Morphological analysis of cardiac chambers of san and vtn mutant embryos. Hematoxylin- and eosin-stained sagittal sections through 72 hpf hearts were used to assess cardiac histology (A,C,E). Double-staining using an atrial-specific antibody (S46) and an in-situ RNA probe to the ventricular myosin heavy chain (vMHC) were used to distinguish the atrial and ventricular chambers (B,D,F). The myocardial wall (indicated by arrows) in wild-type hearts (A) is several cell layers thick. However, the myocardium in both san (C') and vtn (E') mutant hearts does not thicken and remains a single cell layer. Both mutants are shown at a reduced magnification in C and E to illustrate the dramatic dilation of the cardiac chambers. The hearts of (B) wild-type, (D) san^-/-, and (F) vtn^-/- embryos at 48 hpf express markers specific for the two cardiac chambers although the hearts of both mutants are enlarged. Scale bars: 25 μm. a, atrium; e, endocardium; m, myocardium; v, ventricle.

In-situ analysis of san mRNA expression. The san mRNA is expressed in the ventricular zone (black arrow) and diffusely throughout the brain at 28 hpf (A,B). Expression is also detectable at this stage in the posterior cardinal vein (green arrow) as illustrated by whole-mount (C) and in section (D). At 48 hpf, san expression is detectable in the notochord with patchy expression in the vein (E). The staining in the vein is clearly visible from sections through the trunk (F). The position of the dorsal aorta is indicated by the red arrow. The boxed region in C and E represent the region shown in the sections. nc, notochord; nt, neural tube.

In-situ analysis of vtn mRNA expression. The vtn mRNA is robustly expressed in the ventricular zone (black arrow) at 28 hpf (A-C), also shown in section in D. Expression is also strong in the intermediate cell mass (A,E,F, white arrow). Lengthened staining also reveals vtn message in the vein (E, green arrow) confirmed by sectioning (G). At 48 hpf, vtn is expressed in the brain and diffusely in the branchial arches (H). Expression in the vein is detectable by whole-mount (I) and from sections (J), but is weaker than expression of san at this stage. The position of the dorsal aorta is indicated by the red arrow. The boxed region in E and I represent the region shown in the sections. nt, neural tube; nc, notochord.

Valve formation is perturbed in santa. The sites of formation of the endocardial cushions in day 2 embryos of the ty219c san allele are indicated by staining with an in situ RNA probe to the neuregulin gene (Milan et al., 2006). (A,A′) Wild-type sibs of the san ty219c allele demonstrate distinct staining at the sites of future valve formation, indicated by the arrow. (B,B′) No staining is observed in the hearts of stage-matched san mutant embryos. The position of the atrial and ventricular chambers are outlined in A′ to better illustrate the location of the neuregulin staining. wt, wild type; −/−, homozygous mutant.

Sarcomeres are present in the hearts of san and vtn mutant embryos. Transmission EM shows sarcomeres (green arrows) in the hearts of wild-type (A-C), san (D,E) and vtn (F,G) embryos, but with some disarray in the mutants (D,F). Sarcomeres are shown in cross-section (indcated by red circles) in wild-type (B), san (E) and vtn (G) mutants. Both san and vtn mutant hearts are still able to contract although circulation is not generated suggesting that actin/myosin arrangement is normal. Intercalated discs (indicated by blue boxes) are detectable in both mutants (E,G) although their structure in wild-type hearts is better defined (C). Comparison with similar EM analyses of cardiac tissue suggests the electron-dense material along and between the myofilaments is glycogen.