counted over the RXRγ-positive cells detected in the basal and apical-central retina. Error bars correspond to SEM. DAPI, nuclear staining (blue); Ba, basal; Ap, apical; n = 4. Scale bars, 20 μm (A–CII) and 8 μm (magnified pictures in insets).

    Journal: Molecular Therapy

    Article Title: Cone Genesis Tracing by the Chrnb4-EGFP Mouse Line: Evidences of Cellular Material Fusion after Cone Precursor Transplantation

    doi: 10.1016/j.ymthe.2016.12.015

    Figure Lengend Snippet: EGFP Expression in Newly Born Post-mitotic RXRγ-Positive Cones of E15.5 Retinas (A–CII) Transversal sections of E15.5 retinas from Chrnb4- EGFP mice. (A–AII) OTX2 detection is shown in EGFP-positive photoreceptors at the apical retinal side. (B–BII) Double RXRγ- EGFP-positive cones localized at both the apical and basal sides of the retina. (C–CII) EGFP co-expression with PAX6-positive cells adjacent to the basal retinal side is shown. (D) Percentage of double EGFP- RXRγ-positive cells was counted over the EGFP-positive cells detected in the whole, apical, and basal retina. (E) Percentage of RXRγ + -EGFP + was counted over the RXRγ-positive cells detected in the basal and apical-central retina. Error bars correspond to SEM. DAPI, nuclear staining (blue); Ba, basal; Ap, apical; n = 4. Scale bars, 20 μm (A–CII) and 8 μm (magnified pictures in insets).

    Article Snippet: The following antibodies were used: PAX6 (Covance, rabbit, 1:300), OTX2 (Abcam, rabbit, 1:300), S-Opsin (Merck Millipore, rabbit, 1:2,000), ML-Opsin (Merck Millipore, rabbit, 1:2,000), RXRγ (Santa Cruz Biotechnology, rabbit, 1:200), GNAT2 (Santa Cruz Biotechnology, rabbit, 1:200), GNAT1 (Santa Cruz Biotechnology, rabbit, 1:1,000), Zo1 (Invitrogen-Thermo Fisher Scientific, rabbit, 1:2,000), GFAP (Dako Schweiz, rabbit, 1:500), Ki67 (Becton Dickinson, mouse, 1:75), CHRNB4 (Covalab, goat, 1:100), and CHRNB4 (Santa Cruz Biotechnology, goat, 1:100).

    Techniques: Expressing, Mouse Assay, Staining

    EGFP Co-expression with the Post-mitotic Cone Marker RXRγ at E12 (A–CII) Transversal sections of E12 retinas from Chrnb4- EGFP reporter mice. (A–AII) OTX2, essential for photoreceptor differentiation, is detected in elongated EGFP-positive cells adjacent to the apical retinal region. (B–BII) Double RXRγ- EGFP-positive cones localized at both the apical and basal side of the retina. (C–CII) EGFP co-expression with PAX6-positive cells adjacent to the basal retinal region is shown. (A–C) Dashed lines delimit the Ap from the Ba retinal region used for counting in (D). (D) Percentage of double EGFP- RXRγ-positive cells were counted over the EGFP-positive cells detected in the whole, apical-central, and basal retina. Error bars correspond to SEM. (AII, BII, and CII) High-magnification pictures show the corresponding white squares. DAPI, nuclear staining (blue); Ba, basal; Ap, apical; n = 3. Scale bars, 50 μm (A–CI) and 15 μm (magnified insets).

    Journal: Molecular Therapy

    Article Title: Cone Genesis Tracing by the Chrnb4-EGFP Mouse Line: Evidences of Cellular Material Fusion after Cone Precursor Transplantation

    doi: 10.1016/j.ymthe.2016.12.015

    Figure Lengend Snippet: EGFP Co-expression with the Post-mitotic Cone Marker RXRγ at E12 (A–CII) Transversal sections of E12 retinas from Chrnb4- EGFP reporter mice. (A–AII) OTX2, essential for photoreceptor differentiation, is detected in elongated EGFP-positive cells adjacent to the apical retinal region. (B–BII) Double RXRγ- EGFP-positive cones localized at both the apical and basal side of the retina. (C–CII) EGFP co-expression with PAX6-positive cells adjacent to the basal retinal region is shown. (A–C) Dashed lines delimit the Ap from the Ba retinal region used for counting in (D). (D) Percentage of double EGFP- RXRγ-positive cells were counted over the EGFP-positive cells detected in the whole, apical-central, and basal retina. Error bars correspond to SEM. (AII, BII, and CII) High-magnification pictures show the corresponding white squares. DAPI, nuclear staining (blue); Ba, basal; Ap, apical; n = 3. Scale bars, 50 μm (A–CI) and 15 μm (magnified insets).

    Article Snippet: The following antibodies were used: PAX6 (Covance, rabbit, 1:300), OTX2 (Abcam, rabbit, 1:300), S-Opsin (Merck Millipore, rabbit, 1:2,000), ML-Opsin (Merck Millipore, rabbit, 1:2,000), RXRγ (Santa Cruz Biotechnology, rabbit, 1:200), GNAT2 (Santa Cruz Biotechnology, rabbit, 1:200), GNAT1 (Santa Cruz Biotechnology, rabbit, 1:1,000), Zo1 (Invitrogen-Thermo Fisher Scientific, rabbit, 1:2,000), GFAP (Dako Schweiz, rabbit, 1:500), Ki67 (Becton Dickinson, mouse, 1:75), CHRNB4 (Covalab, goat, 1:100), and CHRNB4 (Santa Cruz Biotechnology, goat, 1:100).

    Techniques: Expressing, Marker, Mouse Assay, Staining

    Activation of Otx2 target genes is required at early and late stages of lens placode formation. (A), (B) Otx 2 -EnR inhibits Pax 6 in the lens at stage 17 (50%, n =26). This is rescued by co-injection of Otx 2 ((B); inhibition reduced to 15%, n =20). (C)–(F) Activation of Otx 2 -EnR-GR at stage 10 reduces lens-specific Pax 6 at stage 18 ((D) 52%, n =25); without DEX Pax 6 expression is normal ((C) 0% affected, n =23). FoxE 3 at stage 25 is normal without DEX ((E) 0% affected, n =30), while addition of DEX at stage 18 leads to reduction ((F) 64%, n =22). (G) Otx 2 -E 1 A activates Otx2 targets but does not affect lens Pax 6 (0% affected, n =25). All embryos were injected into the A3 blastomere at the 32-cell stage and are shown in frontal view with dorsal to the top. High magnifications of the lens region are shown below each panel; dotted lines demarcate placodal Pax 6. Turquoise staining reveals the lineage tracer FDX.

    Journal: Developmental Biology

    Article Title: Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning

    doi: 10.1016/j.ydbio.2012.04.025

    Figure Lengend Snippet: Activation of Otx2 target genes is required at early and late stages of lens placode formation. (A), (B) Otx 2 -EnR inhibits Pax 6 in the lens at stage 17 (50%, n =26). This is rescued by co-injection of Otx 2 ((B); inhibition reduced to 15%, n =20). (C)–(F) Activation of Otx 2 -EnR-GR at stage 10 reduces lens-specific Pax 6 at stage 18 ((D) 52%, n =25); without DEX Pax 6 expression is normal ((C) 0% affected, n =23). FoxE 3 at stage 25 is normal without DEX ((E) 0% affected, n =30), while addition of DEX at stage 18 leads to reduction ((F) 64%, n =22). (G) Otx 2 -E 1 A activates Otx2 targets but does not affect lens Pax 6 (0% affected, n =25). All embryos were injected into the A3 blastomere at the 32-cell stage and are shown in frontal view with dorsal to the top. High magnifications of the lens region are shown below each panel; dotted lines demarcate placodal Pax 6. Turquoise staining reveals the lineage tracer FDX.

    Article Snippet: Immunostaining on cryosections was performed ( ) using antibodies against Otx2 (Abcam; 1:50) and Pax3 (Developmental Hybridoma Bank; 1:10) and appropriate secondary antibodies (Invitrogen; 1:1000).

    Techniques: Activation Assay, Injection, Inhibition, Expressing, Staining

    Activation of Otx2 target genes is required at early and late stages of olfactory placode formation. (A), (B) Otx 2 -EnR inhibits the olfactory placode marker Dmrt 4 at stage 21 (59%, n =29). This is rescued by co-injection of Otx 2 ((B) inhibition reduced to 12%, n =33). (C–F) Otx 2 -EnR-GR injections: in the absence of DEX Dmrt 4 expression is normal ((C) 5% affected, n =20); when DEX is added at stage 10 Dmrt 4 expression is lost at stage 18 ((D) 59%, n =44). At stage 25 Dmrt 4 is normal in absence of DEX ((E) 8% affected, n =26), while activation at stage 18 strongly reduces Dmrt 4 ((F); 63%, n =24). (G) Otx 2 -E 1 A has no effect on Dmrt 4 (0% affected, n =14). (H)–(I) Otx 2 mRNA reduces Pax 2 ((H) 72%, n =25), but does not change Eya 1 ((I) 0% affected, n =44). All embryos were injected into the A3 blastomere at the 32-cell stage and are shown in frontal view with dorsal to the top. High magnifications of the olfactory region are shown in small panels; dotted lines demarcate placodal Dmrt 4. Turquoise staining reveals the lineage tracer FDX.

    Journal: Developmental Biology

    Article Title: Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning

    doi: 10.1016/j.ydbio.2012.04.025

    Figure Lengend Snippet: Activation of Otx2 target genes is required at early and late stages of olfactory placode formation. (A), (B) Otx 2 -EnR inhibits the olfactory placode marker Dmrt 4 at stage 21 (59%, n =29). This is rescued by co-injection of Otx 2 ((B) inhibition reduced to 12%, n =33). (C–F) Otx 2 -EnR-GR injections: in the absence of DEX Dmrt 4 expression is normal ((C) 5% affected, n =20); when DEX is added at stage 10 Dmrt 4 expression is lost at stage 18 ((D) 59%, n =44). At stage 25 Dmrt 4 is normal in absence of DEX ((E) 8% affected, n =26), while activation at stage 18 strongly reduces Dmrt 4 ((F); 63%, n =24). (G) Otx 2 -E 1 A has no effect on Dmrt 4 (0% affected, n =14). (H)–(I) Otx 2 mRNA reduces Pax 2 ((H) 72%, n =25), but does not change Eya 1 ((I) 0% affected, n =44). All embryos were injected into the A3 blastomere at the 32-cell stage and are shown in frontal view with dorsal to the top. High magnifications of the olfactory region are shown in small panels; dotted lines demarcate placodal Dmrt 4. Turquoise staining reveals the lineage tracer FDX.

    Article Snippet: Immunostaining on cryosections was performed ( ) using antibodies against Otx2 (Abcam; 1:50) and Pax3 (Developmental Hybridoma Bank; 1:10) and appropriate secondary antibodies (Invitrogen; 1:1000).

    Techniques: Activation Assay, Marker, Injection, Inhibition, Expressing, Staining

    Activation of Otx2 target genes is required for trigeminal placode specification. (A), (B) Otx 2 -EnR inhibits Runx 3 at stage 23 ((A); 59%, n =26; arrowhead: trigeminal placode on uninjected side). This is rescued by co-injection of Otx 2 ((B) inhibition reduced to 17%, n =36). Inserts show higher magnification of the trigeminal region. (C), (D) At stage 28 the profundal and trigeminal placodes can be distinguished; both are reduced after Otx 2 -EnR injection (78%, n =18). Compare control (C) and injected side (D). (E), (F) Injection of Gbx 2 splice and translation blocking morpholinos ((F) 0% affected, n =25) does not affect Runx 3; compare uninjected (E) and injected side (F). (G), (H). Injection of Gbx 2 -EnR does not affect Runx 3 ((H); 3% affected, n =31); compare control (G) and injected side (H). (I)–(K) Activation of Otx 2 -EnR-GR at stage 10 inhibits Pax 3 the profundal placode ((J) 49%, n =33); no change is observed without DEX ((I) 5% affected, n =61; PR: profundal placode) or when added at stage 14 ((K) 5% affected, n =42). Magnifications show profundal region (dotted outline) on the uninjected (top) and injected side (bottom; FDX: turquoise). All embryos were injected into the A3 blastomere at the 32-cell stage.

    Journal: Developmental Biology

    Article Title: Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning

    doi: 10.1016/j.ydbio.2012.04.025

    Figure Lengend Snippet: Activation of Otx2 target genes is required for trigeminal placode specification. (A), (B) Otx 2 -EnR inhibits Runx 3 at stage 23 ((A); 59%, n =26; arrowhead: trigeminal placode on uninjected side). This is rescued by co-injection of Otx 2 ((B) inhibition reduced to 17%, n =36). Inserts show higher magnification of the trigeminal region. (C), (D) At stage 28 the profundal and trigeminal placodes can be distinguished; both are reduced after Otx 2 -EnR injection (78%, n =18). Compare control (C) and injected side (D). (E), (F) Injection of Gbx 2 splice and translation blocking morpholinos ((F) 0% affected, n =25) does not affect Runx 3; compare uninjected (E) and injected side (F). (G), (H). Injection of Gbx 2 -EnR does not affect Runx 3 ((H); 3% affected, n =31); compare control (G) and injected side (H). (I)–(K) Activation of Otx 2 -EnR-GR at stage 10 inhibits Pax 3 the profundal placode ((J) 49%, n =33); no change is observed without DEX ((I) 5% affected, n =61; PR: profundal placode) or when added at stage 14 ((K) 5% affected, n =42). Magnifications show profundal region (dotted outline) on the uninjected (top) and injected side (bottom; FDX: turquoise). All embryos were injected into the A3 blastomere at the 32-cell stage.

    Article Snippet: Immunostaining on cryosections was performed ( ) using antibodies against Otx2 (Abcam; 1:50) and Pax3 (Developmental Hybridoma Bank; 1:10) and appropriate secondary antibodies (Invitrogen; 1:1000).

    Techniques: Activation Assay, Injection, Inhibition, Blocking Assay

    Otx2 and Gbx2 mutually repress each other in the PPR. (A) and (B) Injection of Otx 2 mRNA into the D1 blastomere of 8-cell stage embryos (A) shifts En- 1 posteriorly on the injected side (50%. n =28; FDX: turquoise), while injection into the A3 blastomere of a 32-cell stage embryo has little effect ((B); 5% affected, n =36). (C) and (D) Injection of Gbx 2 mRNA into the D1 blastomere at 8-cell stage (C) shifts En- 1 anteriorly on the injected side (68%; n =64; FDX: turquoise), while injection into the A3 blastomere at 32-cell stage has no effect ((D); 0% affected, n =31). Dorsal view, anterior to the top. (E) and (F) Injection of Otx 2 (E; 68% affected, n =31) or Otx 2 -EnR ((F); 77% affected, n =17) into A3 at the 32-cell stage inhibits Gbx 2 in the PPR. Compare bracket in the injected (FDX: turquoise) and uninjected side. (G) and (H) Injection of Gbx 2 into A3 at the 32-cell stage leads to Otx 2 repression (68%; n =26); compare brackets (G) on the injected ((H): FDX, turquoise) and uninjected side. (I) and (J) Co-injection of splice and translation blocking Gbx2 morpholinos into A3 at 32-cell stage leads to Otx 2 expansion (73%; n =33); compare brackets (I) on the injected ((J) FDX, turquoise) and uninjected side. (K) and (L) Injection of Gbx 2 -EnR into A3 at the 32-cell stage leads to a loss of Otx 2 in 62% of embryos ( n =52); compare brackets (K) on the injected ((L) FDX, turquoise) and uninjected side. (E)–(L) Frontal view, dorsal to the top.

    Journal: Developmental Biology

    Article Title: Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning

    doi: 10.1016/j.ydbio.2012.04.025

    Figure Lengend Snippet: Otx2 and Gbx2 mutually repress each other in the PPR. (A) and (B) Injection of Otx 2 mRNA into the D1 blastomere of 8-cell stage embryos (A) shifts En- 1 posteriorly on the injected side (50%. n =28; FDX: turquoise), while injection into the A3 blastomere of a 32-cell stage embryo has little effect ((B); 5% affected, n =36). (C) and (D) Injection of Gbx 2 mRNA into the D1 blastomere at 8-cell stage (C) shifts En- 1 anteriorly on the injected side (68%; n =64; FDX: turquoise), while injection into the A3 blastomere at 32-cell stage has no effect ((D); 0% affected, n =31). Dorsal view, anterior to the top. (E) and (F) Injection of Otx 2 (E; 68% affected, n =31) or Otx 2 -EnR ((F); 77% affected, n =17) into A3 at the 32-cell stage inhibits Gbx 2 in the PPR. Compare bracket in the injected (FDX: turquoise) and uninjected side. (G) and (H) Injection of Gbx 2 into A3 at the 32-cell stage leads to Otx 2 repression (68%; n =26); compare brackets (G) on the injected ((H): FDX, turquoise) and uninjected side. (I) and (J) Co-injection of splice and translation blocking Gbx2 morpholinos into A3 at 32-cell stage leads to Otx 2 expansion (73%; n =33); compare brackets (I) on the injected ((J) FDX, turquoise) and uninjected side. (K) and (L) Injection of Gbx 2 -EnR into A3 at the 32-cell stage leads to a loss of Otx 2 in 62% of embryos ( n =52); compare brackets (K) on the injected ((L) FDX, turquoise) and uninjected side. (E)–(L) Frontal view, dorsal to the top.

    Article Snippet: Immunostaining on cryosections was performed ( ) using antibodies against Otx2 (Abcam; 1:50) and Pax3 (Developmental Hybridoma Bank; 1:10) and appropriate secondary antibodies (Invitrogen; 1:1000).

    Techniques: Injection, Blocking Assay

    The Otx 2 /Gbx 2 boundary separates otic and trigeminal precursors. (A) Diagram showing HH 7 stage chick embryo: the distance from the center of Hensen's node to the anterior tip of prechordal plate (hn-pc) and from the midline to the edge of the neural plate (ml-np) were set to 100%, respectively. The position of DiI label was measured and expressed as percentage of each distance. (A′). The position of the Gbx2/Otx2 boundary was measured using the same landmarks. In total 9 embryos were measured with the boundary on average at 35±7%; most anterior position measured: 42%, most posterior position: 24.5%. (B) Diagram combining labels from this study and published fate maps ( Streit, 2002; Xu et al., 2008 ); gray: labels contributing to the trigeminal placode; blue: labels contributing to the otic placode. Circles: labels from published fate maps; squares: labels with dual fate from the current study; stars: labels from the current study. 35% indicates the average position of the Otx2/Gbx2 boundary (dotted line)±standard deviation (small arrow); note: mixed trigeminal and otic fates mostly locate near this boundary. (C) HH7 embryo with DiI labeled cells posterior to the average position of the Otx 2/ Gbx 2 boundary (white line). (D) and (E) At HH12 their descendants contribute to the otic placode as shown in whole mount (D) and in transverse sections (E) and (F). HH7 embryo with DiI label anterior to the average position of the Otx2/Gbx2 boundary (black line). (G) and (H) At HH11 their descendants overlap with Pax3 protein (green) in the trigeminal placode. In total, 21 labels were placed into the Otx2 + and 12 into the Gbx2 + domain. (I) Diagram showing the experimental design: blastomeres were injected at the 64-cell stage in Xenopus and their position scored at stage 14. Arrows show the orientation of all embryos. (J)–(L) Neighboring blastomeres were injected with nGFP and nRFP and grown until stage 14. Descendants from injected cells are intermingled as indicated by red and green outlines in L (100%, n =10). (M)–(O) When injected with nGFP/ Otx 2 and nRFP/ Gbx 2 descendants from adjacent blastomeres do not mix (boundary in 79% of embryos, n =14). Red and green outlines in O show the distribution of cells.

    Journal: Developmental Biology

    Article Title: Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning

    doi: 10.1016/j.ydbio.2012.04.025

    Figure Lengend Snippet: The Otx 2 /Gbx 2 boundary separates otic and trigeminal precursors. (A) Diagram showing HH 7 stage chick embryo: the distance from the center of Hensen's node to the anterior tip of prechordal plate (hn-pc) and from the midline to the edge of the neural plate (ml-np) were set to 100%, respectively. The position of DiI label was measured and expressed as percentage of each distance. (A′). The position of the Gbx2/Otx2 boundary was measured using the same landmarks. In total 9 embryos were measured with the boundary on average at 35±7%; most anterior position measured: 42%, most posterior position: 24.5%. (B) Diagram combining labels from this study and published fate maps ( Streit, 2002; Xu et al., 2008 ); gray: labels contributing to the trigeminal placode; blue: labels contributing to the otic placode. Circles: labels from published fate maps; squares: labels with dual fate from the current study; stars: labels from the current study. 35% indicates the average position of the Otx2/Gbx2 boundary (dotted line)±standard deviation (small arrow); note: mixed trigeminal and otic fates mostly locate near this boundary. (C) HH7 embryo with DiI labeled cells posterior to the average position of the Otx 2/ Gbx 2 boundary (white line). (D) and (E) At HH12 their descendants contribute to the otic placode as shown in whole mount (D) and in transverse sections (E) and (F). HH7 embryo with DiI label anterior to the average position of the Otx2/Gbx2 boundary (black line). (G) and (H) At HH11 their descendants overlap with Pax3 protein (green) in the trigeminal placode. In total, 21 labels were placed into the Otx2 + and 12 into the Gbx2 + domain. (I) Diagram showing the experimental design: blastomeres were injected at the 64-cell stage in Xenopus and their position scored at stage 14. Arrows show the orientation of all embryos. (J)–(L) Neighboring blastomeres were injected with nGFP and nRFP and grown until stage 14. Descendants from injected cells are intermingled as indicated by red and green outlines in L (100%, n =10). (M)–(O) When injected with nGFP/ Otx 2 and nRFP/ Gbx 2 descendants from adjacent blastomeres do not mix (boundary in 79% of embryos, n =14). Red and green outlines in O show the distribution of cells.

    Article Snippet: Immunostaining on cryosections was performed ( ) using antibodies against Otx2 (Abcam; 1:50) and Pax3 (Developmental Hybridoma Bank; 1:10) and appropriate secondary antibodies (Invitrogen; 1:1000).

    Techniques: Standard Deviation, Labeling, Injection

    Immunohistochemistry reveals that both predicted activin receptors (Acvr2a and Acvr2b) are expressed in photoreceptors. To validate the cytokine receptor prediction by receptoR, sections of post-natal day 4 mouse retina (without RPE) were immunostained for activin type 2 receptors and well-known photoreceptor/retina markers. (A) Co-localization of rhodopsin (green) and Acvr2a (magenta) indicates the expression of this activin receptor in photoreceptor cells. (B) In contrast, Acvr2b (green) shows weaker, less specific staining throughout the retina, similar to the expression of Otx2 (magenta). Sections are 20 μm thick and were counterstained with DAPI (gray).

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Automated Hypothesis Generation to Identify Signals Relevant in the Development of Mammalian Cell and Tissue Bioprocesses, With Validation in a Retinal Culture System

    doi: 10.3389/fbioe.2020.00534

    Figure Lengend Snippet: Immunohistochemistry reveals that both predicted activin receptors (Acvr2a and Acvr2b) are expressed in photoreceptors. To validate the cytokine receptor prediction by receptoR, sections of post-natal day 4 mouse retina (without RPE) were immunostained for activin type 2 receptors and well-known photoreceptor/retina markers. (A) Co-localization of rhodopsin (green) and Acvr2a (magenta) indicates the expression of this activin receptor in photoreceptor cells. (B) In contrast, Acvr2b (green) shows weaker, less specific staining throughout the retina, similar to the expression of Otx2 (magenta). Sections are 20 μm thick and were counterstained with DAPI (gray).

    Article Snippet: The following antibodies were used to detect the various epitopes: activin receptor type 2A (Abcam, cat. no. ab96793), Activin receptor type 2B (Abcam, cat. no. ab76940), OTX2 (Abcam, cat. no. ab114138), and Rhodopsin (Abcam, cat. no. 98887).

    Techniques: Immunohistochemistry, Expressing, Staining

    Hmga2 is involved in the regulation of Otx2 target genes upon the exit of embryonic stem cells (ESCs) from the pluripotent ground state. a ChIP-qPCR analysis of the association of Hmga2 to gene targets of Otx2 in wildtype (wt) epiblast-like stem cells (EpiLCs). The negative control (neg ctrl) corresponds to the region 10.1 kb upstream of the Hmga2 transcriptional start site. qPCR data represent means of three independent experiments (n = 3) ± SEM. b ChIP-qPCR analysis of the association of Hmga2 to gene targets of Otx2 in wt induced pluripotent stem cells (iPSCs) induced to differentiate. qPCR data represent means of two independent experiments (n = 2) ± SD. c The relative level of the indicated mRNAs was measured by qPCR in EpiLCs derived from ESCs transfected with si Ctrl and si Hmga2. qPCR data represent mean of three independent experiments (n = 3) ± SEM. * P

    Journal: BMC Biology

    Article Title: Hmga2 is necessary for Otx2-dependent exit of embryonic stem cells from the pluripotent ground state

    doi: 10.1186/s12915-016-0246-5

    Figure Lengend Snippet: Hmga2 is involved in the regulation of Otx2 target genes upon the exit of embryonic stem cells (ESCs) from the pluripotent ground state. a ChIP-qPCR analysis of the association of Hmga2 to gene targets of Otx2 in wildtype (wt) epiblast-like stem cells (EpiLCs). The negative control (neg ctrl) corresponds to the region 10.1 kb upstream of the Hmga2 transcriptional start site. qPCR data represent means of three independent experiments (n = 3) ± SEM. b ChIP-qPCR analysis of the association of Hmga2 to gene targets of Otx2 in wt induced pluripotent stem cells (iPSCs) induced to differentiate. qPCR data represent means of two independent experiments (n = 2) ± SD. c The relative level of the indicated mRNAs was measured by qPCR in EpiLCs derived from ESCs transfected with si Ctrl and si Hmga2. qPCR data represent mean of three independent experiments (n = 3) ± SEM. * P

    Article Snippet: The following primary antibodies were used: mouse Oct3/4 (1:1000 Santa Cruz Biotechnology, #sc-5279), rabbit Nanog (1:1000 Calbiochem-EMD Biosciences, #SC1000), mouse GAPDH (1:1000 Santa Cruz Biotechnology, #sc-32233), goat Sox1 (1:100 Santa Cruz Biotechnology, #sc-17318), rabbit Otx2 (1:500 Abcam, #ab114138), rabbit HMGA2 (1:500 Cell Signaling, #5269), and rabbit HMGA2 (1:500).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Derivative Assay, Transfection

    Hmga2 assists Otx2 in the activation of the Hmga2 gene. a The association of Hmga2 to the genomic region upstream of its own gene was measured by ChIP-qPCR in epiblast-like stem cells (EpiLCs). qPCR data represent mean of independent biological replicates (n = 5) ± SEM. b Otx2 binding to the genomic region upstream of Hmga2 gene (left panel) was measured by ChIP-qPCR in EpiLCs derived from embryonic stem cells transfected with the indicated siRNAs. Data are presented as means of independent experiments (n = 4) ± SEM. * P

    Journal: BMC Biology

    Article Title: Hmga2 is necessary for Otx2-dependent exit of embryonic stem cells from the pluripotent ground state

    doi: 10.1186/s12915-016-0246-5

    Figure Lengend Snippet: Hmga2 assists Otx2 in the activation of the Hmga2 gene. a The association of Hmga2 to the genomic region upstream of its own gene was measured by ChIP-qPCR in epiblast-like stem cells (EpiLCs). qPCR data represent mean of independent biological replicates (n = 5) ± SEM. b Otx2 binding to the genomic region upstream of Hmga2 gene (left panel) was measured by ChIP-qPCR in EpiLCs derived from embryonic stem cells transfected with the indicated siRNAs. Data are presented as means of independent experiments (n = 4) ± SEM. * P

    Article Snippet: The following primary antibodies were used: mouse Oct3/4 (1:1000 Santa Cruz Biotechnology, #sc-5279), rabbit Nanog (1:1000 Calbiochem-EMD Biosciences, #SC1000), mouse GAPDH (1:1000 Santa Cruz Biotechnology, #sc-32233), goat Sox1 (1:100 Santa Cruz Biotechnology, #sc-17318), rabbit Otx2 (1:500 Abcam, #ab114138), rabbit HMGA2 (1:500 Cell Signaling, #5269), and rabbit HMGA2 (1:500).

    Techniques: Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Derivative Assay, Transfection

    Hmga2 is necessary for the pioneering activity of Otx2 in the engagement of new enhancers during the transition from embryonic stem cells (ESCs) into epiblast-like stem cells (EpiLCs). a Otx2 association with the indicated genes upon Hmga2 silencing. ESCs transfected with si Ctrl or si Hmga2 were induced to differentiate into EpiLCs and the Otx2 binding to the enhancers of indicated genes was measured by ChIP-qPCR. Data are presented as mean ± SEM of independent biological replicates (Plekha1, FGF5, Hells, n = 3; Slc16a3, Myrf, n = 4). * P

    Journal: BMC Biology

    Article Title: Hmga2 is necessary for Otx2-dependent exit of embryonic stem cells from the pluripotent ground state

    doi: 10.1186/s12915-016-0246-5

    Figure Lengend Snippet: Hmga2 is necessary for the pioneering activity of Otx2 in the engagement of new enhancers during the transition from embryonic stem cells (ESCs) into epiblast-like stem cells (EpiLCs). a Otx2 association with the indicated genes upon Hmga2 silencing. ESCs transfected with si Ctrl or si Hmga2 were induced to differentiate into EpiLCs and the Otx2 binding to the enhancers of indicated genes was measured by ChIP-qPCR. Data are presented as mean ± SEM of independent biological replicates (Plekha1, FGF5, Hells, n = 3; Slc16a3, Myrf, n = 4). * P

    Article Snippet: The following primary antibodies were used: mouse Oct3/4 (1:1000 Santa Cruz Biotechnology, #sc-5279), rabbit Nanog (1:1000 Calbiochem-EMD Biosciences, #SC1000), mouse GAPDH (1:1000 Santa Cruz Biotechnology, #sc-32233), goat Sox1 (1:100 Santa Cruz Biotechnology, #sc-17318), rabbit Otx2 (1:500 Abcam, #ab114138), rabbit HMGA2 (1:500 Cell Signaling, #5269), and rabbit HMGA2 (1:500).

    Techniques: Activity Assay, Transfection, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Otx2 controls Hmga2 expression in epiblast-like stem cells (EpiLCs). a Phenotypic analysis of Otx2 knockout (KO) and wildtype (wt) differentiated embryonic stem cells (ESCs). The indicated markers were analyzed by qPCR at 4 days of differentiation as serum-free embryoid bodies (SFEBs) or into EpiLCs. qPCR data represent the means of independent biological replicates (Oct4, n = 4; Nanog, Rex1, and Pax6, n = 3; Klf4, n = 5) ± SEM. * P

    Journal: BMC Biology

    Article Title: Hmga2 is necessary for Otx2-dependent exit of embryonic stem cells from the pluripotent ground state

    doi: 10.1186/s12915-016-0246-5

    Figure Lengend Snippet: Otx2 controls Hmga2 expression in epiblast-like stem cells (EpiLCs). a Phenotypic analysis of Otx2 knockout (KO) and wildtype (wt) differentiated embryonic stem cells (ESCs). The indicated markers were analyzed by qPCR at 4 days of differentiation as serum-free embryoid bodies (SFEBs) or into EpiLCs. qPCR data represent the means of independent biological replicates (Oct4, n = 4; Nanog, Rex1, and Pax6, n = 3; Klf4, n = 5) ± SEM. * P

    Article Snippet: The following primary antibodies were used: mouse Oct3/4 (1:1000 Santa Cruz Biotechnology, #sc-5279), rabbit Nanog (1:1000 Calbiochem-EMD Biosciences, #SC1000), mouse GAPDH (1:1000 Santa Cruz Biotechnology, #sc-32233), goat Sox1 (1:100 Santa Cruz Biotechnology, #sc-17318), rabbit Otx2 (1:500 Abcam, #ab114138), rabbit HMGA2 (1:500 Cell Signaling, #5269), and rabbit HMGA2 (1:500).

    Techniques: Expressing, Knock-Out, Real-time Polymerase Chain Reaction

    Abnormal neuronal migration and distribution of neurons in ak/ak mesencephalon. ( A-M ) The migrational profile of E11-labeled neuronal progenitors was analyzed in WT (A-C,G) and ak/ak mutant (D-F,H-M) mice at E17. White asterisks indicate areas free of BrdU-labeled cells in WT mesencephalon (A-C,G) and blue asterisks indicate abnormal cell clusters in the red nucleus of the ak/ak mutant (D-F,H). (G,H) Higher magnifications of C and F displaying failed perpendicular migration (arrowheads) in the ak/ak mutant. (I-M) Stalled cells in the ak/ak mutant are double positive for BrdU/Otx2 (arrowheads, I), BrdU/Lmx1b (J,K) and BrdU/Foxa2 (L,M). The boxed regions in J and L are magnified in K and M, respectively, and white arrows indicate double-positive cells. ( N ) Quantification of E11 BrdU-labeled cells distributed in the red nucleus (both hemispheres) of WT and ak/ak mutant (mean density of BrdU + cells ± s.d.). A significant increase in BrdU + cells was observed in the ak/ak mutant. * P

    Journal: Development (Cambridge, England)

    Article Title: Dopaminergic neurons modulate GABA neuron migration in the embryonic midbrain

    doi: 10.1242/dev.078394

    Figure Lengend Snippet: Abnormal neuronal migration and distribution of neurons in ak/ak mesencephalon. ( A-M ) The migrational profile of E11-labeled neuronal progenitors was analyzed in WT (A-C,G) and ak/ak mutant (D-F,H-M) mice at E17. White asterisks indicate areas free of BrdU-labeled cells in WT mesencephalon (A-C,G) and blue asterisks indicate abnormal cell clusters in the red nucleus of the ak/ak mutant (D-F,H). (G,H) Higher magnifications of C and F displaying failed perpendicular migration (arrowheads) in the ak/ak mutant. (I-M) Stalled cells in the ak/ak mutant are double positive for BrdU/Otx2 (arrowheads, I), BrdU/Lmx1b (J,K) and BrdU/Foxa2 (L,M). The boxed regions in J and L are magnified in K and M, respectively, and white arrows indicate double-positive cells. ( N ) Quantification of E11 BrdU-labeled cells distributed in the red nucleus (both hemispheres) of WT and ak/ak mutant (mean density of BrdU + cells ± s.d.). A significant increase in BrdU + cells was observed in the ak/ak mutant. * P

    Article Snippet: Other antibodies used were anti-TH (1:200, Millipore), anti-Otx2 (1:200, Neuromics), anti-GAD65/67 (Gad2/1 – Mouse Genome Informatics) (1:400, Millipore), anti-Lmx1b (1:100; Drs Carmen Birchmeier and Thomas Muller, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany), anti-Foxa2 (1:100, Santa Cruz), anti-Lmx1a (1:100, Millipore), anti-Ki67 (1:30, Sigma), anti-Pax6 (1:30, Sigma), anti-DAT (Slc6a3 – Mouse Genome Informatics) (1:200, Millipore), anti-Helt (1:30, Sigma) and anti-calbindin (1:100, Swant).

    Techniques: Migration, Labeling, Mutagenesis, Mouse Assay

    Controls for perturbation experiments. ( A ) mCerulean fluorescence level correlates with increasing total Sox2 levels, validating the use of mCerulean fluorescence as a measure for Sox2 overexpression. (Left) As a control, we show that Sox2 levels do not increase when only mCerulean is overexpressed. ( B ) Histogram of Otx2 expression (as measured by immunostaining and flow cytometry) following 24 hr of Sox2 overexpression in either naïve ES C 0 cells (Lif2i; yellow) or epiblast-like C 1 cells (D2 PD0; orange). In determining the effects of Sox2 overexpression in the epiblast-like C 1 state ( Figure 5A ), we excluded cells that showed Otx2 expression less than two standard deviations above the mean of Otx2 levels in Lif2i (threshold shown in dotted line). ( C ) Overexpression of only mCerulean does not show any effect on Oct4 levels in both C 0 (left; Lif2i) and C 1 (right; D2 PD0) cell states. ( D ) At day 3 of differentiation using CHIR99021 and Activin A (Materials and methods), populations consist of cells in C 1 (FoxA2-low, T-low) C 2 (FoxA2-low, T-high) and C 4 (FoxA2-high, T-high) states. The fraction of cells in C 4 is 17%. FoxA2 and T levels in undifferentiated C 0 state cells (Lif2i) are shown as a reference (left). ( E ) At day 2.5 of differentiation using PD0325901 (Materials and methods), populations consist of cells in C 1 (Oct4-high) and C 3 (Oct4-low) states. At this point cells have not yet upregulated Pax6 (left two panels) or Slug (right two panels), showing that cells have not yet transitioned to either state C 5 or C 6 . DOI: http://dx.doi.org/10.7554/eLife.20487.021

    Journal: eLife

    Article Title: Dynamics of embryonic stem cell differentiation inferred from single-cell transcriptomics show a series of transitions through discrete cell states

    doi: 10.7554/eLife.20487

    Figure Lengend Snippet: Controls for perturbation experiments. ( A ) mCerulean fluorescence level correlates with increasing total Sox2 levels, validating the use of mCerulean fluorescence as a measure for Sox2 overexpression. (Left) As a control, we show that Sox2 levels do not increase when only mCerulean is overexpressed. ( B ) Histogram of Otx2 expression (as measured by immunostaining and flow cytometry) following 24 hr of Sox2 overexpression in either naïve ES C 0 cells (Lif2i; yellow) or epiblast-like C 1 cells (D2 PD0; orange). In determining the effects of Sox2 overexpression in the epiblast-like C 1 state ( Figure 5A ), we excluded cells that showed Otx2 expression less than two standard deviations above the mean of Otx2 levels in Lif2i (threshold shown in dotted line). ( C ) Overexpression of only mCerulean does not show any effect on Oct4 levels in both C 0 (left; Lif2i) and C 1 (right; D2 PD0) cell states. ( D ) At day 3 of differentiation using CHIR99021 and Activin A (Materials and methods), populations consist of cells in C 1 (FoxA2-low, T-low) C 2 (FoxA2-low, T-high) and C 4 (FoxA2-high, T-high) states. The fraction of cells in C 4 is 17%. FoxA2 and T levels in undifferentiated C 0 state cells (Lif2i) are shown as a reference (left). ( E ) At day 2.5 of differentiation using PD0325901 (Materials and methods), populations consist of cells in C 1 (Oct4-high) and C 3 (Oct4-low) states. At this point cells have not yet upregulated Pax6 (left two panels) or Slug (right two panels), showing that cells have not yet transitioned to either state C 5 or C 6 . DOI: http://dx.doi.org/10.7554/eLife.20487.021

    Article Snippet: Antibodies and dilutions used in this study: Klf4 (Abcam ab129473, 1:400); Nanog (eBiosciences 14–5761, 1:800); Oct4 (Santa Cruz sc-8628, 1:800; Cell Signaling 2840, 1:400); Sox2 (eBiosciences 14–9811, 1:800); Otx2 (Neuromics GT15095, 1:400); T (Brachyury ) (Santa Cruz sc-17745, 1:200); FoxA2 (Cell Signaling 8186, 1:400); Gata4 (eBiosciences 14–9980, 1:400); Sox1 (Cell Signaling 4194, 1:200); Pax6 (DSHB Pax6, 1:200); Msx1 +2 (DSHB 4G1, 1:200); Slug (Cell Signaling 9585, 1:200), Snai1 (Cell Signaling 2879, 1:200).

    Techniques: Fluorescence, Over Expression, Expressing, Immunostaining, Flow Cytometry, Cytometry

    DAPT induces retinal progenitor differentiation and inhibits proliferation. hESCs were differentiated toward retinal neuronal fates. (A) Brightfield and immunostaining of control and DAPT-treated cells after 5 days. A decrease in the number of rosettes, LHX2 and PAX6 expression, along with decreased PH3 staining of mitotic cells was observed in DAPT-treated cells. (B) DAPT treatment resulted in increased TUJ1- and HuC/D-expressing retinal cells. (C) Immunostaining reveals retinal neurons expressing photoreceptor markers, recoverin, and OTX2 ( arrows ). (D) RT-PCR analysis demonstrated decreased PAX6 and increased BRN3B and RCVRN expression in DAPT-treated cells compared to DMSO controls. Mean ± SEM, * P

    Journal: Translational Vision Science & Technology

    Article Title: Transplantation of Human Embryonic Stem Cell-Derived Retinal Cells into the Subretinal Space of a Non-Human Primate

    doi: 10.1167/tvst.6.3.4

    Figure Lengend Snippet: DAPT induces retinal progenitor differentiation and inhibits proliferation. hESCs were differentiated toward retinal neuronal fates. (A) Brightfield and immunostaining of control and DAPT-treated cells after 5 days. A decrease in the number of rosettes, LHX2 and PAX6 expression, along with decreased PH3 staining of mitotic cells was observed in DAPT-treated cells. (B) DAPT treatment resulted in increased TUJ1- and HuC/D-expressing retinal cells. (C) Immunostaining reveals retinal neurons expressing photoreceptor markers, recoverin, and OTX2 ( arrows ). (D) RT-PCR analysis demonstrated decreased PAX6 and increased BRN3B and RCVRN expression in DAPT-treated cells compared to DMSO controls. Mean ± SEM, * P

    Article Snippet: Primary antibodies used include GFP (1:250, Santa Cruz and Acris, Atlanta, GA) Calbindin (D-28K; 1:1000, Swant, Marly, Switzerland), Ki-67 (1:250, Abcam, Eugene, OR), PH3 (1:500, Novus), NeuN1 (1:200, Millipore), TUJI (1:1000, Covance, Princeton, NJ), STEM121 (1:500, Takara/Clontech, Mountain View, CA), HuC/D (1:100, Invitrogen), S-Opsin (1:250, Santa Cruz), Recoverin (1:1000–1:2000, Chemicon, Billerica, MA), and OTX2 (1:200, R & D Systems).

    Techniques: Immunostaining, Expressing, Staining, Reverse Transcription Polymerase Chain Reaction

    Data from immunocytochemistry and flow cytometry-based phagocytosis assays further suggest that the pigmented cells in the SRS are human RPE. (a and b) Pigmented clumps of cells from the subretinal space were isolated using RPE primary culture techniques (a). Some of the isolated cells are human RPE (labeled with antibodies that only recognize human OTX2—a molecular marker for RPE; DAPI = red; OTX2 = green (b)). (c) Some of the isolated cells internalized FITC-labeled porcine outer segments after five hours (labeled as phagocytes; green) in histograms (top panels) and scatter plots (bottom panels). DRAQ5 is a nuclear marker for living cells. Scale bars = 100 μ m.

    Journal: Stem Cells International

    Article Title: iPSC-Derived Retinal Pigment Epithelium Allografts Do Not Elicit Detrimental Effects in Rats: A Follow-Up Study

    doi: 10.1155/2016/8470263

    Figure Lengend Snippet: Data from immunocytochemistry and flow cytometry-based phagocytosis assays further suggest that the pigmented cells in the SRS are human RPE. (a and b) Pigmented clumps of cells from the subretinal space were isolated using RPE primary culture techniques (a). Some of the isolated cells are human RPE (labeled with antibodies that only recognize human OTX2—a molecular marker for RPE; DAPI = red; OTX2 = green (b)). (c) Some of the isolated cells internalized FITC-labeled porcine outer segments after five hours (labeled as phagocytes; green) in histograms (top panels) and scatter plots (bottom panels). DRAQ5 is a nuclear marker for living cells. Scale bars = 100 μ m.

    Article Snippet: Anti-Tra-I-85 (Abcam) [ ], anti-OTX2 (R & D Systems), and anti-Iba1 (Wako) antibodies were used.

    Techniques: Immunocytochemistry, Flow Cytometry, Cytometry, Isolation, Labeling, Marker

    Delayed PV+ cell maturation and visual cortical plasticity in Otx2 +/AA mice. ( a ) A GAG-binding motif is found between the N-terminal (Nter) and the homeodomain of Otx2. The ‘RK’ doublet is mutated to ‘AA’ in the resulting AA mutant. ( b ) Representative images of WFA (staining for PNN) and Otx2 co-labeling in primary visual cortex (V1) layer IV (L4) at P30 (scale bar: 100 µm), comparing +/+ (WT) and +/AA (heterozygous Otx2 +/AA ). ( c-e ) Quantification of Otx2 immunostaining intensity (arbitrary unit, a.u.) in V1 L4 WFA+ cells from P20 to P100 ( c , N=3-5 mice per group), total number of Otx2+ cells at P60 ( d , N=9-12 mice per group) and percentage of Otx2+ cells not stained with WFA at P60 ( e , N=9-12 mice per group). ( f, g ) WFA staining intensity (a.u.) per pixel ( f ) and PV staining intensity (a.u.) per cell ( g ) in V1 L4, quantified from P14 to P200 (N=3-10 mice per group). Shaded area indicates WT ocular dominance critical period. ( h ) Visual acuity measurements of Otx2 +/AA at P30, P100 and P200, with or without short-term (4-day) monocular deprivation (MD; N=4-5 mice per group). (All values: mean ± SEM; t-test; *p

    Journal: Molecular psychiatry

    Article Title: Genetic Otx2 mis-localization delays critical period plasticity across brain regions

    doi: 10.1038/mp.2017.1

    Figure Lengend Snippet: Delayed PV+ cell maturation and visual cortical plasticity in Otx2 +/AA mice. ( a ) A GAG-binding motif is found between the N-terminal (Nter) and the homeodomain of Otx2. The ‘RK’ doublet is mutated to ‘AA’ in the resulting AA mutant. ( b ) Representative images of WFA (staining for PNN) and Otx2 co-labeling in primary visual cortex (V1) layer IV (L4) at P30 (scale bar: 100 µm), comparing +/+ (WT) and +/AA (heterozygous Otx2 +/AA ). ( c-e ) Quantification of Otx2 immunostaining intensity (arbitrary unit, a.u.) in V1 L4 WFA+ cells from P20 to P100 ( c , N=3-5 mice per group), total number of Otx2+ cells at P60 ( d , N=9-12 mice per group) and percentage of Otx2+ cells not stained with WFA at P60 ( e , N=9-12 mice per group). ( f, g ) WFA staining intensity (a.u.) per pixel ( f ) and PV staining intensity (a.u.) per cell ( g ) in V1 L4, quantified from P14 to P200 (N=3-10 mice per group). Shaded area indicates WT ocular dominance critical period. ( h ) Visual acuity measurements of Otx2 +/AA at P30, P100 and P200, with or without short-term (4-day) monocular deprivation (MD; N=4-5 mice per group). (All values: mean ± SEM; t-test; *p

    Article Snippet: The following primary antibodies were used: anti-Otx2 (mouse monoclonal, in house), anti-cFos (rabbit polyclonal, 1/500, Santa Cruz), anti-Cux2 (rabbit polyclonal, 1/200, Sigma), anti-Somatostatin (rat monoclonal, 1/500, Millipore), anti-Calbindin (rabbit polyclonal, 1/200, Millipore), anti-VIP (rabbit polyclonal, 1/200, Genetex), anti-Calretinin (rabbit polyclonal, 1/200, Swant) and anti-PV (rabbit polyclonal, 1/500, Swant).

    Techniques: Mouse Assay, Binding Assay, Mutagenesis, Staining, Labeling, Immunostaining

    PNN turnover and ectopic Otx2 accumulation in Otx2 +/AA mice. ( a-b ) PNN genes whose expression is changed between Otx2 +/AA and WT mice in V1 at P100 ( a ) and in PFC at P60 ( b ). Results are represented by the percentage change from WT mice (N=5 mice per group). Acan, aggrecan; Bcan, brevican; Hapln, hyaluronan and proteoglycan binding link protein; Has, hyaluronan synthase; Adamts, a disintegrin and metalloproteinase with thrombospondin motifs; RTN4R, reticulon 4 receptor; MMP, matrix metalloproteinase; PTPσ, receptor-type protein tyrosine phosphatase σ. ( c-e ) Ectopic Otx2 accumulation in Calretinin (CR)+ cells. ( c ) Representative images of Otx2 and CR co-labeling in V1 layer IV (L4) at P60 (scale bar: 100µm). ( d ) Percentage of CR+ cells accumulating Otx2 in V1 L4 at P60. ( e ) Total number of CR+ cells in V1 L4 at P60 (N=3 mice per group). ( f ) CR gene expression in V1 (N=6 mice per group). (All values: mean ± SEM; t-test; *p

    Journal: Molecular psychiatry

    Article Title: Genetic Otx2 mis-localization delays critical period plasticity across brain regions

    doi: 10.1038/mp.2017.1

    Figure Lengend Snippet: PNN turnover and ectopic Otx2 accumulation in Otx2 +/AA mice. ( a-b ) PNN genes whose expression is changed between Otx2 +/AA and WT mice in V1 at P100 ( a ) and in PFC at P60 ( b ). Results are represented by the percentage change from WT mice (N=5 mice per group). Acan, aggrecan; Bcan, brevican; Hapln, hyaluronan and proteoglycan binding link protein; Has, hyaluronan synthase; Adamts, a disintegrin and metalloproteinase with thrombospondin motifs; RTN4R, reticulon 4 receptor; MMP, matrix metalloproteinase; PTPσ, receptor-type protein tyrosine phosphatase σ. ( c-e ) Ectopic Otx2 accumulation in Calretinin (CR)+ cells. ( c ) Representative images of Otx2 and CR co-labeling in V1 layer IV (L4) at P60 (scale bar: 100µm). ( d ) Percentage of CR+ cells accumulating Otx2 in V1 L4 at P60. ( e ) Total number of CR+ cells in V1 L4 at P60 (N=3 mice per group). ( f ) CR gene expression in V1 (N=6 mice per group). (All values: mean ± SEM; t-test; *p

    Article Snippet: The following primary antibodies were used: anti-Otx2 (mouse monoclonal, in house), anti-cFos (rabbit polyclonal, 1/500, Santa Cruz), anti-Cux2 (rabbit polyclonal, 1/200, Sigma), anti-Somatostatin (rat monoclonal, 1/500, Millipore), anti-Calbindin (rabbit polyclonal, 1/200, Millipore), anti-VIP (rabbit polyclonal, 1/200, Genetex), anti-Calretinin (rabbit polyclonal, 1/200, Swant) and anti-PV (rabbit polyclonal, 1/500, Swant).

    Techniques: Mouse Assay, Expressing, Binding Assay, Labeling

    Altered experience-dependent acoustic preference in adult Otx2 +/AA mice. ( a ) Representative images of Otx2, PV and WFA immunostaining in prefrontal cortex (mPFC) supragranular layers at P60 (scale bar: 100 µm). ( b, c ) Staining intensity of Otx2 and PV ( b , N=4 mice per group) and number of Otx2+ and WFA+ cells ( c , N=4-5 mice per group) in supragranular layers of the infra- and pre-limbic regions of mPFC at P60. ( d ) Typical traces of activity of a mouse inside the arena at the start (first 30 min) and at the end (last 30 min of the 3 h experiment) of the acoustic preference behavior assay. ( e ) Adult (P60) mice were passively exposed to music for 2 weeks and tested for acoustic preference. Cumulative frequency distribution of WT (N=7) and Otx2 +/AA mice (N=16) before (initial) and after (post-music) two-week music exposure. (All values: mean ± SEM; *p

    Journal: Molecular psychiatry

    Article Title: Genetic Otx2 mis-localization delays critical period plasticity across brain regions

    doi: 10.1038/mp.2017.1

    Figure Lengend Snippet: Altered experience-dependent acoustic preference in adult Otx2 +/AA mice. ( a ) Representative images of Otx2, PV and WFA immunostaining in prefrontal cortex (mPFC) supragranular layers at P60 (scale bar: 100 µm). ( b, c ) Staining intensity of Otx2 and PV ( b , N=4 mice per group) and number of Otx2+ and WFA+ cells ( c , N=4-5 mice per group) in supragranular layers of the infra- and pre-limbic regions of mPFC at P60. ( d ) Typical traces of activity of a mouse inside the arena at the start (first 30 min) and at the end (last 30 min of the 3 h experiment) of the acoustic preference behavior assay. ( e ) Adult (P60) mice were passively exposed to music for 2 weeks and tested for acoustic preference. Cumulative frequency distribution of WT (N=7) and Otx2 +/AA mice (N=16) before (initial) and after (post-music) two-week music exposure. (All values: mean ± SEM; *p

    Article Snippet: The following primary antibodies were used: anti-Otx2 (mouse monoclonal, in house), anti-cFos (rabbit polyclonal, 1/500, Santa Cruz), anti-Cux2 (rabbit polyclonal, 1/200, Sigma), anti-Somatostatin (rat monoclonal, 1/500, Millipore), anti-Calbindin (rabbit polyclonal, 1/200, Millipore), anti-VIP (rabbit polyclonal, 1/200, Genetex), anti-Calretinin (rabbit polyclonal, 1/200, Swant) and anti-PV (rabbit polyclonal, 1/500, Swant).

    Techniques: Mouse Assay, Immunostaining, Staining, Activity Assay, Behavioral Assay

    Anxiolysis and recruitment of mPFC circuits following music exposure in Otx2 +/AA mice. ( a-c ) Open field behavior in the first 30 minutes reflecting exploratory anxiety are compared before (Pre) and after (Post) two-week exposure of Otx2 +/AA mice to music. Several parameters are compared: ( a ) duration of time spent at the center of the open field, ( b ) total distance traveled in the field, and ( c ) number of times crossing the center of the open field. All data are normalized to WT littermates conditions (N=17 mice per genotype). ( d-g ) Immunofluorescence staining of Otx2 and cFos in mPFC reveals circuit activation after 1h of music exposure. ( d ) Representative images of Otx2 and cFos staining in mPFC at P60 (scale bar: 100 µm , cortical layers I-V are labeled). cFos signal intensity between genotypes is compared under several parameters: ( e ) cumulative frequency plot, ( inset ) mean intensity (arbitrary unit, a.u.), ( f ) percentage of high intensity cFos+ cells. ( g ) Percentage of Otx2+ cells co-localized with cFos staining (indicated by arrowheads in ( d ) (N=5 mice per genotype). (All values: mean ± SEM; t-test in a-c and f-g , K-S test in e ; *p

    Journal: Molecular psychiatry

    Article Title: Genetic Otx2 mis-localization delays critical period plasticity across brain regions

    doi: 10.1038/mp.2017.1

    Figure Lengend Snippet: Anxiolysis and recruitment of mPFC circuits following music exposure in Otx2 +/AA mice. ( a-c ) Open field behavior in the first 30 minutes reflecting exploratory anxiety are compared before (Pre) and after (Post) two-week exposure of Otx2 +/AA mice to music. Several parameters are compared: ( a ) duration of time spent at the center of the open field, ( b ) total distance traveled in the field, and ( c ) number of times crossing the center of the open field. All data are normalized to WT littermates conditions (N=17 mice per genotype). ( d-g ) Immunofluorescence staining of Otx2 and cFos in mPFC reveals circuit activation after 1h of music exposure. ( d ) Representative images of Otx2 and cFos staining in mPFC at P60 (scale bar: 100 µm , cortical layers I-V are labeled). cFos signal intensity between genotypes is compared under several parameters: ( e ) cumulative frequency plot, ( inset ) mean intensity (arbitrary unit, a.u.), ( f ) percentage of high intensity cFos+ cells. ( g ) Percentage of Otx2+ cells co-localized with cFos staining (indicated by arrowheads in ( d ) (N=5 mice per genotype). (All values: mean ± SEM; t-test in a-c and f-g , K-S test in e ; *p

    Article Snippet: The following primary antibodies were used: anti-Otx2 (mouse monoclonal, in house), anti-cFos (rabbit polyclonal, 1/500, Santa Cruz), anti-Cux2 (rabbit polyclonal, 1/200, Sigma), anti-Somatostatin (rat monoclonal, 1/500, Millipore), anti-Calbindin (rabbit polyclonal, 1/200, Millipore), anti-VIP (rabbit polyclonal, 1/200, Genetex), anti-Calretinin (rabbit polyclonal, 1/200, Swant) and anti-PV (rabbit polyclonal, 1/500, Swant).

    Techniques: Mouse Assay, Immunofluorescence, Staining, Activation Assay, Labeling

    Delayed auditory plasticity in Otx2 +/AA mice. ( a ) Representative images of Otx2, PV and WFA staining in primary auditory cortex (A1) layer IV (L4) at P20 (scale bar: 100 µm). ( b, c ) Staining intensity of Otx2, PV and WFA ( b , N=4-7 mice per group) and number of Otx2+ cells ( c , N=5-8 mice per group) in A1 L4 at P20. ( d ) Illustration of thalamocortical brain slice preparation to study auditory plasticity (representative traces from a WT slice, scale bar: 100 msec, 0.05 ΔF/F). ( e) Normalized (norm.) maximal ΔF/F across L4 loci in response to different ventral medial geniculate body (MGBv) stimulus sites for WT (N=13, p

    Journal: Molecular psychiatry

    Article Title: Genetic Otx2 mis-localization delays critical period plasticity across brain regions

    doi: 10.1038/mp.2017.1

    Figure Lengend Snippet: Delayed auditory plasticity in Otx2 +/AA mice. ( a ) Representative images of Otx2, PV and WFA staining in primary auditory cortex (A1) layer IV (L4) at P20 (scale bar: 100 µm). ( b, c ) Staining intensity of Otx2, PV and WFA ( b , N=4-7 mice per group) and number of Otx2+ cells ( c , N=5-8 mice per group) in A1 L4 at P20. ( d ) Illustration of thalamocortical brain slice preparation to study auditory plasticity (representative traces from a WT slice, scale bar: 100 msec, 0.05 ΔF/F). ( e) Normalized (norm.) maximal ΔF/F across L4 loci in response to different ventral medial geniculate body (MGBv) stimulus sites for WT (N=13, p

    Article Snippet: The following primary antibodies were used: anti-Otx2 (mouse monoclonal, in house), anti-cFos (rabbit polyclonal, 1/500, Santa Cruz), anti-Cux2 (rabbit polyclonal, 1/200, Sigma), anti-Somatostatin (rat monoclonal, 1/500, Millipore), anti-Calbindin (rabbit polyclonal, 1/200, Millipore), anti-VIP (rabbit polyclonal, 1/200, Genetex), anti-Calretinin (rabbit polyclonal, 1/200, Swant) and anti-PV (rabbit polyclonal, 1/500, Swant).

    Techniques: Mouse Assay, Staining, Slice Preparation

    Clinical and genetic heterogeneity among nine subtypes of PFA ependymoma. Nine subtypes of PFA ependymoma displaying variability in: (A) age at diagnosis (years), (B) gender ratio, (C) ratio of pathological grade, (D) progression-free survival (PFS), and (E) overall survival (OS). (F) Elevated OTX2 expression in PFA-2c ependymomas demonstrated by gene expression profiling (2x Affymetrix u133 v2 array probes) and immunohistochemistry (G). Immunonegative PFA ependymoma of another subtype (H). Varying frequencies across subtypes of the four commonest chromosome arm copy number alterations found in PFA ependymomas; 1q gain (I), 6q loss (J), 10q loss (K), 22q loss (L). Scale bars (G/H) = 100μm .

    Journal: Acta neuropathologica

    Article Title: Molecular heterogeneity and CXorf67 alterations in posterior fossa group A (PFA) ependymomas

    doi: 10.1007/s00401-018-1877-0

    Figure Lengend Snippet: Clinical and genetic heterogeneity among nine subtypes of PFA ependymoma. Nine subtypes of PFA ependymoma displaying variability in: (A) age at diagnosis (years), (B) gender ratio, (C) ratio of pathological grade, (D) progression-free survival (PFS), and (E) overall survival (OS). (F) Elevated OTX2 expression in PFA-2c ependymomas demonstrated by gene expression profiling (2x Affymetrix u133 v2 array probes) and immunohistochemistry (G). Immunonegative PFA ependymoma of another subtype (H). Varying frequencies across subtypes of the four commonest chromosome arm copy number alterations found in PFA ependymomas; 1q gain (I), 6q loss (J), 10q loss (K), 22q loss (L). Scale bars (G/H) = 100μm .

    Article Snippet: Di Giovannantonio LG, Di Salvio M, Omodei D, Prakash N, Wurst W, Pierani A, et al. (2014) Otx2 cell-autonomously determines dorsal mesencephalon versus cerebellum fate independently of isthmic organizing activity .

    Techniques: Expressing, Immunohistochemistry

    Generation of highly-pure population of MNPs from hPSCs (A) Schematics showing the time course and small molecule cocktail for hPSC differentiation into MNPs. (B) Representative images of SOX1 + NEPs after 6 days of culture in CHIR+SB+DMH vs. SB+DMH condition. The regional identity ( OTX2 + vs. HOXA3 +) were stained. Scale bars: 50μm. Quantification of SOX1 + NEP percentage and number is shown on right ( > 500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (C) Representative images of pure MNPs at Day12 under different conditions, which express OLIG2 (green) but not NKX2.2 (red). Scale bars: 50μm. Quantification of OLIG2 +, NKX2.2 + and OLIG2 +/ NKX2.2 + cells is shown on right ( > 500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (D) The efficiency of OLIG2 + MNP differentiation from multiple hPSC lines ( > 500 cells from random fields were manually counted in each cell line). The bar graph shows the mean±s.d. (n=3 in each cell line).

    Journal: Nature communications

    Article Title: Generation and Expansion of highly-pure Motor Neuron Progenitors from Human Pluripotent Stem Cells

    doi: 10.1038/ncomms7626

    Figure Lengend Snippet: Generation of highly-pure population of MNPs from hPSCs (A) Schematics showing the time course and small molecule cocktail for hPSC differentiation into MNPs. (B) Representative images of SOX1 + NEPs after 6 days of culture in CHIR+SB+DMH vs. SB+DMH condition. The regional identity ( OTX2 + vs. HOXA3 +) were stained. Scale bars: 50μm. Quantification of SOX1 + NEP percentage and number is shown on right ( > 500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (C) Representative images of pure MNPs at Day12 under different conditions, which express OLIG2 (green) but not NKX2.2 (red). Scale bars: 50μm. Quantification of OLIG2 +, NKX2.2 + and OLIG2 +/ NKX2.2 + cells is shown on right ( > 500 cells from random fields were manually counted in each condition). The bar graph shows the mean±s.d. (n=3 in each condition). (D) The efficiency of OLIG2 + MNP differentiation from multiple hPSC lines ( > 500 cells from random fields were manually counted in each cell line). The bar graph shows the mean±s.d. (n=3 in each cell line).

    Article Snippet: The following primary antibodies were used: SOX1 (gIgG 1:1000, R & D), OTX2 (mIgG, 1:2000, DSHB), HOXA3 (mIgG 1:1000, R & D), OLIG2 (rIgG 1:500, Chemicon), NKX2.2 (mIgG 1:100, DSHB), Ki67 (rIgG 1:200, Chemicon), MNX1 (mIgG 1:50, DSHB), ISL1 (mIgG 1:1000, DSHB), TUJ1 (rIgG 1:5000, Covance), CHAT (gIgG 1:300, Chemicon), MAP2 (mIgG 1:1000, Chemicon), GABA (mIgG 1:1000, Chemicon), FoxP1 (rIgG, 1:1000, Chemicon).

    Techniques: Staining

    Methylation of the OTX2 promoter is sufficient to suppress OTX2 in medulloblastoma. (A) Representative methylation patterns of medulloblastoma samples, with samples organized by row and each CpG location organized in columns. Leftmost column indicates OTX2 expression status (red, OTX2-expressing; green, OTX2-nonexpressing), letters to the right of this column indicate OTX2 copy number status (N, normal; G, gained; HL, heterozygous loss), if known [5] , [19] , [32] , [33] . (B) OTX2 mRNA level, as determined by RT-qPCR, in tumors exhibiting either a methylated or unmethylated OTX2 promoter. ND, not detected. (C) Semi-quantitative RT-PCR of OTX2-nonexpressing cells treated with the indicated concentrations of 5′-aza-2′-deoxycytidine for 5 days. D283 medulloblastoma cells serve as a positive control for OTX2 detection.

    Journal: PLoS ONE

    Article Title: Chromatin Accessibility Mapping Identifies Mediators of Basal Transcription and Retinoid-Induced Repression of OTX2 in Medulloblastoma

    doi: 10.1371/journal.pone.0107156

    Figure Lengend Snippet: Methylation of the OTX2 promoter is sufficient to suppress OTX2 in medulloblastoma. (A) Representative methylation patterns of medulloblastoma samples, with samples organized by row and each CpG location organized in columns. Leftmost column indicates OTX2 expression status (red, OTX2-expressing; green, OTX2-nonexpressing), letters to the right of this column indicate OTX2 copy number status (N, normal; G, gained; HL, heterozygous loss), if known [5] , [19] , [32] , [33] . (B) OTX2 mRNA level, as determined by RT-qPCR, in tumors exhibiting either a methylated or unmethylated OTX2 promoter. ND, not detected. (C) Semi-quantitative RT-PCR of OTX2-nonexpressing cells treated with the indicated concentrations of 5′-aza-2′-deoxycytidine for 5 days. D283 medulloblastoma cells serve as a positive control for OTX2 detection.

    Article Snippet: Chromatin immunoprecipitation Chromatin immunoprecipitation was performed using anti-OTX2 (BAF1979, R & D) or IgG control antibodies using the ChIP Assay Kit (Upstate Cell Signaling).

    Techniques: Methylation, Expressing, Quantitative RT-PCR, Positive Control

    OTX2 regulates its own expression through the DHS 4 enhancer. (A) Luciferase assay for enhancer activity of DHS 4 in OTX2-expressing medulloblastoma cells treated with OTX2 siRNA. (B) Western blotting for ectopic and endogenous OTX2 following transfection of (B) OTX2-expressing or (C) OTX2-nonexpressing medulloblastoma cells with EGFP-tagged OTX2. (D) RT-qPCR demonstrating induction of the OTX2 target gene IL-6 in OTX2-nonexpressing medulloblastoma cells transfected with EGFP-tagged OTX2. (E) Chromatin immunoprecipitation of endogenous OTX2 in OTX2-expressing and -nonexpressing medulloblastoma cells. * p

    Journal: PLoS ONE

    Article Title: Chromatin Accessibility Mapping Identifies Mediators of Basal Transcription and Retinoid-Induced Repression of OTX2 in Medulloblastoma

    doi: 10.1371/journal.pone.0107156

    Figure Lengend Snippet: OTX2 regulates its own expression through the DHS 4 enhancer. (A) Luciferase assay for enhancer activity of DHS 4 in OTX2-expressing medulloblastoma cells treated with OTX2 siRNA. (B) Western blotting for ectopic and endogenous OTX2 following transfection of (B) OTX2-expressing or (C) OTX2-nonexpressing medulloblastoma cells with EGFP-tagged OTX2. (D) RT-qPCR demonstrating induction of the OTX2 target gene IL-6 in OTX2-nonexpressing medulloblastoma cells transfected with EGFP-tagged OTX2. (E) Chromatin immunoprecipitation of endogenous OTX2 in OTX2-expressing and -nonexpressing medulloblastoma cells. * p

    Article Snippet: Chromatin immunoprecipitation Chromatin immunoprecipitation was performed using anti-OTX2 (BAF1979, R & D) or IgG control antibodies using the ChIP Assay Kit (Upstate Cell Signaling).

    Techniques: Expressing, Luciferase, Activity Assay, Western Blot, Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation

    DNase landscape of OTX2-expressing and -nonexpressing cell lines. DNase hypersensitivity map of (A) the OTX2 locus and (B) a region of chromosome 14 exhibiting DHS sites that are shared among the three cell lines shown. From top to bottom, tracks are as follows: Refseq genes, D341 medulloblastoma (Medullo) cells, D721 medulloblastoma cells, H54 glioblastoma (GBM) cells, MultiZ vertebrate alignments, PhyloP vertebrate conservation, CpG islands. Orange bars indicate regions of consistent and robust DNase hypersensitivity in the two medulloblastoma cell lines. Grey bar indicates DHS “R”, also found in retinoblastoma ( Fig. S1A ). Genomic coordinates are indicated below tracks. Carat indicates CpG island subjected to methylation analysis ( Fig. 5 and Table S2 ). See Fig. S1A for extended viewing range.

    Journal: PLoS ONE

    Article Title: Chromatin Accessibility Mapping Identifies Mediators of Basal Transcription and Retinoid-Induced Repression of OTX2 in Medulloblastoma

    doi: 10.1371/journal.pone.0107156

    Figure Lengend Snippet: DNase landscape of OTX2-expressing and -nonexpressing cell lines. DNase hypersensitivity map of (A) the OTX2 locus and (B) a region of chromosome 14 exhibiting DHS sites that are shared among the three cell lines shown. From top to bottom, tracks are as follows: Refseq genes, D341 medulloblastoma (Medullo) cells, D721 medulloblastoma cells, H54 glioblastoma (GBM) cells, MultiZ vertebrate alignments, PhyloP vertebrate conservation, CpG islands. Orange bars indicate regions of consistent and robust DNase hypersensitivity in the two medulloblastoma cell lines. Grey bar indicates DHS “R”, also found in retinoblastoma ( Fig. S1A ). Genomic coordinates are indicated below tracks. Carat indicates CpG island subjected to methylation analysis ( Fig. 5 and Table S2 ). See Fig. S1A for extended viewing range.

    Article Snippet: Chromatin immunoprecipitation Chromatin immunoprecipitation was performed using anti-OTX2 (BAF1979, R & D) or IgG control antibodies using the ChIP Assay Kit (Upstate Cell Signaling).

    Techniques: Expressing, Methylation

    DHS “R” mediates retinoic acid-induced OTX2 repression. (A) DNase sensitivity of DHS “R” in three OTX2-expressing (D283, D341, D721) and two OTX2-nonexpressing (D324, UW228) medulloblastoma cell lines. (B) Luciferase assays measuring enhancer activity of DHS “R” following treatment with 2 µM all- trans retinoic acid (ATRA) for 24 hours. (C) OTX2 mRNA level as determined by qPCR of medulloblastoma cells treated with 2 µM all- trans retinoic acid (ATRA) and/or 35 µM cycloheximide (CYHEX) for 8 hours. * p

    Journal: PLoS ONE

    Article Title: Chromatin Accessibility Mapping Identifies Mediators of Basal Transcription and Retinoid-Induced Repression of OTX2 in Medulloblastoma

    doi: 10.1371/journal.pone.0107156

    Figure Lengend Snippet: DHS “R” mediates retinoic acid-induced OTX2 repression. (A) DNase sensitivity of DHS “R” in three OTX2-expressing (D283, D341, D721) and two OTX2-nonexpressing (D324, UW228) medulloblastoma cell lines. (B) Luciferase assays measuring enhancer activity of DHS “R” following treatment with 2 µM all- trans retinoic acid (ATRA) for 24 hours. (C) OTX2 mRNA level as determined by qPCR of medulloblastoma cells treated with 2 µM all- trans retinoic acid (ATRA) and/or 35 µM cycloheximide (CYHEX) for 8 hours. * p

    Article Snippet: Chromatin immunoprecipitation Chromatin immunoprecipitation was performed using anti-OTX2 (BAF1979, R & D) or IgG control antibodies using the ChIP Assay Kit (Upstate Cell Signaling).

    Techniques: Expressing, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

    Validation of medulloblastoma DHS sites in a cohort of OTX2-expressing and -nonexpressing cells. Nuclei of medulloblastoma cell lines were treated with increasing concentrations of DNase (indicated as Units per reaction), and then the relative proportion of DNA remaining at the indicated regions were determined by qPCR. Open and solid markers indicate OTX2-expressing and -nonexpressing cell lines, respectively. See Fig. S2 for detailed graphs.

    Journal: PLoS ONE

    Article Title: Chromatin Accessibility Mapping Identifies Mediators of Basal Transcription and Retinoid-Induced Repression of OTX2 in Medulloblastoma

    doi: 10.1371/journal.pone.0107156

    Figure Lengend Snippet: Validation of medulloblastoma DHS sites in a cohort of OTX2-expressing and -nonexpressing cells. Nuclei of medulloblastoma cell lines were treated with increasing concentrations of DNase (indicated as Units per reaction), and then the relative proportion of DNA remaining at the indicated regions were determined by qPCR. Open and solid markers indicate OTX2-expressing and -nonexpressing cell lines, respectively. See Fig. S2 for detailed graphs.

    Article Snippet: Chromatin immunoprecipitation Chromatin immunoprecipitation was performed using anti-OTX2 (BAF1979, R & D) or IgG control antibodies using the ChIP Assay Kit (Upstate Cell Signaling).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Primitive neuroectodermal specification of human TiPSCs through EB formation. A : Phase contrast images of an established Tenon’s-derived induced pluripotent stem cell (TiPSC) line at passage P 33, displaying typical undifferentiated human embryonic stem cell (hESC)-like colony morphology. Scale bar = 100 μm. B – D : Confocal immunofluorescent images of undifferentiated TiPSC clones, stained positive for pluripotency associated markers Oct-4, Nanog, and Sox2 but negative for panneural markers Pax6, Otx2, and Nestin. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars = 50 μm. E : Quantitative real-time PCR analysis confirmed pluripotent genes Oct3/4 and Nanog markedly decreased after 6 days of suspension culture (* p

    Journal: Molecular Vision

    Article Title: Stage-specific differentiation of iPSCs toward retinal ganglion cell lineage

    doi:

    Figure Lengend Snippet: Primitive neuroectodermal specification of human TiPSCs through EB formation. A : Phase contrast images of an established Tenon’s-derived induced pluripotent stem cell (TiPSC) line at passage P 33, displaying typical undifferentiated human embryonic stem cell (hESC)-like colony morphology. Scale bar = 100 μm. B – D : Confocal immunofluorescent images of undifferentiated TiPSC clones, stained positive for pluripotency associated markers Oct-4, Nanog, and Sox2 but negative for panneural markers Pax6, Otx2, and Nestin. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Scale bars = 50 μm. E : Quantitative real-time PCR analysis confirmed pluripotent genes Oct3/4 and Nanog markedly decreased after 6 days of suspension culture (* p

    Article Snippet: Primary antibodies Nanog (Cell Signaling, Danvers, MA), Oct3/4, Pax6, Nestin, Tuj, Chx10 (all from Millipore, Temecula, CA), Sox2, ZO1, Rx, calretinin, iSlet1, synaptophysin (all from Abcam, Burlingame, CA), Otx2 (Invitrogen), Chx10 (Santa Cruz, Dallas, TX), and Rx (Santa Cruz) were used at 1:200 dilution.

    Techniques: Derivative Assay, Clone Assay, Staining, Real-time Polymerase Chain Reaction

    Directed derivation of RPCs with the addition of DKK1, Noggin, and Lefty A. A – B : Phase contrast images of representative Tenon’s-derived induced pluripotent stem cell (TiPSC)-derived neuroepithelial colonies ( A , displaying tightly packed colony morphology) and randomly differentiated colonies ( B , displaying flat non-neural morphology) after 10 days of culture on Matrigel. Scale bar = 100 μm. C – F : Confocal images of neuroepithelial colonies stained with early eye field markers Pax6, Chx10, Rx, Otx2, and ZO1. Scale bars = 100 μm. G : Quantitative real-time PCR analysis showed Pax6 and Lhx2 were further upregulated in both groups after attachment for another 10 days, whereas eye field-specific regional genes Rax , Chx10 , Six3 , and Six6 are upregulated substantially in the DNL group when compared with the control group (* p

    Journal: Molecular Vision

    Article Title: Stage-specific differentiation of iPSCs toward retinal ganglion cell lineage

    doi:

    Figure Lengend Snippet: Directed derivation of RPCs with the addition of DKK1, Noggin, and Lefty A. A – B : Phase contrast images of representative Tenon’s-derived induced pluripotent stem cell (TiPSC)-derived neuroepithelial colonies ( A , displaying tightly packed colony morphology) and randomly differentiated colonies ( B , displaying flat non-neural morphology) after 10 days of culture on Matrigel. Scale bar = 100 μm. C – F : Confocal images of neuroepithelial colonies stained with early eye field markers Pax6, Chx10, Rx, Otx2, and ZO1. Scale bars = 100 μm. G : Quantitative real-time PCR analysis showed Pax6 and Lhx2 were further upregulated in both groups after attachment for another 10 days, whereas eye field-specific regional genes Rax , Chx10 , Six3 , and Six6 are upregulated substantially in the DNL group when compared with the control group (* p

    Article Snippet: Primary antibodies Nanog (Cell Signaling, Danvers, MA), Oct3/4, Pax6, Nestin, Tuj, Chx10 (all from Millipore, Temecula, CA), Sox2, ZO1, Rx, calretinin, iSlet1, synaptophysin (all from Abcam, Burlingame, CA), Otx2 (Invitrogen), Chx10 (Santa Cruz, Dallas, TX), and Rx (Santa Cruz) were used at 1:200 dilution.

    Techniques: Derivative Assay, Staining, Real-time Polymerase Chain Reaction