Direct regulation of the Xenopus engrailed-2 promoter by the Wnt signalingpathway, and a molecular screen for Wnt-responsive genes, confirm a role for Wnt signaling during neural patterning in Xenopus

The co-activation of Wnt signaling and concomitant inhibition of BMP signal ing has previously been implicated in vertebrate neural patterning, as evid enced by the combinatorial induction of engrailed-2 and krox-20 in Xenopus. However, screens have not previously been conducted to identify additional potential target genes. Using a PCR-based screening method we determined t hat XA-1, xCRISP, UVS.2, two UVS.2-related genes, and xONR1 are induced in response to Xwnt-3a and a BMP-antagonist, noggin. Two additional genes, con nexin 30 and retinoic acid receptor gamma were induced by Xwnt-3a alone. To determine whether any of the induced genes are direct targets of Wnt signa ling, we focussed on engrailed-2. In the present study we show that the Xen opus engrailed-2 promoter contains three consensus binding sites for LEF/TC F, which are HMG box transcription factors which bind to beta-catenin in re sponse to activation of the Wnt-1 signaling pathway. An engrailed-2 promote r luciferase reporter construct containing these LEF/TCF sites is induced i n embryo explant assays by the combination of Xwnt-3a or beta-catenin and n oggin. These LEF/TCF sites are required for expression of engrailed-2, as a dominant negative Xtcf-3 blocks expression of endogenous engrailed-2 as we ll as expression of the reporter construct. Moreover, mutation of these thr ee LEF/TCF sites abrogates expression of the reporter construct in response to noggin and Xwnt-3a or beta-catenin. We conclude that the engrailed-2 ge ne is a direct target of the Wnt signaling pathway, and that Wnt signaling works with BMP antagonists to regulate gene expression during patterning of the developing nervous system of Xenopus. (C) 1999 Elsevier Science Irelan d Ltd. All rights reserved.