Wnt signaling and transcriptional control of Siamois in Xenopus embryos

Abstract

The Wnt-inducible homeobox gene Siamois is expressed in Xenopus embryos before gastrulation and is necessary for formation of the Spemann organizer. Here we show that 5'-flanking sequences of the Siamois coding region can specifically activate a heterologous reporter gene in dorsovegetal cells, thus mimicking Siamois's endogenous expression. A 245-bp DNA fragment is sufficient for activation by both Wnts and endogenous inducers. A dominant negative form of Xenopus T cell-specific factor 3 (XTCF-3) inhibited promoter activity, indicating that T cell-specific factor (TCF)/lymphocyte enhancer binding factor 1 (LEF-1) signaling is necessary for regulation of Siamois. Mutagenesis of two individual TCF sites in the -245 promoter revealed that the proximal, but not distal, site is necessary for dorsovegetal activation. These observations suggest that Siamois is directly regulated by TCFs during dorsoventral axis determination. Further deletion analysis identified a positive regulatory region that is required for dorsal activation, but not for Wnt inducibility, of the promoter. We also present evidence for autoregulation of Siamois transcription. Furthermore, the Siamois promoter was activated by Wnt signaling in 293T tissue culture cells, demonstrating that regulation of the promoter is functionally conserved.

Figure 1

Upstream regulatory sequence of the Siamois gene. The beginning of the Siamois coding region and of the corresponding cDNA (italic type) are shown. A TATA-like sequence is underlined. Three TCF consensus binding sites are shown in boldface.

Figure 2

Comparison of pSia-Luc constructs in different regions of the embryo. Luciferase activity was measured in embryos microinjected into ventral animal (va), dorsal animal (da), ventral vegetal (vvg), and dorsovegetal (dvg) blastomeres with −833pSia-Luc (A and B), −296pSia-Luc (B and D), −245pSia-Luc (C), and −179pSia-Luc and −135 pSia-Luc (D). Experimental data are expressed as the means from triplicate samples ± SD. One representative experiment is shown in each graph.

Figure 3

TCF signaling is critical for Siamois transcription. (A) Activation of −833pSia-Luc in dorsovegetal blastomeres was suppressed by coinjection of ΔN-XTCF-3 (DN) mRNA. (B) Location of mutated TCF consensus sites (underlined) in the promoter. (C and D) Comparison of mutated constructs in various blastomeres. Abbreviations are as in Fig. 2.

Figure 4

Wnt inducibility of the Siamois promoter. Embryos were microinjected in two ventral animal blastomeres with −833pSia-Luc (A), −245pSia-Luc (B), and −135pSia-Luc and −296pSia-Luc (C). Luciferase activity was measured for embryos injected with promoter constructs alone, or coinjected with Xwnt8 ± DN, tBr, or Xnr3 mRNAs as shown.

Figure 5

Activity of different pSia-Luc constructs in dorsovegetal cells and their inducibility by Wnt signaling. Positions of the three TCF consensus sites are shown. N.D. = not determined.

Figure 6

Autoregulation of Siamois transcription. (A) Induction of −833pSia-Luc by Siamois mRNA in ventral animal blastomeres. (B and C) The effect of SE mRNA on activity of −296, −245, and −179pSia-Luc constructs (B) and −135pSia-Luc (C) in dorsovegetal blastomeres.

Figure 7

−833pSia-Luc is activated by Wnt signaling in mammalian cells. 293T cells were transfected with a control pcDNA3 plasmid, or with Xwnt8-pCS2, Xdsh-pCS2, or β-catenin-pcDNA3 plasmids. Luciferase activity is expressed as the means from triplicate samples ± SD.