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Fig. 4 Loss of Grem2 leads to increased BMP signaling and cardiac defects that can be reversed by the BMP inhibitor dorsomorphin. (A) Whole mount immunohistochemistry using antibodies recognizing the phosphorylated forms of Smads1/5/8 shows stronger pSmad protein staining in grem2 morphants than in controls. Arrowhead marks somite boundaries. (B) Frontal close up views of the heart after pSmad1/5/8 antibody staining at 48 hpf shows sharply increased nuclear staining in cardiomyocytes of Grem2-depleted embryos. The heart is outlined by dotted lines; arrowheads mark pSmad-positive nuclei. (C) Wild-type (WT) embryos and grem2 morphants were incubated with dorsomorphin or its vehicle DMSO between 16 and 48 hpf and stained at 48 hpf with cmlc2 and amhc probes to visualize the ventricle (V) and atrium (A). Dorsomorphin treatment restored atrial patterning and differentiation (arrowheads point to the atrioventricular boundary in wild-type hearts; dotted lines demarcate ventricle-atrium boundary in grem2 morphants). (D) Quantification of heart size (Heart Area) and atrial bulb restoration (Presence of Atrial Bulb) in DMSO-treated controls (WT), DMSO-treated grem2 morphants (MO) and grem2 morphants treated with dorsomorphin (MO+DM). Dorsomorphin treatment restores atrial development. Error bars represent s.d. P-values and number of embryos analyzed (n) are indicated. b, brain; e, eye; m, mouth; y, yolk.