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( A ) Schematic describing embryonic stages of mouse limb bud formation and the main cell populations involved in limb morphogenesis. ( B ) Schematic of the protocol generating cells with AER, surface ectoderm, and mesoderm properties from mESCs. ( C ) Representative confocal image of generated AER-like cells in day 7 cultures. Cyan: Hoechst; white: phalloidin; magenta: TP63; yellow: Fgf8:tdTomato . Scale bar, 100 μm. ( D ) Representative confocal image of day 7 heterogeneous cultures stained for immature surface ectoderm <t>TFAP2C</t> and basal epithelial stem cell marker TP63. Cyan: Hoechst; magenta: TP63; white: TFAP2C. Scale bar, 400 μm. ( E ) Bar plot showing flow cytometry quantification of epithelial cell marker EpCAM+ cells in day 7 cultures. Each dot represents one biological replicate. N = 9. ( F ) Bar plot showing flow cytometry quantification of Fgf8:tdTomato + cells in day 7 cultures. Each dot represents one biological replicate. N = 4. ( G ) UMAP representation of scRNA-seq data of day 7 cultures displaying main cell clusters. See (H) color-coding. ( H and I ) Plots of the scRNA-seq cluster–based proportions of (H) generated cell types, with color-coding for AER-like, mesoderm-like, and surface ectoderm cells, and (I) lineages on day 7. For full information, see fig. S2E. Rep denotes replicate. ( J ) Dot plot showing surface ectoderm, AER, and limb bud mesoderm marker gene expression in the scRNA-seq clusters. Dot size represents the percentage of cells expressing each marker. ( K ) Representative confocal image of day 7 heterogeneous cultures generated from the Prrx1 Enh :mVenus reporter mESC line. White: phalloidin; magenta: TP63; green: Prrx1 Enh :mVenus . Scale bar, 500 μm. ( L ) Representative sequential z -stack confocal images of domes in day 7 cultures. Magenta: TP63; green: Prrx1 Enh :mVenus . Images from left to right show the dome from bottom to top, denoted by z numbers. Scale bars, 200 μm.
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( A ) Schematic describing embryonic stages of mouse limb bud formation and the main cell populations involved in limb morphogenesis. ( B ) Schematic of the protocol generating cells with AER, surface ectoderm, and mesoderm properties from mESCs. ( C ) Representative confocal image of generated AER-like cells in day 7 cultures. Cyan: Hoechst; white: phalloidin; magenta: TP63; yellow: Fgf8:tdTomato . Scale bar, 100 μm. ( D ) Representative confocal image of day 7 heterogeneous cultures stained for immature surface ectoderm <t>TFAP2C</t> and basal epithelial stem cell marker TP63. Cyan: Hoechst; magenta: TP63; white: TFAP2C. Scale bar, 400 μm. ( E ) Bar plot showing flow cytometry quantification of epithelial cell marker EpCAM+ cells in day 7 cultures. Each dot represents one biological replicate. N = 9. ( F ) Bar plot showing flow cytometry quantification of Fgf8:tdTomato + cells in day 7 cultures. Each dot represents one biological replicate. N = 4. ( G ) UMAP representation of scRNA-seq data of day 7 cultures displaying main cell clusters. See (H) color-coding. ( H and I ) Plots of the scRNA-seq cluster–based proportions of (H) generated cell types, with color-coding for AER-like, mesoderm-like, and surface ectoderm cells, and (I) lineages on day 7. For full information, see fig. S2E. Rep denotes replicate. ( J ) Dot plot showing surface ectoderm, AER, and limb bud mesoderm marker gene expression in the scRNA-seq clusters. Dot size represents the percentage of cells expressing each marker. ( K ) Representative confocal image of day 7 heterogeneous cultures generated from the Prrx1 Enh :mVenus reporter mESC line. White: phalloidin; magenta: TP63; green: Prrx1 Enh :mVenus . Scale bar, 500 μm. ( L ) Representative sequential z -stack confocal images of domes in day 7 cultures. Magenta: TP63; green: Prrx1 Enh :mVenus . Images from left to right show the dome from bottom to top, denoted by z numbers. Scale bars, 200 μm.
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( A ) Schematic describing embryonic stages of mouse limb bud formation and the main cell populations involved in limb morphogenesis. ( B ) Schematic of the protocol generating cells with AER, surface ectoderm, and mesoderm properties from mESCs. ( C ) Representative confocal image of generated AER-like cells in day 7 cultures. Cyan: Hoechst; white: phalloidin; magenta: TP63; yellow: Fgf8:tdTomato . Scale bar, 100 μm. ( D ) Representative confocal image of day 7 heterogeneous cultures stained for immature surface ectoderm <t>TFAP2C</t> and basal epithelial stem cell marker TP63. Cyan: Hoechst; magenta: TP63; white: TFAP2C. Scale bar, 400 μm. ( E ) Bar plot showing flow cytometry quantification of epithelial cell marker EpCAM+ cells in day 7 cultures. Each dot represents one biological replicate. N = 9. ( F ) Bar plot showing flow cytometry quantification of Fgf8:tdTomato + cells in day 7 cultures. Each dot represents one biological replicate. N = 4. ( G ) UMAP representation of scRNA-seq data of day 7 cultures displaying main cell clusters. See (H) color-coding. ( H and I ) Plots of the scRNA-seq cluster–based proportions of (H) generated cell types, with color-coding for AER-like, mesoderm-like, and surface ectoderm cells, and (I) lineages on day 7. For full information, see fig. S2E. Rep denotes replicate. ( J ) Dot plot showing surface ectoderm, AER, and limb bud mesoderm marker gene expression in the scRNA-seq clusters. Dot size represents the percentage of cells expressing each marker. ( K ) Representative confocal image of day 7 heterogeneous cultures generated from the Prrx1 Enh :mVenus reporter mESC line. White: phalloidin; magenta: TP63; green: Prrx1 Enh :mVenus . Scale bar, 500 μm. ( L ) Representative sequential z -stack confocal images of domes in day 7 cultures. Magenta: TP63; green: Prrx1 Enh :mVenus . Images from left to right show the dome from bottom to top, denoted by z numbers. Scale bars, 200 μm.
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( A ) Schematic describing embryonic stages of mouse limb bud formation and the main cell populations involved in limb morphogenesis. ( B ) Schematic of the protocol generating cells with AER, surface ectoderm, and mesoderm properties from mESCs. ( C ) Representative confocal image of generated AER-like cells in day 7 cultures. Cyan: Hoechst; white: phalloidin; magenta: TP63; yellow: Fgf8:tdTomato . Scale bar, 100 μm. ( D ) Representative confocal image of day 7 heterogeneous cultures stained for immature surface ectoderm <t>TFAP2C</t> and basal epithelial stem cell marker TP63. Cyan: Hoechst; magenta: TP63; white: TFAP2C. Scale bar, 400 μm. ( E ) Bar plot showing flow cytometry quantification of epithelial cell marker EpCAM+ cells in day 7 cultures. Each dot represents one biological replicate. N = 9. ( F ) Bar plot showing flow cytometry quantification of Fgf8:tdTomato + cells in day 7 cultures. Each dot represents one biological replicate. N = 4. ( G ) UMAP representation of scRNA-seq data of day 7 cultures displaying main cell clusters. See (H) color-coding. ( H and I ) Plots of the scRNA-seq cluster–based proportions of (H) generated cell types, with color-coding for AER-like, mesoderm-like, and surface ectoderm cells, and (I) lineages on day 7. For full information, see fig. S2E. Rep denotes replicate. ( J ) Dot plot showing surface ectoderm, AER, and limb bud mesoderm marker gene expression in the scRNA-seq clusters. Dot size represents the percentage of cells expressing each marker. ( K ) Representative confocal image of day 7 heterogeneous cultures generated from the Prrx1 Enh :mVenus reporter mESC line. White: phalloidin; magenta: TP63; green: Prrx1 Enh :mVenus . Scale bar, 500 μm. ( L ) Representative sequential z -stack confocal images of domes in day 7 cultures. Magenta: TP63; green: Prrx1 Enh :mVenus . Images from left to right show the dome from bottom to top, denoted by z numbers. Scale bars, 200 μm.
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( A ) Schematic describing embryonic stages of mouse limb bud formation and the main cell populations involved in limb morphogenesis. ( B ) Schematic of the protocol generating cells with AER, surface ectoderm, and mesoderm properties from mESCs. ( C ) Representative confocal image of generated AER-like cells in day 7 cultures. Cyan: Hoechst; white: phalloidin; magenta: TP63; yellow: Fgf8:tdTomato . Scale bar, 100 μm. ( D ) Representative confocal image of day 7 heterogeneous cultures stained for immature surface ectoderm TFAP2C and basal epithelial stem cell marker TP63. Cyan: Hoechst; magenta: TP63; white: TFAP2C. Scale bar, 400 μm. ( E ) Bar plot showing flow cytometry quantification of epithelial cell marker EpCAM+ cells in day 7 cultures. Each dot represents one biological replicate. N = 9. ( F ) Bar plot showing flow cytometry quantification of Fgf8:tdTomato + cells in day 7 cultures. Each dot represents one biological replicate. N = 4. ( G ) UMAP representation of scRNA-seq data of day 7 cultures displaying main cell clusters. See (H) color-coding. ( H and I ) Plots of the scRNA-seq cluster–based proportions of (H) generated cell types, with color-coding for AER-like, mesoderm-like, and surface ectoderm cells, and (I) lineages on day 7. For full information, see fig. S2E. Rep denotes replicate. ( J ) Dot plot showing surface ectoderm, AER, and limb bud mesoderm marker gene expression in the scRNA-seq clusters. Dot size represents the percentage of cells expressing each marker. ( K ) Representative confocal image of day 7 heterogeneous cultures generated from the Prrx1 Enh :mVenus reporter mESC line. White: phalloidin; magenta: TP63; green: Prrx1 Enh :mVenus . Scale bar, 500 μm. ( L ) Representative sequential z -stack confocal images of domes in day 7 cultures. Magenta: TP63; green: Prrx1 Enh :mVenus . Images from left to right show the dome from bottom to top, denoted by z numbers. Scale bars, 200 μm.

Journal: Science Advances

Article Title: Specialized signaling centers direct cell fate and spatial organization in a mesodermal organoid model

doi: 10.1126/sciadv.ady7682

Figure Lengend Snippet: ( A ) Schematic describing embryonic stages of mouse limb bud formation and the main cell populations involved in limb morphogenesis. ( B ) Schematic of the protocol generating cells with AER, surface ectoderm, and mesoderm properties from mESCs. ( C ) Representative confocal image of generated AER-like cells in day 7 cultures. Cyan: Hoechst; white: phalloidin; magenta: TP63; yellow: Fgf8:tdTomato . Scale bar, 100 μm. ( D ) Representative confocal image of day 7 heterogeneous cultures stained for immature surface ectoderm TFAP2C and basal epithelial stem cell marker TP63. Cyan: Hoechst; magenta: TP63; white: TFAP2C. Scale bar, 400 μm. ( E ) Bar plot showing flow cytometry quantification of epithelial cell marker EpCAM+ cells in day 7 cultures. Each dot represents one biological replicate. N = 9. ( F ) Bar plot showing flow cytometry quantification of Fgf8:tdTomato + cells in day 7 cultures. Each dot represents one biological replicate. N = 4. ( G ) UMAP representation of scRNA-seq data of day 7 cultures displaying main cell clusters. See (H) color-coding. ( H and I ) Plots of the scRNA-seq cluster–based proportions of (H) generated cell types, with color-coding for AER-like, mesoderm-like, and surface ectoderm cells, and (I) lineages on day 7. For full information, see fig. S2E. Rep denotes replicate. ( J ) Dot plot showing surface ectoderm, AER, and limb bud mesoderm marker gene expression in the scRNA-seq clusters. Dot size represents the percentage of cells expressing each marker. ( K ) Representative confocal image of day 7 heterogeneous cultures generated from the Prrx1 Enh :mVenus reporter mESC line. White: phalloidin; magenta: TP63; green: Prrx1 Enh :mVenus . Scale bar, 500 μm. ( L ) Representative sequential z -stack confocal images of domes in day 7 cultures. Magenta: TP63; green: Prrx1 Enh :mVenus . Images from left to right show the dome from bottom to top, denoted by z numbers. Scale bars, 200 μm.

Article Snippet: The samples were then incubated with primary antibodies [TP63 (Abcam, ab735), TFAP2C (Cell Signaling, 2320S), LEF1 (Abcam, ab137872)], diluted in blocking buffer overnight at 4°C.

Techniques: Generated, Staining, Marker, Flow Cytometry, Gene Expression, Expressing