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First published online July 27, 2006
doi: 10.1242/10.1242/dev.02469

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santa and valentine pattern concentric growth of cardiac myocardium in the zebrafish

John D. Mably^*, Lesley P. Chuang, Fabrizio C. Serluca, Manzoor-Ali P. K. Mohideen, Jau-Nian Chen and Mark C. Fishman^,*

Cardiovascular Research Center, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA.

View larger version (148K): [in a new window]   Fig. 1. 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.

View larger version (47K): [in a new window]   Fig. 2. The san and vtn loci encode the zebrafish homologs of CCM1 and CCM2. (A) The zebrafish (Dr) San (CCM1) protein sequence is aligned with the C. elegans (Ce), mouse (Mm) and human (Hs) homologs, demonstrating strong identity among the three vertebrate genes. The N-terminal NPxY motif indicated by the single brown line (NPAY, residues 191-194 of the zebrafish protein and residues 192-195 of the mouse and human protein) is altered in the C. elegans homolog to NPAF. This motif is associated with interaction of san with both ICAP1 and vtn (Zawistowski et al., 2002; Zawistowski et al., 2005; Zhang et al., 2001). The other two NPxF/Y motifs, indicated by brown double lines, were not required for interaction of san with vtn (Zawistowski et al., 2005). The three blue double lines indicate the location of the ankyrin repeats and the single green line denotes the FERM domain. The red asterisk indicates the position of the Ystop mutation in the zebrafish san ty219c allele. The protein motifs are shown schematically below the sequence. The red asterisk again denotes the san ty219c mutation and the region of the protein shown in red corresponds to exon 14, deleted in the san m775 allele. (B) Real-time PCR analysis was used to examine the level of san mRNA message containing exon 14. mRNA isolated from m775 wild-type siblings, and both wild-type and mutant ty219c embryos show levels of exon 14-containing message 10-fold greater than in the san m775 mutant embryos (m775 -/- level set to 1 for comparison). (C) The Vtn (CCM2) protein is very well conserved among vertebrates [the zebrafish (Dr) Vtn protein sequence is aligned with the mouse (Mm) and human (Hs) homologs, no C. elegans homolog is detectable]. The single blue line indicates the sequence corresponding to the PTB domain and the red asterisk indicates the position of the Ystop mutation in the zebrafish vtn m201 allele. The Vtn protein shown schematically below the sequence illustrates the position of the PTB domain and site of the vtn m201 mutation.

View larger version (124K): [in a new window]   Fig. 3. 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.

View larger version (76K): [in a new window]   Fig. 4. 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.

View larger version (34K): [in a new window]   Fig. 5. Morpholino co-injections reveal evidence of interactions between san, vtn and heg. Injection of low doses (10 and 15 µmol/l) of each morpholino alone is unable to produce large number of embryos with the characteristic enlarged heart and thin-walled myocardium. However, by injection of combinations of any two of the san, vtn and heg morpholinos, a dramatic increase in the level of phenocopy is observed (indicated by the increase in the yellow bars) with a concomitant decrease in the level of wild-type embryos (blue bars). A number of embryos (indicated by the red bars) display a phenotype intermediate between wild type and mutant, characterized by a less dramatic dilation of the heart and some with weak circulation. heg, heg exon 11 donor morpholino injected; san, san exon 1 donor morpholino injected; vtn, vtn exon 2 morpholino injected.