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Fig. 3. Effects of Fgf misexpression. (A,B) Two-color in situ hybridization of three-somite stage. Wild-type (A) and fgf3 (B) plasmid-injected embryos showing pax8 expression (blue) and krox-20 expression (red). The left preotic domain is enlarged significantly and a region with ectopic upregulation of pax8 (arrowhead) is evident. (C,D) Two-color in situ hybridization of three-somite stage embryo injected with fgf3 plasmid showing fgf3 expression (blue) and pax8 expression (red). (C) Brightfield and (D) fluorescent images showing relationship between ectopic fgf3-expressing cells (arrow) and ectopic pax8-expressing cells (arrowhead). The boxed area in C is enlarged in D. The endogenous preotic domain on the left is enlarged in the vicinity of misexpressed fgf3. The r4 domain of fgf3 is faintly visible in C. (E,F) foxi1 expression at three-somite stage in wild-type (E) and fgf8 plasmid-injected embryo (F). Ectopic expression in anterior region of the injected embryo is indicated (arrowhead). (G) Ectopic pax2.1 expression (arrowhead) adjacent to the midbrain of a four-somite stage embryo injected with fgf3-plasmid. (H) Low magnification view of a 30 hpf wild-type embryo injected with fgf8-plasmid showing that overall axial development is essentially normal, although development of anterior sensory structures is perturbed. (I) Higher magnification of the embryo shown in H. Ectopic otic vesicles are indicated (arrowheads). Development of adjacent eye tissue is perturbed, but general features of brain development, such as the epiphysis and midbrain-hindbrain boundary are produced. (J) Frontal/lateral view of an embryo injected with fgf3-plasmid. An ectopic otic vesicle is indicated (arrowhead). Development of adjacent nasal and eye tissue is severely perturbed. (K) Lateral view of a wnt8 morphant injected with fgf8-plasmid. Ectopic otic vesicles (arrowheads) are seen next to the midbrain-hindbrain boundary. Abbreviations: e, epiphysis; mhb, midbrain-hindbrain border; op, endogenous otic placode; r3, rhombomere 3. (A-E,G) Dorsal view with anterior upward. (F) Dorsal-anterior view. (H,I,K) Lateral views with anterior to the left. (J) Frontal-lateral view with anterior to the left. Scale bars: A-C,E-G, 150 µm; D, 60 µm; H, 175 µm; I-K, 75 µm.





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