Embryos were collected at the shield stage, fixed in 4% paraformaldehyde overnight, washed with PBS containing 0.1% Tween 20 for 30 min, blocked with 1% bovine serum albumin for 1 hour at room temperature, and then incubated with anti–phospho-Smad1/5/8 (1:200; 9511, Cell Signaling Technology) for 24 hours at 4°C. After washing with PBS three times for 5 min each, the samples were incubated with a donkey anti-rabbit secondary antibody conjugated to DyLight 594 (1:500; 711-585-152, Jackson ImmunoResearch) overnight at 4°C. All immunofluorescence images were captured using a Nikon A1R+ confocal microscope with the same settings for all samples within each experiment.

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