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Fig. 3 tbx5b expression and confirmation of morpholino knockdown using green fluorescent protein (GFP) -tagged mRNA containing the tbx5b morpholino (MO) target site or reverse transcriptase-polymerase chain reaction (RT-PCR). A,B: tbx5b in situ hybridization of 36 hpf embryos, showing (A) a ventral view with cardiac expression and (B) a dorsal view with signal in the pectoral fins. C,C′: The efficacy of tbx5b morpholino knockdown was confirmed using a Tester mRNA constructed by inserting the tbx5b MO target site in front of EGFP coding sequences. At 36 hpf, 100% of control embryos injected with 1,000 ng/μl Tester mRNA displayed normal body morphology and expressed GFP (n = 30). D,D′: Embryos co-injected with 100 μM morpholino and 1,000 ng/μl Tester mRNA displayed normal body axes and central nervous system development, but (D′) 87% failed to detectably express GFP (n = 30), indicating that the tbx5bMO efficiently targeted the expected binding site. E: RT-PCR–amplified RNA from buffer-injected control (wild-type [WT]) or tbx5bSD-MO (MO) injected embryos, with eF1a loading controls. Expected amplicon sizes are 513 bp for wild-type transcripts and 417 bp for transcripts lacking exon 2. M, marker.