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Fig. 5. XPIASy inhibits abnormal mesoderm induction at the animal side. (A) Design of two XPIASy morpholino (Mo-1 and Mo-2) and a mutated Mo-1 (Mo-mut). Mutated positions of Mo-mut are indicated as capital letters. (B) The XPIASy morpholino-1 (Mo-1) specifically decreases the XPIASy protein level. The indicated concentrations of Mo-1 or control morpholino and Flag-tagged XPIASy (1 ng) were injected into both blastomeres of two-cell stage embryos. The protein was analyzed at stage 10.5 by western blotting against Flag. (C) The ventralized phenotype caused by XPIASy is rescued by co-injection with Mo-1. The mRNAs of 1 ng XPIASy alone (a) or together with 10 ng of Mo-1 (b) were injected into the DMZ of four-cell stage embryos. The phenotype was monitored at stage 28. (c) Uninjected embryo. (D) Secondary axis was induced by Mo-1 injection into VMZ. (E) Mo-1 slightly induces elongation of animal caps. Mo-1 (40 ng) was injected into both blastomeres of two-cell stage embryos, and its effect on elongation was analyzed. (a) Uninjected caps, (b) morpholino injected caps and (c) the sibling embryo. (F) Both XPIASy morpholinos (Mo-1 and Mo-2) induce the expression of Chordin as does Smad2. The mRNAs of ß-gal (0.5 ng) and 0.25 ng XSmad2 (a), 0.5 ng XPIASy (b), 20 ng Mo-1 (c), 20 ng Mo-2 (d) or 20 ng control morpholino (e) were injected in one side of the two-cell stage embryos. At stage 10, embryos were subjected to ß-gal staining followed by in situ hybridization against Chordin. (G) Mo-1 induces transcription of XSmad2 targets but not targets of XSmad1 and ß-catenin. Different concentrations of the indicated morpholino were injected into both blastomeres of two-cell stage embryos. The caps were collected as described in the Materials and methods for RT-PCR analysis. (H) Mo-2 also induces transcription of XSamd2 targets but Mo-mut does not affect transcription of XSmad2 targets. (I) XPIASy inhibits transcription of mesoderm genes induced by Mo-2.





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