Mapping of ruby mutation. (a) Mapping interval derived from exon capture and BSA. The number of recombinants for each RFLP marker, the genetic distance and the location of the marker in v4.1 and v7.1 genome assemblies are shown. The 220 kb interval contains 7 genes: tmem150a, c2orf68, usp39, sftpb, pax8, psd4 and a portion of il1b. Genes in red have mutations causing amino acid or splice site changes based on the exon capture data. (b) Genomic, cDNA and protein sequences of WT and mutant pax8. The mutation of two contiguous nucleotides (nt) in intron 2 (arrowhead) shifts the acceptor splice site 1 nt, causing the inclusion of an extra G in the transcript (cDNA) (arrowhead). The protein sequence shows a change in frame and shortly after a premature STOP codon (*) abrogating the entire paired box domain. (c) Pax2 expression in wt and mutant ruby embryos by Whole Mount in situ hybridization showing clear impairment of pronephric development (arrowhead). (d) Pax8 morpholino phenocopies ruby phenotype. Pax8 morpholino was injected at one or two-cell stage (single cell injected). Embryos were fixed at st. 36–37 and pax2 expression was used to assess pronephric development. The embryo shown for two-cell stage injection was injected on the right side (based on fluorescent dye tracer [not shown]). Arrowheads indicate abnormal pronephros. Embryos in (c,d) are lateral views with dorsal to the top and anterior to the left (in left columns) or right (in right columns).