November 1, 2004;
Analysis of the Tcf-3 promoter during early development of Xenopus.
XTcf-3 functions as a transcriptional regulator in the canonical Wnt signaling cascade and can repress or activate downstream target genes. Expression of XTcf-3 is differentially regulated in time and place during development (Molenaar et al.  Mech Dev. 75:151-154), but little is known about the mechanisms that control transcriptional activation and repression. A 15-kb genomic fragment of Tcf-3 sequences from Xenopus tropicalis was cloned, including the 5'' untranslated region; exons 1, 2, and 3; and intron sequences. We used 5'' deletion constructs for transgenesis and episomal luciferase assays in Xenopus to examine temporal and spatial regulation of the promoter during early development. A -3054/+34-bp Tcf-3 upstream region was identified that drives a green fluorescent protein (GFP) reporter transgene in a pattern similar to endogenous expression of XtTcf-3 from gastrula
bud stages. At stage 12, expression of the reporter is restricted to the middle and posterior neurectoderm
. At stage 22, expression is strongest in the neural plate, the eye
anlagen and branchial arches. At stage 35/36, expression is found in the head mesenchyme
, the branchial arches, the heart
, the mesencephalon
, otic vesicles, notochord
and the lateral
. Part of the cis-acting elements driving this GFP reporter transgene expression map between -372 and -95 bp of the transcription start site. Furthermore, two TCF/LEF sites are necessary for full activity of the promoter during gastrula
stages in episomal luciferase assays.
[+] show captions
Figure 2. Expression of the (-3054/+71)-XtTcf-3::GFP reporter in transgenic X. laevis embryos. A-H: Transcripts of the green fluorescent protein (GFP) reporter (E-H) or endogenous XlTcf-3 (A-D) were detected by whole-mount in situ hybridization with GFP or XlTcf-3 antisense RNA probes, respectively. The expression pattern of the XtTcf-3::GFP transgene overlapping that of endogenous XTcf-3 is indicated by white arrowheads; differences in expression levels are indicated by black arrowheads (for further details see text). I-N: Sections of stage 36 embryos (I-K for endogenous XTcf-3, L-N for transgene expression). Arrowheads in I and L indicate endogenous and transgene expression in the (sub)ependymal layer of the mesencephalon, in the eye, and head mesenchyme. Arrowheads in J and M indicate expression in the epithelium of the otic vesicle. Arrowheads in K and N indicate expression in the lateral plate mesoderm and in the myocardium and endocardium.
Figure 4. Deletion analysis of the XTcf-3 promoter in representative transient transgenic Xenopus laevis. A series of 5 prime deletion constructs was analyzed for green fluorescent protein (GFP) expression in situ. A-C: The expression pattern of the GFP transgenes -619/+34::GFP (A), -372/+34::GFP (B), and -206/+34::GFP (C) is similar to that of the endogenous Tcf-3 expression at stage 35/36 with expression found in specific parts of the brain, including the mesencephalon, eyes, head mesenchyme, branchial arches, heart, otic vesicles, notochord, somites, and lateral plate mesoderm. D: The -95/+34::GFP construct showed only some expression around the eye and tail bud but none of the other structures. E: Further delineation of the construct to -45/+34 bp showed similar loss of expression (for further details, see text and Table 1). F: As a control, in situ hybridization of wild-type embryos with GFP-AS probe showed no staining after 2 days of incubation. G: A representative X. tropicalis transgenic embryo displays an expression pattern of the -372/+34::GFP transgene comparable to its expression in transgenic X. laevis, although expression in the ventral area appears higher and more extended. H: This expression is lost at stage 40. Results indicate that transcriptional regulation of XtTcf-3 is similar in both Xenopus species and that X. laevis can be used for transient analysis of regulatory sequences.
Fig. 3. A 5′ deletion and mutation analysis of the XtTcf-3 promoter. A:Xenopus laevis embryos were injected with 100 pg of supercoiled DNA at the one-cell stage and allowed to develop to stage 11.5. Five embryos were pooled and lysed; an equivalent of one embryo was measured. B: Mutagenesis of the TCF/LEF sites. X. laevis embryos were injected with 100 pg of supercoiled DNA of construct with both sites mutated, one site mutated or intact TCF/LEF consensus binding sites. Injections were done at the one-cell stage, and embryos were allowed to develop to stage 10.5 and stage 12. Five embryos were pooled and lysed; an equivalent of one embryo was measured. Mutation of both TCF/LEF consensus sites gave a strong reduction of luciferase activity at stage 10.5 (74%) and stage 12 (72%). A construct with only one of the binding sites mutated showed 14% reduction in luciferase activity.