Figure 6
Figure 6. Etsrp-expressing precursor cells give rise to both vascular endothelial and myeloid lineages. (A) Diagram of transplantation experiment. Donor embryos were injected at the 1-cell stage with etsrp RNA and TRITC-dextran (B-D) or FITC-dextran (E-H), while recipient embryos were injected with 7.5 ng etsrp MO1/MO2 mixture. Cells were transplanted at the beginning of epiboly. (B-D) flk1-GFP–expressing cells are a subset of etsrp RNA/TRITC-labeled cells. The embryo is at the 8-somite stage, lateral view, anterior to the left. (B) TRITC-filter image. Only transplanted cells are visible. (C) GFP-filter image. Only flk1-GFP–expressing cells are visible. (D) Overlay of the TRITC, GFP, and transmitted light DIC images. Cells where GFP and TRITC fluorescence overlaps are in yellow ( point to some of these cells). Note that every GFP-expressing cell has also TRITC fluorescence. (E-H) pu.1-expressing cells originate from etsrp-expressing cells, transplanted from etsrp RNA-overexpressing embryos into etsrp morphants. Etsrp RNA was coinjected with fluorescein-labeled dextran; pu.1 expression and fluorescein presence was analyzed by 2-color fluorescent (E-G) or conventional (H) in situ hybridization at the 8- to 10-somite stages. (E-G) Anterior-lateral view of the same embryo, dorsal is up. (E) FITC-filter image. Only transplanted cells are visible. (F) pu.1 expression as detected by tyramide-Cy3 amplification, visualized through the rhodamine channel filter. Note the ectopically located pu.1-expressing cells () and 3 remaining endogenous pu.1-expressing cells that are located bilaterally within the anterior lateral mesoderm (). (G) Overlay of FITC, Cy3, and transmitted light images. Note that all 3 ectopic pu.1-expressing cells contain FITC label () while the endogenous pu.1 cells do not (). (H) A posterior region from an embryo containing multiple pu.1 and fluorescein-positive cells. Embryo has been flat-mounted to show dorsal, lateral, and ventral tissues. (1) Endogenous pu.1-expressing cells in the posterior lateral mesoderm. (2) Fluorescein-labeled transplanted cells. (3-5) Double pu.1 and fluorescein-positive cells. Average color for each cell group is shown in the boxes below the figure (“Methods”). Images were taken using Axioplan2 and 10×/0.30 NA (A;C-G) (Zeiss), Axiocam color camera (Zeiss, model 412-312) (H) or monochrome C4742-95 camera (B-G) (Hamamatsu Photonics, Hamamatsu City, Japan) and Openlab 4.0 software (Improvision). Magnification: 100× (B-G); 300× (H).

Etsrp-expressing precursor cells give rise to both vascular endothelial and myeloid lineages. (A) Diagram of transplantation experiment. Donor embryos were injected at the 1-cell stage with etsrp RNA and TRITC-dextran (B-D) or FITC-dextran (E-H), while recipient embryos were injected with 7.5 ng etsrp MO1/MO2 mixture. Cells were transplanted at the beginning of epiboly. (B-D) flk1-GFP–expressing cells are a subset of etsrp RNA/TRITC-labeled cells. The embryo is at the 8-somite stage, lateral view, anterior to the left. (B) TRITC-filter image. Only transplanted cells are visible. (C) GFP-filter image. Only flk1-GFP–expressing cells are visible. (D) Overlay of the TRITC, GFP, and transmitted light DIC images. Cells where GFP and TRITC fluorescence overlaps are in yellow ( point to some of these cells). Note that every GFP-expressing cell has also TRITC fluorescence. (E-H) pu.1-expressing cells originate from etsrp-expressing cells, transplanted from etsrp RNA-overexpressing embryos into etsrp morphants. Etsrp RNA was coinjected with fluorescein-labeled dextran; pu.1 expression and fluorescein presence was analyzed by 2-color fluorescent (E-G) or conventional (H) in situ hybridization at the 8- to 10-somite stages. (E-G) Anterior-lateral view of the same embryo, dorsal is up. (E) FITC-filter image. Only transplanted cells are visible. (F) pu.1 expression as detected by tyramide-Cy3 amplification, visualized through the rhodamine channel filter. Note the ectopically located pu.1-expressing cells () and 3 remaining endogenous pu.1-expressing cells that are located bilaterally within the anterior lateral mesoderm (). (G) Overlay of FITC, Cy3, and transmitted light images. Note that all 3 ectopic pu.1-expressing cells contain FITC label () while the endogenous pu.1 cells do not (). (H) A posterior region from an embryo containing multiple pu.1 and fluorescein-positive cells. Embryo has been flat-mounted to show dorsal, lateral, and ventral tissues. (1) Endogenous pu.1-expressing cells in the posterior lateral mesoderm. (2) Fluorescein-labeled transplanted cells. (3-5) Double pu.1 and fluorescein-positive cells. Average color for each cell group is shown in the boxes below the figure (“Methods”). Images were taken using Axioplan2 and 10×/0.30 NA (A;C-G) (Zeiss), Axiocam color camera (Zeiss, model 412-312) (H) or monochrome C4742-95 camera (B-G) (Hamamatsu Photonics, Hamamatsu City, Japan) and Openlab 4.0 software (Improvision). Magnification: 100× (B-G); 300× (H).

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