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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. |
