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Fig. 1. Wnt/β-catenin signaling is active in the ventral blood island of Xenopus and in the yolk sac mesoderm of mouse embryo. (A–D) Expression of the d2EGFP protein in transgenic embryos carrying the Wnt/β-catenin reporter construct. (A) GFP is expressed in the DLP mesoderm and the VBI, from which definitive and primitive blood cells originate, respectively. (B) Transverse section of a stage-32 embryo (dashed line in E) shows high levels of Wnt activity in the VBI (indicated by mes) and also in the overlying ectoderm (ect). (Inset) DAPI staining of the section. (C) Stage 37 embryos show GFP-expressing blood cells leaving the VBI through the vitelline veins and circulating in the posterior cardinal vein (arrowhead) and throughout the whole body. (D) GFP-expressing and DAPI-stained individual erythrocytes collected from stage-37 embryos and viewed with confocal microscope. (E) Detection of Wnt/β-catenin signaling in E8.5 embryo of BAT-Gal Wnt-reporter mouse. Strong β-gal activity is visible in the embryo proper (lower left half) and in the extraembryonic mesoderm that forms the blood islands in the yolk sac (arrowhead). No activity is seen in the surrounding visceral endoderm (arrow).
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Fig. 2. WISH analysis of Wnt4 mRNA expression in the VBI and DLP in Xenopus embryos. (A–D) Wnt4 transcripts are observed in the neural plate and in the VBI and DLP mesoderm in stage-21 embryos. (B) Transverse section at site indicated by dashed line in A. (C) Closer view of dorsolateral and ventral regions (D). Wnt4 signal is clearly excluded from overlying ectoderm (C and D). Wnt4 transcripts in DLP are sustained in embryos at stage 25 (E) and restricted to pronephric region in embryos at stage 28 (F) and stage 34 (G). Wnt4 is clearly expressed in VBI (E–H) and specifically in the mesodermal layers (arrowhead in H), but not in overlying ectoderm (arrow in H). Wnt4 is also expressed in the brain, branchial arches, and lateral plate mesoderm (arrowhead in C). dlp, dorsal lateral plate, dlpm, dorsal lateral plate mesoderm; ecto, ectoderm; endo, endoderm; hb, hindbrain; hend, hepatic endoderm; mb, midbrain; mhb, midbrain–hindbrain boundary; meso, mesoderm; np, neural plate; nt, neural tube; pt, pronephric tubules; vbi, ventral blood island.
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Fig. 3. Depletion of Wnt4 affects formation of VBI. (A) Wnt-reporter embryos were injected at two-cell stage with 10 ng Co MO or Wnt4 MO in marginal zones, cultured to stage 32, and hybridized in situ with probes for dGFP and blood marker T3-globin (T3). Wnt4 morphant lost most of the GFP and T3-globin signals at the VBI. GFP transcripts were totally abolished at the DLP in Wnt4-depleted embryos (Right, arrowhead). Note that the GFP signal in the morphant embryo was also abrogated in the branchial arches (Upper Right, arrows) and lateral plate mesoderm area, which normally show abundant Wnt4 expression (Fig. 2G, arrow and arrowhead). (B) Targeted depletion of Wnt4 at CD1 or CD4 blastomeres (Upper, highlighted) by injection of Wnt4 morpholino ablates expression of hematopoietic cell markers SCL and T3-globin (violet blue) in corresponding VBI compartments. Arrowheads and arrows indicate aVBI and pVBI, respectively. Cytoplasmic β-gal RNA was coinjected as a lineage tracer (cyan blue).
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Fig. 4. Wnt/β-catenin signaling is required in both ectoderm and mesoderm for formation of VBI. (A) Global inhibition of Wnt/β-catenin signaling reduced expression of the hematopoietic marker gene SCL in VBI. Embryos were injected at the two-cell stage with 0.5 ng RNA of Wnt-inhibiting construct EnR and cultured in the absence or presence of Dex from stage 13 onward, and then assayed for SCL expression by WISH at stage 20 or 24. Ratio in lower right corner indicates proportion of embryos that had the phenotype shown in the picture. (B) Global inhibition of Wnt signaling just before differentiation of erythrocytes still reduces erythrocyte differentiation or survival. Embryos were injected as above and cultured in the absence or presence of Dex from stage 20 onward. Embryos were assayed by WISH for SCL and T3-globin (T3) at stages 25 and 32, respectively. (C) Embryos were injected at the 16-cell stage in CD1, CD4, or A4 blastomeres (as indicated on top) and with 100 pg RNA of EnR and 10 pg RNA of cytoplasmic β-gal as lineage tracer. Embryos were treated with Dex during gastrulation (stage 11.5). SCL was completely abrogated in Dex-treated embryos, regardless of injection site. CD4-injected embryos lost T3 expression completely at pVBI (arrows), whereas CD1-injected embryos show only partial loss of T3 gene expression in aVBI (arrowheads). Domain of T3 down-regulation in both cases was extended to neighboring compartment, i.e., beyond β-gal boundary. Inhibition of Wnt signaling in ventral ectoderm (A4 descendants) during gastrulation resulted in overall down-regulation of T3 expression.
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wnt4 (wingless-type MMTV integration site family, member 4) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 21, lateral view, anterior right, dorsal up.
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wnt4 (wingless-type MMTV integration site family, member 4) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 21, transverse sections, anterior trunk region, dorsal up.
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wnt4 (wingless-type MMTV integration site family, member 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior right, dorsal up.
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wnt4 (wingless-type MMTV integration site family, member 4) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.
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tal1 (T-cell acute lymphocytic leukemia 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
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hba3 ( hemoglobin alpha 3 subunit) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
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Figure S2: Expression of the d2EGFP protein (A-O) in transgenic embryos carrying the Wnt/β-catenin reporter construct. (A) Dorsal view of a stage-11 gastrulating embryo when GFP signals first become visible. (B-E) Dynamic and diversified expression of GFP signal in reporter embryos as development proceeds. Particularly strong expression was observed in the developing neural folds (nf) (B), and in the posterior presomitic mesoderm (psm), the migrating cranial neural crest (nc), and the olfactory epithelium (oe) (C-E). GFP is expressed in the DLP mesoderm and the VBI, from which definitive and primitive blood cells arise, respectively (C,E). (F) Transverse section of a stage-32 embryo (dashed line in E) shows high levels of Wnt activity in the VBI (indicated by meso, mesoderm) and also in the overlying ectoderm (ect). The inset shows DAPI staining of the section. (G) Stage-37 embryos show GFP-expressing blood cells leaving the VBI through the vittelline veins and circulating in the posterior cardinal vein (arrowhead) and throughout the whole body. (G’) A closer view of a selected area (G; dashed rectangle) shows GFP-positive blood cells (arrowheads) circulating in the vittelline veins. (H) GFP-expressing and DAPI-stained individual erythrocytes collected from stage-37 embryos and viewed by confocal microscopy. (I-K) Confocal images of transverse sections of an early tadpole. In the inset in panel I, dashed line 1 refers to panels I and J; dashed line 2 refers to panel K. There is strong expression of GFP in the peripheral retina (cmz, I) and in the differentiating lens fibers (lf in I). Wnt activity was also detected in the lung buds (lb in K), pronephric tubules (pt in K), and the dorsal part of the midbrain- hindbrain boundary (mhb in J). (L-N) Views of a late tadpole stage: dorsal (L), ventral (M) and lateral (N). GFP is continuously observed in the developing liver (lv in L), pronephric tubules (pt in N), and the brain (M,N). Note that GFP is expressed at the boundaries between the rhombomeres of the hindbrain (arrowheads in N). (O-Q) GFP expression is visible in various organs about the time of metamorphosis, especially in the mesencephalon (mesc in O), the pancreas (pc in P), and the growth plates of the developing limbs and digits (arrowhead in Q). Insets correspond to bright field images. (R) Detection of Wnt/β-catenin signaling in an E8.5 embryo of the BAT-Gal Wnt-reporter mouse. Strong β-galactosidase activity is visible in the embryo proper (lower left half) and in the extraembryonic mesoderm that forms the blood islands in the yolk sac (arrowhead). No activity is seen in the surrounding visceral endoderm (arrow).
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Figure S1. Validating the activity and specificity of Wnt-reporter constructs in cell culture and in transgenic Xenopus embryos. (A) The induction of the p8Lef-luciferase plasmid in response to Wnt stimulation is comparable to that of the pTOPflash plasmid. Importantly, the mutant p8mLef-luciferase plasmid exhibited much lower basal activities than pFOPflash. The ratio between the activities of the p8Lef-luciferase and p8mLef-luciferase plasmids is indicated by the number near the standard deviation bars. Significance of differences between means was analyzed by a Student's t-test (* p < 0.05). (B) Schematic overview of pbin8mLef-GFP-EcDsRed (control construct) and pbin8Lef-GFP (Wnt/β-catenin reporter construct). Eca: minimal promoter of the human E-cadherin gene, which drives expression of the DsRed gene in the epidermal tissues. (C) Expression of GFP in 293T cells when the control (top panel) and the reporter (bottom panel) plasmids were contransfected with a stabilized form of β-catenin. (D) Transgenic control (left panel) and reporter embryos (middle and right panel) were treated with 0.3M LiCl for 10 min and processed by WISH using a digoxigenin-labeled d2EGFP probe. Stage 21 Wnt-reporter transgenic X. tropicalis embryos express GFP transcripts ubiquitously in response to LiCl stimulation (right panel). No signal is detected in the transgenic control embryo (inset left panel: DsRed expression used to identify the transgenic embryos). (E) Transgenic Wnt-reporter embryos were injected at the 4-cell stage with 100 pg RNA of a Wnt-inhibiting construct (pEnR-LEF∆N-GR) either in the dorsal (middle panel) or the ventral side (right panel). Rhodamine dextran sulfate was coinjected as a tracer. Embryos were treated with 10 μM Dex at stage 28 and visualized at stage 32. Treated embryos responded to Wnt inhibition and could be directly analyzed in vivo. Arrows: GFP signal at the midbrain-hindbrain boundary; arrowheads: position of GFP expression in the posterior somites.
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Figure S3. In situ hybridization shows expression of the d2EGFP transcripts in transgenic embryos carrying the Wnt/β-catenin reporter construct. (a) Dorsal and (b) vegetal views of a stage-10.5 embryo; the arrowhead indicates the absence of GFP expression at the dorsal blastopore lip. (c) Vegetal and (d) animal views of a stage-11.5 embryo (dorsal side up) show Wnt activity comparable to that observed in vivo. The signal covers the whole ventral and lateral sides, expands toward the central midline as gastrulation proceeds, and concentrates at the neural fold at the end of gastrulation (e, stage 12 posterior view, dorsal side up; f, stage 12, dorsal view, anterior side up). Lateral view of embryos at stages 21 (g) and 28 (h): anterior side to the right, dorsal side up. Note GFP expression in the migrating cranial neural crest (nc) cells. GFP transcripts are also abundant in the DLP and VBI. (i) Stage-32 embryo with Wnt activity in the forebrain-midbrain (fmb) and midbrain-hindbrain (mhb) boundaries, optic vesicle (ev), otic vesicle (ov) and the posterior somites (so). (i-k) Dynamic Wnt activity in the nephric duct (nd); the first signal appears together with the signal in the epithelium of the pronepric tubules (pt) (i) and disappears at stage 32; activity intensifies at the nephric tubules as the second nerphrostome funnel forms (j, rectangle enlarged in k). (l,m) Ventral view of embryos at stages 30 (l) and 32 (m) showing GFP transcripts in the hepatic endoderm (hend) anterior to the VBI (l); the transcripts in the hepatic endoderm disappeared when the VBI formed a V-shape (m). In stage-39 (n) and stage-41 (o) embryos, Wnt activity is detected in heart and liver primodium (n), and in pancreas, liver and anterior part of the intestine (o). ba, branchial arches; bp, blastopore; bpl, blastopore lip; dlp, dorsal lateral plate; ect; ectoderm; endo, endoderm; ev, optic vesicle; fb, forebrain; fmb, forebrain-midbrain boundary; h, heart; hb, hindbrain; hend, hepatic endoderm; ie, inner ear; in, intestine; lv, liver; lvp, liver primodium; mb, midbrain; mhb, midbrain-hindbrain boundary; nc, neural crest; nd, nephric duct; nf, neural fold; ob, olfactory bulb; oe, olfactory epithelium; ov, otic vesicle; pc, pancreas; pt, pronephric tubules; vbi, ventral blood islands.
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Figure S4. Expression of Wnt4 and Wnt reporter (GFP) mRNA in bisected gastrula-stage embryos. (A-C) WISH analysis of stage-10.5 embryos with a GFP probe showing low reporter activity in the future AEM region (dashed box) at the onset of gastrulation (A). At the end of gastrulation in stage-12.5 embryos, GFP transcripts are detected in the leading edge mesoderm (LEM), in the tip of the ventral mesoderm (VM) and in the overlying ventral ectoderm (VE) (the boxed area in B is shown enlarged in C). (D-F) Expression of Wnt4 mRNA in bisected embryos at stages 10.5 and 11.5. At stage 11.5, Wnt4 is expressed in the cells of the dorsal leading edge mesoderm (the boxed area in E is enlarged in F) as well as the tip of the ventral mesoderm. All embryos are shown with animal side up and dorsal to the right. Bp, blastopore; DL, dorsal lip; DLEM, dorsal leading edge mesoderm; VE, ventral ectoderm; VM, ventral mesoderm; VLEM, ventral leading edge mesoderm.
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Figure S5. Depletion of Wnt4 affects the formation of the VBI (see also Fig. 3). Targeted depletion of Wnt4 at CD1 or CD4 blastomeres (highlighted in top panel) by Wnt4 morpholino injection ablates the expression of the hematopoietic cell marker LMO2 (violet blue) in the corresponding VBI compartments. Arrowheads and arrows indicate aVBI and pVBI, respectively. Cytoplasmic β-gal RNA was coinjected as a lineage tracer (cyan blue).
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Figure S6. Effect of Wnt4 depletion on definitive blood formation and rescue experiment. (A) Wnt4 MO injection in V2 blastomeres of embryos at the 8-cell stage depletes the expression of the DLP markers SCL and LMO2 (arrows). (B) Coinjection of 150 pg RNA of Wnt4-HA in the CD1 blastomeres rescues expression of SCL and LMO2 in the corresponding areas (arrows) in Wnt4-depleted embryos; 10 pg RNA of cytoplasmic β-gal was coinjected as lineage tracer. The arrow shows the aVBI. The injected doses of Wnt4 MO were 2.5 ng and 4 ng for embryos at the 16-cell and 8-cell stages, respectively.
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Figure S7. Wnt/β-catenin signaling is required in both ectoderm and mesoderm for the formation of the VBI (see also Fig. 4). Embryos were injected at the 16-cell stage either in the CD1, CD4 or A4 blastomere (as indicated on top) with 100 pg RNA of EnR-LEF∆N-GR and 10 pg RNA of cytoplasmic β-gal as lineage tracer. Embryos were treated with 10 μM Dex during gastrulation (stage 11.5) and fixed at stage 20/21 or 32, stained for β-gal, and then processed for WISH. LMO2 was completely abrogated in Dex-treated embryos, regardless of the injection sites.
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Figure S8. Expression of Xfli1 and R-spondin 3 (Rspo3) in Wnt4-depleted embryos. X. laevis embryos were injected with 12 ng Co MO (A, C, E) or Wnt4 MO (B, D, F) at the 4-cell stage in the dorsal and ventral marginal zones and cultured until stage 29-30. WISH analysis was performed for the hematopoietic marker SCL (A, B), the endothelial marker Xfli-1 (C, D) and for Rspo3 (E, F). In addition to SCL, which was strongly decreased in Wnt4 morphants, also Xfli-1 and Rspo3 were slightly reduced in the region bordering the VBI. The ratio in the right lower corner indicates the number of embryos displaying the phenotype shown in the picture.
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Figure S9. Wnt4 is required in the AEM for the expression of the erythroid marker αT3- globin (T3) in the combined explant assay. AEM of embryos injected with Co MO or Wnt4 MO were explanted and conjugated with ventral ectoderm (VE) of noninjected (NI) embryos at stage 10.5. The combined explants were cultured until stage 32 and processed for WISH. αT3-globin expression in the AEM was completely abolished by Wnt4 depletion.
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Figure S10. Identification of BMP4 as an indirect target gene of Wnt/β-catenin signaling. Influence of the translation inhibitor cycloheximide (CHX) on Wnt-induced expression of BMP4 and Msx1. Embryos were injected at the 4-cell stage in each blastomere with 25 pg inducible Wnt-activating Lef∆N-VP16-GR RNA and treated at tailbud stage with Dex and/or CHX for 2 hours. While both BMP4 and Msx1 are induced by the Dex-mediated activation of Lef∆N-VP16-GR, only Msx1, which is a known Wnt target gene, was induced in the presence of CHX.
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Figure S11. A short (1.5 hours) contact is sufficient for Wnt4 in the AEM to regulate BMP4 expression in the VE. (A) Schematic representation of the experimental setup. Embryos were injected in the marginal zones at the 2-cell stage with 12 ng Co MO or Wnt4 MO. Oregon Green 514 was coinjected in embryos that were used for AEM explants. At stage 10.5, VE and AEM were explanted, combined, and cultured for 1.5 or 3 hours to reach a state equivalent to stage 11.5 or 12.5, respectively. VE was then separated from AEM under the fluorescent microscope and then either assayed immediately by qPCR for BMP4 and epidermal marker Keratin (B) or cultured until stage 18 and assayed for the hematopoietic marker SCL (C). VE of Co MO was cultured alone and assayed the same way. Whole embryos were used as positive control. The separated AEM was cultured until stage 18 and assayed for SCL expression (D). Uncombined AEM and whole embryos at equivalent stages were used as control. Error bars represent standard deviation.
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Figure S12. Spemann organizer genes are not increased in the AEM of Wnt4 morphants. Embryos were injected in the marginal zones at the 2-cell stage with 12 ng Co MO or Wnt4 MO. At stage 10.5, anterior endomesoderm (AEM) was explanted, cultured to stage 11, and assayed for expression of the organizer genes Chordin, Goosecoid (Gsc) and Nodal-related 3 (Xnr3). An endodermal marker (Hex) was assayed as a reference for the total amount of endomesodermal tissue being explanted. Whole embryos at an equivalent stage were used as positive control. Error bars represent standard deviation.
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