Fig 5

(A-F) pnp4a expression in Ib(sp) requires Tfec. At 24 hpf, the number of dorsally located, and of partially fate restricted, migrating chromatoblasts (arrows) expressing pnp4a is reduced in tfec mutants (A,B). Expression overlying the eye is also affected (A,B, insets). This reduction is still prominent at 30 hpf (C,D), when staining is also visibly affected in the lateral patches. pnp4a expression is undetectable in differentiated iridophore positions (E, asterisks) in tfec mutants at 48 hpf (F). pnp4a+ iridophore lineage cells overlying the RPE are noticeably reduced in tfec mutants compared to WT siblings (A-F, insets). (G-L) Generation of tfec-positive Ib(sp) from late Cbls requires Tfec. At 24 hpf, tfec mutants present with anterior expansion of the tfec+ premigratory NC domain (G,H, arrowheads) and lack of medially migrating, Ib(sp) progenitors (arrows). At 36 hpf, the number of dorsal and migrating tfec+ Ib(df) (arrows), as well as those located in the lateral patches is reduced in mutants, compared to WT or heterozygous siblings (I,J). At 48 hpf, a reduced number of cells (asterisks) express tfec along the dorsal stripe of mutant embryos, compared to WT or heterozygous siblings (K,L). tfec mutant embryos were distinguishable after whole-mount in situ hybridisation by the lack of RPE melanisation (I-L, insets). tfec+ iridophore lineage cells overlying the RPE are noticeably reduced in tfec mutants compared to WT siblings (G-L, insets). (M-R) Mb(sp) generation from the late Cbl is delayed in tfec mutants. At 24 hpf, mitfa marker expression is restricted to an increased number of premigratory Cbls (arrowheads) and is undetectable in migrating Mb(sp) (arrows) in tfec mutants (M,N). At 30 hpf, the numbers and distributions of mitfa-positive late Cbls (arrowheads) are indistinguishable between tfec mutants and WT or heterozygous siblings (O,P), but the delay in Mb(sp) migration (arrows) remains distinguishable towards the tail. tfec mutant embryos lack RPE melanisation (O,P, insets). At 48 hpf, mitfa expression in mature melanocytes (asterisks) is indistinguishable between tfec mutants and WT or heterozygous siblings (Q,R). (S, T) tfec mutants lack ltk expression in premigratory late Cbls (arrowhead), in migrating Ib(sp) (arrow) and in iridoblasts of the eye (insets). (U-X) Xanthoblast (Xbl(sp)) specification from Cbls is delayed, as indicated by examinations of two lineage markers, gch2 (U,V) and aox5 (W,X). At 24 hpf, laterally migrating Xbl(sp) (arrows) are restricted to more anterior regions and precursors located in the head are reduced in tfec mutants (V,X) compared to their WT siblings (U,W). All tfec mutant panels show the tfec^ba6 allele except (V,X) which show the tfec^vc60 allele. LP, lateral patches; m, melanisation. (A-J,M-X): lateral views. (K-L, insets of Q-R): dorsal views. Head towards the left. Scale bars: 100 μm.