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FIGURE 1 Alignment of amino acid sequences of XCXCLh, human CXCL10, and human CXCL11. The conserved amino acids are represented by asterisks (*). Red indicates the CXC- type motif. XCXCLh had a 32.6% and 33.1% sequence homology with human CXCL10 and CXCL11, respectively
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FIGURE 2. Expression of XCXCLh. (a) Temporal expression of XCXCLh detected by RT- PCR. “U” indicates unfertilized eggs, and the numbers indicate the developmental stages. Ornithine decarboxylase (ODC) was used as an internal control. (b–d) Whole mount in situ hybridization. Fixed embryos were bisected. Dorsal side is on the right side of the figure. Arrowheads indicate the XCXCLh signals. The arrow indicates the blastopore lip. (b) 32- cell stage. (c) Stage 8 embryo. (d) Stage 10 embryo. (e) Spatial expression patterns of XCXCLh detected by RT- PCR. Embryos were dissected at the indicated stages, and dissections were performed as indicated by the bottom panels
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FIGURE3 Phenotypes of XCXCLh- and XCXCLh- MO- injected embryos. (a) The specificity of the antisense morpholino oligonucleotides for XCXCLh. Western blot analysis of the injected embryos at stage 10. XCXCLh- MO inhibited the translation of FLAG- tagged mRNA containingthetargetedsite.ThetranslationoftheFLAG-taggedmRNAcontaining5-mismatchedsequencesatthetargetedsitewasnot inhibited by the XCXCLh- MO. β- globin is used as a loading control. (b) XCXCLhmRNA(500pg/blastomere)orXCXCLh- MO (10 ng/blastomere) was injected into the marginal region of two dorsal blastomeres of 4- cell embryos. Gastrula, stage 12 (vegetal view). Neurula, stage 16 (dorsal view). Tailbud, stage 30 (lateral view); left, weak phenotypes; right, severe phenotypes). WISH of Chordin, whole mount in situ hybridization of bisected embryos (stage 11) with using Chordin probes. Arrowheads indicate expression of Chordin. (c, d) Effects on phenotype by increasing doses of XCXCLh (c) and XCXCLh-MO(d).Theratioofeachphenotypeintheinjectedembryos(45embryos/bar)was indicated in the graph. Black bars represent ratio of severe phenotypes as shown in B, right panels. Gray bars represent ratio of weak phenotypes as shown in B, left panels. White bars represent normal embryos
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FIGURE4 The role of XCXCLh in chemotaxis. (a) Tissue convergence in three notochord explants. XCXCLh mRNA(500pg/blastomere)orXCXCLh- MO (10 ng/blastomere) with green tracer (EGFP mRNA, 100 pg/blastomere) or red tracer (mCherry mRNA, 100 pg/blastomere) was co- injected into two dorsal blastomeres of 4- cell embryos. Each notochord sector (without epithelium) was dissected from the injected embryos at stage 10. Three notochord sectors were grafted together and cultured on bovine serum albumin (BSA)- coated cover glasses. The elapsed time is indicated at the bottom. The average convergence index for three explants is indicated on the right (n = 3). The far right columns are high magnifications of the last frames (arrowheads). (b) Migration of scattered lateral mesendodermal cells. Lateral mesendodermal cells were dissected and dissociated at stage 10, and placed on fibronectin- coated cover glasses 0.2 to0.5mmfromtheXCXCLh- expressing ventral mesodermal explants or XCXCLh- MO-injected dorsal mesodermal explants. Mesodermal explants were placed on the left side. The elapsed time is indicated at the bottom. (c) Left, schematics of experiments. Right, the ratio of mesendodermal cell migration (n = 10). The ratio of the distance between mesodermal explants and lateral mesendodermal cells was measured every 30 min, and the average ratio is shown by the blue line for the control and red line for the XCXCLh-expressing or XCXCLh- MO- injected explants. (d) The wound healing/scratch assay using MCF- 7 cells. The empty vector (control) or XCXCLh was transfected in MCF- 7 cells, and the confluent monolayer of MCF- 7 cells was scratched with a pipette tip. The elapsed time is indicated at the bottom. (e) Left, schematics of experiments. Right, the ratio of the gap in (d). The gap was measured every 12 hrs, and the average ratio (20 positions) is shown by the blue line for the control and red line for XCXCLh-transfected cells. (f) Growth curves of MCF- 7 cells
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FIGURE5 The role of XCXCLh in differentiation. (A and B) Phenotypes of XCXCLh- injected embryos at stage 23. (a) Control. (b) XCXCLhmRNA(500pg/blastomere) was injected into the dorsal animal blastomeres of 8- cell embryos. (c and d) Animal caps of the injected embryos. (c) Control. (d) XCXCLh mRNA (500pg/blastomere)wasinjectedintoall animal blastomeres of 8- cell embryos, and animal caps were dissected at stage 8andcultureduntilstage25.(e)RT-PCR analysis of the animal caps of the XCXCLh- injected embryos. Total RNA was extracted from the animal caps of stage 10(leftpanels)andstage25(rightpanels)embryos. (f) RT- PCR analysis of the XCXCLh- MO- injected embryos. XCXCLh- MO (10 ng/blastomere) was injected into the vegetal region of all blastomeres of 4- cell embryos. Total RNA was extracted from the embryos (stage 16)
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FIGURE6 Effects of XCXCLh on the anterior- posterior axis formation. (a–c) Phenotypes of tBR and XCXCLh co- injected embryos. (A) Control, stage 40 embryo. (b) The tBRmRNA(500pg/blastomere) was injected into the ventral blastomeres of 4- cell embryos. (c) The tBR (500pg/blastomere)andXCXCLh(500pg/blastomere) mRNAs were co- injected. (d) RT- PCR analysis of animal caps at the neurula stage (stage 16). The tBR(500pg/blastomere) and different dose of XCXCLh (+,125pg;++,250pg;+++,500pg/blastomere) mRNAs were co- injected into the animal blastomeres of 8- cell embryos. Total RNA was extracted from the animal caps (stage 16). (e) RT- PCR analysis of animal caps at the gastrula stage (stage 10). The indicated Xenopus CXC- type receptormRNAs(500pg/blastomere)were injected into the animal blastomeres of 8- cell embryos. Total RNA was extracted at stage 10. (f) The different dose of XCXCLh(+,125pg;++,250pg;pg/blastomere) mRNAs were co- injected with indicated Xenopus CXC- type receptor (+,125pg;++,250pg;pg/blastomere)mRNAs into the animal blastomeres of 8- cell embryos. Total RNA was extracted at stage 10
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FIGURE7 Induction of XCXCLh. (a–d) RT- PCR analysis of animal caps and whole embryos. PCR cycles for XCXCLh were performed with35cycles,whichwasfewercyclesthanthatusedtodetectendogenousXCXCLh in Figure 2. (a) The time after the dissection of the animalcaps(fromstage9.5to10)isindicated.Animalcapswereculturedin1×MBS,andembryoswereculturedin0.15×MBS.TotalRNAwasextractedevery15minfromstage9.5.(b)Wholeembryoswereculturedin1×MBSduringtheindicatedtime(min)untilstage10.TotalRNAwasextractedatstage10.(c)Animalcapsweredissectedatstage9.5andculturedin1×MBSincludingtheindicatedcalciumion during 60 min until stage 10. Total RNA was extracted at stage 10. (d) Xwnt-8(0.5pg/blastomere),β-catenin (1 pg/blastomere), VegT (500pg/blastomere),Mixer(500pg/blastomere),Xnr5 (10 pg/blastomere), or Xnr6 (20 pg/blastomere) mRNAs were injected into all animal blastomeres of 8- cell embryos, and animal caps were dissected at stage 10 and immediately suspended in lysis buffer (lanes 1- 7). The animal capsweredissectedatstage9.5andculturedin1×MBSwithcalciumions(0.74mmol/L)during60min(lane8).TotalRNAwasextractedatstage 10. (e) ΔN-XNF-AT, CREB +VP16, or SRF + VP16 mRNA (100 pg/blastomere) was injected into all animal blastomeres of 8- cell embryos, and animal caps were dissected at stage 10 and immediately suspended in lysis buffer
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