Fig. 5

The s6 and c99 mutations disrupt hand2 genomic DNA. (A) Genomic structure of zebrafish hand2. The single intron is represented by a thick horizontal line; 5′ and 3′ UTRs are represented by thin rectangles and the coding regions of the two exons are represented by thick rectangles. A gray rectangle indicates the basic region and a black rectangle indicates the helix-loop-helix domain. (B) No fragment of the hand2 gene can be amplified by PCR from hans6 genomic DNA. Amplifications of a fragment from exon 1, a fragment from exon 2 and a control fragment from the end of a hand2-containing PAC are shown; alternating lanes represent reactions performed with wild-type and han^s6 genomic DNA templates. (C) The han^c99 mutation is a ~5 kb insertion between bases 346 and 347 of the 5′ UTR. The insertion point is shown with a vertical line; the insertion is not drawn to scale. This insertion can lead to the missplicing of hand2 mRNA at cryptic splice sites at bases 233 and 578. The new ‘intron’ in this splice variant is represented by a thick line. Some 5′ UTR sequence and some coding sequence are spliced out in this variant. (D) han^c99 linkage testing was performed using a combination of three PCR primers (shown as red arrows in C, not drawn to scale). When the insertion is absent, the two primers flanking the insertion site amplify a small fragment, as in the case of a homozygous wild-type embryo (first lane). In the presence of the insertion, the flanking primers are ineffective using standard PCR conditions, but the 5′ primer and the primer complementary to the insertion can amplify a slightly larger fragment, as in homozygous han^c99 mutants (second-sixth lanes). (E-G) Dorsal views of cmlc2 expression in a wild-type embryo (E), a han^s6 mutant sibling (F) and a hand2-injected hans6 mutant sibling (G). All panels are shown at the same magnification. Injection of hand2 mRNA can partially rescue the production of cmlc2-expressing myocardial precursors in hans6 mutants. Injection of hand2 mRNA does not seem to affect the production of myocardial precursors in wild-type embryos (data not shown).