Positional cloning of the zebrafish kto locus. (A) Genetic mapping of the kto locus on chromosome 14, not precisely to scale. Below the cM scale, markers that are polymorphic in a map cross with the TL strain are shown in black, and markers polymorphic in the WIK map cross are shown in red. Recombinants per number of meioses tested are listed below. Note that we have repositioned simple sequence length polymorphism marker z22128 based on these results. Bacterial artificial chromosome (BAC) clones from this region are shown below. A portion of the kto/trap230 gene is located at the end of BAC clone zC119P14. (B) TRAP230 protein domain structure and point mutations in two kto alleles. L, leucine-rich domain; LS, leucine-serine rich domain; PQL, proline-glutamine-leucine-rich domain; OPA, opposite-paired domain. The ktoy81 allele carries a stop codon. A point mutation in a splice donor site in ktoy82 leads to splicing from a cryptic site four nucleotides downstream, which results in a frame shift and premature termination. (C) Expression of trap230 as seen by in situ hybridization. Lateral views except dorsal view in d.(a) Maternal expression as seen in eight-cell-stage embryo; (b) widespread expression at 90% epiboly; (c) in kto embryo at 90% epiboly, expression is strongly reduced; (d) expression of trap230 in the head at 24 hpf; arrowheads point to presumptive cartilage primordia; (e) at 48 hpf, trap230 expression is limited to the head (arrow). di, diencephalon; e, eye; h, hindbrain; m, midbrain; mhb, mid-hindbrain boundary; ov, otic vesicle; te, telencephalon.
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