Functional differences between Tcf1 isoforms in early Xenopus development.

In Xenopus gastrula stage embryos, four isoforms of Tcf1 (B, C, D and E) are present with high amino acid sequence conservation compared to fish, mice and human. We studied possible functional differences between these Tcf1 isoforms during early Xenopus development. After overexpression of single Tcf1 isoforms, two distinct phenotypes were observed. Overexpression of the B or D isoforms of Tcf1, which both lack a C-clamp, enhances early canonical Wnt signaling and induces ectopic dorsal mesoderm at the expense of ventrolateral mesoderm prior to gastrulation, causing severe antero-dorzalization of embryos. Overexpression of the E-isoform, which contains a complete C-clamp, does not induce ectopic dorsal mesoderm, but rather leads to severe caudal truncation. Overexpression of the C-isoform, which contains a partial C-clamp, induces a similar phenotype. Mutation of a single amino acid in the C-clamp, known to produce a hypomorphic mutant in D. melanogaster, led to a gain of function in inducing ectopic organizer tissue, as observed after overexpression of the B or D isoforms of Tcf1. Depletion of the C-clamp exon from the zygotic mRNA pool, by injection of a morpholino oligo that targets the splice acceptor site of the exon containing the C-clamp, caused a severe shortening of the AP-axis. Furthermore, embryos showed poor development of the CNS, paraxial mesoderm and primary blood vessels. In situ hybridization analysis showed that Lef1 expression was downregulated at the mid-hindbrain boundary, in the otic vesicles and the branchial arches. The results indicate that in post-gastrula stage Xenopus embryos, the E-tail of Tcf1 is required for expression of Lef1 and for blood vessel formation.

The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated ß-catenin by sequestering TCF4 to the nuclear membrane.

Given its fundamental role in development and cancer, the Wnt-ß-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of ß-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathway.

The Wnt signaling mediator tcf1 is required for expression of foxd3 during Xenopus gastrulation.

TCF1 belongs to the family of LEF1/TCF transcription factors that regulate gene expression downstream of Wnt/ß-catenin signaling, which is crucial for embryonic development and is involved in adult stem cell regulation and tumor growth. In early Xenopus embryos, tcf1 plays an important role in mesoderm induction and patterning. Foxd3 emerged as a potential tcf1 target gene in a microarray analysis of gastrula stage embryos. Because foxd3 and tcf1 are coexpressed during gastrulation, we investigated whether foxd3 is regulated by tcf1. By using morpholino-mediated knockdown, we show that during gastrulation foxd3 expression is dependent on tcf1. By chromatin immunoprecipitation, we also demonstrate direct interaction of ß-catenin/tcf complexes with the foxd3 gene locus. Hence, our results indicate that tcf1 acts as an essential activator of foxd3, which is critical for dorsal mesoderm formation in early embryos.

T-cell factor 4 (tcf7l2) is the main effector of Wnt signaling during zebrafish intestine organogenesis.

The Wnt pathway orchestrates cell fate decisions during embryonic development, organogenesis, and adult tissues homeostasis. T-cell factor (Tcf )/lymphoid enhancer-binding factor (Lef) transcription factors are the downstream effectors of canonical Wnt signaling. Upon Wnt signal activation, beta-catenin stabilizes and translocates to the nucleus, where it interacts with Tcfs activating the transcription of Wnt target genes. In the absence of Wnt, levels of stable beta-catenin are reduced by the action of adenomatous polyposis coli (Apc) and other cytoplasmic proteins. Mutations in Apc cause constitutive accumulation of beta-catenin and inappropriate activation of the Wnt pathway. apc(mcr/mcr) fish embryos show absence of expression of tissue-specific differentiation markers in the intestine, suggesting that inappropriate activation of Wnt signaling abrogates gut organogenesis. Which Tcf transcription factor mediates Wnt signaling during zebrafish gut organogenesis remains unclear. We studied the combined effect of loss of Tcf family members and Apc in the developing embryo. Tcf4 (tcf7l2) loss rescues the apc(mcr/mcr) phenotype in the intestine. Single depletion of Tcf1 (tcf7) and Tcf3 (tcf7l1a) function in an Apc mutant background had no effect on endoderm development. This study reveals that Tcf4 (tcf7l2) is the major effector of Wnt signaling in the intestine during zebrafish organogenesis.

Distinct roles for Xenopus Tcf/Lef genes in mediating specific responses to Wnt/beta-catenin signalling in mesoderm development.

Tcf/Lef transcription factors and beta-catenin mediate canonical Wnt signalling, which plays remarkably diverse roles in embryonic development, stem cell renewal and cancer progression. To investigate the molecular mechanisms allowing for these diverse yet specific functions, we studied the several distinct roles for Wnt/beta-catenin signalling in early Xenopus development: establishing the dorsal body axis; regulating mesoderm induction; and subsequent ventrolateral patterning. Our previous experiments and the expression patterns of Tcf/Lef factors during these embryonic stages led us to examine whether different Tcf/Lef factors mediate these distinct events downstream of canonical Wnt/beta-catenin signalling. By manipulating gene expression with morpholino-driven gene knockdown and capped RNA-mediated rescue, we show that genes encoding different Tcf/Lef transcription factors mediate distinct responses to Wnt signalling in early Xenopus development: Tcf1 and Tcf3 genes are non-redundantly required in mesoderm induction for mediating primarily transcriptional activation and repression, respectively; while ventrolateral patterning requires both Tcf1 and Lef1 genes to express sufficient levels of transcription-activating Tcf factors. Our investigation further identifies that motifs within their central domain, rather than their C-terminus, determine the particular molecular function of Tcf/Lef factors. These findings suggest that Tcf/Lef genes encode factors of different activities, which function together in antagonistic or synergistic ways to modulate the intensity and outcome of Wnt/beta-catenin signalling and to trigger tissue-specific responses.

Tcf-1 expression during Xenopus development.

We report the cloning and expression of Xenopus Tcf-1. The amino acid sequence of Tcf-1 of Xenopus laevis and Xenopus tropicalis is closely related to that of chicken, mouse and man. Thus, the family of Tcf/Lef proteins in the amphibian Xenopus comprises four members as in higher vertebrates. RT-PCR analysis revealed that Tcf-1 RNA encoding a beta-catenin binding isoform is maternally present as well as throughout early development. Different transcripts are expressed by alternative splicing. In cleavage and blastula stage embryos, Tcf-1 RNA is present at high levels in the animal hemisphere. During gastrulation Tcf-1 is differentially expressed with high levels in the animal cap and most of the marginal zone except for a narrow domain around the blastopore. At neurula stages expression is predominant in the neural plate. At tailbud stages expression is localized in specific areas of the brain, in the eyes, the otic vesicle, branchial arches and head mesenchyme, somites, tailbud, pronephros and pronephric duct.