A Cell-Free Assay System for β-Catenin Signaling That Recapitulates Direct Inductive Events in the Early Xenopus laevis Embryo

Abstract

In vertebrate embryos, signaling via the β-catenin protein is known to play an essential role in the induction of the dorsal axis. In its signaling capacity, β-catenin acts directly to affect target gene transcription, in concert with transcription factors of the TCF/LEF family. We have developed a cell-free in vitro assay for β-catenin signaling activity that utilizes transcriptionally active nuclei and cytoplasm from cleavage-blocked Xenopus laevis embryos. Under these assay conditions, we demonstrate that either addition of β-catenin protein or upstream activation of the β-catenin signaling pathway can induce the expression of developmentally relevant target genes. Addition of exogenous β-catenin protein induced expression of Siamois, XTwin, Xnr3, and Cerberus mRNAs in a protein synthesis independent manner, whereas a panel of other Spemann organizer-specific genes did not respond to β-catenin. Lithium induction of the β-catenin signaling pathway, which is thought to cause β-catenin accumulation by inhibiting its proteasome-dependent degradation, caused increased expression of Siamois in a protein synthesis independent fashion. This result suggests that β-catenin derived from a preexisting pool can be activated to signal, and that accumulation of this activated form does not require ongoing synthesis. Furthermore, activation of the signaling pathway with lithium did not detectably alter cytoplasmic β-catenin levels and was insensitive to inhibition of the proteasome- dependent degradation pathway. Taken together, these results suggest that activation of β-catenin signaling by lithium in this system may occur through a distinct activation mechanism that does not require modulation of levels through regulation of proteasomal degradation.