Supplementary Figure 3. A. xA2 MO coinjected with Xnr1 RNA (1 pg) in left PM. B., C. Xnr1 RNA does not rescue expression of Lefty in the left LPM of xA2-depleted embryos. Double in situ hybridization for Lefty (purple) and injected LacZ RNA (red). Lefty is absent in the left LPM but increased in the midline. B is a left lateral view, C is a dorsal view. Anterior is to the left. D-I. Xnr1 RNA and xA2 MO synergize to increase MyoD expression at stage 18. Double In situ hybridization for MyoD (purple) and LacZ (red) as tracer. D., F., H are dorsal views, anterior to the left. E, G, I are posterior fragments of the same embryos sectioned transversally. xA2 MO alone increased MyoD expression on the injected side stained with Fast Red in addition to the markers stain (n = 25, D, E). Xnr1 RNA alone (1 pg) had no effect (n = 32, F, G), while the combination of RNA and MO significantly increased the lateral expansion of MyoD (n = 29, H, I) and produced a blastopore closure defect (H, right side). Bars in D, F, H indicate the plane of the transversal section. J. Depletion of xA2 protein reduces expression of Xnr1 in posterior mesoderm. RT-PCR for the indicated genes in dorsal posterior fragments of stage 18 embryos. 10 ng xA2MO were injected at the 4 cell stage and targeted at the paraxial mesoderm. K. Expression of the triple mutant xA2 and wild-type tagged xA2 proteins were equal. Embryos (stage 24) were injected with 4 ng RNA and collected at stage 10. L. xA2 does not inhibit the Wnt pathway. Embryos were injected with Siamois reporter gene and Wnt8 RNA (50 pg)+/- xA2 RNA (2ng). Statistical significance was calculated with Student’s t test. M. xA2 is an inhibitor specific for Derrière. The ARE reporter gene (100 pg) was activated by coinjected derrière (100 pg), Xnr2 (5 pg), Xnr4 (50 pg), Xnr5 (0.1 pg), or Activin (100pg) RNA, with or without xA2 RNA (2 ng) at the 4 cell stage and measured at stage 10.5. xA2 overexpression had a significant effect only on Derrière activity (Student’s t test).
Image published in: Vonica A et al. (2011)
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