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Gastrulation is the first obligatory morphogenesis during vertebrate development, by which the body plan is established. Nodal signaling is a key player in many developmental processes, including gastrulation. XPAPC has been found to exert its biological function through modifying the adhesion property of cells and interacting with other several important molecules in embryos. In this report, we show that nodal signaling is necessary and sufficient for XPAPC expression during Xenopus gastrulation. Furthermore, we isolated 4.8 kb upstream DNA sequence of Xenopus XPAPC, and proved that this 4.8-kb genomic contig is sufficient to recapitulate the expression pattern of XPAPC from gastrula to tail bud stage. Transgene and ChIP assays indicate that Activin/nodal signaling participates in regulation of XPAPC expression through a Smad binding element within the XPAPC promoter. Concomitant investigation suggests that the canonical Wnt pathway-activated XPAPC expression requires nodal signaling.
Figure 1. Nodal signaling directly controls the expression of XPAPC in Xenopus embryos. A-J: Embryos were collected at stage indicated and in situ hybridized with indicated probe. Embryos were viewed from the vegetal side, with dorsal side up. A: Induction of XPAPC (marked with white triangle) in embryos injected with Xnr2 mRNA at 4-cell stage at the ventral midline. B: LacZ mRNA was injected as lineage tracing and specificity control. C,F: Perturbing nodal signaling with SB-431542 inhibited the expression of XPAPC. C,E: Embryos were treated with SB-431542. D,F: Embryos were treated with DMSO as specificity control. G-J: Gene expression after SB-431542 treatment in Xenopus embryos. In DMSO treated embryos, Xbra expression formed a ring around the margin zone (G), but expression on the dorsal side was lost in SB-431542-treated embryos (H). In contrast, Sizzled expression was not affected (I and J). K: Nodal signaling could induce XPAPC expression directly in animal caps. Induction of XPAPC was not inhibited by CHX treatment, but the induction of Chordin was. Con cap, control animal cap without treatment; WE, whole embryo (Stage 12); No RT, no-reverse-transcriptase control; ODC, ornithine decarboxylase, as loading control.
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.
Figure 4. A Smad binding site is indispensable to conferring nodal signaling responsiveness and the expression of XPAPC in Xenopus gastrulaembryo. 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.
Figure 6. The canonical Wnt signaling activates the expression of XPAPC via nodal signaling. A-H: Embryos were collected at stage 9.5 and in situ hybridized with XPAPC probe. Embryos were viewed from the vegetal side, with dorsal side up. C-H: LacZ mRNA was injected as lineage tracer and microinjection controls. A: Enhancing the canonical Wnt signaling by LiCl treatment expanded the expression of XPAPC to the whole marginal zone. B: The enhancement was blocked by perturbing nodal signaling through SB-431542 treatment which followed LiCl treatment. C-E: Blocking canonical Wnt pathway through LefEnR injection inhibited the expression of XPAPC (C), activating nodal signaling through Xnr2 injection rescued the expression (D). F-H: Activating the canonical Wnt signaling through LefVP16 injection induced the expression of XPAPC at ventral side of embryos (F), SB-431542 treatment blocked the endogenous and ectopic expression of XPAPC (G).
