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Wnt signaling pathways play essential roles in patterning and proliferation of embryonic and adult tissues. In many organisms, this signaling pathway directs axis formation. Although the importance of intracellular components of the pathway, including beta-catenin and Tcf3, has been established, the mechanism of their activation is uncertain. In Xenopus, the initiating signal that localizes beta-catenin to dorsal nuclei has been suggested to be intracellular and Wnt independent. Here, we provide three lines of evidence that the pathway specifying the dorsal axis is activated extracellularly in Xenopus embryos. First, we identify Wnt11 as the initiating signal. Second, we show that activation requires the glycosyl transferase X.EXT1. Third, we find that the EGF-CFC protein, FRL1, is also essential and interacts with Wnt11 to activate canonical Wnt signaling.
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.
Figure 6. FRL1 Binds Wnt11 and Acts Upstream of β-Catenin
(A) The coinjection of Wnt11 and FRL1 oligo enhances ventralization compared to each oligo alone.
(B) Sibling embryos to those shown in (A) were frozen at the early gastrula stage and assayed for the relative expression of Wnt target genes.
(C) Coinjection of 150 pg FRL1 mRNA with 150 pg Wnt11 mRNA increases the dorsalized phenotype.
(D) Sibling embryos to those shown in (C) were frozen at the early gastrula stage and assayed for the relative expression of Wnt target genes.
(E) The ventralization of FRL1-depleted embryos is partially rescued by the injection of 40 pg b-catenin mRNA (F).
(G) Sibling embryos to those shown in (E) and (F) were frozen at the early gastrula stage and assayed for the relative expression of Wnt
target genes.
(H) One nanogram of each mRNA (FRL1-Flag, FRL1-Myc, and Wnt11-HA) was injected into two different animal cells at the four to eight cell
stage and embryos were harvested at st11 for coimmunoprecipitation. Wnt11 is immunoprecipitated by both FRL1-Flag and Myc. Arrow =
Wnt11; star = IgG.
(I) One nanogram of each mRNA (FRL1-Flag, Wnt5A-Myc, and Wnt8-Myc) was injected into two different animal cells at four to eight cell
stage embryos that were frozen at stage 11. FRL1 interacts with Wnt5A and not Wnt8. Green arrow = Wnt5A-myc; red arrow = Wnt8-Myc;
star = IgG.
(J) One nanogram of each mRNA (Wnt11-HA, Cripto-Flag, FRL1-Flag, and the mutant forms diagramed) was injected into two different animal
cells at four to eight cell stage embryos which were frozen at stage 11 for coimmunoprecipitation. FRL1, Cripto, and -CFC interact with
Wn11; -EGF has reduced binding, and -C-term does not. Arrowhead = wild-type FRL1 (which also has two smaller forms); arrow = Cripto.
References :
Jessen,
Axis formation--beta-catenin catches a Wnt.
2005, Pubmed,
Xenbase
Jessen,
Axis formation--beta-catenin catches a Wnt.
2005,
Pubmed
,
Xenbase