The canonical Wnt/β-catenin signaling pathway classically functions through the activation of target genes by Tcf/Lef-β-catenin complexes. In contrast to β-catenin-dependent functions described for Tcf1, Tcf4 and Lef1, the known embryonic functions for Tcf3 in mice, frogs and fish are consistent with β-catenin-independent repressor activity. In this study, we genetically define Tcf3-β-catenin functions in mice by generating a Tcf3ΔN knock-in mutation that specifically ablates Tcf3-β-catenin. Mouse embryos homozygous for the knock-in mutation (Tcf3(ΔN/ΔN)) progress through gastrulation without apparent defects, thus genetically proving that Tcf3 function during gastrulation is independent of β-catenin interaction. Tcf3(ΔN/ΔN) mice were not viable, and several post-gastrulation defects revealed the first in vivo functions of Tcf3-β-catenin interaction affecting limb development, vascular integrity, neural tube closure and eyelid closure. Interestingly, the etiology of defects indicated an indirect role for Tcf3-β-catenin in the activation of target genes. Tcf3 directly represses transcription of Lef1, which is stimulated by Wnt/β-catenin activity. These genetic data indicate that Tcf3-β-catenin is not necessary to activate target genes directly. Instead, our findings support the existence of a regulatory circuit whereby Wnt/β-catenin counteracts Tcf3 repression of Lef1, which subsequently activates target gene expression via Lef1-β-catenin complexes. We propose that the Tcf/Lef circuit model provides a mechanism downstream of β-catenin stability for controlling the strength of Wnt signaling activity during embryonic development.

The heterochromatin barrier must be overcome to generate induced pluripotent stem cells and cell fusion-mediated reprogrammed hybrids. Here, we show that the absence of T-cell factor 3 (Tcf3), a repressor of β-catenin target genes, strikingly and rapidly enhances the efficiency of neural precursor cell (NPC) reprogramming. Remarkably, Tcf3(-/-) ES cells showed a genome-wide increase in AcH3 and decrease in H3K9me3 and can reprogram NPCs after fusion greatly. In addition, during reprogramming of NPCs into induced pluripotent stem cells, the silencing of Tcf3 increased AcH3 and decreased the number of H3K9me3-positive heterochromatin foci early and long before reactivation of the endogenous stem cell genes. In conclusion, our data suggest that Tcf3 functions as a repressor of the reprogramming potential of somatic cells.

The co-occupancy of Tcf3 with Oct4, Sox2 and Nanog on embryonic stem cell (ESC) chromatin indicated that Tcf3 has been suggested to play an integral role in a poorly understood mechanism underlying Wnt-dependent stimulation of mouse ESC self-renewal of mouse ESCs. Although the conventional view of Tcf proteins as the β-catenin-binding effectors of Wnt signalling suggested Tcf3-β-catenin activation of target genes would stimulate self-renewal, here we show that an antagonistic relationship between Wnt3a and Tcf3 on gene expression regulates ESC self-renewal. Genetic ablation of Tcf3 replaced the requirement for exogenous Wnt3a or GSK3 inhibition for ESC self-renewal, demonstrating that inhibition of Tcf3 repressor is the necessary downstream effect of Wnt signalling. Interestingly, both Tcf3-β-catenin and Tcf1-β-catenin interactions contributed to Wnt stimulation of self-renewal and gene expression, and the combination of Tcf3 and Tcf1 recruited Wnt-stabilized β-catenin to Oct4 binding sites on ESC chromatin. This work elucidates the molecular link between the effects of Wnt and the regulation of the Oct4/Sox2/Nanog network.

Delineating the signaling pathways that underlie ESC pluripotency is paramount for development of ESC applications in both the research and clinical settings. In culture pluripotency is maintained by leukemia inhibitory factor (LIF) stimulation of two separate signaling axes: Stat3/Klf4/Sox2 and PI3K/Tbx3/Nanog, which converge in the regulation of Oct4 expression. However, LIF signaling is not required in vivo for self-renewal, thus alternate signaling axes likely mediate these pathways. Additional factors that promote pluripotency gene expression have been identified, including the direct regulation of Oct4 by liver receptor homolog-1 (Lrh-1) and β-catenin regulation of Nanog. Here, we present genetic, molecular, and pharmacological studies identifying a signaling axis in which β-catenin promotes pluripotency gene expression in an Lrh-1-dependent manner. Furthermore, Lrh-1 was identified as a novel β-catenin target gene, and Lrh-1 regulation is required for maintaining proper levels of Oct4, Nanog, and Tbx3. Elucidation of this pathway provides an alternate mechanism by which the primary pluripotency axis may be regulated in vivo and may pave the way for small molecule applications to manipulate pluripotency or improve the efficiency of somatic cell reprogramming.

Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon-exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.

A combination of cell intrinsic factors and extracellular signals determine whether mouse embryonic stem cells (ESC) divide, self renew, and differentiate. Here, we report a new interaction between cell intrinsic aspects of the canonical Wnt/Tcf/beta-catenin signaling pathway and extracellular Lif/Jak/Stat3 stimulation that combines to promote self renewal and proliferation of ESC. Mutant ESC lacking the Tcf3 transcriptional repressor continue to self renew in the absence of exogenous Lif and through pharmacological inhibition Lif/Jak/Stat3 signaling; however, proliferation rates of TCF3-/- ESC were significantly decreased by inhibiting Jak/Stat3 activity. Cell mixing experiments showed that stimulation of Stat3 phosphorylation in TCF3-/- ESC was mediated through secretion of paracrine acting factors, but did not involve elevated Lif or LifR transcription. The new interaction between Wnt and Lif/Jak/Stat3 signaling pathways has potential for new insights into the growth of tumors caused by aberrant activity of Wnt/Tcf/beta-catenin signaling.

Single-layered embryonic skin either stratifies to form epidermis or responds to Wnt signaling (stabilized beta-catenin) to form hair follicles. Postnatally, stem cells continue to differentially use Wnt signaling in long-term tissue homeostasis. We have discovered that embryonic progenitor cells and postnatal hair follicle stem cells coexpress Tcf3 and Tcf4, which can act as transcriptional activators or repressors. Using loss-of-function studies and transcriptional analyses, we uncovered consequences to the absence of Tcf3 and Tcf4 in skin that only partially overlap with those caused by beta-catenin deficiency. We established roles for Tcf3 and Tcf4 in long-term maintenance and wound repair of both epidermis and hair follicles, suggesting that Tcf proteins have both Wnt-dependent and Wnt-independent roles in lineage determination.

Two manuscripts published recently in Cell Stem Cell (Lluis et al., 2008 [this issue]; Marson et al., 2008) show that Wnt-beta-catenin signaling stimulates nuclear reprogramming. These two studies, using distinct reprogramming methods, offer insights into the mechanisms underlying acquisition and maintenance of pluripotency.

Elucidating the underlying transcriptional control of pluripotent cells is necessary for the development of new methods of inducing and maintaining pluripotent cells in vitro. Three transcription factors, Nanog, Oct4, and Sox2, have been reported to form a feedforward circuit promoting pluripotent cell self renewal in embryonic stem cells (ESC). Previously, we found that a transcriptional repressor activity of Tcf3, a DNA-binding effector of Wnt signaling, reduced Nanog promoter activity and Nanog levels in mouse ESC (mESC). The objective of this study was to determine the scope of Tcf3 effects on gene expression and self renewal beyond the regulation of Nanog levels. We show that Tcf3 acts broadly on a genome-wide scale to reduce the levels of several promoters of self renewal (Nanog, Tcl1, Tbx3, Esrrb) while not affecting other ESC genes (Oct4, Sox2, Fgf4). Comparing effects of Tcf3 ablation with Oct4 or Nanog knockdown revealed that Tcf3 counteracted effects of both Nanog and Oct4. Interestingly, effects of Tcf3 were more strongly correlated with Oct4 than with Nanog, despite the normal levels of Oct4 in TCF3-/- mESC. The deranged gene expression allowed TCF3-/- mESC self renewal even in the absence of leukemia inhibitory factor (Lif) and delayed differentiation in embryoid bodies. These findings identify Tcf3 as a cell-intrinsic inhibitor of pluripotent cell self renewal that functions by limiting steady-state levels of self renewal factors.______________________________________________________________________________ Author contributions: F.Y.: Conception and Design, Collection of data, Data analysis and interpretation, Manuscript writing; L.P.: Conception and design, Provision of study materials; B.M.: Concept and design, Financial support, Data analysis and interpretation, Manuscript writing.

The Wnt signal transduction pathway has been shown to stimulate stem cell self renewal and has been shown to cause cancer in humans. One interesting aspect of Wnt signaling is that it utilizes downstream DNA-binding transcription factors, called Tcf proteins, which can activate transcription of target genes in the presence of a Wnt signal and repress the expression of target genes in the absence of a Wnt signal. Since Tcf proteins are present in Wnt-stimulated and unstimulated stem cells, understanding how Tcf proteins regulate target gene expression in each state offers the potential to understand how stem cells regulate their self-renewal, differentiation, and proliferation. In this article, we will review recent work elucidating the roles Tcf-protein interactions in the context of stem cells and cancer.