Embryos and adult shhact fish were genotyped using genomic PCR. The primers are listed in Supplementary file 1. Embryos were generated by intercrossing shhact/+ fish, crossing tcf21:CreER; shhact/+ with shhact/+ fish, or crossing tcf21:CreER; shhact/+ with tcf21:DsRed2; shhact/+ fish. Embryos were genotyped individually by PCR with template DNAs prepared from single embryos as described subsequently (sample preparation for embryo genotyping and analysis). The WT allele was detected by PCR using F1 and R1 primers (Figure 1B). Amplification by F1/R1 primers was intervened in the conditional trap allele with the inserted Zwitch (4448 bp; Figure 3—figure supplement 1A). The conditional trap allele was detected by PCR using F2 and R2 primers for assessing germline transmission (Figure 1D) or F1 and R2 primers for genotyping (Figure 3—figure supplement 1A). The inverted, hence mutagenic, allele was detected by PCR using F4 and F5 primers (Figure 2B). PCR using Cre-scr-F/R primers was performed to confirm tcf21:CreER and ubb:Cre-GFP transgenes (Figure 3—figure supplements 1C and 2B). Examples of the genotyping results are shown in Figure 3—figure supplements 1 and 2.