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Figure 4. A Smad binding site is indispensable to conferring nodal signaling responsiveness and the expression of XPAPC in Xenopus gastrula embryo. A: Luciferase assay of episomal reporter gene injection. The transcription activity of XPAPC 5 prime contig was enhanced to about 8 folds by nodal signaling and the elevation could be blocked by SB-431542 treatment. The construct in which the Smad binding site was mutated displayed a lower transcription activity and lower responsiveness to nodal signaling. RLU, relative light units, normalized to internal standard. B-G: Whole mount in situ hybridization was performed with GFP antisense probes. Embryos were vegetal view, with dorsal side up. SB-431542 treatment blocked the expression of GFP mRNA in pFLGFP transgenic embryos (B and E). In contrast, DMSO could not affect the expression (C and F). D,G: GFP mRNA could not be detected in transgenic embryos with mutated construct at early gastrulation (D) and faint signal could be observed in the mid-gastrulation embryos (G). B-D: Stage 9.5. E,F: Stage 10.5. H: The percentages of transgenic embryos that show the dorsal expression (white), non-specific expression (brown), and no detected signal (purple) are shown. |
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Figure 3. Promoter-driven GFP mRNA expression recapitulates endogenous XPAPC expression in spatial and temporal-specific manner. Transcripts of the endogenous XPAPC or the GFP reporter gene were detected by whole mount in situ hybridization with XPAPC or GFP antisense probes, respectively. A-E: Expression of endogenous XPAPC RNA at the stages indicated. F-J: Expression of the pFLGFP in transgenic embryos at the same stages. The expression pattern of GFP mRNA was reminiscent of the endogenous XPAPC expression pattern. A, B and F-J: Vegetal views of gastrula with the dorsal side up; (C and H) dorsal views of neurulae with anterior side up; (D and I) lateral views of neurulae, anterior to the left; (E and J) lateral views of tadpoles, anterior to the left. |
