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Fig. 2 unm_t30922t30922 mutants carry a homozygous splice acceptor site mutation within the wnt10a gene, representing a likely wnt10a amorph. (A) Meiotic mapping placed the MFF collapse-causing mutation into a 0.429 cM interval of chromosome 9 between SSLP markers z25735 (2/1400 recombinants/meioses) and NZ79 (4/1400 recombinants/meioses). (B, C) Whole-genome sequencing (WGS) of DNA from 20 pooled mutant embryos revealed a region of homozygosity exclusively on chromosome 9 (B), which was approximately 4 Mb large (indicated by red lines in (C)) and contains a single point mutation (A>G) in the splice acceptor site before exon 4 of wnt10a (C). (D) Sequencing of cDNA from mutant embryos revealed three mutant splice isoforms, resulting from the usage of cryptic splice acceptor sites within intron 3/4 or exon 4 and leading to C-terminally truncated Wnt10a proteins lacking exon 4-encoded amino acid residues 288–442. (E) Quantitative real-time PCR (qRT-PCR) of whole embryos shows reduced amounts of wnt10a transcripts in unm_t30922t30922 mutants at 2 and 5 dpf.