Fig. 9. Retinoic acid-mediated induction of th-expressing cells requires protein synthesis and coincides with increased tfap2a expression. (A-D) Cycloheximide (CHX; B), retinoic acid (RA; C) or a combination of both treatments (D) does not alter the expression of th in the locus coeruleus (LC) compared with untreated wild-type embryos (A). (E-H) th expression at 33 hpf in the hindbrain of wild-type embryos (H) and embryos exposed to (F) cycloheximide, (G) retinoic acid and (H) a combination of cycloheximide and RA. CHX blocks the RA induced expression of th in medulla at 33 hpf. (H) Brown cells are pigment cells and not NA neurons, which would stain blue from whole-mount in situ hybridization procedure. (I-L) Expression of th in the medulla oblongata of wild-type embryos at 72 hpf. (J) Cycloheximide-treated embryos, (K) retinoic acid-treated embryos, (L) embryos treated with both cycloheximide and RA. (M-P) Expression of th in the region in which sympathetic neurons form in wild-type embryos (M) at 33 hpf. Embryos exposed to cycloheximide (N), retinoic acid (O) and a combination of both (P). CHX blocks the RA-induced expression of th in sympathetic ganglia. (Q,R) Expression of tfap2a in wild-type embryos (Q) and embryos treated with RA between 24 and 33 hpf (R). RA induces the expression of tfap2a in the hindbrain. (S,T) Expression of tfap2a (red) and phox2a (blue) in the hindbrain of wild-type embryos (S) and nls mutant embryos (T). The expression of tfap2a (red) and phox2a (blue) is reduced in the posterior hindbrain (r6,7) of neckless/raldh2-deficient mutants. (A-R) dorsal views, anterior towards the left; (S,T) lateral views, anterior towards the left. ce, cerebellum; hb, hindbrain; sc, spinal cord; tec, tectum; 1-7, rhombomeres r1-7; sympat, region in which sympathetic neurons form.