ret expression becomes restricted to cells associated with opercular muscles. (A-B) ret is expressed in the first (1) and second (2) arches at 25 hours post-fertilisation (hpf; A, white arrowheads); transverse section through the arch (A′) reveals that ret+ cells (black arrowhead) lie medially. ret becomes restricted to medial cells of the arches at 30 hpf (B) relative to GFP+ cells in sox10:GFP animals (C). (C-D) Confocal sections reveal that ret+ cells are surrounded by GFP+ cells (C′) and transverse sections fail to show colocalisation of ret and GFP (C′′). At similar stages, ret expression is detected in myoblasts of the first arch that show En2 immunoreactivity (D). (E) ret expression in the arches becomes restricted to discrete populations of cells, including some associated with the developing opercular muscles (arrowheads). (F,F′) ret and myod do not appear to be expressed in the same cells of the opercular muscles (F) and transverse sections fail to show colocalisation (F′). (G-G′′) ret and gfra3 are both expressed in opercular muscles at 55 hpf (G) and transverse sections (G′) and ventral views (G′′) show that they colocalise in the ao and lo, but not in the ah. (H) Differentiating muscle fibres (brown) in alpha-actin:GFP fish do not express ret (blue) at 55 hpf, but ret+ cells lie adjacent to fibres (arrowheads). (I,J) Lateral view of the arches in 72 hpf embryos following in situ hybridisation and immunolabelling (I) shows that ret expressing cells (blue) are associated with the forming do, ao and lo muscles (brown). Transverse sections reveal that ret is expressed in cells of the do but not the lap (J) at this stage. Lines in C, F and G indicate the level of section in C′′, F′ and G′, respectively. yz and xz planes are shown for C′, C′′, D and F′. ah, adductor hyoideous; ao, adductor operculi; do, dilator operculi; e, eye; lap, levator arcus palatini; lo, levator operculi. Pharyngeal arches 1-7 are shown by numbering in A-C. Scale bars: 100 μm in A-C,E,G,J; 20 μm in A′,C′,C′′,F,F′,G′-I; 10 μm in D.