The Pleiotropic Effects of the Canonical Wnt Pathway in Early Development and Pluripotency

Abstract

The technology to derive embryonic and induced pluripotent stem cells from early embryonic stages and adult somatic cells, respectively, emerged as a powerful resource to enable the establishment of new in vitro models, which recapitulate early developmental processes and disease. Additionally, pluripotent stem cells (PSCs) represent an invaluable source of relevant differentiated cell types with immense potential for regenerative medicine and cell replacement therapies. Pluripotent stem cells support self-renewal, potency and proliferation for extensive periods of culture in vitro. However, the core pathways that rule each of these cellular features specific to PSCs only recently began to be clarified. The Wnt signaling pathway is pivotal during early embryogenesis and is central for the induction and maintenance of the pluripotency of PSCs. Signaling by the Wnt family of ligands is conveyed intracellularly by the stabilization of β-catenin in the cytoplasm and in the nucleus, where it elicits the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors. Interestingly, in PSCs, the Wnt/β-catenin-TCF/LEF axis has several unrelated and sometimes opposite cellular functions such as self-renewal, stemness, lineage commitment and cell cycle regulation. In addition, tight control of the Wnt signaling pathway enhances reprogramming of somatic cells to induced pluripotency. Several recent research efforts emphasize the pleiotropic functions of the Wnt signaling pathway in the pluripotent state. Nonetheless, conflicting results and unanswered questions still linger. In this review, we will focus on the diverse functions of the canonical Wnt signaling pathway on the developmental processes preceding embryo implantation, as well as on its roles in pluripotent stem cell biology such as self-renewal and cell cycle regulation and somatic cell reprogramming.

Keywords: Wnt/β-catenin pathway; cell cycle; embryonic stem cells; pre-implantation development; somatic cell reprogramming.

Figure 1

Molecular overview of the Wnt/β-catenin pathway. (A) In absence of Wnt ligands (Wnt OFF conditions), the β-catenin destruction complex phosphorylates β-catenin, which is ubiquitinated by β-TrCP and sent to the proteasome, where it is degraded. Thus, absence of nuclear β-catenin enables repression of Wnt target genes through the T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors. (B) When Wnt ligands bind to the receptor complex, the destruction complex is disassembled allowing the stabilization of β-catenin, which is then able to translocate to the nucleus. Nuclear β-catenin is then able to elicit gene expression changes through the TCF/LEF family of transcription factors. APC: adenomatous polyposis coli; CK1: casein kinase 1; DVL: dishevelled; FZD: frizzled; GSK3β: glycogen synthase kinase 3 beta; LRP5/6: lipoprotein receptor-related protein 5/6; Ub: ubiquitin.

Figure 2

Tracing Wnt activity across early mouse embryogenesis. (A) A schematic overview of the mouse pre-implantation and post-implantation developmental stages, from zygote formation (E0.5) until the pre-gastrulation stage (E6.5). After fertilization, the zygote undergoes a series of mitotic divisions together with progressive cell fate acquisition. At the end of the morula stage, the first segregation event occurs giving rise to the trophectoderm and the inner cell mass (ICM). At E4.5–E5.0, after the ICM segregates into the epiblast and primitive endoderm, the blastocyst implants in the uterus. Around E6.5, the egg cylinder is formed, and anterior–posterior axis patterning is established, along with the first mesendodermal progenitors at the primitive streak. (B) This chart provides information about the main molecular changes in the Wnt/β-catenin signaling pathway. The bimodal Wnt target gene expression is represented in grey stripes and is (at transcript level) already detected at the two- and four-cell stages [27]. In red, active nuclear β-catenin expression [28]. In blue, the Axin2:LacZ reporter is found only at the blastocyst stage [29]. In green, detection of the TCF/Lef:Histone 2B-green fluorescent protein (H2B-GFP) reporter occurs only after implantation stages [30]. (C) Longitudinal and transversal sections of a pre-gastrulating mouse embryo (E6.5) showing in yellow the distribution of the TCF/Lef:β-galactosidase reporter activity in the posterior region [30].

Figure 3

Wnt/β-catenin modulation correlates with different pluripotency and cell cycle stages. In mouse embryonic stem cells (mESCs), activation of the Wnt signaling pathway maintains naive pluripotency. Naive pluripotent conditions with Wnt induction or ERK inhibition correlate with a slow-down in cell cycle progression. Once the naive state is maintained without these inhibitors (i.e., serum + Leukemia Inhibitor Factor (LIF) conditions), mESCs start to proliferate faster. The inhibition of Wnt induces the cells to differentiate towards a primed pluripotency state (epiblast stem cells (EpiSCs)). For further differentiation commitment, activation is crucial to induce mesendoderm cell fate, while an acute repression of the pathway will generate neuroectoderm cells. In addition, cells differentiating show a classic slow cell cycle.

Figure 4

Temporal perturbation of the Wnt signaling pathway modulates somatic cell reprogramming efficiency. The levels of Wnt pathway activation during somatic cell reprogramming need to be precisely modulated in order to reach complete reprogramming. Somatic cells showing low Wnt activity (Wnt OFF state) at early stages of reprogramming and activation of the Wnt pathway (Wnt ON state) at late reprogramming stages, respectively, will result in completely reprogrammed induced pluripotent stem cells (iPSCs). In contrast, somatic cells with Wnt ON at early reprogramming stages or Wnt OFF at late stages produce partial or non-reprogrammed cells. It has also been demonstrated that high or aberrant levels of activation of the Wnt pathway have an inhibitory effect on somatic cell reprogramming.