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Files in this Data Supplement:
Fig. S1. 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.
Fig. S2. 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.
Fig. S3. Positional cloning of the san and vtn genes. (A) Initial bulk segregant analysis established linkage to SSR marker z5183 on zebrafish chromosome 19. The san mutation was fine mapped to the region between SSR markers z9512 and z5183. Another SSR in the interval (z5583) was identified from the zebrafish RH map and was used to initiate a walk to create a physical map over the san gene consisting of yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones. A BAC, 92i12, was isolated using recombinant markers on either side of the mutation and partially sequenced. Another BAC not shown (184d07) was sequence to obtain the complete sequence of the san gene. (B) Initial bulk segregant analysis established linkage to SSR marker z3824 (not shown on this high-resolution map) on zebrafish chromosome 20. Other SSR markers in the region were tested for linkage and the vtn interval was defined to the region between z21067/z29926 and z22659. Genome walks were initiated from both directions, but because of lack of BAC and YAC representation, only the walk from the one direction (z22659) was continued. Although multiple BACs and YACs were isolated from this direction, another gap in clone representation prevented further progress. With the availability of the complete fugu genome sequence, a more effective analysis of conserved synteny became possible. Several of the closest BACs had been partially shotgun sequenced to provide more information for the analysis of synteny and generation of genetic markers. A single non-chimeric BAC contained the zebrafish homologs of at least three genes, SOX7 (SRY (sex determining region Y)-box 7), PINX1 (PIN2-interacting protein 1), and TDH (L-threonine dehydrogenase). (BAC 8m8 was nonchimeric based on mapping of subcloned regions on both the RH panel and the vtn map cross). These three genes also mapped to a single scaffold on the fugu genome map. When these genes were compared back to their location within sequenced zebrafish BACs placed on the zebrafish BAC fingerprint (fpc) map, a single fpc contig was identified that contained two of the three genes (PINX1 and TDH). Sequence analysis of both the fugu scaffold and the zebrafish fpc contig revealed another closely linked gene, MGC4607, which was tested as a candidate and confirmed as the vtn gene by both morpholino antisense oligomer phenocopy of the mutation and determination of the genetic lesion in the m201 allele of vtn. y, YAC clone; b, BAC clone.
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