Fig. 3. Gene expression analyses. Dorsal views are shown with anterior towards the top. (A-C) her1 expression in control (A) and SU5402-treated (B) embryos. The embryos were treated with SU5402 for 8 minutes at the two-somite stage and fixed at the six-somite stage when the prospective large somites are being specified. For comparison, the left half of the control embryo shown in A and the right half of the treated embryo shown in B are shown as a composite (C). In treated embryos, the anterior her1 stripe is missing. Arrows indicate the level of the anterior her1 stripe in a control embryo. (D-F) her1 expression in fused somite (fss) mutants after treated with control (D) or SU5402 (E) medium. A composite (F), the left half of SU5402-treated wild-type embryo shown in B and the right half of SU5402-treated fss embryo in E, shows that the her1 stripe in the intermediate PSM is not maintained in fss–/– background after SU5402 treatment. The arrows in F indicate the level of the intermediate (anteriormost in this case) her1 stripe in the embryo shown in B. As fss embryos show no segmentation, the embryos were treated with SU5402 for 8 minutes when their siblings reached the two-somite stage, and they were fixed when their siblings reached the six-somite stage. (G,H) mesp-a expression at the six-somite stage in control (G) and SU5402-treated (H) embryos. The embryos were treated with SU5402 for 8 minutes at the two-somite stage, fixed at the six-somite stage and followed by in situ hybridization with mesp-a (blue) probe. To make clear the relationship between the mesp-a stripe and the tailbud (indicated by double-headed arrows), the embryos were also stained with the antibody against No tail (Ntl). The notochord and the tailbud is positive for the antibody (yellow). The anterior mesp-a expression domain, which normally demarcates the somite primordium at the position of –II (G) (Durbin et al., 2000; Sawada et al., 2000), posteriorly shifts to the intermediate PSM (H) after treatment of SU5402. (I) A composite picture of papc expression at the six-somite stage in control (left) and SU5402-treated (right) embryos. papc is expressed in four bilateral pairs of stripes in the paraxial mesoderm during the segmentation period (Yamamoto et al., 1998). The first stripe is located in the anterior border of the newest somite formed (I) and the second in the forming somite (0). The two posterior, stronger and broader stripes seem to be located in successive somite primordia (-I and -II). In treated embryos, the positions of the posterior two stripes are posteriorly displaced when compared with control embryos with a wider interval between them. (J-M) her1 expression in the PSM exposed to exogenous Fgfs. The embryos were transplanted with Fgf beads at the two-somite stage, and fixed at the two-somite (J), five-somite (K) and eight-somite (L,M) stages. Red arrowheads indicate the location of the beads in the PSM. The intermediate her1 stripe is anteriorly expanded around the Fgf bead with the posterior border unchanged (L); the anterior her1 stripe is located in more anterior region than on the control side (arrow in L). In fss embryo, the transplanted Fgf-bead exerts the same effect on the intermediate her1 stripe (M; compare with L). Note that the anterior her1 stripes are always missing in fss embryos on both sides of the PSM. (N-P) her1 expression in after eight (aei) mutant after treatment with control (N) or SU5402 (O) medium. A composite (P) shows that the anterior her1 expression shifts posteriorly to the intermediate PSM after treatment with SU5402. The arrows in P indicate the level of the anteriormost her1 expression in control and treated aei embryos. In aei mutants, the first eight to ten somites are normally formed and, thereafter, defects in boundary formation are evident. Thus, the embryos were treated with SU5402 at the four-somite stage, and fixed at the eight-somite stage (later stages than other experiments). The genotyping of the embryos was carried out using PCR (Holley et al., 2000). Scale bar: 100 µm.
