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Fig. 2. Eng-XMeis3 RNA encodes an antimorph protein; VP16-XMeis3 RNA encodes a caudalizing protein. (A) One-cell stage embryos were injected in the animal hemisphere with 1.0 ng of XMeis3 RNA (lane 4), 1.6 ng of Eng-XMeis3 RNA (XMeis3-AM; lane 5) or both (lane 6). Eighteen animal cap explants were removed from uninjected (lane 3) and injected groups (lanes 4-6) of blastula embryos (stage 8-9). Explants from each group were grown to stage 20 and total RNA was isolated. RT-PCR analysis was performed with the markers: Krox20, HoxD1 and HoxB9. EF1{alpha} 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 embryos. (B) One-cell stage embryos were injected in the animal hemisphere with 1.6 ng of VP16-XMeis3 RNA (lane 4). Eighteen animal cap explants were removed from uninjected (lane 3) and injected groups (lane 4) of blastula embryos (stage 8-9). Explants from each group were grown to stage 20 and total RNA was isolated. RT-PCR analysis was performed with the markers: Krox20, HoxB3, HoxD1 and RAR{alpha}2.2. EF1{alpha} 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 embryos. (C) Embryos at the one-cell stage were injected with 1.3 ng of in vitro transcribed VP16-XMeis3. The upper embryo serves as an uninjected control. Injected embryos had anterior truncations and highly reduced cement gland formation (lower panel). The dorsal anterior index (DA) (Kao and Elinson, 1988) was 2.5 (n=39); over 20% of the embryos completely lacked cement glands and another 80% had extreme posterior truncations, with partial cement gland formation (lower panel). Embryos are oriented posterior to anterior: left to right. Embryos were fixed for photography at stages 35/36. (D) Embryos at the one-cell stage were injected with 1.0 ng of XMeis3-AM antimorph RNA. The top embryo serves as an uninjected control. In this representative experiment, over 80% of the XMeis3-AM injected displayed phenotypes. Embryos are oriented posterior to anterior: left to right. Embryos were fixed for photography at stages 30/31.