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Figure 1. Loss of Function of the Dorsally Expressed Protein ONT1 Causes Dorsalization of the Embryo(A) Structure comparison of ONT1 with other Olfactomedin-class proteins. SP, signal peptide; CC, coiled-coil domain; OLF, Olfactomedin domain.(B–D) Whole-mount in situ hybridization of ONT1 expression at the early gastrula ([B]; sagittal section), midgastrula ([C]; sagittal section), and neurula ([D]; cross-section) stages. Brackets (B and C) show ONT1 expression in the dorsal axial tissues. The arrowhead in (B) indicates the dorsal lip. so, somite; not, notochord.(E and F) Radial injection of ONT1-MO (F) at the four-cell stage.(G–N) Effects of ONT1-MO injection at the four-cell stage on DV markers analyzed in the gastrula. ONT1-MO (25 ng) caused expansion of the dorsal axial markers Gsc ([G and K]; anterior view; stage 13), Chordin ([H and L]; dorsal view; stage 13), and Shh ([I and M]; dorsal view; stage 13), whereas expression of the ventral marker Szl was reduced ([J and N]; vegetal view; stage 12).(O–R) Dorsalization induced by ONT1-MO (25 ng) injection at the four-cell stage was rescued by coinjecting a small amount (4 pg) of MO-resistant ONT1 RNA (ONTa(mut)). The expansion of dorsal markers Gsc (52%, n = 21), Chordin (54%, n = 13), and Shh (79%, n = 14) was reversed by ONTa(mut) (O–Q).(R) The reduction of Szl (70%, n = 20) was rescued by ONTa(mut).(S and T) ONT1-MO was injected specifically into two dorsal (S) or ventral (T) blastomeres at the four-cell stage. Gsc expression is shown in anterior view. Insets show the expression of coinjected GFP in dorsal (S) and ventral (T) blastomere progenies in the corresponding embryos. |
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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). |
