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. 2001 Jul 10;98(15):8668–8673. doi: 10.1073/pnas.151258098

Rescue of a Wnt mutation by an activated form of LEF-1: Regulation of maintenance but not initiation of Brachyury expression

Juan Galceran 1, Shu-Chi Hsu 1, Rudolf Grosschedl 1,*
PMCID: PMC37493  PMID: 11447280

Abstract

Members of the LEF-1/TCF family of transcription factors have been implicated in mediating a nuclear response to Wnt signals by association with β-catenin. Consistent with this view, mice carrying mutations in either the Wnt3a gene or in both transcription factor genes Lef1 and Tcf1 were previously found to show a similar defect in the formation of paraxial mesoderm in the gastrulating mouse embryo. In addition, mutations in the Brachyury gene, a direct transcriptional target of LEF-1, were shown to result in mesodermal defects. However, direct evidence for the role of LEF-1 and Brachyury in Wnt3a signaling has been limiting. In this study, we genetically examine the function of LEF-1 in the regulation of Brachyury expression and in signaling by Wnt3a. Analysis of the expression of Brachyury in Lef1−/−Tcf1−/− mice and studies of Brachyury:lacZ transgenes containing wild type or mutated LEF-1 binding sites indicate that Lef1 is dispensable for the initiation, but is required for the maintenance of Brachyury expression. We also show that the expression of an activated form of LEF-1, containing the β-catenin activation domain fused to the amino terminus of LEF-1, can rescue a Wnt3a mutation. Together, these data provide genetic evidence that Lef1 mediates the Wnt3a signal and regulates the stable maintenance of Brachyury expression during gastrulation.

Signaling by Wnt proteins regulates multiple biological processes that include cell fate decisions, cell proliferation, cell polarisation, and morphogenetic movements (1, 2). Genetic analysis of Wnt signaling in the mouse by targeted inactivation of Wnt genes has revealed a role of this signaling pathway in the patterning of tissues, such as the isthmic organizer of the midbrain/hindbrain boundary and the formation of organs and tissues, including kidney and placenta (3–6). In addition, a targeted null mutation in Wnt3a has been show to impair the formation of paraxial mesoderm in the early gastrulating mouse embryo (7). Wnt3a mutant mice form extra neural tubes at the expense of mesoderm, suggesting that Wnt3a regulates the balance between mesodermal and neural fates (8). A naturally occurring hypomorphic mutation of Wnt3a, termed vestigial tail (vt), which is allelic with Wnt3a, decreases the level of Wnt3a expression and abrogates tail formation in homozygous mice (9). The vt mutation also impairs the generation of caudal vertebra, consistent with the mutant phenotype of the Wnt3a null mutation.

Biochemical experiments and cell transfection assays have been used to show that the transcriptional effects of signaling by Wnt proteins are mediated by LEF-1/TCF transcription factors (10–15). LEF-1 has no transcriptional activation potential by itself and can stimulate transcription only in collaboration with other proteins (16). In the absence of Wnt signals, LEF-1 can associate either with coactivators, such as ALY (17), or with corepressors, such as Groucho (18), to activate or repress specific target genes, respectively (17, 18). In the presence of Wnt signals, LEF-1/TCF proteins associate with β-catenin and activate transcription from synthetic enhancers containing multimerized LEF-1/TCF binding sites or from natural Wnt-responsive enhancers or promoters in transfected tissue culture cells (11, 13, 19–21). The association of β-catenin with LEF-1/TCF proteins is mediated through evolutionarily conserved amino acids at the amino terminus of the transcription factors (19, 22, 23). β-catenin contains amino- and carboxy-terminal transcriptional activation domains and a covalent fusion of either domain with the amino terminus of LEF-1 generates a fusion protein that functions constitutively and activates transcription independently of a Wnt signal (19).

The number of direct target genes for Wnt signals in the mouse has been limiting. Brachyury, one of the earliest genes expressed at the onset of gastrulation during embryonic stage 6.5 (E6.5), has been shown to be regulated by Wnt3a signaling in the posterior mesoderm (7, 24, 25). Likewise, the transcription factor genes Lef1 and Tcf1 are expressed in the primitive streak during gastrulation in a pattern that overlaps that of Wnt3a (26, 27). Although targeted mutation of Lef1 or Tcf1 genes alone did not generate any phenotype that resembled that of the Wnt3a mutation, inactivation of both Lef1 and Tcf1 resulted in a Wnt3a−/−-like phenotype (26, 28, 29). Thus, these transcription factors act redundantly to regulate mesoderm formation in the gastrulating mouse embryo, presumably by mediating a nuclear response to a Wnt3a signal (26). A role of LEF-1/TCF transcription factors in the regulation of Brachyury has been implicated from experiments demonstrating that the Brachyury promoter contains binding sites for LEF-1 that are functionally important for expression of a transgene in early mouse embryos (24, 25). Taken together, these experiments suggest that LEF-1 and Brachyury form a transcriptional cascade in the transduction of Wnt3a signaling in the early mouse embryo. However, no formal genetic proof for the role of LEF-1/TCF proteins in Wnt signaling has yet been obtained.

In this study, we demonstrate that LEF-1 is required specifically for the maintenance, but not the initiation of Brachyury expression. In addition, the rescue of the tail deficiency in Wnt3avt/vt embryos with a constitutive β-catenin-LEF-1 fusion protein provides direct genetic evidence that LEF-1 is a downstream effector of signaling by Wnt3a.

Materials and Methods

Transgene Construction.

The XbaI-BglII fragment from pEVRF0 (30) containing the splice donor, IVS2, and exon 3 with the polyadenylation site from rabbit β globin gene was subcloned into Bluescript II KS+ to generate pBSpA. This gene construct was used for insertion of the murine Brachyury promoter (nucleotides −429 to +221 in ref. 31; coordinates are taken from ref. 32). The resulting gene construct, pBrapA, was used for subsequent cloning of the catC-Lef1 fusion gene or the bacterial lacZ gene. The catC-Lef1 fusion gene, in which the C-terminal activation domain of human β-catenin (amino acids 696–781) is linked to the amino terminus of LEF-1, was obtained from pCMV-catCLEF-1 (19). The lacZ DNA fragment was derived from hsp68lacZpA (33). In the resulting plasmids, pBra-catCLef1 and pBra-lacZ, the 5′ untranslated region and the initial methionine of Brachyury were fused in frame to the catC-Lef1 and lacZ genes, respectively. Both gene constructs were sequenced to confirm the reading frame. pmutBra-lacZ was generated by site-directed mutagenesis, using single-stranded DNA from pBra-lacZ and the mutant oligonucleotide 5′-TGCTCGGTACTTGAATTCGTGTCCCGC. DNA constructs were digested with SacII and KpnI, and the inserts were purified to remove vector sequences and used for pronuclear injection following standard protocols (34).

Genotyping and Wnt3a-Vestigial-Tail Analysis.

Transgenic animals carrying the wild-type Bra-lacZ or mutant mutBra-lacZ transgenes were dissected at the indicated times of gestation and the yolk sacs were used for PCR genotyping with primers derived from the Brachyury promoter 5′-GAAGTGAAGGTGGCTGTTGG and the lacZ gene 5′-CGATCGGTGCGGGCCTCTTCGCTAT. To identify animals carrying the catC-Lef1 transgene, genotyping was performed by PCR using primers for the human β-catenin gene 5′-CGTTCTTTTCACTCTGGTGGA and the murine Lef1 gene 5′-GTTAACCAAAGATGACTTGATG. Lef1−/− Tcf1−/− embryos were generated and genotyped as described (26).

The vestigial tail mouse stock B6C3H-a/A-wa2-vt was purchased from Jackson Laboratory (Bar Harbor, ME) and maintained by backcrossing with C57BL6/J mice. Genotyping was performed by simple sequence length polymorphism (SSLP) analysis as described (9).

Electrophoretic Mobility Shift Assay.

DNA binding assays were performed as described (19). The binding reaction contained 100 ng of purified His-6-tagged LEF-1 protein and 2 fmol of 5′ end-labeled double-stranded oligonucleotide in 10 mM Hepes (pH 7.9), 50 mM NaCl, 1 mM DTT, 5 mM EDTA, 5% glycerol, 50 μg/ml BSA, and 10 μg/ml poly(dI-dC)⋅poly(dI-dC). The binding reaction was performed in the absence or presence of increasing amounts of unlabeled competitor DNA. The nucleotide sequences of the probes for Brachyury site I (BraSI) and competitor DNA were as follows. Bra-SI wt: 5′-TGCTCGGTACTTCAAAGGGTGTCCCGCTAGCGATC; Bra-SI mut: 5′-TGCTCGGTACTTGAATTCGTGTCCCGCTAGCGATC; competitor wt: 5′-GCACCCTTTGAAGCTCGCTAGCGATC; competitor mut: 5′-GCACCAATTCAAGCTCGCTAGCGATC.

Xgal Staining and Whole Mount in Situ Hybridization.

Dissected embryos were fixed with 4% paraformaldehyde in PBS for 10 min on ice and stained for β-galactosidase activity by incubation in PBS containing 2 mM MgCl2, 5 mM K4Fe(CN)6, 5 mM K3Fe(CN)6, and 0.4 mg/ml X-gal at 37°C for 2 to 24 h. After staining, the embryos were washed in PBS and further fixed in 4% paraformaldehyde in PBS at 4°C. The procedure and the probes for in situ hybridizations were the same as described (26).

Results

A role of LEF-1/TCF factors in the regulation of Brachyury was inferred from recent experiments in which mutations of binding sites for LEF-1 in the Brachyury promoter, linked to a lacZ transgene, were found to result in the loss of transgene expression in E9.5 embryos (24). Therefore, the question arose as to whether LEF-1 and TCF-1 regulate the expression of Brachyury throughout early mouse development. Toward this end, we examined the expression pattern of Brachyury in wild-type and Lef1−/−Tcf1−/− mice at different stages of development (Fig. 1). As anticipated, Brachyury expression could not be detected in compound mutant embryos at E9.5 (Fig. 1i). However, expression of Brachyury was detected in Lef1−/−Tcf1−/− mice at E8.5, albeit at a reduced level relative to wild-type embryos (Fig. 1f). At E7.5, expression of Brachyury in compound mutant embryos was almost indistinguishable from that in wild-type embryos (Fig. 1 b and c). Because other members of the Lef1/Tcf family are not expressed at significant levels in the posterior region of the early mouse embryo (26, 35), this result suggests that LEF-1 and TCF-1 act specifically in the maintenance, but not in the initiation of Brachyury expression.

Figure 1.

Figure 1

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Maintenance but not initiation of Brachyury expression is regulated by LEF-1 and TCF-1 during early mouse development. Whole mount in situ hybridizations of wild-type and Lef1−/−Tcf1−/− embryos between days 7.5 and 9.5 of gestation (E7.5–E9.5) to detect Brachyury or Lef1 transcripts. At E7.5, Brachyury expression can be detected in the primitive streak (PS) of both wild-type and compound mutant embryos (b and c). In E8.5 mutant embryos, expression in the primitive streak is reduced relative to the wild-type embryo (e and f). Expression of Brachyury is specifically reduced in the anterior part of the primitive streak, similar to Wnt3a−/− embryos (Insets in e and f; ref. 24). At E9.5, compound mutant embryos contain Brachyury transcripts only in the notochord (NC), whereas wild-type embryos express Brachyury in both the tailbud (TB, arrow) and the notochord. For comparison, Lef1 shows a temporal and spatial expression pattern that overlaps the expression pattern of Brachyury in wild-type embryos (a, d, and g). In addition, Lef1 expression is observed in the forelimbs (FL).

To examine whether this temporal regulation of Brachyury expression is mediated by direct binding of LEF-1 to the Brachyury promoter, we generated transgenes in which the wild-type or a mutated Brachyury promoter was fused to the bacterial lacZ gene (Bra:lacZ). The Brachyury promoter contains three binding sites for LEF-1 (24, 25). We introduced a triple point mutation in the proximal LEF-1 binding site (−117/−110) and confirmed by competition in electrophoretic mobility shift assays that mutation of this binding site significantly impaired the interaction with LEF-1 (Fig. 2 A and B). Analysis of E9.5–E10.5 embryos carrying the wild-type transgene (Bra:lacZ) revealed that lacZ is expressed in the tailbud of 13 out of 16 different transgenic mice (Fig. 2C Left). In contrast, none of 13 embryos carrying the mutant transgene (mutBra:lacZ) showed lacZ expression (Fig. 2C Middle). Thus, mutation of a single LEF-1 binding site abrogates the activity of the Brachyury promoter at E10.5.

Figure 2.

Figure 2

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Brachyury is a direct target of LEF-1. (A) Electrophoretic mobility shift assays to detect binding of purified recombinant LEF-1 to sequences in the Brachyury promoter. Specific binding to the promoter-proximal LEF-1 recognition sequence (5′-TTCAAAGG, nucleotides −117/−110) is shown by a competition experiment in which the protein⋅DNA complex is sensitive to the addition of an excess of a LEF-1 binding site consensus oligonucleotide (5′-TTCAAAGG), but is resistant to the addition of a corresponding mutated oligonucleotide (5′-TTGAATTC). The positions of the bound and free DNA probes are indicated. (B) Structure of a Brachyury:lacZ transgene in which the wild-type or mutated Brachyury (Bra) promoter is linked to the bacterial β-galactosidase (lacZ) gene. To allow for stable transcript accumulation, intron and polyadenylation sequences from the human β-globin gene were included. The nucleotide sequences of the wild-type (wt) and mutated (mut) proximal LEF-1 binding site are indicated. (C) Expression of the Bra:lacZ and mutBra:lacZ transgenes in E10.5 embryos. Thirteen of 16 transgenic embryos expressed the wild-type transgene in the tailbud (arrow), whereas none of 13 embryos expressed the mutant transgene. In E7.5 embryos, the mutant transgene is expressed in the primitive streak (PS). (Scale bar, 800 μm for the Left and Center and 225 μm for the Right.)

We also examined whether the Brachyury promoter mediates the transient expression of the endogenous Brachyury gene in early Lef1−/−Tcf1−/− embryos. Toward this end, we analyzed the expression of the mutBra:lacZ transgene at E7.5. At this early developmental stage, the mutant transgene was expressed, indicating that the Brachyury promoter can establish gene expression in a LEF-1-independent manner (Fig. 2C Right). Taken together, these data suggest that LEF-1 is dispensable for activation of the Brachyury promoter, but is required for maintenance of Brachyury expression during gastrulation.

Two lines of evidence suggest that LEF-1 and TCF-1 mediate the nuclear response to a Wnt3a signal. First, activation of synthetic LEF-1/TCF enhancers or Wnt-responsive enhancers/promoters in tissue culture cells depends on the association of LEF-1/TCF proteins with β-catenin (13, 19). Second, the targeted inactivation of both Lef1 and Tcf1 genes results in a mutant phenotype that is very similar to that of the null mutation of the Wnt3a gene (7, 26). In particular, the specific down-regulation of Brachyury in the anterior streak of Lef1−/−Tcf1−/− embryos at early somite stages supports the similarity with the phenotype of the Wnt3a mutation (Fig. 1 e and f; ref. 24). To obtain direct genetic evidence for the role of LEF-1 in Wnt signaling, we attempted to rescue a mutation in the Wnt3a gene by a constitutively active form of LEF-1. For this experiment, we used a covalent fusion of the carboxy-terminal activation domain of β-catenin with the amino terminus of LEF-1, which can activate a synthetic LEF-1 enhancer in the absence of a Wnt signal (19). In addition, the initial expression of Brachyury in Lef1−/−Tcf1−/− embryos enabled us to use the Brachyury promoter for the expression of the LEF-1-β-catenin fusion protein (catC-LEF-1) in Wnt3a-deficient embryos. For the rescue, we chose the naturally occurring hypomorphic allele of Wnt3a, termed vestigial tail (vt). In homozygous mice, this mutant allele results in a reduced level of Wnt3a expression and a severe defect in tail formation (9).

We introduced the Brachyury:Lef1-β-catenin gene (catC-Lef1, Fig. 3A) into the mouse germ line and found by whole mount in situ hybridization that the transgene is expressed in the primitive streak of E8.5 embryos (Fig. 4A). To examine whether the catC-Lef1 transgene could rescue the mutant Wnt3avt/vt phenotype, we crossed the transgene into vt/vt mice. A cross between a nontransgenic vt/vt male with two vt/+ females carrying the transgene generated three vt/vt offsprings carrying the transgene (Fig. 3B and data not shown). The genotyping of the vt allele was performed by PCR amplification of a nucleotide sequence polymorphism that is associated with the vt allele (9) and the presence of the transgene was determined by genomic PCR (Fig. 3 C and D). Newborn vt/vt offsprings showed the characteristic truncation of the tail, whereas vt/vt mice carrying the transgene all had normal tails (Fig. 3E). Wild type and +/vt mice carrying the transgene had normal tails (Fig. 3E and data not shown). This result was confirmed by additional crosses, which produced a total of 50 offspring. From these crosses, only 13 newborns, instead of the expected 25, were tail-less and all of them lacked the transgene (data not shown). Thus, the catC-Lef1 transgene rescues the defect of the Wnt3avt/vt mutation, providing direct evidence that LEF-1 mediates the Wnt3a signal.

Figure 3.

Figure 3

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Rescue of a Wnt3a mutation by a β-catenin-LEF-1 fusion protein. (A) Structure of the β-catenin-Lef1 transgene (catC-Lef1), encoding a protein in which the carboxy-terminal activation domain of β-catenin is fused to the amino terminus of full-length LEF-1 (19). The coding sequence is linked to the Brachyury promoter [nucleotides −429 to + 221 (31, 32) and the rabbit β-globin splice and polyA site (30)]. (B) Pedigree of a cross between a vt/vt male (filled square) and +/vt female (half-filled circle) carrying the transgene. The offspring are represented by filled (vt/vt) or half-filled (+/vt) diamonds. (C) Analysis of the wild-type and vt allele by simple sequence length polymorphism (SSLP). PCR amplification of Wnt3a genomic sequences by using SSLP to detect differences between the wild-type and vt alleles (9). (D) Detection of the catC-Lef1 transgene (Tg) by genomic PCR amplification. (E) Tail phenotypes of newborn littermates from the cross between a vt/vt male and a +/vt female that carries the catC-Lef1 transgene. vt/vt offsprings have a truncated tail, whereas vt/vt mice carrying the catC-Lef1 transgene have a normal tail.

Figure 4.

Figure 4

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Molecular analysis of the rescue of the Wnt3avt/vt mutation by expression of a constitutively active form of LEF-1. (A) Whole-mount in situ hybridization to detect the expression of the catC-Lef1 transgene, encoding a protein in which the C-terminal activation domain is fused to the amino terminus of LEF-1, in E8.5 and E10.5 embryos (a and b). Expression of the transgene is detected in the primitive streak (PS) and tailbud (arrow) in a pattern that overlaps with that of endogenous Brachyury (c and d). In addition, Brachyury is expressed in the notochord (NC). The distal portion of the tails of the E10.5 embryos is shown at a higher magnification in the inset. (B) Expression of Wnt3a, Brachyury, and Notch1 in the tailbud of wild-type, Wnt3avt/vt (vt/vt), and Wnt3avt/vtcatC-Lef1 (vt/vt catC-Lef1) embryos at E10.5. Whole mount in situ hybridization showing that the levels of Wnt3a expression in the tailbud (arrow) of a vt/vt embryo, which is markedly reduced relative to the wild-type embryo, is also reduced in vt/vt catC-Lef1 embryos (a, b, and c). In contrast, Wnt3a expression in the neural tube is similar in all three embryos. Expression of Brachyury, which is slightly reduced in the tailbud of vt/vt embryos relative to wild-type embryos, is also affected in the notochord (arrow), presumably because of defective mesoderm formation (d and e). In vt/vt catC-Lef1 embryos, normal Brachyury expression is observed, including a normally formed notochord (f). Expression of Notch1, a marker for the presomitic mesoderm in the region adjacent to the first forming somite (45) is reduced in vt/vt embryos, but is normal in the vt/vt catC-Lef1 embryos (g, h, and i). These patterns of expression were confirmed by whole mount in situ hybridizations of multiple embryos (data not shown).

To obtain molecular evidence for the rescue of the Wnt3avt/vt mutation by the catC-Lef1 transgene, we analyzed transgenic and nontransgenic mice for the expression of Wnt3a, Brachyury, and Notch1. We confirmed the proper expression of the transgene by whole mount in situ hybridization and compared the expression pattern with that of Brachyury at E8.5 and E10.5 (Fig. 4A). A similar pattern of expression was observed, although expression of the transgene was restricted to the more caudal region of the primitive streak and was not detected in the notochord. For the analysis of molecular markers in Wnt3avt/vt and Wnt3avt/vtcatC-Lef1 mice, we used E10.5 embryos because at this developmental stage the expression of endogenous Wnt3a is severely reduced in vt/vt mice, before the loss of somites can be detected (ref. 9 and Fig. 4).

A positive feedback loop between the target gene of LEF-1, Brachyury, and Wnt3a has been proposed (24). However, in both Wnt3avt/vt and in Wnt3avt/vtcatC-Lef1 embryos, the expression of Wnt3a is markedly reduced (Fig. 4B), indicating that the constitutively active form of LEF-1 does not up-regulate the expression of Wnt3a. Expression of Brachyury, which is modestly reduced in the tailbud of Wnt3avt/vt mice is normal in Wnt3avt/vtcatC-Lef1 embryos, consistent with the regulation of the Brachyury promoter by LEF-1. Thus, these data suggest that Wnt3a and Brachyury do not form a positive feedback loop. The expression of Brachyury in the notochord is also restored in the transgenic mice. Moreover, expression of Notch1, a marker for somite formation, is severely impaired in Wnt3avt/vt mice, but is normal in Wnt3avt/vtcatC-Lef1 embryos. Taken together, the expression of the catC-Lef1 transgene rescues both the morphological and the molecular defects of the Wnt3avt/vt mice, providing formal genetic proof that LEF-1 mediates signaling by Wnt3a.

Discussion

One result of our experiments is the finding that LEF-1 and TCF-1 regulate the maintenance, but not the initiation of Brachyury expression. A role of Wnt signaling in the maintenance of gene expression has been found in the Drosophila embryo, in which Wingless signaling in the epidermis is required for the maintenance of engrailed expression (36–38). Likewise, Wnt1 signaling regulates the maintenance of engrailed-1 expression in the developing central nervous system of the mouse (39). Thus, the in vivo analysis of two Wnt target genes in the mouse indicates a role of this signaling pathway in the maintenance rather than initiation of gene expression.

The signals that initiate Brachyury expression independently of LEF-1 and TCF-1 are still unknown. Two possible mechanisms may be involved in the initiation of Brachyury expression in the primitive streak. Firstly, the Brachyury promoter is regulated by FGF and activin signals in Xenopus and by activin signals and Ets transcription factors in transfected mammalian P19 cells (32, 40). These factors may be involved in the initiation of Brachyury expression during early mouse development. Secondly, Wnt3 is expressed before gastrulation at E6 in a domain similar to that of the initial Brachyury expression (41). In addition, Brachyury is not expressed in Wnt3−/− embryos, consistent with the initiation of Brachyury expression by Wnt3 (41). This regulation by Wnt3 could be mediated by the low levels of TCF-3, which is found to be broadly expressed throughout the embryo (35). The observation that the mutant Brachyury promoter–lacZ construct is still expressed at E7.5 would argue against this interpretation; however, we mutated only one of three LEF-1/TCF binding sites in the Brachyury promoter. Therefore, it is possible that the remaining two sites mediate the early activation of the promoter. In addition, we cannot rule out the possibility that signaling by Wnt3 is independent of LEF-1/TCF proteins, similar to Wnt5a, which has been found to elicit cellular responses independently of changes in β-catenin levels (42). Although Wnt5a is also expressed in the primitive streak, it has been shown that Wnt5a is not required for Brachyury expression (43). Thus, Wnt3 or FGF signaling might regulate the initiation of Brachyury expression, whereas Wnt3a signaling, either alone or in combination with other signaling pathways, controls the maintenance of Brachyury expression.

In addition to the potential role of FGF signaling in the initiation of Brachyury expression, Brachyury has been found to directly regulate the expression of the embryonic eFgf gene during mesoderm formation in Xenopus (44). The Xenopus eFgf gene is closely related to the mammalian Ffg4 gene, and the regulation of Fgf4 by Brachyury appears to be conserved in mouse and humans (44). Thus, FGF-4 may act both upstream and downstream of Brachyury, generating a reinforcing feedback loop that would allow for sustained FGF signaling and differentiation of mesoderm. Although Wnt3a itself does not seem to be directly part of this autoregulatory loop, the dependence of Brachyury expression on LEF-1 would indirectly link these signaling pathways.

The transcriptional hierarchy of LEF-1 and Brachyury allows for temporal regulation of genes, which together with the potential collaboration between different signaling pathways may help to implement a complex differentiation program. A notable hallmark of LEF-1 is the marked influence of its transcriptional activity on the presence of other specific transcription factors. Although LEF-1 can activate a synthetic enhancer containing multimerized LEF-1 binding sites in transient transfection assays, such a synthetic LEF-1 enhancer is nonfunctional in transgenic Drosophila embryos, even in regions in which LEF-1 is coexpressed with wingless, the fly orthologue of Wnt (15). In the Drosophila ultrabithorax enhancer, the response to wingless signaling depends on the association of LEF-1 with armadillo and the cooperation of LEF-1/β-catenin with a member of the CREB family that binds to a dpp response element in the vicinity of a LEF-1 binding site (15). A specific context dependence of LEF-1 function has also been shown for the T cell receptor (TCR) α enhancer, which is regulated by LEF-1 in a β-catenin-independent manner (16). Thus, it will be of interest to compare the LEF-1-mediated regulation of the Wnt3a-responsive Brachyury enhancer with that of the β-catenin-independent TCRα enhancer.

Although biochemical experiments and functional studies in tissue culture transfection assays provided strong support for a role of LEF-1 in mediating the response to a Wnt signal, no formal genetic proof has yet been obtained. The rescue of a Wnt3a mutation with a constitutively active form of LEF-1 provides now direct evidence for the role of LEF-1 in Wnt3a signaling in the mouse. A similar rescue of a Wnt1 mutation by an engrailed transgene under the control of a Wnt1 enhancer has shown that engrailed is a critical downstream target of Wnt signaling in formation of the midbrain/hindbrain boundary (39). Similarly, expression of Brachyury and Fgf4 transgenes in Wnt3a-deficient embryos may be used to further examine the transcriptional hierarchy of LEF-1 and Brachyury and to gain insight into the potential network of Wnt3a and FGF signaling in the gastrulating mouse embryo.

Acknowledgments

We are grateful to Roel Nusse and Rudi Balling for gifts of cDNA probes. We thank Hans Clevers for providing us with the Tcf1 knockout mice. We thank Yow-Ning Chang at the University of California, San Francisco Transgenic/Knockout Core Facility for help with the generation of the transgenic animals. We thank Jan Kitajewski for critical reading of the manuscript and members of the Grosschedl lab for discussions. The work has been supported by a grant from the German Science Foundation (DFG).

Abbreviations

En

embryonic stage n

vt

vestigial tail

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