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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. |
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Figure 5. Excessive Doses of ONT1 Cause Dorsalization in the Embryo(A and B) The dorsalized phenotype was observed following the injection of ONT1 mRNA (200 pg; [B]).(C–F) Microinjection of ONT1 mRNA (200 pg) increased the dorsal markers Gsc ([C and D]; anterior view) and Chordin (not shown) and suppressed the ventral marker Szl ([E and F]; vegetal view).(G) Embryos were classified by morphological inspection of (H)–(L).(H–L) Stage 23 embryos received injections of BMP1 mRNA (200 pg) or ONT1 mRNA (400 pg) into the two dorsal blastomeres at the four-cell stage. Overexpression of ONT1 reversed the ventralization induced by BMP1 (I and J) but not a constitutively active form of BMPR (CA/BMPR; 6.25 pg; [K and L]).(M–O) The dorsalizing effect of ONT1 overexpression requires Chordin. In situ hybridization for the indicated markers in a control embryo (M) or in embryos given an injection of Chordin-MO (4 ng each; N) or ONT1 mRNA (200 pg) + Chordin-MO (4 ng each) (O). |
