To validate that shhact was a loss-of-function allele, we obtained shhact/ct embryos from incrosses of shhact/+ fish and individually genotyped the embryos using PCR (Figure 3—figure supplement 1A and B). Quantitative RT-PCR (qRT-CPR) revealed that the expression of shha and Hedgehog (Hh) target genes was strongly decreased in Cre+shhact/ct embryos but not in Cre− shhact/ct embryos, indicating that the shhact allele is functional in the absence of Cre activation (Figure 3A). Furthermore, most Cre+ shhact/ct embryos exhibited truncated pectoral fins and U-shaped somites that lacked clear horizontal myosepta (Figure 3B and C), defects reminiscent of homozygous shha mutant embryos (Schauerte et al., 1998). To confirm this result, we established shhact/+ fish carrying the transgene Tg(ubb:iCRE-GFP) (ubb:Cre-GFP), in which codon-improved Cre (iCRE) DNA was expressed by a strong, ubiquitously expressed ubiquitin B (ubb) promoter (Mosimann et al., 2011) (Figure 2—figure supplement 1A). From incrosses of this line, we obtained embryos carrying the WT and/or mutagenic shha allele and analyzed the phenotype of these embryos (Figure 3—figure supplement 2A and B). We found that pectoral fin development was largely normal in WT and heterozygous mutant embryos but severely hampered in all homozygous mutants examined (Figure 3—figure supplement 2C). We also performed semi-qRT-PCR analysis of shha expression and confirmed that its expression was reduced to nearly 50% of WT levels in the heterozygous mutants and to an undetectable level in the homozygous mutants (Figure 3—figure supplement 2D and E). Together, these results provide clear evidence that Zwitch can inducibly disrupt gene function in zebrafish.10.7554/eLife.24635.009Figure 3.Phenotype of shhact/ct embryos globally expressing Cre.(A) qRT-PCR analysis of 72 hpf Cre+ and Cre− shhact/ct embryos (n = 10 and 9). WT embryos injected with pUbb-iCRE-GFP DNA were used as a control (n = 9). Ten pooled embryos per sample were used for qRT-PCR analysis. The data are presented as the mean ± SEM (***p<0.001, Mann–Whitney U test). (B) Phenotypes of 72 hpf Cre+ and Cre- shhact/ct embryos. Arrows, pectoral fins; arrowheads, somite boundaries; brackets, horizontal myoseptum. Bright field images were captured using an MVX10 microscope. The composite images shown were generated using ImageJ software. Somite defects were observed in all embryos with severe pectoral fin defects. (C) Quantification of pectoral fin phenotypes from the embryos in B (n = 7 [WT, Cre+], n = 12 [shhact/ct, Cre−], and n = 28 [shhact/ct, Cre+]; **p<0.01, Fisher’s exact test). N.S., not significant (p=0.5392). The embryos used in A–C were selected on the basis of their high-level expression of Cre as described in Figure 2—figure supplement 1B (see also Cre DNA and mRNA injection, Materials and methods). A moderate pectoral fin defect was observed in control samples, likely due to injection artifacts.DOI: http://dx.doi.org/10.7554/eLife.24635.00910.7554/eLife.24635.010Figure 3—figure supplement 1.Genotyping PCR of shhact alleles.(A) Schematic of the shha WT allele (+) and conditional trap allele (ct) and primers sites. (B) Genotyping PCR result of single embryos from an incross of shhact/+ fish injected with Cre DNA at single-cell stage. Note that ct/ct genotype was predictable due to the developmental defects observed in shhact/ct embryos after Cre DNA injection. (C) Genotyping PCR result of single embryos from a cross of tcf21:CreER; shhact/+ fish with shhact/+ fish. We did not use embryos exhibiting a faint band for experiments (asterisk). PCR with Cre-Scr-F/R primers occasionally amplified a faint non-specific band at a lower size. ND, not determined. Please see the Genotyping and Sample preparation for embryo genotyping and analysis section in the Materials and methods for details of the screening procedures.DOI: http://dx.doi.org/10.7554/eLife.24635.01010.7554/eLife.24635.011Figure 3—figure supplement 2.Analysis of embryos carrying mutagenic shha alleles.(A) Schematic of the shha WT allele (+), non-mutagenic, conditional-trap allele (ct), and inverted mutagenic allele (m), and primers sites. (B) Genotyping PCR result of single embryos from incrosses of ubb:Cre-GFP; shhact/+ fish. (C) Pectoral fin phenotype of WT and heterozygous and homozygous mutagenic embryos in B. Embryos that did not carry ubb:Cre-GFP transgene were analyzed (Cre-GFP−). Pectoral fin development was normal in all WT embryos (0 abnormal in 5 analyzed) and most heterozygous mutants (1 moderately abnormal in 7 analyzed; p=0.3774, Fisher's exact test), but severely hampered in all homozygous mutants (3 abnormal in 3 analyzed; p<0.01, Fisher’s exact test). (D) Semi-qRT-PCR analysis of shha expression in WT and heterozygous and homozygous mutagenic embryos. The number above each lane in the gel picture indicates the Embryo ID in B. (E) Densitometric quantification of the PCR result in D. The data represent the mean ± SD (**p<0.01; Mann–Whitney U test).DOI: http://dx.doi.org/10.7554/eLife.24635.011