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Beta-catenin is a multifunctional protein involved in cell adhesion and communication. In response to signaling by Wnt growth factors, beta-catenin associates with nuclear TCF factors to activate target genes. A transactivation domain identified at the C-terminus of beta-catenin can stimulate expression of artificial reporter genes. However, the mechanism of target gene activation by TCF/beta-catenin complexes and the physiological relevance of the beta-catenin transactivation domain still remain unclear. Here we asked whether the beta-catenin transactivation domain can generate a Wnt-response in a complex biological system, namely axis formation during Xenopus laevis embryogenesis. We show that a chimeric transcription factor consisting of beta-catenin fused to the DNA-binding domain of LEF-1 induces a complete secondary dorsoanterior axis when expressed in Xenopus. A LEF-1-beta-catenin fusion lacking the C-terminal transactivation domain is impaired in signaling while fusion of just the beta-catenin transactivator to the DNA-binding domain of LEF-1 is sufficient for axis-induction. The latter fusion molecule is blocked by dominant negative LEF-1 but not by excess cadherin indicating that all events parallel or upstream of the transactivation step mediated by beta-catenin are dispensable for Wnt-signaling. Moreover, beta-catenin can be replaced by a heterologous transactivator. Apparently, the ultimate function of beta-catenin in Wnt signaling is to recruit the basal transcription machinery to promoter regions of specific target genes.
Fig. 2. Induction of an ectopic dorsoanterior axis and siamois gene expression by injecting mRNA coding for a direct fusion of the LEF-1 HMG box to the C- terminal transactivation domain of b-catenin. (a) Micrographs showing duplication of the axis in embryos injected with LEFDN-bCTA RNA. Embryos in the left panels are at neurula stage (st17) with forming neural tubes visible as dark pigmented lines (arrows) and the cement glands observable as dark dots (arrowheads). In the right panels, embryos are shown at the tadpole stage. The upper pannels show normal uninjected embryos. Embryos in the lower pannels were injected with LEFDN-bCTA RNA. Note the double cement glands at neurula stage and the complete secondary head, including extra eyes, at the tadpole stage. (b) RT-PCR analysis of ectopic Siamois expression induced in animal cap explants by injection of LEF-1-b-catenin fusions as indicated on the top. As controls, a sample that was not treated with reverse transcriptase (no RT) and a RT-PCR reaction from a whole embryo were included. The ubiquitously expressed elongation factor 1 (EF-1) was used as control for the presence and integrity of mRNA. (c) Expression of LEF-1-b-catenin fusion constructs as detected by immunoblotting with anti-HA antibodies. Mr: relative molecular mass of standard proteins. Open and solid arrowheads indicate the position of LEFDN-bCTA and LEFDNDHMG-bCTA, respectively.
Fig. 3. The VP16 transactivation domain can functionally replace b-catenin. (a) Expression of LEF-1-VP16 fusions in 293 kidney cells. Mr: relative molecular mass of marker proteins. (b) Activation of the TOPFLASH reporter gene by LEF-VP16 and LEFDN-VP16. Calculation of relative luciferase activity (rel. luc. act.) was as in Fig. 1c. (c) Micrographs showing ectopic axis formation in embryos injected with LEFDN-VP16 RNA. Embryos shown are at neurula stage (st17) with forming neural tubes visible as dark pigmented lines and at tadpole stage with a complete secondary head including an extra eye pair. (d) Rescue of axis formation in UV-treated embryos by LEFDN-VP16. Ventralization and dorsalization were scored at the tadpole stage and are expressed as dorso-anterior index (DAI). Normal embryos have an index of 5. Embryos were UV-treated within 40 min after fertilization and were not further treated (shaded boxes) or were injected at 2 cell stage with 500 pg of LEFDN-VP16 RNA (solid boxes). (e) RT-PCR analysis of ectopic Siamois expression induced in animal cap explants by injection of 500 pg LEFDN-VP16 RNA. As a control for RNA integrity, EF-1 expression was analyzed.
