In vitro and in vivo assays showing that the two retinoic acid response elements (RAREs) are required for retinoic acid (RA) inducibility of cyp26a1 gene. A: Sequences of wild-type and mutated RAREs of zebrafish cyp26a1 promoter. The core sequences of wild-type RARE and mutated RAREs are underlined. R1 and R2 represent the proximal and the distal site RARE of cyp26a1 promoter, respectively. B: Schematic diagram showing the different promoter fragments of cyp26a1 gene driving luciferase reporter gene. WT: the 2.5 k fragment with wild-type R1 and R2; R1mut: the 2.5 k fragment with mutated R1 and normal R2; R2mut: the 2.5 k fragment with mutated R2 and normal R1; DF379: truncated fragment with only 379 bp of 2,533 bp. C: Luciferase activity assay showing that R1 and R2 are required for the RA-inducible activity of zebrafish cyp26a1 promoter. X-axis: the different promoter fragments shown in (B) are examined for their RA inducibilities. The activities of WT, R1mut, R2mut, and DF379 fragments were tested at 100 nM RA. No RA CTL represents that the activity of WT is measured when there is no RA treatment. Y-axis: the relative luciferase activity. Units represent ratios of luciferase activity to control renilla luciferase activity for each sample. Mutating either of two RARE sites or truncating promoter fragment with only R1 present results in that the RA-inducible activities of the promoter fragments are significantly lower than that of wild-type 2.5 k promoter fragment (**P < 0.01). D-G,D′-G′: In vivo transient expression assay showing the activities of the fragments with different lengths or mutations of cyp26a1 regulatory sequence. The linearized constructs of p2.5kcyp26a1pr_eYFP (WT) (D, D′), pR1mut_eYFP (E,E′), pR2mut_eYFP (F,F′), and pDF379_eYFP (G,G′) were microinjected into 1- to 4-cell stage embryos, respectively. The injected eggs were grown to 6 hours postfertilization (hpf) and then treated with 100 nM RA for 6 hr for examining the transient eYFP expression at 12 hpf (D′: WT; E′: R1mut; F&prime: R2mut; G′: DF379). The injected embryos incubated at 0.1% dimethyl sulfoxide (DMSO) were used as control (D, WT; E, R1mut; F, R2mut; G, DF379). Embryos are laterally viewed and positioned anterior top. Fluorescent images are photographed by the DP70 digital camera (Olympus, Japan) with 10-sec exposure time. Scale bar = 100 μm.

Expression of enhanced yellow fluorescent protein (eYFP) in embryos from a female transgenic zebrafish. Nontransgenic embryos are produced by a female transgenic zebrafish (heterozygous for the transgene) mated with a male wild-type zebrafish. Images are photographed by the DP70 digital camera (Olympus, Japan) with 5-sec exposure time. Embryos are laterally viewed and positioned animal pole top (A-D) or anterior top (E). A: Fertilized egg at one-cell stage. B: Two-cell. C: 50% epiboly. D: 75% epiboly. E: Three-somite stage (11 hours postfertilization [hpf]). eYFP expressions appearing in these stages are due to maternal loading of eYFP protein (A-C) or both eYFP protein and mRNA (D,E). Scale bar = 100 μm.

Expression pattern of transgenic reporter in zebrafish early development. Transgenic embryos are produced by a female wild-type zebrafish mated with a male transgenic zebrafish. A-K: Fluorescent images are photographed by the DP70 digital camera with 10-sec exposure time. A′-F′,K′: Whole-mount in situ hybridization is performed to reveal the expression pattern of cyp26a1. Embryos are laterally viewed (except that K′ is dorsal viewed) and positioned animal pole top (A,B; A′,B′), anterior top (C,D; C′,D′), or anterior left (E-K; E′-K′). A,B: Enhanced yellow fluorescent protein (eYFP) is initially detected at presumptive neural plate and around the blastoderm margin of the embryo at 75% epiboly (A) and then 95% epiboly (B). The fluorescent expression patterns mimic the expression of cyp26a1 at 75% epiboly (A′) and 95% epiboly (B′), respectively. C,C′: At 11 hours postfertilization (hpf), eYFP is strong at the presumptive brain region and the tail bud (C), corresponding to the cyp26a1 expression at presumptive forebrain, midbrain and the tail bud (C′). D,D′: At 16 hpf, the transgene signals are present in developing eyes, pharyngeal arches and tail bud (D) where the cyp26a1 is also expressed (D′). E,E′: At 24 hpf, the fluorescent signals are mainly present in retina, cells in olfactory vesicle, anterior dorsal spinal cord, proctodeum, and caudal notochord (E) where the cyp26a1 is also expressed (E′). F,F′,K′: At 30 hpf, the expression pattern of the transgene (F) is similar to that of cyp26a1 (F′, K′). G-K: High magnification reveals that the fluorescent signals are mainly present in retina (G), cells in olfactory vesicle (G), anterior dorsal spinal cord (H), proctodeum (I), caudal notochord (J), and pharyngeal arches (K). de, developing eye; dp, developing pharyngeal arch; ep, epidermis; fr, presumptive forebrain; mr, presumptive midbrain; nc, caudal notochord; of, cells in olfactory vesicle; ph, pharyngeal arch; pr, proctodeum; rt, retina; sc, anterior dorsal spinal cord; tb, tail bud. Scale bar = 100 μm.

Retinoic acid (RA) inducibility of enhanced yellow fluorescent protein (eYFP) expression in transgenic zebrafish embryos is similar to that of cyp26a1. B-E′: The transgenic embryos produced by a female wild-type zebrafish mated with a male transgenic zebrafish were treated with 1 μM RA (B,B′) or 10 μM DEAB (C,C′) for 6 hr starting from 18 hours postfertilization (hpf), or 10 nM RA (D,D′), or 5 μM DEAB (E,E′) for 24 hr starting from 0 hpf to examine whether the RA-induced expression of the transgene (B-E) is similar to that of cyp26a1 (B′-E′; revealed by whole-mount in situ hybridization) during zebrafish early development. A,A′: Untreated transgenic embryos were used as controls. Fluorescent images were photographed by a DP70 digital camera with 10-sec exposure time. Embryos are laterally viewed and positioned anterior left. In the embryos that were treated with 1 μM RA for 6 hr (B,B′), the greatly up-regulated expression was mainly seen in forebrain, anterior spinal cord, proctodeum, and whole tail (A,B,A′,B′). In the embryos treated with 10 nM RA for 24 hr treatment (D,D′), the up-regulated expression was almost in whole body but mainly seen in retina, anterior spinal cord and caudal notochord (A,D,A′,D′). When the embryos are treated with 10 μM DEAB for 6 hr (C, C′) or 5 μM DEAB for 24 hr (E,E′), the expressions of the transgene and cyp26a1 both are down-regulated mainly in retina, anterior spinal cord, and caudal notochord compared with wild-type embryos (A,A′). Scale bar = 100 μm.