Artefactual gene induction during preparation of Xenopus laevis animal cap explants

The animal cap assay in Xenopus laevis was used to study the induction and regulation of the mesoderm-specific gene Xegr-1, a homolog of the mammalian egr-1 genes. Egr-1 is an immediate-early gene whose growth factor-stimulated transcriptional induction displays a transient activity profile and occurs independent of protein synthesis. The Xegr-1 promoter contains multiple serum response elements (SREs). In this paper we show that Xegr-1 is induced unspecifically during the process of animal cap preparation. Transcripts of Xegr-1 appear already 30 min after cutting of animal caps. Xfos, another SRE-regulated immediate-early gene, is induced with the same kinetics as Xegr-1. In contrast, the Xbra gene is not induced under the experimental conditions used. Xfos and Xegr-1 transcripts are not rapidly down-regulated after mechanical stimulation, but can be detected for up to 4 h later. Wounding-dependent Xegr-1 induction is reduced by injection of either mRNA coding for the dominant inhibitory forms of both the FGF receptor and the transcription factor Elk-1. Xegr-1 expression can be reinduced by mesoderm-inducing factors. These results led us to develop a new protocol for animal cap preparation, which circumvents the observed undesired artefactual gene activation events.