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Fig. 2. Inhibition of mesoderm formation and Activin signalling by xGalntl-1. (A) Stage 40 uninjected control Xenopus embryo and (B) sibling embryos microinjected radially with 400 pg xGalntl-1 mRNA at 2- to 4-cell stages. (C) In situ hybridisation for the pan-mesodermal marker xbra in gastrula stage embryo microinjected either radially with xGalntl-1 mRNA alone or into single blastomeres together with lacZ mRNA at the 4-cell stage. (D) RT-PCR analysis of dorsal (DMZ) and ventral marginal zone (VMZ) explants at stage 20. The expression of α-globin (blood), gata1 and xvent1 (ventral mesoderm), msx1 (neural crest, epidermis) was reduced in VMZ from embryos microinjected with 200 pg xGalntl-1 mRNA, whereas the expression levels of xag (cement gland), otx2, rx2a (forebrain) and ncam (neural) were increased. The expression of α-actin (muscle) was reduced in DMZ and slightly increased in VMZ. (E) RT-PCR analysis for chordin (dorsal mesoderm), sox17β (endoderm) and xbra (mesoderm) expression using stage 20 animal cap explants from embryos microinjected with mRNA encoding eFGF (100 pg) or Activin (2 pg), in the presence or absence of 200 pg xGalntl-1 mRNA. (F) Western blot analysis of Smad and phospho-Smad proteins from HEK 293T cells that were transiently transfected with Smad2-FLAG and xGalntl-1 expression vectors and cultured for 2 hours in the absence or presence of 2ng/ml recombinant Activin protein. (G) In vitro translation of xGalntl-1 mRNA containing (+5′-UTR) or lacking (-5′-UTR) morpholino target sequences. Two different morpholinos (Galntl-1-MO1 and Galntl-1-MO2, 2.5 μm) as well as a mixture of both (1.25 μm each) specifically interfered with translation of RNA containing the morpholino target sequences. (H) RT-PCR analysis of animal cap explants at stage 20 from embryos microinjected with 0.5 pg Activin mRNA, xGalntl-1 morpholinos (1 ng each) and 20 pg xGalntl-1 mRNA. The morpholino stimulated the induction of myoD (muscle) and xbra expression by Activin. In all RT-PCR experiments, odc served as a loading control. |
