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Figure 6. Double Knockdown of ONT1 and ADMP Induces a Severe Dorsalization of the Embryo(A) Quantitative RT-PCR analysis. A mixture of BMP2-MO (8 ng), BMP4-MO (4 ng), BMP7-MO (4 ng), and ADMP-MO (8 ng) (BMPs-MO; Reversade and De Robertis, 2005) or BMP4 mRNA (12.5 pg) was injected radially at the four-cell stage, and embryos were harvested at stage 13. The level of the control was defined as 1. ODC, ornithine decarboxylase.(B–E) The expression of Gsc (anterior view) was strongly expanded at stage 13 in the ONT1-MO (12.5 ng) and ADMP-MO (3 ng) coinjected embryo.(F) Quantitative RT-PCR analysis of Gsc expression levels under the same conditions as in (B)–(E). ONT1-MO and ADMP-MO synergistically upregulated Gsc expression. The level of the control was defined as 1.(G–J) ONT1-MO (12.5 ng) and ADMP-MO (3 ng) coinjection caused a strong expansion of the anterior neural marker Rx (G and H) and dorsal axial marker Shh (I and J) at stage 13. Dorsal views.(K and L) Coinjection of Chordin-MO reversed the hyperdorsalization induced by the double knockdown of ONT1 and ADMP. The strong expansion of Gsc (anterior view) or Rx (dorsal view) induced by the double knockdown of ONT1-MO (12.5 ng) and ADMP-MO (3 ng) was reversed by Chordin-MO (4 ng each) at stage 13.(M and N) Double knockdown of B1TP (BMP1 and Xlr) and ADMP caused a strong expansion of Gsc expression (anterior view) at stage 13. Embryos were given an injection of BMP1-MO (6 ng), Xlr-MO (6 ng), and ADMP-MO (3 ng). |
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Figure 3. ONT1 Is Required for Robust Resistance to Increased Chordin in DV Patterning of the Early Xenopus Embryo(A) Recombinant Chordin protein (44 fmol/blastocoele) was injected into the blastocoele at stage 9. Effects on DV patterning were examined at stage 13.(B–I) Whole-mount in situ hybridization with the Gsc probe ([B–E]; anterior-dorsal oblique view) and the mixed probes ([F–I]; Rx, Krox20, and MyoD; anterior-dorsal oblique view). Embryos were given an injection of Chordin protein ([D, E, H, and I]; Chd-prot) or control BSA (B, C, F, and G) as shown in (A).(C, E, G, and I) Embryos were injected with ONT-MO (25 ng) at the four-cell stage. |
