Direct regulation of the Xenopus engrailed-2 promoter by the Wnt signaling pathway, 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 signaling has previously been implicated in vertebrate neural patterning, as evidenced 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 that 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, connexin 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 signaling, we focussed on engrailed-2. In the present study we show that the Xenopus engrailed-2 promoter contains three consensus binding sites for LEF/TCF, which are HMG box transcription factors which bind to beta-catenin in response to activation of the Wnt- 1 signaling pathway. An engrailed-2 promoter luciferase reporter construct containing these LEF/TCF sites is induced in embryo explant assays by the combination of Xwnt-3a or beta-catenin and noggin. These LEF/TCF sites are required for expression of engrailed-2, as a dominant negative Xtcf-3 blocks expression of endogenous engrailed-2 as well as expression of the reporter construct. Moreover, mutation of these three 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 gene 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.
McGrew
L L
LL
Howard Hughes Medical Institute, Department of Pharmacology and Center for Developmental Biology, University of Washington School of Medicine, Seattle, WA 98195, USA.
Takemaru
K
K
Bates
R
R
Moon
R T
RT
D000818:Animals; D019521:Body Patterning / Q000235:genetics; D002352:Carrier Proteins; D019070:Cell Lineage; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005799:Genes, Dominant; D017930:Genes, Reporter; D024002:HMGB Proteins; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051821:Lymphoid Enhancer-Binding Factor 1; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009420:Nervous System / Q000196:embryology; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D051820:TCF Transcription Factors; D015534:Trans-Activators; D058127:Transcription Factor 3; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D051153:Wnt Proteins; D051155:Wnt1 Protein; D014981:Xenopus; D029867:Xenopus Proteins; D029961:Zebrafish Proteins; D051176:beta Catenin; D000099176:Noggin Protein; D000099266:Engrailed 2 Protein
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D024002:HMGB Proteins; D018398:Homeodomain Proteins; D051821:Lymphoid Enhancer-Binding Factor 1; D009419:Nerve Tissue Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D051820:TCF Transcription Factors; D015534:Trans-Activators; D058127:Transcription Factor 3; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D051153:Wnt Proteins; D051155:Wnt1 Protein; D029867:Xenopus Proteins; D029961:Zebrafish Proteins; D051176:beta Catenin; D000099176:Noggin Protein; D000099266:Engrailed 2 Protein; C495266:CTNNB1 protein, Xenopus; C495270:CTNNB1 protein, human; C544912:TCF7L1 protein, human; C544917:Tcf3 protein, Xenopus; C495249:WNT1 protein, Xenopus; C495247:WNT1 protein, human