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Dev Dyn
2004 Nov 01;2313:510-7. doi: 10.1002/dvdy.20150.
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Analysis of the Tcf-3 promoter during early development of Xenopus.
Spieker N
,
Peterson J
,
Reneman S
,
Destrée O
.
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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. [1998] 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 to tail 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, eyes, otic vesicles, notochord, somites and the lateral plate mesoderm. 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.
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 headmesenchyme. 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, headmesenchyme, 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.
