FIGURE 2

Identification of mastl as the gene responsible for the kt441b mutation. (A) A summary of genetic mapping of kt441b on chromosome 24. Numbers of recombinants and calculated distances for microsatellite markers are indicated. Gray bars indicate BAC clones, and a dotted line indicates a contig. (B)In situ hybridization showing expression of mastl in siblings and kt441b homozygous embryos at the 24-somite stage. For each, 10 embryos identified by their morphology, were stained at the same time. (C) Comparison of genomic sequences around a mutated nucleotide with the corresponding sequence of the wild-type allele. In kt441b mutation, a nucleotide in the splice donor site at the 3′ end of exon 4 of zebrafish mastl is changed from T to G. (D) PCR products of mastl cDNA prepared from 50 embryos of siblings and kt441b homozygous mutant embryos. cDNA was amplified with primers based on exons 3 and 5 of zebrafish mastl. Each PCR fragment from kt441b homozygotes was cloned and sequenced. The size of PCR fragment #3 is matched to the predicted one with proper splicing. Bands indicated with asterisks appear due to contamination of genomic DNA because they contain entire introns 3 and 4 in addition to exons 3,4 and 5 of zebrafish mastl. Note that siblings were a mixture of wild-type and heterozygote embryos. ex: exon, in: intron. (E) Schematic representation of zebrafish Mastl proteins with positions corresponding to exon-intron boundaries. Gray regions indicate the kinase domain, which is separated by a non-conserved region. One CRISPR mutant allele mastl^kt3002 in which 11 bp are deleted in the mastl gene caused frameshifts upstream of the conserved kinase domain (Supplementary Figure S1). (F) Morphologies of mastl^kt3002 homozygotes in comparison with mastl^kt441b homozygotes. mastl^kt3002 homozygous mutants exhibited similar morphology to mastl^kt441b homozygotes and did not complement mastl^kt441b. Arrows indicate the end of the yolk tube and the tip of the tailbud. (G) Expression of mastl mRNA at different stages of wild-type embryos. Expression of mastl mRNA was detectable even at the 64-cell stage, indicating that it is maternally expressed. Also, mastl mRNA was ubiquitously expressed from the 64-cell stage to the 24-somite stage. Scale bars, 100 μm.