Cloning and Characterization of ThreeXenopus Slug Promoters Reveal Direct Regulation by Lef/β-Catenin Signaling

Jérôme Vallin,Raphaël Thuret,Emiliana Giacomello,Marisa M Faraldo,Jean P Thiery,Florence Broders

doi:10.1074/jbc.m103167200

Abstract

In amphibians and birds, one of the first steps of neural crest cell (NCC) determination is expression of the transcription factor Slug. This marker has been used to demonstrate that BMP and Wnt molecules play a major role in NCC induction. However, it is unknown whether Slug expression is directly or indirectly regulated by these signals. We report here the cloning and characterization of three Xenopus Slug promoters: that of the Xenopus tropicalis slug gene and those of two Xenopus laevis Slug pseudoalleles. Although the three genes encode proteins with almost identical amino acid sequences and are expressed with similar spatiotemporal patterns, their 5'-flanking regions are quite different. A striking difference is a deletion in the X. tropicalis gene located precisely at the transcription initiation site that results in the X. tropicalis promoter being inefficient in X. laevis. Additionally, we identified two regions common to the three promoters that are necessary and sufficient to drive specific expression in NCCs. Interestingly, one of the common regulatory regions presents a functional Lef/beta-catenin-binding site necessary for specific expression. As the Lef.beta-catenin complex is a downstream effector of Wnt signaling, these results suggest that Xenopus Slug is a direct target of NCC determination signals.

Highlights

In vertebrates, neural crest cells (NCCs)1 form a unique population of cells
As the Lef1⁄7␤-catenin complex is a downstream effector of Wnt signaling, these results suggest that Xenopus Slug is a direct target of NCC determination signals
Recent studies have suggested that Wnt is a direct signal required for NCC determination, whereas fibroblast growth factor-dependent XSlug induction would be a consequence of an indirect effect mediated by Wnt (10)

Summary

EXPERIMENTAL PROCEDURES

Slug Gene Isolation and Sequence Analyses—The X. tropicalis gene was isolated from a genomic DNA library A 500-bp fragment of previously described X. laevis Slug cDNA (7) was used as a probe. Each construct was inserted in the Sleeping Beauty plasmid between the two inverted terminal repeats (14) These inverted terminal repeats enable genomic integration if transposase is co-injected, which was not the case here. Since this plasmid is remarkably stable and nontoxic, it can be used alone for ectopic expression in Xenopus embryos. To detect Slug transcripts, the downstream primer 5Ј-TCGGAAAGTTTGGTTTGGAGTC was combined with 5Ј-CGGGCGAGGACGAAACCAT for slug ␣, with 5Ј-AGGCCGGGGAGGAAACATA for slug ␤, and with 5Ј-CTGTGCTACCCCAGCCTGACAT for the amplification of both Slug genes. For each X. laevis Slug gene, the 5Ј-UTR was used as a short specific probe. Protein1⁄7DNA complexes were subjected to electrophoresis on 4.5% native acrylamide gels and visualized by autoradiography

RESULTS

Characterization of Xenopus Slug Promoters

Region B

DISCUSSION