(A) Schematic of Cre-dependent conversion of Zwitch from the non-mutagenic orientation to the mutagenic orientation. Cre activation induces an inversion between loxP or lox5171 sites and the subsequent excision of loxP or lox5171-flanking DNA sequences (Schnütgen and Ghyselinck, 2007), thereby permanently converting Zwitch into the mutagenic form and inducing aberrant shha splicing. (B) PCR analysis of the Zwitch inversion. Genomic DNA from 72 hpf Cre+ and Cre− shhact/+ embryos was analyzed using PCR. (C) RT-PCR analysis of shha expression in 72 hpf Cre+ and Cre− shhact/+ embryos. (D) Schematic of Flp-mediated excision of the FRT-flanked LG tag in the shhact allele. (E) Genomic PCR analysis of Flp-injected (+) or uninjected (−) embryos from a cross of shhact/+ adults. PCR using F3 and R1 primers detected shhact alleles in both samples. Flp mRNA was synthesized from linearized pCS2-FLPo (Materials and methods). (F) Representative image of embryos injected with Flp mRNA. Arrows indicate the lens. (G) Quantification of phenotypes of the embryos analyzed in F. A total of 87 Flp-injected (+) and 99 uninjected embryos (−) were analyzed (****p<1.0 × 10−8 Fisher’s exact test). dpf, days post-fertilization.