(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.