The Wnt catenin pathway can also activate somatic cell repro
The Wnt/β-catenin pathway can also activate somatic cell reprogramming to pluripotency. Mouse embryonic fibroblasts (MEFs) transduced with retroviruses carrying Oct4, Klf4, and Sox2 and cultured in medium containing Wnt3a can generate induced pluripotent stem cell (iPSC) colonies with enhanced efficiency in absence of c-Myc (Marson et al., 2008). Furthermore, activation of the Wnt pathway in ESCs enables them to reprogram neural precursor Metabolism Compound Library after fusion (Lluis et al., 2008). Finally, the deletion of Tcf3 greatly enhances cell-fusion-mediated reprogramming, as well as the production of induced pluripotent stem cells (iPSCs) (Lluis et al., 2011; Ombrato et al., 2012).
In addition to the Wnt-mediated control of ESC pluripotency and somatic cell reprogramming, Wnt signaling is also a driver of differentiation during early developmental phases (Tam and Loebel, 2009). Anterior-posterior axis specification in the mouse embryo occurs through the activity of Wnt signaling (Merrill et al., 2004; Sokol, 2011). In particular, Wnt signaling activity is essential for establishment of the primitive streak and anterior-posterior polarity, i.e., for epithelial-to-mesenchymal transition of epiblast cells in the primitive streak (Kalluri and Weinberg, 2009; Murry and Keller, 2008; Tanaka et al., 2011; ten Berge et al., 2008).
These apparently opposite roles of the Wnt signaling pathway are therefore a conundrum; on one hand, Wnt activity controls ESC pluripotency, and on the other hand, it regulates early developmental differentiation events. To reconcile these opposite functions, one reasonable hypothesis is based on the level of activation of the Wnt pathway in time. It is well known that Wnt signaling oscillates during development and that its target genes have an oscillatory behavior (Sokol, 2011; van Amerongen and Nusse, 2009). At the same time, cyclic activation of the Wnt/β-catenin pathway is essential for enhancing somatic cell reprogramming (Lluis and Cosma, 2009; Lluis et al., 2008). If β-catenin activity is either high or very low, reprogramming does not take place. We therefore wondered whether the activation of Wnt signaling activity mediated by TCF factors is essential in a specific phase of the reprogramming of MEFs into iPSCs.
TCF3 and TCF1 share a similar DNA binding domain, and they represent the most highly expressed TCF family factors in ESCs (Lluis et al., 2011; Pereira et al., 2006). TCF3 acts as a repressor of Wnt target genes, and in contrast TCF1 can activate or repress Wnt-targets via its association with β-catenin (Brantjes et al., 2001; Hikasa et al., 2010). However, little is known about TCF1 function in ESCs, and here we investigated the role of TCF1 in the reprogramming process of MEFs into iPSCs. Surprisingly, in this context, we found that the activity of the Wnt/β-catenin pathway needs to be switched off during the first days and that the cells undergoing reprogramming have low levels of stabilized β-catenin. Remarkably, sorted Wnt “off” MEFs generate a very high number of NANOG-positive iPSCs. Interestingly, during the early phases of four-factor-induced reprogramming, TCF1 functions as a repressor of Wnt signaling, and this activity correlates with downregulation of senescent genes, such as p21, p19, and p16, and activation of mesenchymal-to-epithelial transition (MET) genes. The activity of the Wnt/β-catenin pathway is instead necessary during the late phases of the reprogramming process.
Discussion Earlier we showed that cyclic activation of the Wnt signaling pathway is necessary to enhance cell-fusion-mediated reprogramming (Lluis et al., 2008). Also, we showed that finely tuned levels of the Wnt pathway are essential for enhancing somatic cell reprogramming. Too high or too low levels of β-catenin activity result in an impaired reprogramming efficiency (Lluis et al., 2008). Here, we demonstrate that, to achieve reprogramming, Wnt signaling needs to be repressed during the early phase of the process and to be active at the late steps; indeed, in a recent interesting study, a similar conclusion was reached (Ho et al., 2013). However, whereas Ho et al. studied prevalently the function of TCF3 in the regulation of reprogramming and focused on the analysis of the whole MEFs population, here we have extensively studied the function of TCF1, demonstrating its essential role during the reprogramming process. In addition, we have characterized the activity of TCF1 in the THY-negative cells, which are the cells that undergo reprogramming. We have shown that TCF1 acts as a repressor of the Wnt signaling pathway at the onset of reprogramming. Furthermore, it promotes repression of senescent genes and activation of MET (by inhibition of transcription of mesenchymal genes and by activation of epidermal genes) in the THY-negative cells, whereas TCF1 acts as an activator in the THY-positive cells that do not undergo reprogramming.