Fig. 1

Fig. 1 med10 is the gene for tennismatch (ten). (A) Positional cloning of the ten locus. ten was mapped between SSLP markers Z7058 and Z11872 for chromosome 19. The chromosomal walk was initiated from EST9, which has zero recombinants out of 6040 meioses. A physical contig was constructed consisting of two overlapping BACs covering the ten locus, as each end of the contig picks up two recombinants on either side. Two genes were predicted using computational tools after sequencing these two BACs. The genomic structure of med10 is shown at the bottom (GenBank Accession Number AY698065). EST9 is 4 kb away from med10 and is transcribed in the opposite direction. A 1.2 kb sequence (GenBank Accession Number AY698066) was identified to be inserted before the transcriptional start site of med10 in ten mutant zebrafish. (B) Expression of med10 mRNA was abolished in ten mutant embryos. Shown are Northern blots of total RNA extracted from embryos at 42 hpf. (C) Expression of med10 mRNA was quantified by real-time RT–PCR analysis in embryos collected at 36 hpf, when ten mutant embryos start to exhibit phenotypes, and at 72 hpf. Data shown here are the means ± SD from three independent experiments, expressed as a percentage of wild-type transcripts. (D) Injection of morpholino antisense oligonucleotides against med10 phenocopies ten mutant embryos. Injected embryos were evaluated at day 3 based on two visible phenotypes of ten mutants: cardiac malformation and stunted pectoral fin bud growth. Shown are the percentages of embryos that had the corresponding phenotype. (E–G) Injection of med10 mRNA rescues ten mutant phenotypes. Shown are wild-type (E), ten/ten mutant (F), and ten/ten mutant plus med10 mRNA (2 ng) injection (G) at 72 hpf. Anterior is to the right and dorsal is up. The heart is located ventral to the yolk. The brackets indicate the pectoral fin bud. V, ventricle; A, atrium.