Fig. 1.

The lof^t2 mutation causes cis-ectopic expression of kcnh2a. (A,B) Bright-field images of adult TL (Tüpfel longfin: leo^t1/t1; lof^t2/t2) and wild-type AB zebrafish. Scale bars: 1 cm. (C) Schematic diagram of 1.5 Mb region of zebrafish chromosome 2 (chr2). The putative location of the lof^t2 mutation is outlined with a dashed red line and the region deleted in the suppressed lof^jg1 is highlighted by a green box, as determined by Iovine and Johnson (2002). (D) Left: schematic of RNA-Seq experimental design to identify genes mis-expressed in regenerated lof^t2. Right: RNA-Seq data showing gene expression at chr2:22,931,376-24,880,659 from lof^t2/+ relative to clutchmate lof^+/+ controls. The asterisks indicate the two differentially expressed genes (***P<10⁻⁴). ns, not significant. Inset: expression of kcnh2a in lof^t2/+ (in red) and lof^+/+ (in blue) clutchmates determined by RT-qPCR using 4 dpa fin cDNA. Data were normalized to rpl8 reference expression levels and are presented as fold change relative to lof^t2/+. runx2b expression levels are shown for comparative purposes and were not significantly changed between the two genotypes. Expression of kcnh2a was below limits of detection in lof^+/+ fish (indeterminate, ND). Each point represents a cohort of three animals. (E) RT-qPCR on 4 dpa caudal fin cDNA from lof^t2 kcnh2a^WIK-TL; lof⁺ kcnh2a^AB fish to detect chromosome-specific expression of kcnh2a. Sequences of non-coding kcnh2a polymorphisms that specifically amplify either kcnh2a^WIK-TL, which is located on the lof^t2 mutant chr2 (red squares), or kcnh2a^AB, which is located on AB chr2 (green triangles). Data were normalized to total kcnh2a levels (blue circles) determined using primers that amplify both alleles indiscriminately. Each data point represents a result from an individual fish. RT-qPCR statistical analyses used one-way ANOVA with Tukey's multiple comparisons tests.