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. 2006 Dec 4;175(5):791–802. doi: 10.1083/jcb.200606017

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Copyright © 2006, The Rockefeller University Press

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Figure 7. — Endogenous subcellular sites of Wnt11 and Fz7 activity marked by Dsh-YFP recruitment to the plasma membrane. (A–C) Epiblast cells in germ ring of fixed gastrulating embryos (shield stage, 6 hpf) expressing dsh-yfp (75 pg mRNA) and lyn-cfp (75 pg mRNA). Dsh-YFP (green) localized in puncta (arrowheads) at the plasma membrane (red) in wild-type (A), slb/wnt11 mutant (B), and “rescued” slb/wnt11 mutant embryos (C; coinjected with 20 pg wnt11 mRNA). (D) Number of Dsh-YFP puncta at the plasma membrane (for rescue condition, column represents both epiblast and hypoblast cells). Quantification performed in 3D by counting the number of Dsh-YFP puncta at the plasma membrane of a chosen cell in all z sections (step size = 1.5 μm). Number of cells analyzed was as follows (epiblast/hypoblast): 50/43 (wild type); 43/43 (slb/wnt11 mutants); 83 (slb/wnt11 rescue; epiblast + hypoblast) out of 6 embryos per condition. Error bars represent the SEM. (E–G′) Epiblast cells in the germ ring of living embryos (6 hpf) expressing dsh-cfp (100 pg mRNA), and mosaic wnt11-yfp mRNA (50–100 pg mRNA injection at the 16-cell–stage). Plasma membranes stained (E′–G′; blue) with FM464 by intercellular injection at 5 hpf. Wnt11-YFP (E, E′, G, and G′; green) and Dsh-CFP (F, F′, G, and G′; red) colocalize in puncta (arrowheads) at the plasma membrane (blue) of both Wnt11-producing (green cytoplasm) and receiving cells. (H–L) As diagramed (H and I), cells expressing dsh-yfp (60 pg mRNA; red) were transplanted into the animal pole of host embryos (4 hpf) coexpressing dsh-cfp (100 pg mRNA; green) and wnt11 (50 pg mRNA). FM464 marks plasma membrane (blue). Epiblast cells imaged 3 h after transplantation within the germ ring of a living host embryo (6 hpf). (J–L) Dsh-YFP of a transplanted cell and Dsh-CFP in adjacent host cell colocalize to the same site (arrowheads). Bars, 10 μm.