Motomura E , Narita T , Nasu Y , Kato H , Sedohara A , Nishimatsu S , Sakai M .

	Fig. 1. Permanent blastula-type embryo (PBE) as a test system for Wnt8 distribution. (A) Schematic drawing of the cytoplasmic contents of the PBE (upper area). PBEs do not contain both the dorsal determinant and the VegT (endomesodermal determinant). (B–D) Deletion of the vegetal cytoplasm (>60% of the egg surface) with a glass rod results in the formation of the PBE. (B) Just after placing the glass rod. (C) Just after the separation into an animal egg fragment (PBE) and a vegetal egg fragment. (D) A PBE at the neurula stage. The PBE does not form dorsal or endomesodermal structures. (E) A normal embryo at the same stage.
	Fig. 2. Wnt8 protein is localized in the endoplasmic reticulum (ER) of mRNA-injected cells. (A) A stage-17 permanent blastula-type embryo (PBE) injected with VegT (15 pg) and Wnt8-HA (3 pg) into a blastomere at the 4-cell stage. Co-injection of VegT mRNA is necessary for proboscis formation (Katsumoto et al. 2004), which is a marker of dorsalization. Formation of the proboscis (34 out of 34) indicates dorsalization. (B) Reverse transcription–polymerase chain reaction (RT–PCR) analyses for dorsalization markers siamois and Xnr3 expression in control PBEs and PBEs injected with the two mRNAs. ODC serves as loading control. Control, normal embryos; No injection, PBEs with no mRNA injection; W8+V, PBEs injected with Wnt8-HA (3 pg) and VegT (15 pg) mRNAs; -RT, PCR with cDNA synthesized without reverse transcriptase. (C,D) A transverse section of a PBE injected with 250 pg Wnt8-HA and 15 pg VegT mRNAs containing 0.1% Alexa Fluor 647 dextran into an 8-cell stage blastomere. The sample was fixed and processed for immunostaining 2 h before stage 10. (E–L) Low-magnification (E–H) and high magnification (I–L) images showing the same region of a section. (E,I) Subcellular distribution of Wnt8-HA protein (green). (F,J) PDI immunostaining (red) showing subcellular distribution of ER. (G,K) Alexa Fluor 647 staining (magenta) showing the lineage of the mRNA-injected cells. (H,L) Merged images of Wnt (E, I) and PDI staining (F, J). Yellow regions indicate the overlap of these two molecules. Note the strong fluorescence of Alexa Fluor 647 in the nucleus (G,K), which is connected to the cytosol via the nucleopore, whereas HA and PDI staining was absent in the nucleus (I,J and L). (M) A PBE injected with VegT (15 pg) and Wnt8-HA-KDEL (10 pg). Proboscises (12 out of 12) formed as shown in (A). (N) RT-PCR for siamois and Xnr3. (O) An embryo with duplicated axis: This embryo was injected with 15 pg Wnt8-HA-KDEL mRNA into a ventral vegetal blastomere of 8-cell stage embryo. (P) A control embryo at the same stage (stage 17). (Q–T) Wnt8-HA-KDEL protein also overlapped with ER staining. Staining was the same as for the above-mentioned panels for Wnt8-HA (I–L). Bar in A and M, 1 mm. Bar in C for C and D, 100 lm. Bar in E for E–H, 100 lm. Bar in I for I–L, 50 lm. Bar in O for O-R, 50 lm. Bar in O for O and P, 1 mm.
	Fig. 3. Wnt8-HA-KDEL protein is retained in the cell in cell culture. Plasmid constructs of Wnt8-HA or Wnt8-HA-KDEL were transfected into HEK293 cells. Proteins (40 lg) from total cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and examined by Western blotting using the primary anti-HA antibody. b-actin was used as a loading control (cell lysates). Coomassie brilliant blue (CBB) staining of acrylamide gels was used as a loading control (cell lysates and culture media).
	Fig. 4. Wnt8 lacking a signal peptide (DSP-Wnt8-HA) did not cause dorsalization. (A) DSP-Wnt8-HA-injected PBEs do not form dorsal structures. Proboscises were not observed (0 out of 10). (B) Reverse transcription–polymerase chain reaction (RT–PCR) analyses for dorsalization markers siamois and Xnr3 expression in control PBEs (no injection) and PBEs injected with DSP-Wnt8- HA and VegT. (C–F) DSP-Wnt8-HA (green) was broadly expressed in the cytosol of the injected cells. Note that strong HA staining (C) was observed in the nucleus, whereas ER staining was not found in the nucleus (D), suggesting that DSP-Wnt8- HA is in the cytosol and not in the ER.
	Fig. 5. Wnt8 is localized at the cell boundaries in the absence of methanol treatment, but is restricted within the tracer injected domain. Wnt8-HA or Wnt8-HA-KDEL proteins were detected by HA-immunostaining (green), and the lineage of mRNA-injected cells was shown by Alexa Fluor 647 dextran (magenta) as in Figure 2. Samples were not treated with methanol in these experiments. (A–F) A Wnt8-HA injected sample. (A) Low magnification image of Wnt8-HA staining. Note that weak but substantial staining within the cell, likely in the endoplasmic reticulum (ER), was also detected. (B) Alexa Fluor 647 staining (magenta) showing the lineage of the mRNA-injected cells. (C) Composite image of A and B. Alexa Fluor 647 (lineage tracer) is indicated in the red and blue channel (thus shown in magenta) whereas Wnt8-HA is represented in the green channel. (D–F) High magnification view of A–C. (G–L) Wnt8-HA-KDEL injected sample. Staining within the cell is stronger as compared to Wnt8-HA (A–F). (G) A Wnt8-HA-KDEL injected sample. (H) Alexa Fluor 647 staining (magenta) (I) Composite image of G and H. (J–L) High magnification view of G–I. White arrowheads indicate the border of the lineagelabeled/ negative cells. Bar in A for A–C and G–I, and bar in D for D–F and J–L: 100 lm.
	Fig. 6. Co-injection of Frzb (500 pg) and Wnt8-HA (250 pg) or Wnt8-HA-KDEL (250 pg) mRNAs. Frzb and Wnt8-HA stains the cell boundaries, outside of the mRNA-injected domain while Wnt8-HA-KDEL is restricted in the lineage positive domain. (A–F) Co-injection of Frzb with Wnt8-HA. Methanol was used for immunohistochemistry (see Materials and methods). (A) Alexa Fluor 647 staining (magenta) showing the lineage of the mRNA-injected cells. (B) Frzb staining is seen predominantly at the cell boundaries, even outside of the mRNA-injected domain. (C) Staining for Wnt8-HA. Wnt8-HA staining was also at the cell boundaries but it diffused less extensively as compared to Frzb. (D) Composite image of A and B. Alexa647 is in the red and blue channel (thus shown in magenta) and originally red Frzb staining is represented in the green channel. Note that green Frzb staining is observed at the cell boundaries of both the lineage-injected domain and the lineage-negative domain, but not overlapped with the lineage tracer which represents inside of the cell. (E) Composite image of A and C. Note some cell boundaries outside of the tracer-positive region are stained in green. (F) Composite image of B and C. These images were from methanol-treated samples. These results indicate that methanol treatment does not eliminate secreted proteins. Wnt8 can be detected on the cell surface when it is secreted. (G–L) Co-injection of Frzb with Wnt8-HAKDEL. (G) Alexa Fluor 647 staining (magenta). (H) Frzb is seen both within the lineage positive cells and cell boundaries throughout the PBE. (I) Wnt8-HA-KDEL staining (green) is restricted in the lineage positive cells. (J) Composite image of G and H. (K) Composite image of G and I. Note that the outer region of the lineage positive cells is colored magenta. (L) Composite image of H and I. Note that the outer region of the lineage positive cells is colored red, suggesting that Frzb exists on the surface while Wnt8-HA-KDEL is absent on the cell surface. Bar in A for A–L, 500 lm.
	Fig. 7. Chordin is expressed only in Wnt8 mRNA-injected cells. (Top) Schematic drawing of the injection of Wnt8-HA mRNA and Oregon Green dextran. Normal embryos with a typical cleavage pattern were selected for injection. Wnt8-HA mRNA and Oregon Green dextran were injected into a C4 blastomere. The embryo was fixed 30–60 min before the onset of gastrulation and processed for in situ hybridization combined with histochemistry for lineage tracing (see Materials and methods). (A) Lateral view of an embryo showing the injected area. Expression of chordin is shown as dark blue dots, whereas lineage tracing by immunostaining of Oregon Green dextran is shown in red. (B) Enlarged image of A. (C) Counterstaining with Hoechst 33258. Note that the distribution pattern of nuclei precisely matches that of chordin expression. (D) The nuclei pattern can be superimposed on the image and therefore can be used as a single-cell level map. Bar in A, 500 lm. Bar in D for B–D, 200 lm.
	Fig. 8. Two models for Wnt signaling. (A) General model. Wnt (light blue) is secreted on the plasma membrane where it interacts with the Wnt receptors (red and green). (B) Our new model. Wnt signaling occurs cell autonomously. It may acts within the cell and/or on the cell surface in an autocrine manner.
	Figure 1. Permanent blastula-type embryo (PBE) as a test system for Wnt8 distribution. (A) Schematic drawing of the cytoplasmic contents of the PBE (upper area). PBEs do not contain both the dorsal determinant and the VegT (endomesodermal determinant). (B–D) Deletion of the vegetal cytoplasm (>60% of the egg surface) with a glass rod results in the formation of the PBE. (B) Just after placing the glass rod. (C) Just after the separation into an animal egg fragment (PBE) and a vegetal egg fragment. (D) A PBE at the neurula stage. The PBE does not form dorsal or endomesodermal structures. (E) A normal embryo at the same stage.
	Figure 2. Wnt8 protein is localized in the endoplasmic reticulum (ER) of mRNA-injected cells. (A) A stage-17 permanent blastula-type embryo (PBE) injected with VegT (15 pg) and Wnt8-HA (3 pg) into a blastomere at the 4-cell stage. Co-injection of VegTmRNA is necessary for proboscis formation (Katsumoto et al. 2004), which is a marker of dorsalization. Formation of the proboscis (34 out of 34) indicates dorsalization. (B) Reverse transcription–polymerase chain reaction (RT–PCR) analyses for dorsalization markers siamois and Xnr3 expression in control PBEs and PBEs injected with the two mRNAs. ODC serves as loading control. Control, normal embryos; No injection, PBEs with no mRNA injection; W8+V, PBEs injected with Wnt8-HA (3 pg) and VegT (15 pg) mRNAs; -RT, PCR with cDNA synthesized without reverse transcriptase. (C,D) A transverse section of a PBE injected with 250 pg Wnt8-HA and 15 pg VegTmRNAs containing 0.1% Alexa Fluor 647 dextran into an 8-cell stage blastomere. The sample was fixed and processed for immunostaining 2 h before stage 10. (E–L) Low-magnification (E–H) and high magnification (I–L) images showing the same region of a section. (E,I) Subcellular distribution of Wnt8-HA protein (green). (F,J) PDI immunostaining (red) showing subcellular distribution of ER. (G,K) Alexa Fluor 647 staining (magenta) showing the lineage of the mRNA-injected cells. (H,L) Merged images of Wnt (E, I) and PDI staining (F, J). Yellow regions indicate the overlap of these two molecules. Note the strong fluorescence of Alexa Fluor 647 in the nucleus (G,K), which is connected to the cytosol via the nucleopore, whereas HA and PDI staining was absent in the nucleus (I,J and L). (M) A PBE injected with VegT (15 pg) and Wnt8-HA-KDEL (10 pg). Proboscises (12 out of 12) formed as shown in (A). (N) RT-PCR for siamois and Xnr3. (O) An embryo with duplicated axis: This embryo was injected with 15 pg Wnt8-HA-KDEL mRNA into a ventral vegetal blastomere of 8-cell stage embryo. (P) A control embryo at the same stage (stage 17). (Q–T) Wnt8-HA-KDEL protein also overlapped with ER staining. Staining was the same as for the above-mentioned panels for Wnt8-HA (I–L). Bar in A and M, 1 mm. Bar in C for C and D, 100 μm. Bar in E for E–H, 100 μm. Bar in I for I–L, 50 μm. Bar in O for O-R, 50 μm. Bar in O for O and P, 1 mm.
	Figure 3. Wnt8-HA-KDEL protein is retained in the cell in cell culture. Plasmid constructs of Wnt8-HA or Wnt8-HA-KDEL were transfected into HEK293 cells. Proteins (40 μg) from total cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and examined by Western blotting using the primary anti-HA antibody. β-actin was used as a loading control (cell lysates). Coomassie brilliant blue (CBB) staining of acrylamide gels was used as a loading control (cell lysates and culture media).
	Figure 4. Wnt8 lacking a signal peptide (ΔSP-Wnt8-HA) did not cause dorsalization. (A) ΔSP-Wnt8-HA-injected PBEs do not form dorsal structures. Proboscises were not observed (0 out of 10). (B) Reverse transcription–polymerase chain reaction (RT–PCR) analyses for dorsalization markers siamois and Xnr3 expression in control PBEs (no injection) and PBEs injected with ΔSP-Wnt8-HA and VegT. (C–F) ΔSP-Wnt8-HA (green) was broadly expressed in the cytosol of the injected cells. Note that strong HA staining (C) was observed in the nucleus, whereas ER staining was not found in the nucleus (D), suggesting that ΔSP-Wnt8-HA is in the cytosol and not in the ER.
	Figure 5. Wnt8 is localized at the cell boundaries in the absence of methanol treatment, but is restricted within the tracer injected domain. Wnt8-HA or Wnt8-HA-KDEL proteins were detected by HA-immunostaining (green), and the lineage of mRNA-injected cells was shown by Alexa Fluor 647 dextran (magenta) as in Figure2. Samples were not treated with methanol in these experiments. (A–F) A Wnt8-HA injected sample. (A) Low magnification image of Wnt8-HA staining. Note that weak but substantial staining within the cell, likely in the endoplasmic reticulum (ER), was also detected. (B) Alexa Fluor 647 staining (magenta) showing the lineage of the mRNA-injected cells. (C) Composite image of A and B. Alexa Fluor 647 (lineage tracer) is indicated in the red and blue channel (thus shown in magenta) whereas Wnt8-HA is represented in the green channel. (D–F) High magnification view of A–C. (G–L) Wnt8-HA-KDEL injected sample. Staining within the cell is stronger as compared to Wnt8-HA (A–F). (G) A Wnt8-HA-KDEL injected sample. (H) Alexa Fluor 647 staining (magenta) (I) Composite image of G and H. (J–L) High magnification view of G–I. White arrowheads indicate the border of the lineage-labeled/negative cells. Bar in A for A–C and G–I, and bar in D for D–F and J–L: 100 μm.
	Figure 6. Co-injection of Frzb (500 pg) and Wnt8-HA (250 pg) or Wnt8-HA-KDEL (250 pg) mRNAs. Frzb and Wnt8-HA stains the cell boundaries, outside of the mRNA-injected domain while Wnt8-HA-KDEL is restricted in the lineage positive domain. (A–F) Co-injection of Frzb with Wnt8-HA. Methanol was used for immunohistochemistry (see Materials and methods). (A) Alexa Fluor 647 staining (magenta) showing the lineage of the mRNA-injected cells. (B) Frzb staining is seen predominantly at the cell boundaries, even outside of the mRNA-injected domain. (C) Staining for Wnt8-HA. Wnt8-HA staining was also at the cell boundaries but it diffused less extensively as compared to Frzb. (D) Composite image of A and B. Alexa647 is in the red and blue channel (thus shown in magenta) and originally red Frzb staining is represented in the green channel. Note that green Frzb staining is observed at the cell boundaries of both the lineage-injected domain and the lineage-negative domain, but not overlapped with the lineage tracer which represents inside of the cell. (E) Composite image of A and C. Note some cell boundaries outside of the tracer-positive region are stained in green. (F) Composite image of B and C. These images were from methanol-treated samples. These results indicate that methanol treatment does not eliminate secreted proteins. Wnt8 can be detected on the cell surface when it is secreted. (G–L) Co-injection of Frzb with Wnt8-HA-KDEL. (G) Alexa Fluor 647 staining (magenta). (H) Frzb is seen both within the lineage positive cells and cell boundaries throughout the PBE. (I) Wnt8-HA-KDEL staining (green) is restricted in the lineage positive cells. (J) Composite image of G and H. (K) Composite image of G and I. Note that the outer region of the lineage positive cells is colored magenta. (L) Composite image of H and I. Note that the outer region of the lineage positive cells is colored red, suggesting that Frzb exists on the surface while Wnt8-HA-KDEL is absent on the cell surface. Bar in A for A–L, 500 μm.
	Figure 7. Chordin is expressed only in Wnt8mRNA-injected cells. (Top) Schematic drawing of the injection of Wnt8-HAmRNA and Oregon Green dextran. Normal embryos with a typical cleavage pattern were selected for injection. Wnt8-HAmRNA and Oregon Green dextran were injected into a C4 blastomere. The embryo was fixed 30–60 min before the onset of gastrulation and processed for in situ hybridization combined with histochemistry for lineage tracing (see Materials and methods). (A) Lateral view of an embryo showing the injected area. Expression of chordin is shown as dark blue dots, whereas lineage tracing by immunostaining of Oregon Green dextran is shown in red. (B) Enlarged image of A. (C) Counterstaining with Hoechst 33258. Note that the distribution pattern of nuclei precisely matches that of chordin expression. (D) The nuclei pattern can be superimposed on the image and therefore can be used as a single-cell level map. Bar in A, 500 μm. Bar in D for B–D, 200 μm.
	Figure 8. Two models for Wnt signaling. (A) General model. Wnt (light blue) is secreted on the plasma membrane where it interacts with the Wnt receptors (red and green). (B) Our new model. Wnt signaling occurs cell autonomously. It may acts within the cell and/or on the cell surface in an autocrine manner.

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