Figure 4. Nodal-expressing explants exhibit asymmetric Nodal signaling activity.

(A) Representative confocal images of immunofluorescent staining for phosphorylated Smad2 and DAPI-labeled nuclei in 8hpf explants from WT embryos injected with 10 pg ndr2. DAPI z-stacks were used to create a three-dimensional mask from which nuclear pSmad intensities were detected and measured in an automated fashion. (B, C) Axis position of pSmad2-positive nuclei (blue) and all nuclei (gray) in explants from WT embryos injected with 10 pg ndr2 (B) or uninjected (C) at the time points indicated. Each dot represents a single nucleus, pink bars are median values among pSmad2⁺ nuclei. N indicates the number of explants in each condition from five independent trials. Kolmogorov-Smirnov tests were used to compare the distribution of pSmad⁺ nuclei to all nuclei; ****, p<0.0001; **, p<0.01. (D) Representative images of WISH for lefty1 in uninjected (top) and ndr2-injected (bottom) explants fixed at the time points indicated. Scale bars are 100 μm.

Figure 4—figure supplement 1. Nodal signaling activity in intact embryos.

(A) Representative confocal z-projections of immunofluorescent (IF) staining for phosphorylated Smad2 (bottom) overlaid with DAPI-labeled nuclei (top) in WT embryos fixed at the time points indicated. (B) Representative confocal z-projections of pSmad2 IF and DAPI staining in 5.5 hpf WT embryos treated with DMSO (top) or SB505124 (bottom), starting at 3 hpf. Panels to the right are magnified views of the regions within the white squares. Fractions indicate the number of embryos with the depicted phenotype over the total number of embryos examined for each condition and/or timepoint. The animal pole is at the top of all images. Scale bars are 50 μm.