The unlimited potential of the fertilized egg to contribute all differentiated cell types becomes progressively restricted in cell progeny as embryogenesis proceeds. Transplantation experiments and genetic lineage tracing have shown that the cells of the blastocyst inner cell mass (ICM) and their progeny in postimplantation epiblast maintain lineage potential to differentiate into each of the cell types in the adult organism, a property termed pluripotency (38
). Remarkably, the pluripotency and unlimited proliferative capacity of mouse ICM cells were discovered to be maintained during the in vitro culture of so-called embryonic stem cells (ESC) (15
). While in vitro self-renewal of ESC initially required coculture with feeder cells or conditioned media (15
), identification of the leukemia inhibitory factor (LIF) cytokine as the factor secreted by feeders allowed feeder-independent ESC self-renewal in LIF-conditioned media (60
A combination of cell culture and gene ablation studies in mouse embryos has been used to identify intracellular determinants of ICM and ESC pluripotency. Ablation of the gene encoding the homeobox-containing Oct4 (MGI name, POU5F1) transcription factor in mouse embryos prevented proliferation of ICM cells and promoted differentiation into trophectoderm (43
). Reducing Oct4 expression in ESC to 50% promoted trophectoderm differentiation, and increasing Oct4 expression to 150% promoted endoderm differentiation (45
). Ablation of the Sox2 HMG domain-containing protein produced embryos that failed to form an epiblast and ESC that failed to self-renew in vitro (2
). A third transcription factor, the homeodomain-containing Nanog protein, has proven to be both necessary and sufficient for promoting ESC self-renewal (9
). Unlike Oct4, overexpression of Nanog did not induce differentiation but instead was sufficient to maintain pluripotency in the absence of LIF (LIF−
). Gene ablation studies showed that Nanog was required for the proliferation and pluripotency of mouse ICM and ESC (9
). Moreover, differentiation displayed by Nanog+/−
ESC and RNA interference (RNAi)-mediated knockdown of Nanog to 30% of its normal levels showed that reduction of Nanog promoted differentiation in vitro (22
). Thus, although the levels of Oct4 must be maintained between a high and low threshold, Nanog protein must be maintained only above a threshold to promote ESC self-renewal.
Elucidation of extensive molecular genetic interactions between these three intrinsic determinants of ESC characteristics (Oct4, Nanog, and Sox2) has led to the hypothesis that these factors regulate a core transcriptional circuit for ESC self-renewal (4
). This model was supported by the heterodimerization of Oct4 and Sox2 proteins on conserved DNA elements in promoter regulatory regions of several target genes expressed in ESC, including Fgf4, Oct4, Sox2, Nanog, Utf1, Opn, and Fbx15 (3
). Analysis of the promoter regions for each Oct4, Sox2, and Nanog gene showed that the presence of an Oct4-Sox2 DNA-binding site was required for the activation of each promoter in ESC or embryonic carcinoma cells (11
). In addition, small interfering RNA-mediated knockdown of Oct4 or Sox2 reduced the activity of Nanog, Oct4, and Sox2 promoters in ESC (11
). A genome-wide assessment of chromatin binding sites for Oct4, Sox2, and Nanog in human ESC revealed that they not only bound one another's promoter region but also bound a highly overlapping set of target gene promoters (4
). Taken together, these data suggested the existence of a feed-forward circuit whereby Oct4, Sox2, and Nanog all positively regulate one another's promoter activity, thus promoting self-renewal. Given that the embryonic function of ICM and epiblast cells is to differentiate into lineage-committed cells according to spatiotemporal constraints and molecular stimuli, the existence of such a feed-forward circuit would predict inherent stabilizing mechanisms limiting levels of these core transcription factors. In particular, Nanog's ability to promote self-renewal when overexpressed in ESC could require negative feedback to prevent differentiation defects; such a mechanism has not yet been described as functioning within self-renewing ESC.
Tcf proteins (Tcf1, Tcf3, Tcf4, and Lef1 in mammals) are the DNA-binding transcriptional regulators of the canonical Wnt signaling pathway. Through a highly conserved HMG domain and an amino-terminal β-catenin interaction domain, each Tcf protein can promote transcription of downstream targets when Wnt-stabilized β-catenin accumulates intracellularly (6
). In the absence of stabilized β-catenin, Tcf proteins have been shown to function as transcriptional repressors by interacting with corepressor proteins, such as Groucho, CtBP, and HIC-5 (5
). Direct relationships between the biochemical properties of Tcf proteins and their physiological effects have been demonstrated by several studies expressing mutated forms of the proteins in model organisms (28
Although a role for Tcfs in ESC has not yet been identified, the effects of alterations to Wnt signaling on ESC characteristics have suggested that these downstream components of the pathway could play an important role in regulating stem cell characteristics. Treatment of mouse ESC (mESC) with Wnts or Wnt pathway stimulators, such as the glycogen synthase kinase 3-β inhibitor, BIO, promoted self-renewal under LIF−
). Similarly, activation of the Wnt pathway by mutations affecting the adenomatous polyposis coli protein inhibited in vitro mESC differentiation and neural differentiation in teratoma assays (27
). In contrast, Wnt3a stimulation or β-catenin overexpression promoted neural differentiation in mESC grown at high densities (47
). In addition, treatment with Wnts provided a transient stimulation of human ESC proliferation, but it did not prevent differentiation during long-term culture (14
). The contradictory nature of these observations has suggested that Wnt signaling could have multiple and/or complex effects on ESC characteristics and that elucidation of the molecular mechanisms affecting Wnt signaling in ESC could provide a better understanding of how these effects occur. It remains unknown whether the Wnt signaling pathway functionally affects transcription factors Nanog, Oct4, and Sox2 in regulating stem cell self-renewal.
The purpose of this study was to determine whether Tcf factors affect the process of self-renewal and differentiation of ESC. Although expression of all four Tcf mRNAs was detected in mESC, Tcf3 accounted for nearly two thirds of total Tcf expression. Removal of Tcf3 by genetic ablation caused ESC to delay differentiation in favor of self-renewal. These defects were associated with elevated levels of Nanog protein, mRNA, and promoter activity in self-renewing ESC. The mechanism causing increased Nanog required Tcf3 repressor activity and Tcf3 binding to the Nanog promoter DNA. The effects of Tcf factors on Nanog promoter activity link Wnt signaling to the proposed feed-forward circuit regulating ESC self-renewal. By limiting the level of Nanog in self-renewing ESC, Tcf3 functions in ESC to allow appropriate and efficient responses to differentiation-promoting cues.