Fig. 6

Loss of RA activity leads to the posterior expansion of the Fgf signaling domain. Duration of the inhibitor treatments and timing of fixations are indicated on the top-left for A-G, H-N and O-V, although the embryos shown in (O-T) and ones in (U, V) were fixed at different timing. Developmental stages for fixation for O-V are shown in the top-right corners. After drug treatment embryos were immediately fixed and subjected to in situ hybridization<. Drugs for treatment (0.5% DMSO or 10 µM DEAB) are shown on the left. Numbers in the bottom-left indicate numbers of embryos showing the phenotype. (A-F, H-M) Flat-mounted embryos at 14 hpf stained with hmx3+pax2a (A, B, H, I), stained with pea3 (C, D, J, K) and stained with lef1 (E, F, L, M). pax2a marks the otic vesicles in red. Anterior is to the left. (G, N) Quantifications of pea3 expression domains in (C, D, J, K) (e.g. black bar in C). DEAB treatment from the 80% epiboly stage causes significant (p<0.001) elongation of the pea3 domains (G), whilst treatment starting at 10 hpf does not (N). Numbers of pea3 expression domains measured are indicated below each bar. (O-R) The hindbrain area of flat-mounted embryos at 11 hpf stained with fgf3+eya1 (O, P) and fgf8a+eya1 (Q, R). eya1 marks the PPR in red in these panels. Anterior is to the left. (S-V) Lateral views of 11 hpf (S, T) and 14 hpf (U, V) embryos stained with hoxb1b and hmx3, respectively. Anterior is oriented to the top in S, T, whilst anterior is to the left in U, V. Blue arrowheads mark elongated part of pea3 expression domains. Black arrowheads indicates the anterior edge of the neural hoxb1b expression, and black arrows mark the position of the ear. hmx3 expression in the ear is posteriorly expanded as also observed in B. Colored bars indicate the positions of the aLLp, otic vesicle and pLLp. Abbreviations: aLLp: anterior lateral line placode, ov: otic vesicle, pLLp: posterior lateral line placode, r5: rhombomere 5. Scale bars: 100 µm.