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Fig. 2. Localized expression of Wnts induces a polar AP neural pattern at long range in animal caps. (A) Experimental design: at the eight-cell stage, albino embryos were injected with 0.25 ng/blastomere noggin mRNA and pigmented embryos were injected with Xwnt3A mRNA (B) or pCSKA-Xwnt8 (C) into the four animal blastomeres. Animal caps were explanted at late blastula stage and sandwiched to yield pigmented/unpigmented conjugates. The conjugates were cultured until control siblings reached stage 15 and marker gene expression was analysed by in situ hybridization. Between 12-40 conjugates were analysed in B,C, of which representative samples are shown with albino halves of the conjugates always pointing to the top. (B) Animal cap conjugates from uninjected or Noggin-injected albino embryos and pigmented embryos injected with nil, 0.1 (+) or 1 ng/blastomere (++) Xwnt3A mRNA were analysed for the expression of Bf1, En2 and Krox20 as indicated. Note that in d′′ an organized AP pattern is generated with the more posterior Krox20 closer to the pigmented cap than En2. (C) Animal cap conjugates from uninjected or Noggin-injected albino embryos and pigmented embryos which were uninjected or injected with 0.05 ng/blastomere pCSKA-Xwnt8 were analysed for the expression of Bf1 and En2 by in situ hybridization.
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Fig. 3. Wnt signalling regulates AP patterning in gastrula ectoderm. (A) Albino embryos were uninjected (co) or injected at the eight-cell stage into the four animal blastomeres with 0.05 ng/blastomere pCSKA-Xwnt8, 0.05 ng/blastomere (+) or 0.2 ng/blastomere (++) Xdkk1 mRNA. The embryos were cultured until late gastrula stage (stage 12) and analysed by in situ hybridization for the expression of HoxD1, Gbx2, Otx2 and Xanf1. Lateral views are shown, dorsal points to the right. 12-18 embryos were analysed per sample in three independent experiments. (B) Summary of the results shown in A. Colour code of gene expression domains is on the left. The approximate orientation of the prospective ectodermal AP axis is indicated on the left (P→A). WT, wild-type embryo with normal Wnt levels.
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Fig. 4. Regulation of AP neural patterning by Wnt signalling in neurulae. (A) Albino embryos were uninjected or injected at the eight-cell stage into the four animal blastomeres with 0.05 ng/blastomere pCSKA-Xwnt8, 0.05 ng/blastomere or 0.2 ng/blastomere Xdkk1 mRNA, 0.1 ng/blastomere or 0.4 ng/blastomere NXfz8 mRNA, 0.8 ng/blastomere or 1.6 ng/blastomere Xfrzb1 mRNA. Embryos were cultured until neural plate stage (stage 15) and analysed by triple in situ hybridization for the expression of Krox20, Bf1 (black) and Otx2 (red). Frontal views are shown, dorsal points towards the top. In a, the expression domains of Bf1, Otx2 and Krox20 (rhombomeres 3 and 5) are indicated. Note residual Krox20 expression for highest doses of XFrzb1 (arrowheads in e′) but not XDkk1 (c′) or NXFz8 (d′). 25-35 embryos were analysed per sample in three independent experiments. (B) Summary of the results shown in A. Colour code of gene expression is on the left. WT, wild-type embryo with normal Wnt levels.
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Fig. 5. Wnt signalling is necessary and sufficient to pattern neuroectoderm in a long-range fashion. (A) Experimental design: pigmented 8-cell stage embryos (donors) were injected into the four animal blastomeres with mRNAs encoding Wnt pathway activators or Wnt antagonists. At late blastula stage, small groups of pigmented animal cap cells were grafted into the presumptive neural plate of early gastrula albino embryos (hosts). The hosts were cultured until neurula stages and analysed for marker gene expression by in situ hybridization. (B) Grafted donor caps were uninjected (a) or injected with 0.25 ng/blastomere Xwnt3A mRNA (b) and placed in the presumptive anterior host neural plate. Embryos were analysed at neural plate stage for expression of Krox20 (black) and En2 (red) and are shown in lateral view, dorsal side upwards. Grafts are indicated by asterisks. Note ectopic induction of En2 and Krox20 with reversed AP polarity by the Xwnt3A expressing graft (arrowheads in b). (C) Grafted donors were injected with 0.25 ng/blastomereXdkk1 mRNA and placed in the presumptive posterior (a) or anterior (b) host neural plate, respectively. Embryos were analysed at neural plate stage for expression of Bf1 (a) and Krox20 (b) and are shown in dorsofrontal view. Grafted tissue is outlined in red in panel a. Note posterior expansion of Bf1 expression in a and downregulation of Krox20 (arrowheads in b). (D) Grafted donors were uninjected (a,a′) or injected with 1.5 ng/blastomereXgsk3β mRNA (b,b′) and placed in the presumptive posterior host neural plate. Embryos were analysed at neural plate stage for expression of HoxD1 and are shown in dorsal view, anterior down. Grafted tissue is outlined in red in panels a,b. The same embryos as in panels a,b are shown following bleaching in panels a′,b′. Note holes in HoxD1 expression in panel b′ compared to continuous expression in panel a′.
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Fig. 6. Long-range patterning of neuroectoderm by XWnt3A is direct. (A) Titration of Xwnt3A and Xβcat* mRNAs. For every mRNA concentration (in pg/nl), 15 embryos were injected at four- to eight-cell stages vegetally into two opposite blastomeres (2.5 nl/blastomere), cultured until tailbud stage and scored for the induction of secondary body axes. Comparable results were obtained in four independent experiments. (B) RT-PCR analysis of expression of the indicated marker genes at (a) early gastrula stage (stage 10+) and (b) neural plate stage (stage 15) in whole embryos (we) and animal caps cut from embryos injected at the eight-cell stage into the four animal blastomeres with no (co), 0.25 ng/blastomere Xwnt3A or 2.5 ng/blastomere Xβcat* mRNAs. H4, histone4 for normalization; –RT, negative control without reverse transcriptase. (C) Pigmented donors uninjected (co) or injected with 0.25 ng/blastomere Xwnt3A or 2.5 ng/ blastomere Xβcat* mRNA were grafted as depicted in Fig. 5A into the presumptive host anterior neural plate. Embryos were analysed at neural plate stage (stage 15) for expression of Bf1, En2 and Krox20. Frontal views are shown (dorsofrontal in b′′,c′′). Between 21 and 63 embryos were analysed for every type of transplant in six independent experiments. Note that Xβcat*-expressing grafts do not induce changes in host marker gene expression unlike Xwnt3A (arrows), even at 5 ng/blastomere (not shown).
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Fig. 8. An AP gradient of nuclear β-catenin in the presumptive neural plate during gastrulation. (A) Mid-gastrula embryos were cut sagittally and left and right halves were analysed by in situ hybridization for Gbx2 expression and by immunostaining for β-catenin, respectively. Eight consecutive regions of the presumptive neuroectoderm of immunostained halves were selected from posterior to anterior positions, according to the gastrula fate map. Corresponding windows are shown in B for one representative embryo. Note prominent nuclear staining in windows 1 and 2, and absence of nuclear staining in 7 and 8. The AP axis is indicated (P→A) and the approximate position of the Gbx2 expression domain is given in relation to the windows. Data in C represent mean normalized densities of the nuclear regions of all cells within a window.
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Fig. 9. Expression of various Wnts and Wnt antagonists establishes a Wnt activity gradient in the gastrulating Xenopus embryo. (A) Expression of Xanf1, Xwnt8 (a) in whole-mount and Xwnt3A (b) in sagittal section at late gastrula stage by in situ hybridization. Black arrowheads, dorsal blastopore lip; red arrowheads, leading edge. Note expression of Xwnt8 in proximity to presumptive posterior neuroectoderm (compare with fate map in Fig. 8A). Xwnt3A is expressed in chordamesoderm and in a ring surrounding the blastopore (not visible). (B) Expression domains in the late Xenopus gastrula of Wnts (a) and Wnt antagonists (b). Only ectodermal expressions are shown in a. The circumblastoporal expression of Xwnt3A is not depicted for simplicity. Note expression of Wnt antagonists in the anterior of the gastrulating embryo. (C) Simplified model for AP patterning of neuroectoderm by a Wnt activity gradient in Xenopus. Wnts (red) and Wnt antagonists (green) are expressed in the mesendoderm (ME, ochre) underlying the neuroectoderm (NE, blue). The expression of Wnts and Wnt antagonist in the neuroectoderm is not shown for simplicity (see B). Their combined activities result in the formation of a Wnt signalling gradient (dark orange) that patterns the AP neuraxis. The AP axis is indicated (A→P). Formation of the posterior nervous system also requires other factors (FGFs, RA) which are not shown here. (D) The AP and DV axes in the gastrulating Xenopus embryo are patterned by gradients of Wnts and BMPs, respectively (a). The Drosophila wing imaginal disc is patterned by gradients of Wingless (Wg) and Decapentaplegic (Dpp), which are secreted along the DV and AP compartment boundaries, respectively (b).
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