Phenotypic analysis of nlsu11 mutant and sibling embryos and larvae. A–H: Scanning electron micrographs (A,B) and stereomicroscopic images (C–H). Dorsal views, anterior is to the left. A: At 3 days postfertilization (dpf), wild-type siblings show pectoral fins apposed to the yolk. B: No pectoral fins are seen in most nlsu11 mutants (arrows in A and B). C:tbx5 expression marks the initially contiguous heart (arrowhead) and pectoral fin (asterisk) primordia in wild-types at the 10s stage. E: The fin precursor specific tbx5 expression domain lateral to somites 1–3 (asterisk) has separated from the heart field domain (arrowhead). G: Pectoral fin buds retain tbx5 expression at 32 hr (asterisk). D,F: A fin precursor specific tbx5 expression domain is lacking at all stages in nlsu11 embryos (asterisks). H: This prefigures the later observed lack of pectoral fin buds (asterisk).

Application of 10-5M diethylaminobenzaldehyde (DEAB) to zebrafish embryos. A:tbx5 positive fin precursors are absent at 24 hpf in embryos treated from shield, 75% epiboly and tail bud stage but are detectable if treatments are initiated at the 6-somite (s) stage; dorsal view; anterior is to the left. B: DEAB treatments throughout somitogenesis lead to loss of tbx5 expression and fin bud development at 32 hours postfertilization (hpf). Later treatments lead to reduction of tbx5 expression and fin bud size at 32 hpf; lateral view, anterior is to the left. C: DEAB treatments during early to midsomitogenesis reduce tbx5 expression and fin bud size at 32 hpf; lateral view, anterior is to the left. D: Schematics of inhibitor treatments and results.

A–K: Perturbation of cyp26a1 (A–F) and aldh1a2 (G–K) expression domains in the gastrula hypoblast upon 10-5M diethylaminobenzaldehyde (DEAB) treatments from 30% epiboly until the stage of fixation. Animal is to the top and dorsal to the right (A,B,D,E,G,H) or shown in dorsal view (J,K) and in parasagittal sections (C,F). A–F: DEAB treatments lead to an absence of the paraxial cyp26a1 domain at 75% and to a notable reduction of the marginal hypoblast expression domain (compare expression domains in A,D,C to B,E,F, arrowheads). G,H,J,K: DEAB treatments from 30% epiboly on reveal an enhanced signal intensity of the aldh1a2 expression domain ventrolaterally at 90% epiboly and tail bud stage (compare G,J to H,K). I: The graph shows the relative enhancement of marginal aldh1a2 expression at 90% epiboly of 10 experimental embryos (red) compared to 10 control embryos (blue) in one experiment; asterisks show significant differences in staining in controls vs. experimental embryos.

Kinetics of diethylaminobenzaldehyde (DEAB) effects on retinoic acid (RA) target gene transcription after incubation of embryos with 10-5M DEAB for different periods of time during gastrulation (A), somitogenesis (B), and after inhibitor wash out (C). Stages of inhibitor incubation are given on the lower left, duration in hours (h) on the lower right side of each frame. A: During gastrulation, inhibitor treatments for 2.75 h, 2 h, and 1 h reduce the ventral marginal domain and the paraxial domain of cyp26a expression (arrowheads; dorsal is to the right). B: Application of DEAB for 8 h, 4 h, or 1 h before the 10s stage strongly down-regulate hoxb5a expression (dorsal view, anterior is to the left). Lack of RA synthesis manifests itself at least 1 h after DEAB application (A,B). C: Wash-out of DEAB at 75% epiboly after 2 h of treatment does not affect hoxb5a expression at 10s (dorsal view, anterior is to the left).

Application of 10-5M diethylaminobenzaldehyde (DEAB) to zebrafish embryos before the 10-somite stage; myoD, tbx5 double-stainings at the10s stage; dorsal views, anterior is to the left. A: DEAB is applied from successively later stages onward; early fin precursors are absent if treatments include gastrula stages but are present if treatments start after gastrulation. B: Two-hour inhibitor treatments: Fin precursors are absent if treatments are conducted during gastrulation but are present if treatment are conducted during early somitogenesis. C: Schematics of inhibitor treatments and results. Red boxes indicate the time window of fin precursor specification.

Response of the lateral plate upon a delayed endogenous retinoic acid (RA) signal; lateral views. A: Lack of fin buds in embryos at 32 hours postfertilization (hpf) upon application of 10-5M DEAB from 30% epiboly to tail bud stage. B: In situ hybridization reveals some tbx5 expression in the mesoderm of the presumptive fin bud territory at the same time.

Acknowledgments
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