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Fig. 7. (A) Western blot of late gastrula embryos injected into the two blastomeres at the two-cell stage with 300 pg of MEF2DGRF, MEF2CGRF or ScleraxisF synthetic mRNA. Proteins were detected with anti-flag antibody. Molecular weight markers are indicated in kilodalton. (B) Xscleraxis expression at stage 31. (C) Left: detection of Xscleraxis mRNA by whole mount in situ hybridization after unilateral injection of MEF2CGRF synthetic mRNA (+ MEF2CGRF) at the two-cell stage and induction by dexamethasone at stage 20. Right: Detection of Xscleraxis mRNA by real-time PCR after bilateral injection of mock (mock), MEF2CGRF or MEF2DGRF synthetic mRNA at the two-cell stage and induction by dexamethasone at stage 20. (D) Betaig-h3 expression at stage 31 (left) and comparison of betaig-h3 with XMRF4 expression (betaig-h3 + MRF4) indicates that betaig-h3 is expressed in intersomitic space (right, arrows). (E) Embryos were injected unilaterally at the two-cell stage, at the marginal zone level with the quantities of mRNA used for western blot. Embryos were injected by MEF2CGRF synthetic mRNA (+ MEF2CGRF), scleraxisF synthetic mRNA (+ scleraxisF) or by a mix of MEF2CGRF and scleraxisF synthetic mRNA (+ MEF2CGRF + scleraxisF). Embryos were induced by 10 μM dexamethasone at stage 20, fixed at stages 30–31 and submitted to whole-mount in situ hybridization to detect betaig-h3. (F) Tenascin C expression at stage 32 (left) and comparison of tenascin C with XMEF2C expression (tenascin C + MEF2C) indicates that tenascin C is expressed in somitic cells (right, arrows). (G) Embryos were injected unilaterally at the two-cell stage as described in (E) and analyzed for tenascin C expression. mRNA encoding the lineage tracer β-galactosidase (blue) was used to identify the injected side in panels C, E and G. Inj., injected side; uninj., uninjected side. In panels C, E and G, injected side at the top, non-injected side in the middle, and dorsal view at the bottom. No variations were observed in uninduced embryos injected by MEF2CGRF synthetic mRNA. |
