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Fig. 2 Generation and testing of a conditional (“floxed”) tbx20 allele. a. Experimental design. b. Genotyping of adult fish for intron 2 loxP site. c. Results of nested PCR screening for loxP integration into intron 1. d. Sequence of intron 1 loxP integration in recovered tpl145 floxed tbx20 allele. e. Genotyping of “F1” adults. Primer binding sites are shown as black arrows, loxP sites as red or blue triangles, exon as an open box. f, g. Induction of tbx20 loss of function by injection of Cre mRNA. f. One quarter of embryos obtained by in-crossing tbx20tpl145 heterozygotes and injected with Cre mRNA display a consistent, severe heart development defect. g. Genotyping of embryos with severe heart defects (lanes 1–3) and phenotypically normal siblings (lanes 4–8). L, GeneRuler DNA Ladder (ThermoFisher Scientific). Genotyping lanes 1 and 5 correspond to images in f. h, i. Induction of tbx20 deletion by 4-hydroxytamoxifen. tbx20tpl145 heterozygote was crossed to Tg(ubi:CreERT2) line. GFP-positive embryos were collected at 2dpf and incubated with 5μM 4-HT for 24 hours. j. Adults raised from Cre-injected tbx20tpl145/+ embryos were incrossed, resulting in approximately 1/4 of embryos (36/128, 28%) with severe heart defects. k. Confirmation of Cre-mediated excision of the second intron of tbx20 by sequence analysis. l, m. Analysis of excision efficiency by qPCR. l. Excision efficiency was assessed by qPCR in embryos treated with 4-HT at various concentrations at different time points and in two different ubb:CreERT2 driver lines. The y-axis indicates un-excised tpl145 normalized to the untreated control. m. Images of tbx20 phenotypes shown after treatment with 4-HT. Individuals 1, 2, and 3 from l correspond to images labeled 1, 2, and 3.