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Figure S4. Amer2 affects neuroectodermal patterning. A, The xAmer2-MO sequence specifically targets xAmer2. Embryos were injected in both blastomeres at the 2-cell-stage with 0.8 pmol of xAmer2-MO or control-MO along with either 100 pg RNA of the Amer2 pCS-MOsite-EGFP construct or pCS-EGFP alone. The Amer2-MOsite construct contains the xAmer2 sequence targeted by the Amer2-MO fused to EGFP. Injected embryos and uninjected controls were analyzed by western blotting at NF stage 12.5 as indicated. B, Depletion of xAmer2 by Amer2-MO injection affects anterior-posterior patterning of the neuroectoderm as shown by expression of En-2 and krox-20. Embryos were injected in one anterior-dorsal blastomere at the 8-cell-stage with 100pg LacZ DNA plus 0.2 or 0.4 pmol of Amer2-MO or control-MO. For rescue experiments 500 pg of LefδBD RNA were co-injected as indicated below the panels. Embryos were stained for LacZ to label the injected side, probed by in situ hybridization for expression of En-2 and krox-20 at NF stage 20 and scored for reduced or expanded expression of the markers on the injected side as compared to the uninjected side of the same embryo. Images on the top show embryos representative for observed phenotypes, injected sides are oriented to the right in all images. Images on the top show embryos representative for observed phenotypes. The colored insets represent phenotypes as following: blue=enhanced/posteriorly shifted markers at injected side, green=equal/normal expression of markers, yellow=markers reduced/anteriorly shifted at injected side, orange=markers lost at injected side, red=markers lost at injected and uninjected side of the embryo. The graph shows the statistics of at least three independent experiments. Numbers below the graph represent analyzed embryos. C, Depletion of xAmer2 by Amer2-MO injection does not affect neural induction as revealed by Sox2. Whole mount in situ hybridization with the pan-neural marker Sox2 at NF stage 20 as indicated. Embryos were injected in both dorsal blastomeres at the 4-cell-stage with 0.8 pmol of xAmer2-MO or control-MO and 100 pg LacZ DNA. |
