Fig. 6. Antagonism of Nodal signaling alters myocardial fate assignment in tier 1 blastomeres. (A-D) Whole-mount immunofluorescence with the anti-myosin heavy chain monoclonal antibodies MF20 (TRITC) and S46 (FITC) at 36 hpf. (A,B,D) Lateral views, anterior towards the left. (C) Dorsal view, anterior towards the top. (A) In wild-type (wt) embryos, double exposure indicates MF20 staining in the ventricle (red, V), and overlap of MF20 and S46 staining in the atrium (yellow, A). In embryos injected with small amounts of lefty1 mRNA (B-D), we observe variable reduction of myocardium, ranging from a moderately reduced ventricle and a subtly affected atrium (B) to severe reductions of ventricular tissue together with significant reductions of atrial tissue (C,D). In addition, ectopic expression of lefty1 can disrupt endoderm formation, causing cardia bifida as shown in C (Yelon, 2001). (E,F) Examples of myocardial contributions by tier 1 blastomeres in embryos injected with lefty1. Labeled myocardial cells (blue precipitate) are examined with reference to an atrium-specific marker, amhc (pink precipitate) (Berdougo et al., 2003). (E) Frontal view at 36 hpf, dorsal towards the top. The amount of myocardium present resembles that in the embryo depicted in B. In this embryo, blastomeres located 65-150° from dorsal in tier 1 contributed to both atrial myocardium (arrows) and ventricular myocardium (below eyes and out of focal plane). (F) Dorsal view at 36 hpf, anterior towards the top. The amount of myocardium present resembles that in the embryo depicted in C. In this embryo, blastomeres located 90-135° from dorsal in tier 1 contributed to atrial myocardium (arrow). (G) Fate map of myocardial chamber progenitors in embryos injected with lefty1. Data are depicted as described for the myocardial fate map (Fig. 2B), except that experiments were performed only in tier 1. In this map, bar colors correspond to the myocardial contribution of labeled cells: red, contribution to ventricular myocardium; yellow, contribution to atrial myocardium; cyan, contribution to both ventricular and atrial myocardium; black, no myocardial progeny. When Nodal signaling is antagonized, both ventricular and atrial myocardial progenitors reside in tier 1. The dorsoventral organization of these progenitors roughly resembles that observed in tiers 2 and 3 of the wild-type fate map (Fig. 2), in that blastomeres located more dorsally than 125° give rise to ventricular myocardium and blastomeres located more ventrally than 90° give rise to atrial myocardium.