anti-ap2α Search Results


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  • 90
    Developmental Studies Hybridoma Bank anti ap2α
    Fate mapping of NEC/NCC and otic placode ectoderm populations. ( A-D ) Lateral views of whole-mount embryos from E8.5 to E9.5. GFP reporter expression is activated by Wnt1-Cre, Pax3 Cre/+ , Hoxb1 Cre/+ or Pax2-Cre . Otic tissue is outlined in yellow; boxes indicate regions that are magnified in adjacent images. ( E , F ) NEC derivatives (green) express <t>Ap2α</t> (red). (E) Transverse sections through the trunk neural tube (NT) and dorsal root ganglion (DRG). Images on the right are higher magnification demonstrating expression of GFP with Ap2α in the DRG. (F) Lateral whole-mount views (left column) of the first and second branchial arches (BA1, BA2). Sagittal sections through the branchial arches (middle column) with magnified views of the BA2 mesoderm (right column). Nuclei are stained with DAPI (blue). ss, somite stage.
    Anti Ap2α, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 90/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher anti ap2α
    Fate mapping of NEC/NCC and otic placode ectoderm populations. ( A-D ) Lateral views of whole-mount embryos from E8.5 to E9.5. GFP reporter expression is activated by Wnt1-Cre, Pax3 Cre/+ , Hoxb1 Cre/+ or Pax2-Cre . Otic tissue is outlined in yellow; boxes indicate regions that are magnified in adjacent images. ( E , F ) NEC derivatives (green) express <t>Ap2α</t> (red). (E) Transverse sections through the trunk neural tube (NT) and dorsal root ganglion (DRG). Images on the right are higher magnification demonstrating expression of GFP with Ap2α in the DRG. (F) Lateral whole-mount views (left column) of the first and second branchial arches (BA1, BA2). Sagittal sections through the branchial arches (middle column) with magnified views of the BA2 mesoderm (right column). Nuclei are stained with DAPI (blue). ss, somite stage.
    Anti Ap2α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology ap2α
    Notch drives Schwann cell generation from SCPs. ( a,b ) Top and middle panels, longitudinal sections of E14 hind limb nerves immunolabeled for TUJ1 (inset; red), <t>AP2α</t> or S100β (DAB). Insets, spinal cord (S), DRG (arrowheads). Bottem panels,
    Ap2α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Becton Dickinson ap2α
    Notch drives Schwann cell generation from SCPs. ( a,b ) Top and middle panels, longitudinal sections of E14 hind limb nerves immunolabeled for TUJ1 (inset; red), <t>AP2α</t> or S100β (DAB). Insets, spinal cord (S), DRG (arrowheads). Bottem panels,
    Ap2α, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GeneTex ap2α
    Notch drives Schwann cell generation from SCPs. ( a,b ) Top and middle panels, longitudinal sections of E14 hind limb nerves immunolabeled for TUJ1 (inset; red), <t>AP2α</t> or S100β (DAB). Insets, spinal cord (S), DRG (arrowheads). Bottem panels,
    Ap2α, supplied by GeneTex, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Developmental Studies Hybridoma Bank mouse anti ap2α
    Prdm13 marks a subset of amacrines in the adult retina. (A–D ‴ ) P30 Prdm13-GFP/+ heterozygous animals co-stained with GFP (green) and amacrine cell markers (grey/red). (A–A ‴ ) A small subset of Prdm13-GFP+ amacrine cells co-express <t>AP2α</t> (grey, arrows) and Bhlhb5 (red, arrowheads). (B–B ‴ ) Nearly half of GFP+ cells co-express calretinin (grey, arrows), while less than 1% of GFP+ cells co-express calbindin (red). No intensely calretinin+ horizontal cells co-express GFP. (C–C ‴ ) A large fraction of GFP+ cells co-express the glycinergic amacrine marker GlyT (grey, arrows), but few GFP+ cells co-express the GABAergic marker GAD65/67 and these were sometimes GlyT+ as well. (D–D ‴ ) The majority of GFP+ cells co-express Ebf3 (grey, arrows), but all Ebf3+ cells in the INL are GFP+. A minority of GFP+ cells co-express Bhlhb5 (red, arrowheads) and cells that co-express Bhlhb5, Ebf3, and GFP are rarely seen. (E) Plot of the percentage of GFP+ cells that co-express a given marker. GFP does not overlap with Brn3, TH, or vGlut3 (not shown). (F) Plot showing the percentage of Ebf3+ amacrines, Pax6+ INL nuclei, and AP2α+ INL cells that co-express Prdm13-GFP. Sample size was 3 mice per condition. Statistical significance determined by unpaired two-sample t tests: * P
    Mouse Anti Ap2α, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 90/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Developmental Studies Hybridoma Bank anti ap2α tcfap2a
    Prdm13 marks a subset of amacrines in the adult retina. (A–D ‴ ) P30 Prdm13-GFP/+ heterozygous animals co-stained with GFP (green) and amacrine cell markers (grey/red). (A–A ‴ ) A small subset of Prdm13-GFP+ amacrine cells co-express <t>AP2α</t> (grey, arrows) and Bhlhb5 (red, arrowheads). (B–B ‴ ) Nearly half of GFP+ cells co-express calretinin (grey, arrows), while less than 1% of GFP+ cells co-express calbindin (red). No intensely calretinin+ horizontal cells co-express GFP. (C–C ‴ ) A large fraction of GFP+ cells co-express the glycinergic amacrine marker GlyT (grey, arrows), but few GFP+ cells co-express the GABAergic marker GAD65/67 and these were sometimes GlyT+ as well. (D–D ‴ ) The majority of GFP+ cells co-express Ebf3 (grey, arrows), but all Ebf3+ cells in the INL are GFP+. A minority of GFP+ cells co-express Bhlhb5 (red, arrowheads) and cells that co-express Bhlhb5, Ebf3, and GFP are rarely seen. (E) Plot of the percentage of GFP+ cells that co-express a given marker. GFP does not overlap with Brn3, TH, or vGlut3 (not shown). (F) Plot showing the percentage of Ebf3+ amacrines, Pax6+ INL nuclei, and AP2α+ INL cells that co-express Prdm13-GFP. Sample size was 3 mice per condition. Statistical significance determined by unpaired two-sample t tests: * P
    Anti Ap2α Tcfap2a, supplied by Developmental Studies Hybridoma Bank, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology rabbit anti ap2α
    Dbx1 expression and tracing. (A–J) Dbx1 expression analysed by whole-mount in situ hybridisation. B and D are dorsal views of the embryos presented in A and C respectively, rostral is up. F, H and J are frontal views of the embryos shown in E, G and I respectively. Antero-posterior subdivisions of the neural plate/tube are indicated. Mb: midbrain, Fb: Forebrain, Mes: mesencephalon, Di: diencephalon, Tel: telencephalon. Arrows in E, G and I point to the vDi, dDM and FE subsets respectively; the arrowhead in I shows the ANR subset. (K, L) Coronal sections through the forebrain of a 12ss embryo showing d bx1 expression in the ANR (K) and facial ectoderm (L). (M) Coronal section collected at the midbrain level of a 3ss embryo illustrating dbx1 mRNA expression; the neural plate is delineated by dashed lines. (N) Coronal section collected at the level of the midbrain of a 6ss Dbx1 Cre ;R26 YFP embryo and stained for YFP (green) and DAPI (blue). (O) Quantification of dbx1 expression by qPCR. Asterisks indicate stages at which dbx1 could not be amplified. The relative expression values (normalised to 1 at 2ss and expressed in arbitrary units) are indicated on top of each bar as mean ± s.e.m. (P, Q) Coronal sections of a 20ss (E9.5) Dbx1 Cre ;R26 YFP embryo at the level of the telencephalon (P) and di/mesencephalon (Q), stained for YFP (green) and DAPI (blue). YFP is detected in regions that express dbx1 at earlier stages: ANR (red arrowhead in P), FE (white arrowheads in P), vDi and dDM (red and white arrowheads in P respectively) but also in cells located in the head mesenchyme (arrows in P). (R–W) Examples of YFP + cells found in the head mesenchyme of Dbx1 Cre ;R26 YFP embryos at 8ss (R–T) and 11ss (U–W) revealed by DAB immunostaining. Sections in R, S and U were collected at telencephalic levels, T, V and W at diencephalic levels. Neural tissues, surface ectoderm and endoderm are surrounded by black, blue and green dash lines respectively. (X) Coronal sections of a 8ss Dbx1 Cre ;R26 YFP embryo immunostained for YFP (green) and the neural crest cells marker <t>AP2α</t> (red); cell nuclei are stained by DAPI (blue). Some of the YFP + cells found in the neural plate are AP2α − (arrowhead in X 1 ) whereas YFP + cells located at the most lateral aspect of the neural plate or in the mesenchyme (arrows in X 1 –X 3 ) are AP2α + . Images X 1 –X 3 correspond to single confocal planes. (Y) Schematic drawing of dbx1 expression pattern. Scale bars: A: 200 µm; K, M, N, R, X: 50 µm; P: 100 µm.
    Rabbit Anti Ap2α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Proteintech rabbit anti ap2α
    HBx activates the SPHK1 promoter by upregulating the transcription factor <t>AP2α.</t> (A, B) The activity of the SPHK1 promoter (pGL3-320) was measured by luciferase reporter gene assays in HepG2 (or HepG2-X) cells that were co-transfected with pGL3-320 and pcDNA3.1-HBx (or psi-HBx). (C) A model demonstrates the predicted conserved AP2α binding site at nucleotides -20/-15 of the SPHK1 promoter. The generated mutant site at the SPHK1 promoter region is indicated. The luciferase activities of pGL3-320 and pGL3-320-mut were examined by luciferase reporter gene assays in HepG2 (or LO2) cells. (D) The expression of SPHK1 was detected using Western blot analysis in HepG2-X cells transfected with si-AP2α. (E) The interaction of AP2α with SPHK1 promoter was determined by ChIP assays in HepG2-X cells. (F) The relative activity of pGL3-320 was measured in HepG2 cells that were co-transfected with pcDNA3.1-HBx and si-AP2α. (G) The interfering efficiency of the siRNA pool targeting AP2α (si-AP2α) was validated by Western blot analysis in HepG2 cells. The data are shown as the mean±SD of three independent experiments. b P
    Rabbit Anti Ap2α, supplied by Proteintech, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bioworld Antibodies ap2α
    Retina-like tissue in the subretinal space of a 2-week-old P RPE65 -ngn3 mouse. A–C: Low magnification images to show the relative position of retina-like tissue (arrow) in a cross-section of the eye, with the anterior to the left and the posterior to the right. A: Bright-field (BF) view. B: Epi-fluorescence of DAPI staining of the nuclei. C: Epi-fluorescence of anti-recoverin (Rcv) immunostaining to mark photoreceptor cells. Boxes outline the corresponding regions in A–C, or similar locations on serial sections (D–O). D–G: A serial section under bright-field view (D), after DAPI nuclear staining (E), and after double immunostaining for Otx2 (in green, F) to identify bipolar cells and for Pax6 (in red, G) to label amacrine cells in the INL and in the GCL (misplaced amacrine). H–K: A serial section under bright-field view (H), with DAPI nuclear staining (I), and with double immunostaining for <t>AP2α</t> (in green, J) to label horizontal (albeit weakly) and amacrine cells in the INL and in the GCL (misplaced amacrine), and for Brn3A (in red, K) to identify a subset of ganglion cells. L–O: A serial section under bright-field view (L), with DAPI nuclear staining (M), and with double immunostaining for photoreceptor protein recoverin (in green, N) and for RPE protein RPE65 (in red, O). RPE-L, RPE-like; ONL-L, outer nuclear layer-like; INL-L inner nuclear layer-like; GCL-L, ganglion cell layer-like. Scale bar (100 μm) in A also applies to B, C. Scale bar (100 μm) in D also applies to E–O.
    Ap2α, supplied by Bioworld Antibodies, used in various techniques. Bioz Stars score: 91/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson mouse anti ap2α
    Retina-like tissue in the subretinal space of a 2-week-old P RPE65 -ngn3 mouse. A–C: Low magnification images to show the relative position of retina-like tissue (arrow) in a cross-section of the eye, with the anterior to the left and the posterior to the right. A: Bright-field (BF) view. B: Epi-fluorescence of DAPI staining of the nuclei. C: Epi-fluorescence of anti-recoverin (Rcv) immunostaining to mark photoreceptor cells. Boxes outline the corresponding regions in A–C, or similar locations on serial sections (D–O). D–G: A serial section under bright-field view (D), after DAPI nuclear staining (E), and after double immunostaining for Otx2 (in green, F) to identify bipolar cells and for Pax6 (in red, G) to label amacrine cells in the INL and in the GCL (misplaced amacrine). H–K: A serial section under bright-field view (H), with DAPI nuclear staining (I), and with double immunostaining for <t>AP2α</t> (in green, J) to label horizontal (albeit weakly) and amacrine cells in the INL and in the GCL (misplaced amacrine), and for Brn3A (in red, K) to identify a subset of ganglion cells. L–O: A serial section under bright-field view (L), with DAPI nuclear staining (M), and with double immunostaining for photoreceptor protein recoverin (in green, N) and for RPE protein RPE65 (in red, O). RPE-L, RPE-like; ONL-L, outer nuclear layer-like; INL-L inner nuclear layer-like; GCL-L, ganglion cell layer-like. Scale bar (100 μm) in A also applies to B, C. Scale bar (100 μm) in D also applies to E–O.
    Mouse Anti Ap2α, supplied by Becton Dickinson, 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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    99
    Cell Signaling Technology Inc ap2α
    Retina-like tissue in the subretinal space of a 2-week-old P RPE65 -ngn3 mouse. A–C: Low magnification images to show the relative position of retina-like tissue (arrow) in a cross-section of the eye, with the anterior to the left and the posterior to the right. A: Bright-field (BF) view. B: Epi-fluorescence of DAPI staining of the nuclei. C: Epi-fluorescence of anti-recoverin (Rcv) immunostaining to mark photoreceptor cells. Boxes outline the corresponding regions in A–C, or similar locations on serial sections (D–O). D–G: A serial section under bright-field view (D), after DAPI nuclear staining (E), and after double immunostaining for Otx2 (in green, F) to identify bipolar cells and for Pax6 (in red, G) to label amacrine cells in the INL and in the GCL (misplaced amacrine). H–K: A serial section under bright-field view (H), with DAPI nuclear staining (I), and with double immunostaining for <t>AP2α</t> (in green, J) to label horizontal (albeit weakly) and amacrine cells in the INL and in the GCL (misplaced amacrine), and for Brn3A (in red, K) to identify a subset of ganglion cells. L–O: A serial section under bright-field view (L), with DAPI nuclear staining (M), and with double immunostaining for photoreceptor protein recoverin (in green, N) and for RPE protein RPE65 (in red, O). RPE-L, RPE-like; ONL-L, outer nuclear layer-like; INL-L inner nuclear layer-like; GCL-L, ganglion cell layer-like. Scale bar (100 μm) in A also applies to B, C. Scale bar (100 μm) in D also applies to E–O.
    Ap2α, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology mouse monoclonal anti ap2α
    Loss of Msx2 activity does not alter Pax6 and <t>Ap2α</t> expression. Protein expression in WT and Msx2 -deficient embryos, respectively, was examined by immunofluorescence for Pax6 on E9.5 ( A and B ) and E11.5 ( C and D ), Ap2-α ( E – F ), and crystallin on E11.5 ( G and H ) and E12.5 ( I and J ). At the lens vesicle stage of eye development, the intensity and the spatial domain of immune-reactivity are not altered significantly. Immunofluorescence of WT and Msx2 -deficient embryonic eyes at E11.5 and E12.5 show significant overexpression of α- and β-crystallin in the Msx2 -deficient lens vesicle ( G–J ). Scale bars: 50 μm ( A and B ), 100 μm ( C–J ). Re, retina; Le, lens; OV, optic vesicle.
    Mouse Monoclonal Anti Ap2α, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit polyclonal anti ap2α antibody
    Loss of Msx2 activity does not alter Pax6 and <t>Ap2α</t> expression. Protein expression in WT and Msx2 -deficient embryos, respectively, was examined by immunofluorescence for Pax6 on E9.5 ( A and B ) and E11.5 ( C and D ), Ap2-α ( E – F ), and crystallin on E11.5 ( G and H ) and E12.5 ( I and J ). At the lens vesicle stage of eye development, the intensity and the spatial domain of immune-reactivity are not altered significantly. Immunofluorescence of WT and Msx2 -deficient embryonic eyes at E11.5 and E12.5 show significant overexpression of α- and β-crystallin in the Msx2 -deficient lens vesicle ( G–J ). Scale bars: 50 μm ( A and B ), 100 μm ( C–J ). Re, retina; Le, lens; OV, optic vesicle.
    Rabbit Polyclonal Anti Ap2α Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti ap2α antibody
    Loss of Msx2 activity does not alter Pax6 and <t>Ap2α</t> expression. Protein expression in WT and Msx2 -deficient embryos, respectively, was examined by immunofluorescence for Pax6 on E9.5 ( A and B ) and E11.5 ( C and D ), Ap2-α ( E – F ), and crystallin on E11.5 ( G and H ) and E12.5 ( I and J ). At the lens vesicle stage of eye development, the intensity and the spatial domain of immune-reactivity are not altered significantly. Immunofluorescence of WT and Msx2 -deficient embryonic eyes at E11.5 and E12.5 show significant overexpression of α- and β-crystallin in the Msx2 -deficient lens vesicle ( G–J ). Scale bars: 50 μm ( A and B ), 100 μm ( C–J ). Re, retina; Le, lens; OV, optic vesicle.
    Anti Ap2α Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Fate mapping of NEC/NCC and otic placode ectoderm populations. ( A-D ) Lateral views of whole-mount embryos from E8.5 to E9.5. GFP reporter expression is activated by Wnt1-Cre, Pax3 Cre/+ , Hoxb1 Cre/+ or Pax2-Cre . Otic tissue is outlined in yellow; boxes indicate regions that are magnified in adjacent images. ( E , F ) NEC derivatives (green) express Ap2α (red). (E) Transverse sections through the trunk neural tube (NT) and dorsal root ganglion (DRG). Images on the right are higher magnification demonstrating expression of GFP with Ap2α in the DRG. (F) Lateral whole-mount views (left column) of the first and second branchial arches (BA1, BA2). Sagittal sections through the branchial arches (middle column) with magnified views of the BA2 mesoderm (right column). Nuclei are stained with DAPI (blue). ss, somite stage.

    Journal: Development (Cambridge, England)

    Article Title: Dual embryonic origin of the mammalian otic vesicle forming the inner ear

    doi: 10.1242/dev.069849

    Figure Lengend Snippet: Fate mapping of NEC/NCC and otic placode ectoderm populations. ( A-D ) Lateral views of whole-mount embryos from E8.5 to E9.5. GFP reporter expression is activated by Wnt1-Cre, Pax3 Cre/+ , Hoxb1 Cre/+ or Pax2-Cre . Otic tissue is outlined in yellow; boxes indicate regions that are magnified in adjacent images. ( E , F ) NEC derivatives (green) express Ap2α (red). (E) Transverse sections through the trunk neural tube (NT) and dorsal root ganglion (DRG). Images on the right are higher magnification demonstrating expression of GFP with Ap2α in the DRG. (F) Lateral whole-mount views (left column) of the first and second branchial arches (BA1, BA2). Sagittal sections through the branchial arches (middle column) with magnified views of the BA2 mesoderm (right column). Nuclei are stained with DAPI (blue). ss, somite stage.

    Article Snippet: Primary antibodies were: anti-NeuroD (SCBT sc-1084, 1:500), anti-islet 1 (DSHB 39.4D5, 1:100), anti-neurofilament (DSHB 2H3, 1:100), anti-P75 (Promega G3231, 1:500), anti-S100 (DAKO Z0311, 1:500), anti-MyoVIIA (DSHB MYO7A 138-1, 1:500), anti-Sox10 (Santa Cruz sc-17342, 1:100), anti-Pax3 (DSHB, 1:50), anti-Pax2 (Invitrogen 71-6000, 1:50), anti-AP2α (DSHB 3B5, 1:100), anti-Snai1 (Antonio García de Herreros, IMIM Hospital del Mar Research Institute, Barcelona, Spain, 1:5) and anti-laminin (Millipore AB2034, 1:200).

    Techniques: Expressing, Staining

    Differentiation occurs at the edge of hESC colonies. ( A ) Phase and ( B ) immunostaining images of hESC colonies when they are undifferentiated (top) and after 3 days BMP4 treatment (bottom). ( C ) Analysis of expression of pluripotency marker, SOX2, and differentiation marker, AP2α, after 3 days BMP4 treatment. Fluorescent intensity is plotted as a function of distance from the colony edge and normalized to the maximum intensity of each colony [ n = 20 colonies, **** p

    Journal: Scientific Reports

    Article Title: Edges of human embryonic stem cell colonies display distinct mechanical properties and differentiation potential

    doi: 10.1038/srep14218

    Figure Lengend Snippet: Differentiation occurs at the edge of hESC colonies. ( A ) Phase and ( B ) immunostaining images of hESC colonies when they are undifferentiated (top) and after 3 days BMP4 treatment (bottom). ( C ) Analysis of expression of pluripotency marker, SOX2, and differentiation marker, AP2α, after 3 days BMP4 treatment. Fluorescent intensity is plotted as a function of distance from the colony edge and normalized to the maximum intensity of each colony [ n = 20 colonies, **** p

    Article Snippet: The following primary antibodies were used overnight at 4°C: AP2α (1:50, Developmental Studies Hybridoma Bank), β-actin (1:2,000, Sigma).

    Techniques: Immunostaining, Expressing, Marker

    Cellular forces are predominantly on the edge of colonies. ( A ) Representative image of an hESC colony after 3 days of BMP4 treatment, grown on PDMS of 3 KPa stiffness. Nuclei are shown by DAPI staining and differentiation at the edge shown by immunostaining against AP2α and SOX2. ( B ) Bar graph of average differentiation band width on PDMS substrates of different stiffnesses. Soft is measured as 3 KPa, medium is estimated to be 30 KPa and stiff to be 100 KPa. ( C ) The differentiation band width of hESC colonies plated on matrigel- coated 3 KPa PDMS substrates (gray), overlaid on the data points from matrigel-coated glass coverslips or plastic dishes (red, as seen in Fig. 1D ). ( D ) DIC images (top) and corresponding strain energy densities (bottom) of two undifferentiated hESC colonies, measured by traction force microscopy. 1 pN/μm = 10 −6 J/m 2 . ( E ) Quantification of localization of peak regions of strain energy hESC colonies shows maximum strain energy peaks are near the edge of the colony, generally within 50 μm. ( n = 7 colonies). ( F ) Histogram of distance from the edge, normalized by total colony effective radius, for peaks of strain energy within the colony. The dashed vertical line indicates the effective colony center. All measured strain energy maxima are closer to the edge than to this center.

    Journal: Scientific Reports

    Article Title: Edges of human embryonic stem cell colonies display distinct mechanical properties and differentiation potential

    doi: 10.1038/srep14218

    Figure Lengend Snippet: Cellular forces are predominantly on the edge of colonies. ( A ) Representative image of an hESC colony after 3 days of BMP4 treatment, grown on PDMS of 3 KPa stiffness. Nuclei are shown by DAPI staining and differentiation at the edge shown by immunostaining against AP2α and SOX2. ( B ) Bar graph of average differentiation band width on PDMS substrates of different stiffnesses. Soft is measured as 3 KPa, medium is estimated to be 30 KPa and stiff to be 100 KPa. ( C ) The differentiation band width of hESC colonies plated on matrigel- coated 3 KPa PDMS substrates (gray), overlaid on the data points from matrigel-coated glass coverslips or plastic dishes (red, as seen in Fig. 1D ). ( D ) DIC images (top) and corresponding strain energy densities (bottom) of two undifferentiated hESC colonies, measured by traction force microscopy. 1 pN/μm = 10 −6 J/m 2 . ( E ) Quantification of localization of peak regions of strain energy hESC colonies shows maximum strain energy peaks are near the edge of the colony, generally within 50 μm. ( n = 7 colonies). ( F ) Histogram of distance from the edge, normalized by total colony effective radius, for peaks of strain energy within the colony. The dashed vertical line indicates the effective colony center. All measured strain energy maxima are closer to the edge than to this center.

    Article Snippet: The following primary antibodies were used overnight at 4°C: AP2α (1:50, Developmental Studies Hybridoma Bank), β-actin (1:2,000, Sigma).

    Techniques: Staining, Immunostaining, Microscopy

    Plating hESCs in smaller colonies improves their differentiation efficiency. ( A ) Differentiation efficiency after 3 days BMP4 treatment, measured by percent of colony area that is undifferentiated ( n = 175 colonies). Experimental data points (red) are plotted with theoretical curves assuming a constant differentiation band width of 149 μm (blue line) ± 59 μm (S.D.) (cyan lines). ( B ) Differentiation efficiency, measured by the number of AP2α + cells per colony area ( n = 11 colonies). Experimental data points (red) and plotted with theoretical curves assuming a constant number of AP2α + cells per circumference of 43 cells (maroon line) ± 12 cells (S.D.) (magenta lines). ( C ) Schematic of idealized circular colony with radius, R , and differentiation band width, a . If R is close to a , then the radius of the undifferentiated cells in the middle, r undiff , will be small. ( D ) Scheme for controling colony size through single cells. ( E ) Quantification of average colony size shows colonies are smaller 2–3 days after plating than 4–5 days. ( F ) Phase (top) and immunostaining (bottom) images of representative small (left) and large (right) colonies. After 3 days of BMP4 treatment, smaller colonies have a greater percentage of differentiated cells. ( G ) Representative western analysis of AP2α levels with β-actin as loading control. Smaller colonies show greater expression of AP2α compared to larger colonies. Four independent experiments were performed. ( H ) Quantification of differentiation efficiency, measured by percent of colony area. Smaller colonies have greater efficiency and follow a curve similar to that in panels ( A ) and ( B ). Inset: average differentiation efficiency of small (red) versus large (blue) colonies. ( n = 83 colonies, **** p

    Journal: Scientific Reports

    Article Title: Edges of human embryonic stem cell colonies display distinct mechanical properties and differentiation potential

    doi: 10.1038/srep14218

    Figure Lengend Snippet: Plating hESCs in smaller colonies improves their differentiation efficiency. ( A ) Differentiation efficiency after 3 days BMP4 treatment, measured by percent of colony area that is undifferentiated ( n = 175 colonies). Experimental data points (red) are plotted with theoretical curves assuming a constant differentiation band width of 149 μm (blue line) ± 59 μm (S.D.) (cyan lines). ( B ) Differentiation efficiency, measured by the number of AP2α + cells per colony area ( n = 11 colonies). Experimental data points (red) and plotted with theoretical curves assuming a constant number of AP2α + cells per circumference of 43 cells (maroon line) ± 12 cells (S.D.) (magenta lines). ( C ) Schematic of idealized circular colony with radius, R , and differentiation band width, a . If R is close to a , then the radius of the undifferentiated cells in the middle, r undiff , will be small. ( D ) Scheme for controling colony size through single cells. ( E ) Quantification of average colony size shows colonies are smaller 2–3 days after plating than 4–5 days. ( F ) Phase (top) and immunostaining (bottom) images of representative small (left) and large (right) colonies. After 3 days of BMP4 treatment, smaller colonies have a greater percentage of differentiated cells. ( G ) Representative western analysis of AP2α levels with β-actin as loading control. Smaller colonies show greater expression of AP2α compared to larger colonies. Four independent experiments were performed. ( H ) Quantification of differentiation efficiency, measured by percent of colony area. Smaller colonies have greater efficiency and follow a curve similar to that in panels ( A ) and ( B ). Inset: average differentiation efficiency of small (red) versus large (blue) colonies. ( n = 83 colonies, **** p

    Article Snippet: The following primary antibodies were used overnight at 4°C: AP2α (1:50, Developmental Studies Hybridoma Bank), β-actin (1:2,000, Sigma).

    Techniques: Immunostaining, Western Blot, Expressing

    Neuronal and Muller cell genesis in hyperglycemic animals. A–C . Retinal sections of CTL and STZ-treated P6 rat pups were stained with various antibodies specific to neurons of the INL (A), photoreceptors cells (B) and glial cells (C) of the retina. A : INL neuron generation was not affected by hyperglycemia and similar patterns of staining (red) were observed in CTL and STZ animals for PKCα (bipolar cells), Ap2α (amacrine cells) and calretinin (CALR, horizontal cells). B . Photoreceptor generation was not affected by hyperglycemia and similar patterns of staining (red) were observed for peanut agglutinin (PNA, cones), Rho4D2 (R4D2, rods) in STZ P6 animals compared to CTL. C . Muller cells cell genesis was not affected by hyperglycemia when compared to CTL. Nuclei were counterstained with DAPI. Scale bars 50 µm. GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer.

    Journal: PLoS ONE

    Article Title: Neonatal Hyperglycemia Inhibits Angiogenesis and Induces Inflammation and Neuronal Degeneration in the Retina

    doi: 10.1371/journal.pone.0079545

    Figure Lengend Snippet: Neuronal and Muller cell genesis in hyperglycemic animals. A–C . Retinal sections of CTL and STZ-treated P6 rat pups were stained with various antibodies specific to neurons of the INL (A), photoreceptors cells (B) and glial cells (C) of the retina. A : INL neuron generation was not affected by hyperglycemia and similar patterns of staining (red) were observed in CTL and STZ animals for PKCα (bipolar cells), Ap2α (amacrine cells) and calretinin (CALR, horizontal cells). B . Photoreceptor generation was not affected by hyperglycemia and similar patterns of staining (red) were observed for peanut agglutinin (PNA, cones), Rho4D2 (R4D2, rods) in STZ P6 animals compared to CTL. C . Muller cells cell genesis was not affected by hyperglycemia when compared to CTL. Nuclei were counterstained with DAPI. Scale bars 50 µm. GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer.

    Article Snippet: In addition to the antibodies used on retinal flatmounts, the following primary antibodies were used to study retinal cells type on cryosections: anti-Ap2α antibody (3B5, for amacrine and horizontal cells) was purchased to Developmental Studies Hybridoma Bank (DHSB, Iowa city, Iowa); mouse anti-glutamine synthetase antibody (MAB302, for Muller cells) was obtained from Millipore (Saint-Quentin Fallavier, France), and rabbit anti-protein kinase Cα antibody (PKCα) (Sc-208, for bipolar cells) from Santa Cruz (Heidelberg, Germany).

    Techniques: CTL Assay, Staining

    Notch drives Schwann cell generation from SCPs. ( a,b ) Top and middle panels, longitudinal sections of E14 hind limb nerves immunolabeled for TUJ1 (inset; red), AP2α or S100β (DAB). Insets, spinal cord (S), DRG (arrowheads). Bottem panels,

    Journal: Nature neuroscience

    Article Title: Notch controls embryonic Schwann cell differentiation, postnatal myelination and adult plasticity

    doi: 10.1038/nn.2323

    Figure Lengend Snippet: Notch drives Schwann cell generation from SCPs. ( a,b ) Top and middle panels, longitudinal sections of E14 hind limb nerves immunolabeled for TUJ1 (inset; red), AP2α or S100β (DAB). Insets, spinal cord (S), DRG (arrowheads). Bottem panels,

    Article Snippet: The samples were incubated in 0.2% triton in blocking solution (BS: PBS containing 10% FCS, 0.1% lysine and 0.02% sodium azide) followed by overnight incubation at 4 °C with the following primary antibodies: BFABP (rabbit, 1:5000); TUJ1 (mouse, 1:5000, Covance); S100β (rabbit, 1:1000, Dakopatts); AP2α (rabbit, 1:1000; Santa Cruz Biotechnology); MBP (mouse, 1:500, Sternberger Monoclonals); Jagged 1 (rabbit, 1:200, Santa Cruz Biotechnology); Notch 1 (hamster, 1:100, Upstate); and neurofilament (mouse, 1:50).

    Techniques: Immunolabeling

    p73 and AP2 bind to the NEU4 promoter in ChIP assays. ( A ) p73 bound the NEU4 and p21 promoters, but not the open reading frame of NEU4, in HT29 cells. ( B ) AP2α bound the NEU4 promoter, but not the open reading frame of NEU4, in HT29 cells. ( C ) AP2γ bound the NEU4 promoter, but not the open reading frame of NEU4, in DLD1 cells. ( A – C ) Results are displayed as mean ± SD, n = 3 (***p

    Journal: Scientific Reports

    Article Title: Synergistic activation of the NEU4 promoter by p73 and AP2 in colon cancer cells

    doi: 10.1038/s41598-018-37521-7

    Figure Lengend Snippet: p73 and AP2 bind to the NEU4 promoter in ChIP assays. ( A ) p73 bound the NEU4 and p21 promoters, but not the open reading frame of NEU4, in HT29 cells. ( B ) AP2α bound the NEU4 promoter, but not the open reading frame of NEU4, in HT29 cells. ( C ) AP2γ bound the NEU4 promoter, but not the open reading frame of NEU4, in DLD1 cells. ( A – C ) Results are displayed as mean ± SD, n = 3 (***p

    Article Snippet: We used ChIP-grade anti-p73 (ab14430) along with anti-AP2α and -AP2γ (sc-184X and sc-8977x, respectively; Santa Cruz).

    Techniques: Chromatin Immunoprecipitation

    p73 and AP2 affect NEU4 expression. ( A ) sialyl Lewis X was down-regulated under starvation conditions in HT29 cells (orange line, isotype control under full serum culture; blue line, isotype control under starvation; green line, sialyl Lewis X under full serum culture; red line, sialyl Lewis X under starvation). ( B ) Sialyl Lewis A levels did not change under starvation conditions in HT29 cells (orange line, isotype control under full serum culture; blue line, isotype control under starvation; green line, sialyl Lewis A under full serum culture; red line, sialyl Lewis A under starvation). ( C ) NEU4, but not FUT2, mRNA was increased by p73 overexpression in HT29 cells. ( D,E , F ) Knock-down of p73 ( D ) or AP2α ( E ) in HT29 cells or of AP2γ in DLD1 cells ( F ) reduced NEU4 mRNA. ( C–F ) Results are displayed as mean ± SD, n = 3 (***p

    Journal: Scientific Reports

    Article Title: Synergistic activation of the NEU4 promoter by p73 and AP2 in colon cancer cells

    doi: 10.1038/s41598-018-37521-7

    Figure Lengend Snippet: p73 and AP2 affect NEU4 expression. ( A ) sialyl Lewis X was down-regulated under starvation conditions in HT29 cells (orange line, isotype control under full serum culture; blue line, isotype control under starvation; green line, sialyl Lewis X under full serum culture; red line, sialyl Lewis X under starvation). ( B ) Sialyl Lewis A levels did not change under starvation conditions in HT29 cells (orange line, isotype control under full serum culture; blue line, isotype control under starvation; green line, sialyl Lewis A under full serum culture; red line, sialyl Lewis A under starvation). ( C ) NEU4, but not FUT2, mRNA was increased by p73 overexpression in HT29 cells. ( D,E , F ) Knock-down of p73 ( D ) or AP2α ( E ) in HT29 cells or of AP2γ in DLD1 cells ( F ) reduced NEU4 mRNA. ( C–F ) Results are displayed as mean ± SD, n = 3 (***p

    Article Snippet: We used ChIP-grade anti-p73 (ab14430) along with anti-AP2α and -AP2γ (sc-184X and sc-8977x, respectively; Santa Cruz).

    Techniques: Expressing, Over Expression

    p73 or AP2α knock-down rescues starvation-mediated sialyl Lewis X repression. ( A ) Starvation repressed sialyl Lewis X expression in HT29 cells infected with a scrambled shRNA (orange line, isotype control with cells infected with a control scrambled shRNA cultured with full serum; blue line, isotype control with cells infected with a control scrambled shRNA under starvation; green line, sialyl Lewis X staining with cells infected with a control scrambled shRNA cultured with full serum; red line, sialyl Lewis X staining with cells infected with a control scrambled shRNA cultured under starvation). ( B ) p73 knock down (p73 KD1 cells, as in Fig. 2D ) rescued the starvation-mediated sialyl Lewis X repression (orange line, isotype control with p73 KD1 cells cultured with full serum; blue line, isotype control with p73 KD1 cells cultured under starvation; green line, sialyl Lewis X staining with p73 KD1 cells cultured with full serum; red line, sialyl Lewis X staining with p73 KD1 cells cultured under starvation). ( C ) AP2α knock down (AP2α KD1 cells, as in Fig. 2E ) rescued starvation-mediated sialyl Lewis X repression (orange line, isotype control for AP2α KD1 cells cultured with full serum; blue line, isotype control for AP2α KD1 cells cultured under starvation; green line, sialyl Lewis X staining for AP2α KD1 cells cultured with full serum; red line, sialyl Lewis X staining for AP2α KD1 cells cultured under starvation).

    Journal: Scientific Reports

    Article Title: Synergistic activation of the NEU4 promoter by p73 and AP2 in colon cancer cells

    doi: 10.1038/s41598-018-37521-7

    Figure Lengend Snippet: p73 or AP2α knock-down rescues starvation-mediated sialyl Lewis X repression. ( A ) Starvation repressed sialyl Lewis X expression in HT29 cells infected with a scrambled shRNA (orange line, isotype control with cells infected with a control scrambled shRNA cultured with full serum; blue line, isotype control with cells infected with a control scrambled shRNA under starvation; green line, sialyl Lewis X staining with cells infected with a control scrambled shRNA cultured with full serum; red line, sialyl Lewis X staining with cells infected with a control scrambled shRNA cultured under starvation). ( B ) p73 knock down (p73 KD1 cells, as in Fig. 2D ) rescued the starvation-mediated sialyl Lewis X repression (orange line, isotype control with p73 KD1 cells cultured with full serum; blue line, isotype control with p73 KD1 cells cultured under starvation; green line, sialyl Lewis X staining with p73 KD1 cells cultured with full serum; red line, sialyl Lewis X staining with p73 KD1 cells cultured under starvation). ( C ) AP2α knock down (AP2α KD1 cells, as in Fig. 2E ) rescued starvation-mediated sialyl Lewis X repression (orange line, isotype control for AP2α KD1 cells cultured with full serum; blue line, isotype control for AP2α KD1 cells cultured under starvation; green line, sialyl Lewis X staining for AP2α KD1 cells cultured with full serum; red line, sialyl Lewis X staining for AP2α KD1 cells cultured under starvation).

    Article Snippet: We used ChIP-grade anti-p73 (ab14430) along with anti-AP2α and -AP2γ (sc-184X and sc-8977x, respectively; Santa Cruz).

    Techniques: Expressing, Infection, shRNA, Cell Culture, Staining

    Prdm13 marks a subset of amacrines in the adult retina. (A–D ‴ ) P30 Prdm13-GFP/+ heterozygous animals co-stained with GFP (green) and amacrine cell markers (grey/red). (A–A ‴ ) A small subset of Prdm13-GFP+ amacrine cells co-express AP2α (grey, arrows) and Bhlhb5 (red, arrowheads). (B–B ‴ ) Nearly half of GFP+ cells co-express calretinin (grey, arrows), while less than 1% of GFP+ cells co-express calbindin (red). No intensely calretinin+ horizontal cells co-express GFP. (C–C ‴ ) A large fraction of GFP+ cells co-express the glycinergic amacrine marker GlyT (grey, arrows), but few GFP+ cells co-express the GABAergic marker GAD65/67 and these were sometimes GlyT+ as well. (D–D ‴ ) The majority of GFP+ cells co-express Ebf3 (grey, arrows), but all Ebf3+ cells in the INL are GFP+. A minority of GFP+ cells co-express Bhlhb5 (red, arrowheads) and cells that co-express Bhlhb5, Ebf3, and GFP are rarely seen. (E) Plot of the percentage of GFP+ cells that co-express a given marker. GFP does not overlap with Brn3, TH, or vGlut3 (not shown). (F) Plot showing the percentage of Ebf3+ amacrines, Pax6+ INL nuclei, and AP2α+ INL cells that co-express Prdm13-GFP. Sample size was 3 mice per condition. Statistical significance determined by unpaired two-sample t tests: * P

    Journal: Developmental biology

    Article Title: Prdm13 is required for Ebf3+ amacrine cell formation in the retina

    doi: 10.1016/j.ydbio.2017.12.003

    Figure Lengend Snippet: Prdm13 marks a subset of amacrines in the adult retina. (A–D ‴ ) P30 Prdm13-GFP/+ heterozygous animals co-stained with GFP (green) and amacrine cell markers (grey/red). (A–A ‴ ) A small subset of Prdm13-GFP+ amacrine cells co-express AP2α (grey, arrows) and Bhlhb5 (red, arrowheads). (B–B ‴ ) Nearly half of GFP+ cells co-express calretinin (grey, arrows), while less than 1% of GFP+ cells co-express calbindin (red). No intensely calretinin+ horizontal cells co-express GFP. (C–C ‴ ) A large fraction of GFP+ cells co-express the glycinergic amacrine marker GlyT (grey, arrows), but few GFP+ cells co-express the GABAergic marker GAD65/67 and these were sometimes GlyT+ as well. (D–D ‴ ) The majority of GFP+ cells co-express Ebf3 (grey, arrows), but all Ebf3+ cells in the INL are GFP+. A minority of GFP+ cells co-express Bhlhb5 (red, arrowheads) and cells that co-express Bhlhb5, Ebf3, and GFP are rarely seen. (E) Plot of the percentage of GFP+ cells that co-express a given marker. GFP does not overlap with Brn3, TH, or vGlut3 (not shown). (F) Plot showing the percentage of Ebf3+ amacrines, Pax6+ INL nuclei, and AP2α+ INL cells that co-express Prdm13-GFP. Sample size was 3 mice per condition. Statistical significance determined by unpaired two-sample t tests: * P

    Article Snippet: The following primary antibodies were used at the given concentrations: mouse anti-AP2α (1:400; 5E4-c, DSHB Iowa City, Iowa USA); goat anti-Bhlhb5 (1:500; sc-6045, Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA); mouse anti-Brn3a (1:500; sc-6026, Santa Cruz Biotechnology, Inc); rabbit anti-Calbindin (1:400; AB1778, Millipore, Temecula, CA, USA); mouse anti-Calretinin (1:500; MAB1568.

    Techniques: Staining, Marker, Mouse Assay

    Ebf3+ amacrines are lost in adult Prdm13 mutants. (A–A ′ ) Schematics of the two transgenic mice, Prdm13-GFP (A) and Prdm13-Δ115 (A′). The Δ115 allele has an 115bp deletion in the first exon of Prdm13 . Unlike homozygous Prdm13-GFP mice, homozygous Δ115/Δ115 mice and compound heterozygous Prdm13-GFP / Δ115 are viable. (B–B ′ ) Prdm13-GFP/Δ115 mice (B′) have far fewer GFP+ cells (arrows) than Prdm13-GFP/+ control mice (B). (C) Plot showing the reduction in GFP+ cells between the two genotypes. (D–K) Prdm13-GFP/+ and Prdm13-GFP/Δ115 retinas stained for GFP (green) and various amacrine cell markers (red, grey). (D–E ″ ) The number of GlyT (grey) and GAD+ (red) cells is modestly altered in Prdm13-GFP/Δ115 mice (E) versus controls (D). The number of GlyT+/GFP+ cells (arrows) is reduced in Prdm13-GFP/Δ115 mice. (F–G ″ ) Staining for AP2α (grey) and Bhlhb5 (red) show a small decrease in both populations in Prdm13-GFP/Δ115 mice (E) compared to controls (F). Cells expressing GFP, AP2α, and Bhlhb5 are marked by stars. (H–I ″ ) Calbindin (red) staining is similar between genotypes and rarely overlaps with GFP, but calretinin (grey) stains revealed a disrupted IPL in Prdm13-GFP/Δ115 mice. There are fewer calretinin+ cells in the INL and far fewer GFP+/calretinin+ cells in Prdm13-GFP/Δ115 mice compared to Prdm13-GFP controls (arrows). (J–K ″ ) Ebf3+ (grey) amacrine cells are almost entirely absent from the INL in Prdm13-GFP/Δ115 mice. GFP+/Ebf3+ cells (arrows) are rare in Prdm13-GFP/Δ115 mice (K) compared to control (J). Some Bhlhb5+/GFP+ cells are seen in both conditions, some of which are Ebf3+/Bhlhb5+/GFP+ (stars). (L–L ′ ) Close up views of calretinin staining in control (L) and Prdm13-GFP/Δ115 (L′) retinas. Loss of sublamina 3 and a thinning of the IPL are evident. (M) Plot showing the number of marker positive cells in control and Prdm13-GFP/Δ115 mice. (N) Plot showing the number of marker positive cells that co-express GFP. Sample sizes are 3 mice per condition for heterozygotes and 6 mice for mutants. Statistical significance determined by unpaired two-sample t tests: * P

    Journal: Developmental biology

    Article Title: Prdm13 is required for Ebf3+ amacrine cell formation in the retina

    doi: 10.1016/j.ydbio.2017.12.003

    Figure Lengend Snippet: Ebf3+ amacrines are lost in adult Prdm13 mutants. (A–A ′ ) Schematics of the two transgenic mice, Prdm13-GFP (A) and Prdm13-Δ115 (A′). The Δ115 allele has an 115bp deletion in the first exon of Prdm13 . Unlike homozygous Prdm13-GFP mice, homozygous Δ115/Δ115 mice and compound heterozygous Prdm13-GFP / Δ115 are viable. (B–B ′ ) Prdm13-GFP/Δ115 mice (B′) have far fewer GFP+ cells (arrows) than Prdm13-GFP/+ control mice (B). (C) Plot showing the reduction in GFP+ cells between the two genotypes. (D–K) Prdm13-GFP/+ and Prdm13-GFP/Δ115 retinas stained for GFP (green) and various amacrine cell markers (red, grey). (D–E ″ ) The number of GlyT (grey) and GAD+ (red) cells is modestly altered in Prdm13-GFP/Δ115 mice (E) versus controls (D). The number of GlyT+/GFP+ cells (arrows) is reduced in Prdm13-GFP/Δ115 mice. (F–G ″ ) Staining for AP2α (grey) and Bhlhb5 (red) show a small decrease in both populations in Prdm13-GFP/Δ115 mice (E) compared to controls (F). Cells expressing GFP, AP2α, and Bhlhb5 are marked by stars. (H–I ″ ) Calbindin (red) staining is similar between genotypes and rarely overlaps with GFP, but calretinin (grey) stains revealed a disrupted IPL in Prdm13-GFP/Δ115 mice. There are fewer calretinin+ cells in the INL and far fewer GFP+/calretinin+ cells in Prdm13-GFP/Δ115 mice compared to Prdm13-GFP controls (arrows). (J–K ″ ) Ebf3+ (grey) amacrine cells are almost entirely absent from the INL in Prdm13-GFP/Δ115 mice. GFP+/Ebf3+ cells (arrows) are rare in Prdm13-GFP/Δ115 mice (K) compared to control (J). Some Bhlhb5+/GFP+ cells are seen in both conditions, some of which are Ebf3+/Bhlhb5+/GFP+ (stars). (L–L ′ ) Close up views of calretinin staining in control (L) and Prdm13-GFP/Δ115 (L′) retinas. Loss of sublamina 3 and a thinning of the IPL are evident. (M) Plot showing the number of marker positive cells in control and Prdm13-GFP/Δ115 mice. (N) Plot showing the number of marker positive cells that co-express GFP. Sample sizes are 3 mice per condition for heterozygotes and 6 mice for mutants. Statistical significance determined by unpaired two-sample t tests: * P

    Article Snippet: The following primary antibodies were used at the given concentrations: mouse anti-AP2α (1:400; 5E4-c, DSHB Iowa City, Iowa USA); goat anti-Bhlhb5 (1:500; sc-6045, Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA); mouse anti-Brn3a (1:500; sc-6026, Santa Cruz Biotechnology, Inc); rabbit anti-Calbindin (1:400; AB1778, Millipore, Temecula, CA, USA); mouse anti-Calretinin (1:500; MAB1568.

    Techniques: Transgenic Assay, Mouse Assay, Staining, Expressing, Marker

    Sema3c expression is negatively regulated by Tbx1-Fgf8-Erk signaling. ( A ) Sema3c mRNA expression in cNCC explants treated with or without Fgf8 for 3 days. n = 5 per group. ( B ) Sema3c mRNA expression in cNCC explants treated with Fgf8 with or without AKT inhibitor (MK-2206) or ERK1/2 inhibitor (SCH772984). n = 6 per group. ( C ) Immunostaining for phosphor-Erk1/2 (red) and Ap2α (green) of the pharyngeal arch region in wild-type ( Tbx1 +/+ ) and Tbx1 neo/neo mutant embryos at E10.5. Right panels showed hyper-magnified views of area indicated by the white box on left panels. Scale bars, 100 μm. pa, pharynx; da, dorsal aorta; III and IV, 3rd and 4th pharyngeal arch arteries. * p

    Journal: Scientific Reports

    Article Title: Regulation of Sema3c and the Interaction between Cardiac Neural Crest and Second Heart Field during Outflow Tract Development

    doi: 10.1038/s41598-017-06964-9

    Figure Lengend Snippet: Sema3c expression is negatively regulated by Tbx1-Fgf8-Erk signaling. ( A ) Sema3c mRNA expression in cNCC explants treated with or without Fgf8 for 3 days. n = 5 per group. ( B ) Sema3c mRNA expression in cNCC explants treated with Fgf8 with or without AKT inhibitor (MK-2206) or ERK1/2 inhibitor (SCH772984). n = 6 per group. ( C ) Immunostaining for phosphor-Erk1/2 (red) and Ap2α (green) of the pharyngeal arch region in wild-type ( Tbx1 +/+ ) and Tbx1 neo/neo mutant embryos at E10.5. Right panels showed hyper-magnified views of area indicated by the white box on left panels. Scale bars, 100 μm. pa, pharynx; da, dorsal aorta; III and IV, 3rd and 4th pharyngeal arch arteries. * p

    Article Snippet: Frozen sections (8 μm) were blocked with 5% goat serum in PBS with 0.1% Tween20 for 1 hr at room temperature and stained with following primary antibodies: rat monoclonal anti-Sema3 (1:50, R & D MAB1728), mouse monoclonal anti-AP2α clone 3B5 (1:100, Developmental studies hybridoma bank), rabbit polyclonal anti-β-galactosidase (1:1000, Rockland 200–4136) and rabbit polyclonal anti-Isl1 (1:100, Abcam ab20670).

    Techniques: Expressing, Immunostaining, Mutagenesis

    Altered expression of Sema3c and defects of cardiac neural crest migration in the hypomorphic state of Tbx1 ( Tbx1 neo/neo ). ( A ) Sema3c int1/3′- lacZ transgene construct with Sema3c intron1, exon2, and the 3′-flanking sequence including highly conserved regions (indicated by black bar) (from +151 to +2052 followed by the hsp68 basal promoter, lacZ gene, and the 3′ region from +155988 to +157878). ( B ) LacZ expression in Sema3c int1/3′- lacZ : Tbx1 neo/+ (upper, left) and Tbx1 neo/neo (lower, left) double transgenic (tg) embryos at E10.5 reminiscent of endogenous Sema3c mRNA expression. High magnification views of the pharyngeal arch region (white box) are shown in the right lane. Enhanced lacZ expression in the pharyngeal arch region of Sema3c int1/3′- lacZ tg: Tbx1 neo/neo embryo (lower, right) compared to Sema3c int1/3′- lacZ tg: Tbx1 neo/+ embryo (upper, right) Scale bars, 500 μm. ( C ) Coronal section in the pharyngeal arch region of Sema3c int1/3′- lacZ tg: Tbx1 neo/neo or Sema3c int1/3′- lacZ tg: Tbx1 neo/+ embryos. Right panels are high magnification views of boxed areas in the left panels. Scale bars, 100 μm. ( D ) Immunostaining for Sema3c (green) and βGal (red) on coronal sections of the pharyngeal arch region in Sema3c int1/3′- lacZ tg: Tbx1 neo/+ or Sema3c int1/3′- lacZ tg: Tbx1 neo/neo embryos. Scale bars, 100 μm. ( E ) Immunostaining for nucleus (blue), Sema3c (green) and Ap2α (red) on coronal sections of the pharyngeal arch region in wild-type ( Tbx1 +/+ ) and Tbx1 neo/neo mutant embryos at E10.5. Lower panels show hyper-magnified views of areas indicated by the white box in the upper panels. Scale bars, 100 μm. (F) Immunostaining for nucleus (blue), Sema3c (green), Ap2α (red) and Isl1 (cyan) on coronal sections of the pharyngeal arch region in wild-type ( Tbx1 +/+ ) and Tbx1 neo/neo mutant embryos at E10.5. Cut-in panels showed hyper-magnified views of cells indicated by white arrowheads in the upper panels. Scale bars, 50 μm: lower magnification view; 10 μm: hyper magnification view. lv, left ventricle; rv, right ventricle; oft, outflow tract; pa, pharynx; nt, neural tube; da, dorsal aorta; II, III, IV, and VI, 2nd, 3rd, 4th, and 6th pharyngeal arch arteries, respectively.

    Journal: Scientific Reports

    Article Title: Regulation of Sema3c and the Interaction between Cardiac Neural Crest and Second Heart Field during Outflow Tract Development

    doi: 10.1038/s41598-017-06964-9

    Figure Lengend Snippet: Altered expression of Sema3c and defects of cardiac neural crest migration in the hypomorphic state of Tbx1 ( Tbx1 neo/neo ). ( A ) Sema3c int1/3′- lacZ transgene construct with Sema3c intron1, exon2, and the 3′-flanking sequence including highly conserved regions (indicated by black bar) (from +151 to +2052 followed by the hsp68 basal promoter, lacZ gene, and the 3′ region from +155988 to +157878). ( B ) LacZ expression in Sema3c int1/3′- lacZ : Tbx1 neo/+ (upper, left) and Tbx1 neo/neo (lower, left) double transgenic (tg) embryos at E10.5 reminiscent of endogenous Sema3c mRNA expression. High magnification views of the pharyngeal arch region (white box) are shown in the right lane. Enhanced lacZ expression in the pharyngeal arch region of Sema3c int1/3′- lacZ tg: Tbx1 neo/neo embryo (lower, right) compared to Sema3c int1/3′- lacZ tg: Tbx1 neo/+ embryo (upper, right) Scale bars, 500 μm. ( C ) Coronal section in the pharyngeal arch region of Sema3c int1/3′- lacZ tg: Tbx1 neo/neo or Sema3c int1/3′- lacZ tg: Tbx1 neo/+ embryos. Right panels are high magnification views of boxed areas in the left panels. Scale bars, 100 μm. ( D ) Immunostaining for Sema3c (green) and βGal (red) on coronal sections of the pharyngeal arch region in Sema3c int1/3′- lacZ tg: Tbx1 neo/+ or Sema3c int1/3′- lacZ tg: Tbx1 neo/neo embryos. Scale bars, 100 μm. ( E ) Immunostaining for nucleus (blue), Sema3c (green) and Ap2α (red) on coronal sections of the pharyngeal arch region in wild-type ( Tbx1 +/+ ) and Tbx1 neo/neo mutant embryos at E10.5. Lower panels show hyper-magnified views of areas indicated by the white box in the upper panels. Scale bars, 100 μm. (F) Immunostaining for nucleus (blue), Sema3c (green), Ap2α (red) and Isl1 (cyan) on coronal sections of the pharyngeal arch region in wild-type ( Tbx1 +/+ ) and Tbx1 neo/neo mutant embryos at E10.5. Cut-in panels showed hyper-magnified views of cells indicated by white arrowheads in the upper panels. Scale bars, 50 μm: lower magnification view; 10 μm: hyper magnification view. lv, left ventricle; rv, right ventricle; oft, outflow tract; pa, pharynx; nt, neural tube; da, dorsal aorta; II, III, IV, and VI, 2nd, 3rd, 4th, and 6th pharyngeal arch arteries, respectively.

    Article Snippet: Frozen sections (8 μm) were blocked with 5% goat serum in PBS with 0.1% Tween20 for 1 hr at room temperature and stained with following primary antibodies: rat monoclonal anti-Sema3 (1:50, R & D MAB1728), mouse monoclonal anti-AP2α clone 3B5 (1:100, Developmental studies hybridoma bank), rabbit polyclonal anti-β-galactosidase (1:1000, Rockland 200–4136) and rabbit polyclonal anti-Isl1 (1:100, Abcam ab20670).

    Techniques: Expressing, Migration, Construct, Sequencing, Transgenic Assay, Immunostaining, Mutagenesis

    The Ap2 α-IRESCre driver ablates FGF8 in the developing pharyngeal arch (PA) ectoderm. (A) A 12 kb genomic fragment containing exons 6, 7, the 3′ UTR and polyadenylation signal from the Ap2 α locus was used to generate the targeting vector for homologous recombination in ES cells. (B) The targeted allele contains the IRESCre cassette (see Materials and methods), positioned 198 bp 3′ of the translation stop in an engineered Asc I site. (C) AP2 α- IRESCre was tested for Cre activity by crossing to the Rosa26 lacZ reporter strain. 12, 21 and 35 somite stage embryos, with the genotype AP2 α IRESCre/+ ; Rosa26 lacZ/+ , were assayed for β-galactosidase activity (blue staining). Somite stages (ss) are labeled in the lower right corner of each panel and PAs are numbered. Blue staining, and both yellow and red arrowheads denote regions of Cre activity in the ectoderm of the PAs as they develop; Cre activity in the caudal ectoderm that will form PAs 3-6 is highlighted by the large red arrowhead in the 12 and 21 ss embryos. (D) Functionally relevant recombination of the Fgf8 AP conditional reporter allele in the developing PA ectoderm by the AP2 α- IRESCre driver. A whole-mount, 21 ss Fgf8 AP/+ ;AP2 α -IRESCre/+ embryo is shown in the left panel after assaying for alkaline phosphatase activity. The black line indicates the plane of the coronal section shown in the right panel. The ectoderm of developing PA 3, and that of PAs 1 and 2, are stained violet because of Fgf8 APR activity. Although AP2 α- IRESCre is expressed in neural crest, the ectomesenchyme of the PAs is not stained because Fgf8 is not expressed in these cells. (E) AP2 α -IRESCre ablates Fgf8 function throughout its expression domains in the PA ectoderm. Expression of Fgf8 GFPR after recombination with the AP2 α -IRESCre ), was assessed by whole-mount anti-GFP immunohistochemistry of 22 ss, stage-matched embryos (ss in lower right corner). The domains of Fgf8 inactivation resulting from the AP2 α -IRESCre driver are the same ectodermal domains detected with the universal ‘deleter’ Cre. Fgf8 GFPR is expressed in caudal ectoderm that will form PAs 3-6. (F,G) Coronal sections through PAs 1 and 2, and the developing third arch region of a 20 ss embryo. Fgf8 GFP/+ ;AP2α IRESCre/+ embryo (G) reveals Fgf8 GFPR expression throughout the ectoderm of the developing third arch and cleft.

    Journal: Development (Cambridge, England)

    Article Title: Ablation of Specific Expression Domains Reveals Discrete Functions of Ectoderm- and Endoderm-Derived FGF8 During Cardiovascular and Pharyngeal Development

    doi: 10.1242/dev.00850

    Figure Lengend Snippet: The Ap2 α-IRESCre driver ablates FGF8 in the developing pharyngeal arch (PA) ectoderm. (A) A 12 kb genomic fragment containing exons 6, 7, the 3′ UTR and polyadenylation signal from the Ap2 α locus was used to generate the targeting vector for homologous recombination in ES cells. (B) The targeted allele contains the IRESCre cassette (see Materials and methods), positioned 198 bp 3′ of the translation stop in an engineered Asc I site. (C) AP2 α- IRESCre was tested for Cre activity by crossing to the Rosa26 lacZ reporter strain. 12, 21 and 35 somite stage embryos, with the genotype AP2 α IRESCre/+ ; Rosa26 lacZ/+ , were assayed for β-galactosidase activity (blue staining). Somite stages (ss) are labeled in the lower right corner of each panel and PAs are numbered. Blue staining, and both yellow and red arrowheads denote regions of Cre activity in the ectoderm of the PAs as they develop; Cre activity in the caudal ectoderm that will form PAs 3-6 is highlighted by the large red arrowhead in the 12 and 21 ss embryos. (D) Functionally relevant recombination of the Fgf8 AP conditional reporter allele in the developing PA ectoderm by the AP2 α- IRESCre driver. A whole-mount, 21 ss Fgf8 AP/+ ;AP2 α -IRESCre/+ embryo is shown in the left panel after assaying for alkaline phosphatase activity. The black line indicates the plane of the coronal section shown in the right panel. The ectoderm of developing PA 3, and that of PAs 1 and 2, are stained violet because of Fgf8 APR activity. Although AP2 α- IRESCre is expressed in neural crest, the ectomesenchyme of the PAs is not stained because Fgf8 is not expressed in these cells. (E) AP2 α -IRESCre ablates Fgf8 function throughout its expression domains in the PA ectoderm. Expression of Fgf8 GFPR after recombination with the AP2 α -IRESCre ), was assessed by whole-mount anti-GFP immunohistochemistry of 22 ss, stage-matched embryos (ss in lower right corner). The domains of Fgf8 inactivation resulting from the AP2 α -IRESCre driver are the same ectodermal domains detected with the universal ‘deleter’ Cre. Fgf8 GFPR is expressed in caudal ectoderm that will form PAs 3-6. (F,G) Coronal sections through PAs 1 and 2, and the developing third arch region of a 20 ss embryo. Fgf8 GFP/+ ;AP2α IRESCre/+ embryo (G) reveals Fgf8 GFPR expression throughout the ectoderm of the developing third arch and cleft.

    Article Snippet: The Ap2α transcription factor, which is expressed in neural crest and ectoderm, was detected with a mouse monoclonal anti-AP2α-antibody (1:25, 3B5, Developmental Studies Hybridoma Bank) and a FITC-conjugated anti-mouse IgG secondary (1:500, Molecular Probes).

    Techniques: Plasmid Preparation, Homologous Recombination, Activity Assay, Staining, Labeling, Expressing, Immunohistochemistry

    Dbx1 expression and tracing. (A–J) Dbx1 expression analysed by whole-mount in situ hybridisation. B and D are dorsal views of the embryos presented in A and C respectively, rostral is up. F, H and J are frontal views of the embryos shown in E, G and I respectively. Antero-posterior subdivisions of the neural plate/tube are indicated. Mb: midbrain, Fb: Forebrain, Mes: mesencephalon, Di: diencephalon, Tel: telencephalon. Arrows in E, G and I point to the vDi, dDM and FE subsets respectively; the arrowhead in I shows the ANR subset. (K, L) Coronal sections through the forebrain of a 12ss embryo showing d bx1 expression in the ANR (K) and facial ectoderm (L). (M) Coronal section collected at the midbrain level of a 3ss embryo illustrating dbx1 mRNA expression; the neural plate is delineated by dashed lines. (N) Coronal section collected at the level of the midbrain of a 6ss Dbx1 Cre ;R26 YFP embryo and stained for YFP (green) and DAPI (blue). (O) Quantification of dbx1 expression by qPCR. Asterisks indicate stages at which dbx1 could not be amplified. The relative expression values (normalised to 1 at 2ss and expressed in arbitrary units) are indicated on top of each bar as mean ± s.e.m. (P, Q) Coronal sections of a 20ss (E9.5) Dbx1 Cre ;R26 YFP embryo at the level of the telencephalon (P) and di/mesencephalon (Q), stained for YFP (green) and DAPI (blue). YFP is detected in regions that express dbx1 at earlier stages: ANR (red arrowhead in P), FE (white arrowheads in P), vDi and dDM (red and white arrowheads in P respectively) but also in cells located in the head mesenchyme (arrows in P). (R–W) Examples of YFP + cells found in the head mesenchyme of Dbx1 Cre ;R26 YFP embryos at 8ss (R–T) and 11ss (U–W) revealed by DAB immunostaining. Sections in R, S and U were collected at telencephalic levels, T, V and W at diencephalic levels. Neural tissues, surface ectoderm and endoderm are surrounded by black, blue and green dash lines respectively. (X) Coronal sections of a 8ss Dbx1 Cre ;R26 YFP embryo immunostained for YFP (green) and the neural crest cells marker AP2α (red); cell nuclei are stained by DAPI (blue). Some of the YFP + cells found in the neural plate are AP2α − (arrowhead in X 1 ) whereas YFP + cells located at the most lateral aspect of the neural plate or in the mesenchyme (arrows in X 1 –X 3 ) are AP2α + . Images X 1 –X 3 correspond to single confocal planes. (Y) Schematic drawing of dbx1 expression pattern. Scale bars: A: 200 µm; K, M, N, R, X: 50 µm; P: 100 µm.

    Journal: PLoS ONE

    Article Title: Dbx1-Expressing Cells Are Necessary for the Survival of the Mammalian Anterior Neural and Craniofacial Structures

    doi: 10.1371/journal.pone.0019367

    Figure Lengend Snippet: Dbx1 expression and tracing. (A–J) Dbx1 expression analysed by whole-mount in situ hybridisation. B and D are dorsal views of the embryos presented in A and C respectively, rostral is up. F, H and J are frontal views of the embryos shown in E, G and I respectively. Antero-posterior subdivisions of the neural plate/tube are indicated. Mb: midbrain, Fb: Forebrain, Mes: mesencephalon, Di: diencephalon, Tel: telencephalon. Arrows in E, G and I point to the vDi, dDM and FE subsets respectively; the arrowhead in I shows the ANR subset. (K, L) Coronal sections through the forebrain of a 12ss embryo showing d bx1 expression in the ANR (K) and facial ectoderm (L). (M) Coronal section collected at the midbrain level of a 3ss embryo illustrating dbx1 mRNA expression; the neural plate is delineated by dashed lines. (N) Coronal section collected at the level of the midbrain of a 6ss Dbx1 Cre ;R26 YFP embryo and stained for YFP (green) and DAPI (blue). (O) Quantification of dbx1 expression by qPCR. Asterisks indicate stages at which dbx1 could not be amplified. The relative expression values (normalised to 1 at 2ss and expressed in arbitrary units) are indicated on top of each bar as mean ± s.e.m. (P, Q) Coronal sections of a 20ss (E9.5) Dbx1 Cre ;R26 YFP embryo at the level of the telencephalon (P) and di/mesencephalon (Q), stained for YFP (green) and DAPI (blue). YFP is detected in regions that express dbx1 at earlier stages: ANR (red arrowhead in P), FE (white arrowheads in P), vDi and dDM (red and white arrowheads in P respectively) but also in cells located in the head mesenchyme (arrows in P). (R–W) Examples of YFP + cells found in the head mesenchyme of Dbx1 Cre ;R26 YFP embryos at 8ss (R–T) and 11ss (U–W) revealed by DAB immunostaining. Sections in R, S and U were collected at telencephalic levels, T, V and W at diencephalic levels. Neural tissues, surface ectoderm and endoderm are surrounded by black, blue and green dash lines respectively. (X) Coronal sections of a 8ss Dbx1 Cre ;R26 YFP embryo immunostained for YFP (green) and the neural crest cells marker AP2α (red); cell nuclei are stained by DAPI (blue). Some of the YFP + cells found in the neural plate are AP2α − (arrowhead in X 1 ) whereas YFP + cells located at the most lateral aspect of the neural plate or in the mesenchyme (arrows in X 1 –X 3 ) are AP2α + . Images X 1 –X 3 correspond to single confocal planes. (Y) Schematic drawing of dbx1 expression pattern. Scale bars: A: 200 µm; K, M, N, R, X: 50 µm; P: 100 µm.

    Article Snippet: Immunostaining The following primary antibodies were used: chick anti-GFP (AvesLab; 1∶2000), rabbit anti-AP2α (Santa Cruz; H-79; 1∶100), rabbit anti-PH3 (Upstate; 1∶500) and rabbit anti-activated Caspase-3 (Cell Signalling; 5A1; 1∶800).

    Techniques: Expressing, In Situ, Hybridization, Staining, Real-time Polymerase Chain Reaction, Amplification, Immunostaining, Marker

    HBx activates the SPHK1 promoter by upregulating the transcription factor AP2α. (A, B) The activity of the SPHK1 promoter (pGL3-320) was measured by luciferase reporter gene assays in HepG2 (or HepG2-X) cells that were co-transfected with pGL3-320 and pcDNA3.1-HBx (or psi-HBx). (C) A model demonstrates the predicted conserved AP2α binding site at nucleotides -20/-15 of the SPHK1 promoter. The generated mutant site at the SPHK1 promoter region is indicated. The luciferase activities of pGL3-320 and pGL3-320-mut were examined by luciferase reporter gene assays in HepG2 (or LO2) cells. (D) The expression of SPHK1 was detected using Western blot analysis in HepG2-X cells transfected with si-AP2α. (E) The interaction of AP2α with SPHK1 promoter was determined by ChIP assays in HepG2-X cells. (F) The relative activity of pGL3-320 was measured in HepG2 cells that were co-transfected with pcDNA3.1-HBx and si-AP2α. (G) The interfering efficiency of the siRNA pool targeting AP2α (si-AP2α) was validated by Western blot analysis in HepG2 cells. The data are shown as the mean±SD of three independent experiments. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Hepatitis B virus X protein promotes human hepatoma cell growth via upregulation of transcription factor AP2α and sphingosine kinase 1

    doi: 10.1038/aps.2015.38

    Figure Lengend Snippet: HBx activates the SPHK1 promoter by upregulating the transcription factor AP2α. (A, B) The activity of the SPHK1 promoter (pGL3-320) was measured by luciferase reporter gene assays in HepG2 (or HepG2-X) cells that were co-transfected with pGL3-320 and pcDNA3.1-HBx (or psi-HBx). (C) A model demonstrates the predicted conserved AP2α binding site at nucleotides -20/-15 of the SPHK1 promoter. The generated mutant site at the SPHK1 promoter region is indicated. The luciferase activities of pGL3-320 and pGL3-320-mut were examined by luciferase reporter gene assays in HepG2 (or LO2) cells. (D) The expression of SPHK1 was detected using Western blot analysis in HepG2-X cells transfected with si-AP2α. (E) The interaction of AP2α with SPHK1 promoter was determined by ChIP assays in HepG2-X cells. (F) The relative activity of pGL3-320 was measured in HepG2 cells that were co-transfected with pcDNA3.1-HBx and si-AP2α. (G) The interfering efficiency of the siRNA pool targeting AP2α (si-AP2α) was validated by Western blot analysis in HepG2 cells. The data are shown as the mean±SD of three independent experiments. b P

    Article Snippet: The following primary antibodies were used: mouse anti-HBx (Abcam, Cambridge, UK), rabbit anti-SPHK1 (Proteintech, Wuhan, China), rabbit anti-AP2α (Proteintech), mouse anti-β-actin (NeoMarkers, MA, USA), and rabbit anti-β-actin (NeoMarkers, MA, USA).

    Techniques: Activity Assay, Luciferase, Transfection, Binding Assay, Generated, Mutagenesis, Expressing, Western Blot, Chromatin Immunoprecipitation

    Retina-like tissue in the subretinal space of a 2-week-old P RPE65 -ngn3 mouse. A–C: Low magnification images to show the relative position of retina-like tissue (arrow) in a cross-section of the eye, with the anterior to the left and the posterior to the right. A: Bright-field (BF) view. B: Epi-fluorescence of DAPI staining of the nuclei. C: Epi-fluorescence of anti-recoverin (Rcv) immunostaining to mark photoreceptor cells. Boxes outline the corresponding regions in A–C, or similar locations on serial sections (D–O). D–G: A serial section under bright-field view (D), after DAPI nuclear staining (E), and after double immunostaining for Otx2 (in green, F) to identify bipolar cells and for Pax6 (in red, G) to label amacrine cells in the INL and in the GCL (misplaced amacrine). H–K: A serial section under bright-field view (H), with DAPI nuclear staining (I), and with double immunostaining for AP2α (in green, J) to label horizontal (albeit weakly) and amacrine cells in the INL and in the GCL (misplaced amacrine), and for Brn3A (in red, K) to identify a subset of ganglion cells. L–O: A serial section under bright-field view (L), with DAPI nuclear staining (M), and with double immunostaining for photoreceptor protein recoverin (in green, N) and for RPE protein RPE65 (in red, O). RPE-L, RPE-like; ONL-L, outer nuclear layer-like; INL-L inner nuclear layer-like; GCL-L, ganglion cell layer-like. Scale bar (100 μm) in A also applies to B, C. Scale bar (100 μm) in D also applies to E–O.

    Journal: PLoS ONE

    Article Title: Induction of Ectopic Retina-Like Tissue by Transgenic Expression of neurogenin

    doi: 10.1371/journal.pone.0116171

    Figure Lengend Snippet: Retina-like tissue in the subretinal space of a 2-week-old P RPE65 -ngn3 mouse. A–C: Low magnification images to show the relative position of retina-like tissue (arrow) in a cross-section of the eye, with the anterior to the left and the posterior to the right. A: Bright-field (BF) view. B: Epi-fluorescence of DAPI staining of the nuclei. C: Epi-fluorescence of anti-recoverin (Rcv) immunostaining to mark photoreceptor cells. Boxes outline the corresponding regions in A–C, or similar locations on serial sections (D–O). D–G: A serial section under bright-field view (D), after DAPI nuclear staining (E), and after double immunostaining for Otx2 (in green, F) to identify bipolar cells and for Pax6 (in red, G) to label amacrine cells in the INL and in the GCL (misplaced amacrine). H–K: A serial section under bright-field view (H), with DAPI nuclear staining (I), and with double immunostaining for AP2α (in green, J) to label horizontal (albeit weakly) and amacrine cells in the INL and in the GCL (misplaced amacrine), and for Brn3A (in red, K) to identify a subset of ganglion cells. L–O: A serial section under bright-field view (L), with DAPI nuclear staining (M), and with double immunostaining for photoreceptor protein recoverin (in green, N) and for RPE protein RPE65 (in red, O). RPE-L, RPE-like; ONL-L, outer nuclear layer-like; INL-L inner nuclear layer-like; GCL-L, ganglion cell layer-like. Scale bar (100 μm) in A also applies to B, C. Scale bar (100 μm) in D also applies to E–O.

    Article Snippet: Primary, polyclonal antibodies included those against AP2α (1:200 dilution; Bioworld Technologies), cellular retinaldehyde binding protein (1:200 dilution; Proteintech Group), Crx (1:300 dilution; Santa Cruz), glial fibrillary acidic protein (1:100 dilution; Sigma), Otx2 (1:200 dilution; AB9566, Millipore), recoverin (1:700 dilution; Chemicon), and red opsin (1:200 dilution; Chemicon).

    Techniques: Fluorescence, Staining, Immunostaining, Double Immunostaining

    Retina-like tissue exterior to the eyecup and in the vicinity of the optic nerve head of a 5-week-old P RPE65 -ngn3 mouse. A–C: Bright-field (A), DAPI staining (B), and double immunostaining (C) for red opsin (Red) and for rhodopsin (Rho, in green) of a posterior region of the eye. D–F: Bright-field (D), DAPI staining (E), and double immunostaining for AP2α (in green), a marker for horizontal cells and amacrine cells, and for Brn3A (in red), a marker for a subset of ganglion cells, on a serial section of the region. G–I: Bright-field (G), DAPI staining (H), and double immunostaining for a bipolar cell marker Otx2 (in green) and for an amacrine cell marker Pax6 (in red) of a serial section of the region. ON, optic nerve; ONH: optic nerve head; Ret-L: retina-like. Scale bar (100 μm) applies to all panels.

    Journal: PLoS ONE

    Article Title: Induction of Ectopic Retina-Like Tissue by Transgenic Expression of neurogenin

    doi: 10.1371/journal.pone.0116171

    Figure Lengend Snippet: Retina-like tissue exterior to the eyecup and in the vicinity of the optic nerve head of a 5-week-old P RPE65 -ngn3 mouse. A–C: Bright-field (A), DAPI staining (B), and double immunostaining (C) for red opsin (Red) and for rhodopsin (Rho, in green) of a posterior region of the eye. D–F: Bright-field (D), DAPI staining (E), and double immunostaining for AP2α (in green), a marker for horizontal cells and amacrine cells, and for Brn3A (in red), a marker for a subset of ganglion cells, on a serial section of the region. G–I: Bright-field (G), DAPI staining (H), and double immunostaining for a bipolar cell marker Otx2 (in green) and for an amacrine cell marker Pax6 (in red) of a serial section of the region. ON, optic nerve; ONH: optic nerve head; Ret-L: retina-like. Scale bar (100 μm) applies to all panels.

    Article Snippet: Primary, polyclonal antibodies included those against AP2α (1:200 dilution; Bioworld Technologies), cellular retinaldehyde binding protein (1:200 dilution; Proteintech Group), Crx (1:300 dilution; Santa Cruz), glial fibrillary acidic protein (1:100 dilution; Sigma), Otx2 (1:200 dilution; AB9566, Millipore), recoverin (1:700 dilution; Chemicon), and red opsin (1:200 dilution; Chemicon).

    Techniques: Staining, Double Immunostaining, Marker

    Retina-like tissue in the optic nerve (ON) in a P19 P RPE65 -ngn3 mouse. A–C: Bright-field (A), DAPI staining (B), and immunohistochemistry with anti-recoverin (Rcv) immunostaining to mark photoreceptor cells of a posterior region of the eye. D–F: Bright-field (D), DAPI staining (E), and double immunostaining for rhodopsin (Rho) and for red opsin (Red) of a serial section of the region. G–K: Bright-field (G), DAPI staining (H), and double immunostaining for a bipolar cell marker Otx2 (I) and for an amacrine cell marker Pax6 (J) of a serial section of the region. K: A merge of I and J to show the non-overlapping localization of Otx2 + (in green) and Pax6 + (in red) cells in the retinal-like tissue in the optic nerve. L–O: Bright-field (L), DAPI staining (M), and immunostaining for AP2α (N), a marker for horizontal cells and amacrine cells of a serial section of the region. Scale bar (100 μm) in A also applies to B, C. Scale bar (100 μm) in D also applies to E–N.

    Journal: PLoS ONE

    Article Title: Induction of Ectopic Retina-Like Tissue by Transgenic Expression of neurogenin

    doi: 10.1371/journal.pone.0116171

    Figure Lengend Snippet: Retina-like tissue in the optic nerve (ON) in a P19 P RPE65 -ngn3 mouse. A–C: Bright-field (A), DAPI staining (B), and immunohistochemistry with anti-recoverin (Rcv) immunostaining to mark photoreceptor cells of a posterior region of the eye. D–F: Bright-field (D), DAPI staining (E), and double immunostaining for rhodopsin (Rho) and for red opsin (Red) of a serial section of the region. G–K: Bright-field (G), DAPI staining (H), and double immunostaining for a bipolar cell marker Otx2 (I) and for an amacrine cell marker Pax6 (J) of a serial section of the region. K: A merge of I and J to show the non-overlapping localization of Otx2 + (in green) and Pax6 + (in red) cells in the retinal-like tissue in the optic nerve. L–O: Bright-field (L), DAPI staining (M), and immunostaining for AP2α (N), a marker for horizontal cells and amacrine cells of a serial section of the region. Scale bar (100 μm) in A also applies to B, C. Scale bar (100 μm) in D also applies to E–N.

    Article Snippet: Primary, polyclonal antibodies included those against AP2α (1:200 dilution; Bioworld Technologies), cellular retinaldehyde binding protein (1:200 dilution; Proteintech Group), Crx (1:300 dilution; Santa Cruz), glial fibrillary acidic protein (1:100 dilution; Sigma), Otx2 (1:200 dilution; AB9566, Millipore), recoverin (1:700 dilution; Chemicon), and red opsin (1:200 dilution; Chemicon).

    Techniques: Staining, Immunohistochemistry, Immunostaining, Double Immunostaining, Marker

    Extra retina in a 2-month-old P RPE65 -ngn3 mouse. A–C: Low magnification images to show the relative positions of the retinae in a cross-section of the eye, with the anterior facing up. A: Bright-field (BF) view. B: DAPI staining of the nuclei. C: Anti-recoverin (Rcv) immunostaining to mark photoreceptor cells. D–F: High (2.5X) magnification of the boxed region in A–C to show the well-defined 3 nuclear layers of two retinae coexisting at the region. G: Double immunostaining of a serial section for Otx2 (in green) to mark RPE cells and bipolar cells, and for Pax6 (in red) to mark amacrine cells in the INL and in the GCL (misplaced amacrine). H: Double immunostaining of a serial section for AP2α (in green), a marker for horizontal cells and amacrine cells in the INL and misplaced ones in the GCL, and for Brn3A (in red), a marker for a subset of ganglion cells. I–N: Immunostaining of serial sections for RPE65 (K) and for cytokeratin 18 (N), markers of RPE cells. Bright-field (I, L) and DAPI staining (J, M) were provided to show the histology. RPE-L: RPE-like; GCL: ganglion cell layer. Scale bar (100 μm) in A also applies to B, C. Scale bar (100 μm) in D also applies to E–N.

    Journal: PLoS ONE

    Article Title: Induction of Ectopic Retina-Like Tissue by Transgenic Expression of neurogenin

    doi: 10.1371/journal.pone.0116171

    Figure Lengend Snippet: Extra retina in a 2-month-old P RPE65 -ngn3 mouse. A–C: Low magnification images to show the relative positions of the retinae in a cross-section of the eye, with the anterior facing up. A: Bright-field (BF) view. B: DAPI staining of the nuclei. C: Anti-recoverin (Rcv) immunostaining to mark photoreceptor cells. D–F: High (2.5X) magnification of the boxed region in A–C to show the well-defined 3 nuclear layers of two retinae coexisting at the region. G: Double immunostaining of a serial section for Otx2 (in green) to mark RPE cells and bipolar cells, and for Pax6 (in red) to mark amacrine cells in the INL and in the GCL (misplaced amacrine). H: Double immunostaining of a serial section for AP2α (in green), a marker for horizontal cells and amacrine cells in the INL and misplaced ones in the GCL, and for Brn3A (in red), a marker for a subset of ganglion cells. I–N: Immunostaining of serial sections for RPE65 (K) and for cytokeratin 18 (N), markers of RPE cells. Bright-field (I, L) and DAPI staining (J, M) were provided to show the histology. RPE-L: RPE-like; GCL: ganglion cell layer. Scale bar (100 μm) in A also applies to B, C. Scale bar (100 μm) in D also applies to E–N.

    Article Snippet: Primary, polyclonal antibodies included those against AP2α (1:200 dilution; Bioworld Technologies), cellular retinaldehyde binding protein (1:200 dilution; Proteintech Group), Crx (1:300 dilution; Santa Cruz), glial fibrillary acidic protein (1:100 dilution; Sigma), Otx2 (1:200 dilution; AB9566, Millipore), recoverin (1:700 dilution; Chemicon), and red opsin (1:200 dilution; Chemicon).

    Techniques: Staining, Immunostaining, Double Immunostaining, Marker

    Loss of Msx2 activity does not alter Pax6 and Ap2α expression. Protein expression in WT and Msx2 -deficient embryos, respectively, was examined by immunofluorescence for Pax6 on E9.5 ( A and B ) and E11.5 ( C and D ), Ap2-α ( E – F ), and crystallin on E11.5 ( G and H ) and E12.5 ( I and J ). At the lens vesicle stage of eye development, the intensity and the spatial domain of immune-reactivity are not altered significantly. Immunofluorescence of WT and Msx2 -deficient embryonic eyes at E11.5 and E12.5 show significant overexpression of α- and β-crystallin in the Msx2 -deficient lens vesicle ( G–J ). Scale bars: 50 μm ( A and B ), 100 μm ( C–J ). Re, retina; Le, lens; OV, optic vesicle.

    Journal: The American Journal of Pathology

    Article Title: Loss of Msx2 Function Down-Regulates the FoxE3 Expression and Results in Anterior Segment Dysgenesis Resembling Peters Anomaly

    doi: 10.1016/j.ajpath.2012.02.017

    Figure Lengend Snippet: Loss of Msx2 activity does not alter Pax6 and Ap2α expression. Protein expression in WT and Msx2 -deficient embryos, respectively, was examined by immunofluorescence for Pax6 on E9.5 ( A and B ) and E11.5 ( C and D ), Ap2-α ( E – F ), and crystallin on E11.5 ( G and H ) and E12.5 ( I and J ). At the lens vesicle stage of eye development, the intensity and the spatial domain of immune-reactivity are not altered significantly. Immunofluorescence of WT and Msx2 -deficient embryonic eyes at E11.5 and E12.5 show significant overexpression of α- and β-crystallin in the Msx2 -deficient lens vesicle ( G–J ). Scale bars: 50 μm ( A and B ), 100 μm ( C–J ). Re, retina; Le, lens; OV, optic vesicle.

    Article Snippet: The following primary antibodies were used: mouse monoclonal anti-Ap2α (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-Pax6 (Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti-BrdU (Sigma, St Louis, MO), and rabbit polyclonal anti–α-crystallin and anti–β-crystallin antibodies (generously provided by Dr. Samuel Zigler, John Hopkins University, Baltimore, MD).

    Techniques: Activity Assay, Expressing, Immunofluorescence, Over Expression

    Arrest of LFC differentiation following Pax6 loss is not mediated by upregulation of Sox2. ( A ) Sox2 lox targeting vector and somatic deletion allele. The neo selection cassette is flanked by frt sites (blue triangles). The single Sox2 exon is flanked by loxP sites (green triangles). ( B-M ′) Cre-mediated deletion results in the Sox2 Δ allele. Sox2 lox/lox ;Pax6 lox/lox control (B-G) and Sox2 lox/lox ;Pax6 lox/lox ;Mlr10 E14.5 double somatic mutant (H-M′) mouse lenses analyzed by H E staining (B,H) and antibody labeling for Sox2 and Ap2α (C,I, green and red, respectively), phosphohistone H3 (PH3, red in D,J,J′), αA-crystallin (E,K), β-crystallin (F,L) and cleaved caspase 3 (cCas, red in G,M,M′). Counterstaining was with DAPI (D,F,G,J,L,M). Arrows indicate the lens equator, arrowheads to the aberrant cells in the lens posterior. LE, lens epithelium; re, retina. Scale bar: 100 μm.

    Journal: Development (Cambridge, England)

    Article Title: Pax6 is essential for lens fiber cell differentiation

    doi: 10.1242/dev.032888

    Figure Lengend Snippet: Arrest of LFC differentiation following Pax6 loss is not mediated by upregulation of Sox2. ( A ) Sox2 lox targeting vector and somatic deletion allele. The neo selection cassette is flanked by frt sites (blue triangles). The single Sox2 exon is flanked by loxP sites (green triangles). ( B-M ′) Cre-mediated deletion results in the Sox2 Δ allele. Sox2 lox/lox ;Pax6 lox/lox control (B-G) and Sox2 lox/lox ;Pax6 lox/lox ;Mlr10 E14.5 double somatic mutant (H-M′) mouse lenses analyzed by H E staining (B,H) and antibody labeling for Sox2 and Ap2α (C,I, green and red, respectively), phosphohistone H3 (PH3, red in D,J,J′), αA-crystallin (E,K), β-crystallin (F,L) and cleaved caspase 3 (cCas, red in G,M,M′). Counterstaining was with DAPI (D,F,G,J,L,M). Arrows indicate the lens equator, arrowheads to the aberrant cells in the lens posterior. LE, lens epithelium; re, retina. Scale bar: 100 μm.

    Article Snippet: Immunofluorescence analysis was performed on paraffin sections as previously described ( ) using the following primary antibodies: rabbit anti-Pax6 (1:1000, Chemicon), mouse anti-Ap2α (1:50, Santa Cruz), rabbit anti-cleaved caspase 3 (1:100, Cell Signaling), goat anti-αA-crystallin (1:1000, Santa Cruz), goat anti-αB-crystallin (1:100, Santa Cruz), rabbit anti-βB1-crystallin (1:250, Santa Cruz), rabbit anti-γF-crystallin (1:50, Santa Cruz), rabbit anti-cyclin D1 (1:250, Thermo Scientific), rat anti-Ki67 (1:100, Dako), goat anti-p57Kip2 (1:100, Santa Cruz), rabbit anti-Prox1 (1:50, Acris) and rabbit anti-Sox2 (1:500, Chemicon).

    Techniques: Plasmid Preparation, Selection, Mutagenesis, Staining, Antibody Labeling

    Pax6 downregulates Sox2 in the lens equator. ( A , B ) Immunofluorescent detection of Sox2 (green) and Ap2α (red) in E14.5 control (A) and Pax6 lox/lox ;Mlr10 (B) mouse lenses. Arrowheads in B indicate elevated expression of Sox2 at the lens equator and in the posterior lens. ( A ′, B ′) Counterstaining of A,B with DAPI. ( C ) Quantification of Sox2 protein by confocal image analysis ( n =6, ** P

    Journal: Development (Cambridge, England)

    Article Title: Pax6 is essential for lens fiber cell differentiation

    doi: 10.1242/dev.032888

    Figure Lengend Snippet: Pax6 downregulates Sox2 in the lens equator. ( A , B ) Immunofluorescent detection of Sox2 (green) and Ap2α (red) in E14.5 control (A) and Pax6 lox/lox ;Mlr10 (B) mouse lenses. Arrowheads in B indicate elevated expression of Sox2 at the lens equator and in the posterior lens. ( A ′, B ′) Counterstaining of A,B with DAPI. ( C ) Quantification of Sox2 protein by confocal image analysis ( n =6, ** P

    Article Snippet: Immunofluorescence analysis was performed on paraffin sections as previously described ( ) using the following primary antibodies: rabbit anti-Pax6 (1:1000, Chemicon), mouse anti-Ap2α (1:50, Santa Cruz), rabbit anti-cleaved caspase 3 (1:100, Cell Signaling), goat anti-αA-crystallin (1:1000, Santa Cruz), goat anti-αB-crystallin (1:100, Santa Cruz), rabbit anti-βB1-crystallin (1:250, Santa Cruz), rabbit anti-γF-crystallin (1:50, Santa Cruz), rabbit anti-cyclin D1 (1:250, Thermo Scientific), rat anti-Ki67 (1:100, Dako), goat anti-p57Kip2 (1:100, Santa Cruz), rabbit anti-Prox1 (1:50, Acris) and rabbit anti-Sox2 (1:500, Chemicon).

    Techniques: Expressing