Molecular characterization of retinoic acid (RA) signaling functions during amphioxus pharyngeal development. Lateral views of the anterior portions of amphioxus larvae at 36 hours post fertilization (hpf) are shown with the anterior to the left. The 50-μm scale bar in A is applicable to the entire figure. Control larvae are shown as well as those treated with either 10-6 M RA or 2 × 10-6 M BMS009, an RAR antagonist, starting at three different developmental stages (16 hpf, 20 hpf and 24 hpf). The assayed genes are amphioxus Pax1/9 (A), (B), (B’), (B”), (C), (C’), (C”), Six1/2 (D), (E), (E’), (E”), (F), (F’), (F”), Tbx1/10 (G), (H), (H’), (H”), (I), (I’), (I”), Pax2/5/8 (J), (K), (K’), (K”), (L), (L’), (L”), Pitx (M), (N), (N’), (N”), (O), (O’), (O”) and Cyp26-2 (P), (Q), (Q’), (Q”), (R), (R’), (R”). RA treatment at 16 hpf eliminates the expression of Pax1/9, Six1/2, Tbx1/10 and Pax2/5/8 and severely reduces that of Pitx. In contrast, expression of Cyp26-2 is expanded. When RA is applied at progressively later stages, normal expression of the genes is gradually restored. Treatments with BMS009 result in an expansion of the Pax1/9 domain, in abnormal Tbx1/10 expression, in a reduction of the Pax2/5/8 signal in gill slit primordia and in a severe reduction of Cyp26-2 expression. In contrast, the patterns of Six1/2 and Pitx are largely unaffected. The effects of BMS009 are independent of the treatment time point.