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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 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).
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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 7. Triple Knockdown of ONT1, ADMP, and BMP2 Results in a Dramatic Shift toward Dorsalization(A and B) BMP2 expression (dorsal view) was strongly expanded at stage 11.5 in an embryo coinjected with ONT1-MO (12.5 ng) and ADMP-MO (3 ng).(C–H) Triple knockdown of ONT1, ADMP, and BMP2 caused a dramatic dorsalization of the embryo. Embryos were given microinjections of ADMP-MO (3 ng), BMP2-MO (8 ng), and ONT1-MO (8 ng). Whole-mount in situ hybridization of Gsc ([C, E, and G]; anterior view) and Shh ([D, F, and H]; dorsal view) expression at stage 13.
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olfml3 (olfactomedin-like 3) gene expression in bisected Xenopus laevis/ tropicalis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 10.5, dorsal right, anterior up.
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olfml3 (olfactomedin-like 3) gene expression in bisected Xenopus laevis/ tropicalis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 12.5, dorsal right, anterior up.
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olfml3 (olfactomedin-like 3) gene expression in bisected Xenopus laevis/ tropicalis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 18, dorsal right, anterior up.
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A triple in situ hybridization in Xenopus embryo, NF stage 18/19, showing marker gene expression: rax (Rx) identifies optic field and eye primordium; en2 (Krox20) marks midbrain-hindbrain boundary, and myod1 (MyoD) marks presomitic mesoderm.
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