Fig. 2

kitla misexpression induced ectopic melanophores within the myotomes.

(A) kitla was normally expressed within the epidermis and hypodermis (arrow) during post-embryonic development of wild-type fish. sc, spinal cord. m, myotome, e, epidermis. (B) In sibling Tg(hsp70::kitla) larvae, heat shock resulted in increased kitla transgene expression within the epidermis (arrowheads) as well as ectopic expression within the myotomes (arrow), spinal cord, and elsewhere. (C) The late melanophore lineage marker dopachrome tautomerase (dct), encoding an enzyme required for melanin synthesis [96], [97], was not expressed within the myotomes of wild-type fish. (D) However, dct was expressed by scattered cells within the myotomes (arrow) in larvae misexpressing kitla. (E,F) Newly differentiated ectopic melanophores (arrow) were found between myotubes (arrowheads; E) and these cells continued to express sox10 protein (F). (G) Vibratome section revealing ectopic melanophores (arrow) within the myotome of a Tg(hsp70::kitla) larva 48 h after the initiation of kitla misexpression. Melanophores deep within the myotome were found only in Tg(hsp70::kitla), though melanophores were occasionally found within the horizontal myoseptum of both transgenic and wild-type larvae [ectopic melanophores per larva, Tg(hsp70::kitla): mean±SE = 1.3±0.15, range = 0–7 cells, n = 80 larvae; non-transgenic siblings: mean±SE = 0±0, range = 0, n = 69]. Longer durations of kitla misexpression resulted in more ectopic melanophores per larva. Suggesting that ectopic melanophores differentiated in situ rather than migrated into the myotomes from the hypodermis, labeling of hypodermal cells by photoconversion of mitfa::Eos+ [55] failed to reveal movement of cells away from enhanced kitla expression in the epidermis into the myotome (n = 10 larvae, 3–5 cells per individual). (H) In contrast to the wild-type, ectopic melanophores were significantly fewer in erbb3b; Tg(hsp70::kitla) mutants [Wilcoxon test, Z = 7.1, P<0.0001; ectopic melanophores per larva, erbb3b; Tg(hsp70::kitla): mean±SE = 0.04±0.03, range = 0–1, n = 50 larvae; non-transgenic siblings: mean±SE = 0±0, range = 0, n = 70] and in Tg(hsp70::kitla) larvae treated with AG1478 during the ErbB embryonic critical period [Wilcoxon test, Z = 2.9, P<0.005; ectopic melanophores per larva, AG1478-treated Tg(hsp70::kitla): mean±SE = 0.7±0.2, range = 0–4, n = 45 larvae; untreated Tg(hsp70::kitla) siblings: mean±SE = 1.6±0.2, range = 0–5, n = 35].