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Cdx homeodomain transcription factors play important roles in the development of the vertebrate body axis and gut epithelium. Signaling involving FGF, wnt and retinoic acid ligands has been implicated in the regulation of individual Cdx genes. In this study we examine the requirement for FGF-dependent signal transduction pathways in the regulation of Cdx gene expression. In the amphibian Xenopus laevis the earliest expression of Cdx1, Cdx2 and Cdx4 is within the developing mesoderm. We show that a functional FGF signaling pathway is required for the normal expression of all three amphibian Cdx genes during gastrula stages. We show that FGF stimulation activates signaling through both the MAP kinase pathway and the PI-3 kinase pathway in Xenopus tissue explants. However, our analysis of these pathways in gastrula stage embryos indicates that the MAP kinase pathway is required for Cdx gene expression, whereas the PI-3 kinase pathway is not. We show that FGF and wnt signaling can interact in the regulation of Cdx genes and during gastrula stages the normal expression of the Cdx genes requires the activity of both pathways. Furthermore, we show that wnt mediated Cdx regulation is independent of the MAP kinase pathway.
Fig. 1. Inhibition of Cdx gene expression with a dominant negative FGF receptor. Panel A shows whole-mount in situ hybridization analysis for Cdx1, Cdx2 and Cdx4 expression at gastrula stage 11 in control embryos and embryos injected with 4 ng dnFGFR1 mRNA. Panel B is an RNAase protection analysis (RPA) showing the expression of Cdx genes from mid blastula to mid gastrula stages, in control embryos and embryos injected with 4 ng dnFGFR1 mRNA. Ubiquitously expressed ornithine decarboxylase (ODC) was included as a loading control. Panel C is an RPA showing the expression of Cdx genes in control embryos and embryos injected with 4 ng dnFGFR1 or dnFGFR4 mRNA at gastrula stage 10.5. Panel D shows whole-mount in situ hybridization analysis for Brachyury (Bra), Snail (Sna) and FGF8 at gastrula stage 11 in control embryos and embryos injected with 4 ng dnFGFR1.
Fig. 5. Combinatorial activity of FGF and wnt signaling. Panel A shows the phenotype at swimming larva stage 41 of control embryos and embryos injected with 2 ng FrzB mRNA or 2 ng dnFGFR1 mRNA or 2 ng FrzB mRNAÂ +Â 2 ng dnFGFR1 mRNA. Panel B shows whole-mount in situ hybridization analysis for Cdx1, Cdx2 and Cdx4 expression at gastrula stage 11 in control embryos and embryos injected with 2 ng FrzB mRNA or 2 ng FrzB mRNAÂ +Â 2 ng dnFGFR1 mRNA. Panel C is an RPA showing the expression of Cdx genes at gastrula stage 11 in control embryos and embryos injected with 2 ng dnFGFR1 mRNA or 2 ng FrzB mRNA or 2 ng FrzB mRNAÂ +Â 2 ng dnFGFR1 mRNA. Panel D is an RPA showing Cdx gene expression at control stage 13 in control animal cap explants and animal cap explants from embryos injected with 10 pg FGF4 mRNA or 50 pg wnt8 mRNA or 10 pg FGF4 mRNAÂ +Â 50 pg wnt8 mRNA. Panel E is a bar chart showing the relative levels of expression as determined by densitometric analysis of each Cdx gene in the animal caps analyzed in panel D. Expression levels are normalized to the ODC loading control and the expression of each gene is shown relative to expression in untreated control caps. Panel F is a Western blot of control embryos and embryos injected with 2 ng dnFGFR1 mRNA or 2 ng FrzB mRNA or 2 ng FrzB mRNAÂ +Â 2 ng dnFGFR1 mRNA probed for p-S473 Akt, dp-ERK and GAPDH at gastrula stage 10.5. Panel G shows whole-mount in situ hybridization analysis of wnt8 expression at gastrula stage 11 in control embryos and embryos injected with 4 ng dnFGFR1 mRNA.
