Loss of dusp6 and dusp2 does not impact early development. a Offspring from crosses between double heterozygous dusp2 um287/+ ;dusp6 um286/+ males and females were raised to adulthood and genotyped in order to determine the percentage of each possible genotype in surviving fish. A chi-square test indicates no significant statistical difference between the actual and expected Mendelian ratios. b Outline of RNA-seq library production from wildtype and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males) 18hpf embryos. c Diagrams representing the number of differentially expressed up-regulated (left circles) and down-regulated (right circles) genes in dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males) versus wildtype embryos. Inner circles indicate the subset of genes annotated in ZFIN. d 23 genes identified as differentially expressed by RNA-seq were re-examined by qPCR on cDNA derived from wildtype versus dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males) embryos. e-h 48hpf wildtype (e, f) and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; g, h) embryos were assayed for changes in otx5 (e, g) and six7 (f, h) expression by in situ hybridization. i-q Uninjected (i-k) or MO-injected (l-q) dusp6 mutant (derived from crosses between dusp6 um286/um286 females and dusp6 um286/um286 males; i), dusp2 mutant (derived from crosses between dusp2 um287/um287 females and dusp2 um287/um287 males; j), and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; k) embryos were assayed by immunostaining with 3A10 antibody to detect the Mauthner neurons at 48hpf. r-w 12hpf wildtype (r, s, t) and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; u, v, w) embryos were assayed by immunostaining for pERK (green in r, u; red counterstain detects the Valentino transcription factor), as well as by in situ hybridization for expression of pea3 (s, v), and erm (t, w). x-aa Wildtype (x, y) and dusp2/dusp6 double mutant (derived from crosses between dusp2 um287/um287 ;dusp6 um286/um286 females and dusp2 um287/um287 ;dusp6 um286/um286 males; z, aa) embryos were injected with fgf8 mRNA and the expression pattern of pea3 visualized by in situ hybridization. All embryos are in dorsal view with anterior to the top. Numbers in top right corner of each panel indicate the total number of embryos assayed for that condition. Numbers in bottom right corner indicate percent of embryos with the phenotype shown
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