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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 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). |
