(a) SEM of a stage 32 embryo. Placodes (outlined) are visible surface bulges. (b) Schematic summarizing histological serial reconstructions at stage 32 of the three pre-otic (anterodorsal [yellow], anteroventral [turquoise], otic [teal]) and three post-otic lateral line placodes (middle, supratemporal, posterior [green]). (c, d) Transverse sections through the anterodorsal placode at the level of the eye (c) and otic vesicle (d). Blue arrowheads indicate extent of thickened epithelium (i.e., the placode). Red arrowhead indicates a developing neuromast primordium. (e-i) Whole-mount in situ hybridisations for Eya1, Eya4, Six1 and Six4 from stages 32-46; schematics show the general expression pattern at those stages. Lateral views; anterior is left. (e) At stage 32, Eya1, Six1 and Six4 are expressed in multiple placodes including olfactory and otic (purple), epibranchial (pink), and lateral line (dark purple); strong expression of Eya4 is restricted to lateral line placodes and the otic vesicle. By this stage, the lateral line placodes have started to elongate or spread along the head. (f) At stage 36, all genes are expressed in developing neuromast canal lines and presumptive ampullary organ fields flanking those lines. (g) At stage 39, expression of all genes continues in elongating lateral line primordia and neuromast canals. Individual ampullary organ fields start to be identifiable. At (h) stage 42 and (i) stage 46, expression of all genes continues in developing lateral line organs as well as in the migrating posterior lateral line primordium. (j) Transverse sections showing Eya4 expression in the developing anterior ventral placode (avp) from stages 32-46. By stage 46, the hair cell marker Pv3 (grey-scale image) overlaps with Eya4 as shown in the false-colour overlay of Eya4 (green) and Pv3 (pink). Scale bars: 100 μm (a, c, d, j), 200 μm (e-i). Abbreviations: adp, anterodorsal placode; avp, anteroventral placode; e, eye; epi, epibranchial placode region; hb, hindbrain; mb, midbrain; mlp, middle lateral line placode; olf, olfactory; otp, otic lateral line placode; ov, otic vesicle; plp, posterior lateral line placode; S, stage; stp, supratemporal placode.