Fig. 4. XMeis3 antisense morpholino oligonucleotide (AMO) inhibits posterior neural marker expression by blocking translation of XMeis3 RNA. (A) One-cell stage embryos were injected in the animal hemisphere with 15 ng of AMO (lanes 4-5 and 8-9), 15 ng of control morpholino oligonucleotide (CMO; lanes 2-3 and 6-7) and 1.0 ng of XMeis3 RNA (lanes 3, 5, 7 and 9). Eighteen animal cap explants were removed from all injected groups (lanes 6-9) of blastula embryos (stage 8-9). Explants from each group were grown to stage 20 and total RNA was isolated. In parallel, total RNA was also isolated from pools of seven embryos from each injected group (lanes 1-5). RT-PCR analysis was performed with the markers: Krox20, HoxB3 and HoxB9. EF1 served as a control for quantifying RNA levels in the different samples. For controls, RT-PCR (lane 2) and -RT-PCR (lane 1) were performed on total RNA isolated from normal CMO-injected embryos (lane 2). (B) Western analysis of XMeis3-Myc protein. One-cell stage embryos were injected in the animal hemisphere with 1.6 ng of RNA encoding the XMeis3-Myc fusion protein (lanes 2-3) and 16 ng of AMO (lane 3) or 16 ng of CMO (lane 1-2). Protein was isolated from pools of seven embryos per group at stage 12.5: control (lane 1), XMeis3-Myc/CMO (lane 2), XMeis3-Myc/AMO (lane 3). As a positive control, in vitro synthesized XMeis3-Myc protein was also examined on the filter (lane 4). Analysis was performed using the 9E10 Myc antibody. As a positive control, total Erk protein was detected with the p44/p42 antibody. (C) Embryos from the above experiment (Fig. 4B) were grown to late neurula stages. Total RNA was also isolated from pools of seven embryos from the control (lanes 1-2) and injected groups (lanes 3-4). RT-PCR analysis was performed with the markers XMeis3 and Krox20. EF1 served as a control for quantifying RNA levels in the different samples. For controls, RT-PCR (lane 2) and -RT-PCR (lane 1) were performed on total RNA isolated from uninjected control embryos (lane 2).