Trajectories and average velocities of migrating cells within the cardiac cone.

A–G: First frame of time lapse movies taken from a dorsal view of Tg(myl7:eGFP) embryos overlaid with cell trajectories. Yellow tracks: left cells. White tracks: center cells. Red tracks: right cells. A: All cells exhibit trajectories directed toward the anterior and left in WT embryos. B: Bilateral expression of spaw in ntl morphants leads to loss of L/R asymmetry in cardiac cell migrations. C–G: L/R directionality is also absent in embryos C: lacking the Nodal ligand spaw; D: treated with the SB-505124 Nodal inhibitor drug E: lacking spaw and one functional copy of bmp4; F: injected with both bmp4 and spaw morpholinos; and G: homozygous midway mutants. H: Numbers within each bar indicate the number of cells tracked on the left, center and right for all embryos of each genotype. The number of embryos utilized in analysis is indicated (n = ) for each genotype. WT embryos exhibit differences in average cell velocities along the L/R axis, with left cells migrating significantly faster than right cells. Bilateral exposure to Spaw leads to significant increases in average velocities in left and right cells of ntl morphants, while loss of Spaw and global inhibition of Nodal signaling through drug treatment results in significantly decreased rates of migration. In embryos with diminished Bmp signaling along with loss of Spaw, average cell velocities are significantly increased compared with loss of Spaw alone. Error bars in H indicate standard error of the mean. MO: morpholino. Het: Heterozygotes.

Jogging laterality phenotypes.

Embryos were scored for laterality of cardiac jogging between 24 and 30 hpf. A-C: Dorsal views of the heart at 24 hpf showing jogging laterality phenotypes by RNA in situ hybridizations using the myl7 probe D: WT embryos exhibit primarily left-directed cardiac jog, as do bmp4^Y180 mutants (I) lacking both maternal and zygotic bmp4 (MZbmp4). E,F: While WT embryos injected with spaw morpholino [9] and late-zygotic (LZ) oep mutants display randomized jogging laterality, H: MZbmp4^Y180 mutants injected with spaw morpholino lack significant jogging asymmetries. I: Loss of bmp4 alone does not affect jogging laterality and resembles WT (D) suggesting Bmp is dispensable for correct jogging when asymmetric Nodal signals are present. J: Jogging laterality in the absence of Spaw is highly sensitive to the level of Bmp4 present, as loss of a single functional copy of bmp4 in embryos deficient for Spaw is sufficient to result in predominantly midline jogging phenotypes. K: midway mutants homozygous for a nonsense mutation in the Nodal transcription factor FoxH1 display jogging phenotypes similar to what is observed in embryos lacking both Nodal and Bmp signaling. We note that a discrepancy exists between the randomized jogging of spaw morphants and the predominantly midline jogging of SB-505124-treated embryos (G). As described in more detail in the discussion, these phenotypic differences are likely due to the more global inhibition of TGFβ signaling achieved through drug treatment and we believe reflects the involvement of a second, as yet unidentified, TGFβ ligand in establishing jogging laterality. MO: morpholino. Het: Heterozygotes. Mut: Mutant. Asterisk: jogging data presented supplementary to [9].

Quantitation of Bmp pathway activity by average fluorescence intensity and number of p-Smad1/5/8 positive cells.

Immunofluorescence images for activated Smad1/5/8 (A,B,D,E,G,H,J,K,P–P3) in Tg(myl7:eGFP) embryos (myocardial GFP in B,E,H,K,P–P23) or in Tg(kdrl:egfp) embryos (endocardial GFP in Q–Q3). C,F,I,L: Schematics of B, E, H and I using IMARIS surface tool. GFP signal in green; p-Smad1/5/8 positive cells are labeled according to intensity using the red (low) to yellow (high) spectrum ranging from an intensity value of 35–65, respectively. All images are dorsal views except for optical cross sections generated in IMARIS along the anterior/posterior axis (P2, Q2) or across the myocardium (P3, Q3). A–C: p-Smad1/5/8 fluorescence intensity is higher on the left of the WT cardiac cone (n = 6). D–F: p-Smad1/5/8 fluorescence intensity on both sides of the cone in spaw morphants is similar to that observed in cells on the left in WT (n = 6). G–I: midway mutants exhibit reduced fluorescence intensities and numbers of p-Smad1/5/8 positive cells, while p-Smad levels elsewhere in the embryo are seemingly unaffected (data not shown, n = 3). J–L: bmp4 and spaw double morphants exhibit diminished p-Smad1/5/8 compared with WT (n = 6) but not as severe as observed in midway mutants. M: Comparison of the average fluorescence intensities of p-Smad1/5/8 positive cells on the left and right of the cardiac cone. N: Comparison of the average number of p-Smad1/5/8 positive cells on the left and right of the cardiac cone. O: Comparison of the average number of p-Smad1/5/8 positive cells in the entire cardiac field. P–P3: The myocardium shows little to no overlap of p-Smad1/5/8 positive cells (red) with the green of the myocardium. Q–Q3: Conversely, p-Smad1/5/8 (red) is observed to colocalize with the GFP positive endothelial cells in Tg(kdrl:egfp), indicated with arrowheads in Q2 and Q3, indicating that Bmp signals more strongly to the endocardium. Error bars indicate standard error of the mean. L: Low. H: High. MO: Morpholino. Het: heterozygote.