FIGURE 3.

Knockdown of ets1 does not obviously affect NC formation but blocks NC migration. A–J, the expression of foxd3 or snail2 was not inhibited by ets1MOs. ets1MO1 or ets1MO2 was injected either separately into both blastomeres (A–D, 60 ng of ets1MO1/embryo; E–H, 60 ng of ets1MO2/embryo) or together with 100 pg of lacZ mRNA into one blastomere (I and J, 30 ng/embryo) at the two-cell stage. The injected embryos were collected at stage 17 and examined for foxd3 (B, 90%, 18 of 20; F, 96%, 24 of 25; I, 95%, 21 of 22) and snail2 (D, 95%, 22 of 23; H, 92%, 23 of 25; J, 90%, 18 of 20) by whole-mount in situ hybridization. The injected side was traced by red X-Gal staining and is marked with an asterisk (I and J). K, overexpression and knockdown of ets1 repressed NC formation in an animal cap assay. Expression of the indicated genes in animal caps injected with either chordin (Chd) + wnt3a, chordin + wnt3a + ets1, or chordin + wnt3a + ets1MO1 was examined by RT-PCR. L–Q, spatial expression pattern of ets2 as detected by in situ hybridization. st, stage. L, P, and Q, lateral view; M and N, vegetal view; O, dorsal view. The black arrow in N indicates weak expression of ets2. R and S, ets1MO1 (30 ng) and lacZ mRNA (100 pg) were co-injected into one dorsal blastomere of four-cell stage embryos, and the embryos were collected at stage 20. Segmentation and extension of cranial NC were blocked at the ets1MO1-injected side (R, 85%, 17 of 20; S, 83%, 20 of 24). T–W, knockdown of ets1 decreased the expression of twist1 in both neurula (T and U) and tail bud embryos (V and W). T, control embryo at stage 20. U, embryos injected with ets1MO1 at one side. V and W, embryos at stage 25 injected with ets1MO1 at one side. At the injected side (U, 85%, 11 of 13; W, 90%, 9 of 10), the twist1 signal stripes were weaker and did not extend laterally as far as those at the uninjected side (T and V). An asterisk indicates the injected side. U′ and U″, transverse sections of embryos shown in U (5 of 5 embryos). ets1MO1 and lacZ mRNA were co-injected at one side of the embryos. In the injected side, NC cells marked by twist1 staining were concentrated laterally to the neural tube and seemed not to detach from the neural plate. In the uninjected side, NC cells extended out of the neural plate, and signal spots were scattered underneath the mesoderm region, suggesting that NC cells migrated into the arches. An asterisk indicates the injected side. Scale bars in U′ and U″ indicate 100 μm. X and Y, Ets1MO2 (30 ng/embryo) was co-injected with lacZ mRNA (100 pg; used as a lineage tracer) into one blastomere of two-cell stage embryos, and embryos were stained for the expression of twist1 (81%, 17 of 21) and snail2 (79%, 11 of 14). The extension of cranial NC was decreased in the ets1MO2-injected side. WE, uninjected whole embryo; AC, uninjected animal caps; RT−, without reverse transcriptase; con, control.