Fig. 4

ednrb1 is expressed by nonmelanocytic neural crest-derived pigment cell lineages during embryonic pigment pattern development. (A–D) Molecular marker and mutant analyses reveal ednrb1 expression by xanthophore precursors. (A,A′) Corresponding brightfield and epifluorescence views revealing ednrb1+ cells (blue, A) that coexpress fms (red, A7prime;; e.g., arrowhead). Shown is a 12-μm cryosection through the anterior trunk of a 24-h embryo. (B,B′) ednrb1+ cells (blue, B) also coexpress the xanthophore marker xdh (red, B′; e.g., arrowhead). Shown are cells in the dorsolateral neural crest migratory pathway in the middle trunk region of a 24-h embryo. (C,C′) Comparison of ednrb1 expression in wild-type (C) and fms mutant (C′) embryos reveals an abnormal accumulation of ednrb1+ cells at the midbrain–hindbrain junction (arrowhead). Shown are the heads of 26-h embryos. e, eye. (D,D′) In the midtrunk at 32 h, unmelanized ednrb1+ cells are present over the flank, between developing melanocyte stripes in wild-type embryos (D; e.g., arrowhead), but unmelanized cells are not present in this region in fms mutants. In contrast, ednrb1+ melanocytes are present in both wild-type and fms mutants (arrows). (E, F) ednrb1 is expressed by differentiated iridophores. (E) Individual iridophores (arrowheads) can be observed along the dorsal myotomes as highly reflective cells under oblique illumination in the posterior trunk of a living 72-h larva that has been treated with phenylthiourea to inhibit melanin synthesis. Yellow coloration in the dorsal trunk is due to xanthophore pigmentation. (E′) In the same individual processed for in situ hybridization, ednrb1+ cells (arrowheads) are present along the dorsal myotome in a pattern coincident with iridophores observed prior to histological preparation. Additional iridophores in ventral regions of the larva stain as well, but are out of the focal plane in (E′). In comparison with ednrb1 expression by iridophores, ednrb1 expression by xanthophore and melanocyte lineages is dramatically reduced at these stages and cannot be seen here. (F,F′) Comparison of ednrb1 expression between wild-type (F) and shady mutant (F′) embryos also supports the inference that ednrb1 is expressed by iridophores. (F) At 72 h in wild-type, unmelanized cells along the dorsal myotomes exhibit robust ednrb1 expression (arrowheads). (F′) In shady mutants, which lack iridophores, ednrb1 expression by unmelanized cells in the corresponding region is not observed. Scale bars: (A,A′) 60 μm; (B,B′) 40 μm; (C,C′) 40 μm; (D,D′) 100 μm; (E,E′) 60 μm; (F,F′) 50 μm.