Tao Q , Yokota C , Puck H , Kofron M , Birsoy B , Yan D , Asashima M , Wylie CC , Lin X , Heasman J .

Paper published

	Figure 1. Wnt11 Overexpression Causes Dorsalization by a β-Catenin-Dependent Pathway (A) Upper panel: Tailbud control embryos and siblings injected with 150 pg of Wnt11 mRNA vegetally at the times shown. Pre-Mat = prematuration; Post-Fert = postfertilization); middle panel: b-catenin− and b-catenin− + Wnt11 mRNA, siblings of those shown in the upper panel, injected as oocytes; lower panel: 150 pg Wnt11 mRNA + 150 pg Xfz7 mRNA siblings of those in the top panel. (B) Show Wnt target gene expression in embryos from upper, middle, and lower panels respectively from (A) frozen for analysis at the early gastrula stage and assayed by real-time RT-PCR. (E and F) Sibling control, Wnt11 (250 pg) and Wnt8 (25 pg) mRNA injected oocytes at the neurula (E) and late tailbud stages (F). (G) Relative expression of Wnt target genes in siblings of those shown in (E) and (F) at the late blastula (stage 9.5) and early gastrula (stage 10) stages.
	Figure 2. Wnt11 Is Required for Dorsal Axis Formation (A) Embryos derived from Wnt11 oligo 3H-injected (3H) and control uninjected (C) oocytes were frozen as matured oocytes, and at the early blastula (stage 7), late blastula (stage 9), early, mid, and late gastrula (stages 10, 11, and 12) stages and assayed by real-time RT-PCR for the relative expression of Wnt11mRNA. (B) Ventralization of early tailbud embryos from oligo-injected oocytes compared to an untreated control embryo. Arrow points to closed blastopore. (C and D) The relative expression levels of Wnt and VegT target genes in control and Wnt11-depleted embryos assayed by real-time RT-PCR. (E) Ventralization of Wnt11-depleted embryos is partially rescued by the reintroduction of Wnt11 mRNA (F) The coinjection of Wnt11 MO, 50 ng, and phosphodiester 3H oligo, 5 ng, enhances ventralization compared to the effects of either oligo alone. (G) Expression of NCAM, MyoD, Otx2, and Xhex in Wnt11-depleted tailbud stage embryos is partially rescued by the reintroduction of Wnt11 mRNA. (H) Expression of Wnt target genes at the early gastrula stages of sibling embryos to those shown in (F).
	Figure 3. Maternal Wnt11 Acts Upstream of β-Catenin in Axis Formation (A) Embryos derived from control (top row) and Wnt11-depleted oocytes (L = low dose, 4 ng 3H oligo; H = high dose, 6 ng 3H oligo) at the neurula stage. (B) Sibling Wnt11-depleted embryos to those in (A) injected with 50 pg of b-catenin mRNA at the four cell stage. In control embryos, 50 pg b-catenin mRNA injected ventrally causes axis duplication (bottom two embryos in [B]). (C) Sibling embryos to those shown in (A) and (B) were frozen at the early gastrula stage and assayed by real-time RT-PCR for the relative expression of Wnt target genes. (D) TOPflash reporter activation after injection into two dorsal cells of four cell stage control embryos compared to activation occurring after similar injections into Wnt11-depleted embryos (5 ng 4T oligo). Error bars indicate the standard deviation from the mean. (E) A vegetal view of one of the 86 cell stage embryos used in (F). Arrow points to the concentration of pigment that marks the yolk-free area of germ plasm. V = ventral side; dotted line denotes cut. (F) Wild-type embryos at the 86 cell stage were dissected into dorsal and ventral halves (E) and prepared for RT-PCR. In two samples, each representing four embryo halves, the levels of both polyadenylated and total Wnt11 RNA (and not disheveled mRNA) is higher dorsally than ventrally. D = dorsal halves; V = ventral halves.
	Figure 4. X.EXT1 Is Required for Dorsal Axis Formation (A) Relative expression of X.EXT1 mRNA in embryos derived from X.EXT1-depleted and control oocytes frozen at the stages shown. C = control; 3T = antisense oligo-injected. (B) The relative expression level of Wnt target genes in control versus X.EXT1-depleted embryos at the early, mid, and late gastrula stages. (C) The appearance of early tailbud embryos derived from control (top) and X.EXT1-depleted oocytes (3.5 ng 3T oligo). (D) Ventralization of X.EXT1-depleted embryos is partially rescued by the reintroduction of X.EXT1 mRNA. (E) The expression of Wnt target genes is partially rescued by the reintroduction of X.EXT1 mRNA. (F) The expression of Wnt target genes is partially rescued by the reintroduction of b-catenin mRNA in control versus X.EXT1 embryos, while BMP and VegT targets Xvent1 and Xsox17 are not affected. (G and H) The ventralized appearance is partially rescued by the reintroduction of b-catenin mRNA in control versus X.EXT1 embryos. (I) Embryos derived from control, low dose (50 ng MO) Wnt11-depleted, low dose (2.5 ng oligo 3T) X.EXT1-depleted, and Wnt11 + X.EXT1- depleted oocytes. (J) The relative expression level of Wnt target genes in sibling embryos to those shown in (I) frozen at the late blastula stage.
	Figure 5. FRL1 Is Required for Dorsal Axis Formation (A) Embryos derived from FRL1-depleted and control oocytes frozen as matured oocytes and as embryos at the stages shown and assayed by real-time RT-PCR for the relative expression of FRL1 mRNA. C = control; L = 1.5 ng mp9; H = 2 ng mp9. (B) Early tailbud embryos derived from control and FRL1-depleted oocytes (1, 1.5, 2 ng oligo injected). (C) Sibling embryos to those shown in (B) were frozen at the early gastrula stage and assayed by real-time RT-PCR for the relative expression of Wnt target genes. (D) The ventralization of FRL1-depleted embryos is partially rescued by the injection of 50 pg FRL1 mRNA. (E and F) The relative expression level of Wnt and VegT target genes in control, FRL1-depleted, and FRL1-depleted + 50 pg FRL1 mRNA injected sibling embryos (to those shown in [D]) at the stages shown.