The Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency

Abstract

Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

The scramblases VMP1 and TMEM41b are required for primitive endoderm specification by targeting WNT signaling

“…A close analysis of WNT pathway-deregulated genes allowed to identify several well-established WNT-target genes, such as Frzb, Sox17, Wnt3, and Ccdnd1 (Du et al, 2009; Tetsu and McCormick, 1999; Zhang et al, 2009). Their down-regulation strongly suggests a decrease in activity of the WNT pathway in mutant cells which is a plausible explanation for the XEN specification defects (Athanasouli et al, 2023). Interestingly, we detected Ddit3 among the upregulated genes in Double KO cells.…”

“…(Fig.2C). The finding of WNT signaling deregulation in Vmp1 and Tmem41b XEN cells explains the specification delay detected in mutant cells: WNT/TCF7L1 transcriptional response is necessary in vitro and in vivo to promote PE expansion (Athanasouli et al, 2023). Retardation – not impairment – of PE specification induced by Vmp1 or Tmem41b mutations may permit embryo implantation but the accumulation of defects likely blocks development during gastrulation.…”

“…Defects in endoderm specification were also observed in Embryoid bodies derived from single Vmp1 KO or Tmem41b KO cells but with lower consistency than those derived from Double KO cells. The finding of WNT signaling deregulation in Vmp1 and Tmem41b XEN cells, explains the specification delay detected in mutant cells: it has been shown that WNT/TCF7L1 transcriptional response is necessary in vitro and in vivo to promote PE expansion (Athanasouli et al, 2023). Retardation – not impairment – of PE specification induced by Vmp1 or Tmem41b mutations might still lead to embryo implantation but also to the accumulation of defects, which are incompatible with the gastrulation stage.…”

“…A close analysis of these genes allowed to identify several well-established WNT-target genes, including Frzb, Sox17, Wnt3, and Ccdnd1 (Du et al, 2009;Tetsu and McCormick, 1999;Zhang et al, 2009). Their down-regulation strongly suggested a decreased activity of the WNT pathway in Double KO cells and might explain the observed defect in XEN specification (Athanasouli et al, 2023).…”

Common modes of ERK induction resolve into context specific signalling via a combination of signal duration and cell type specific transcriptional repression

“…ESCs and RSCs are cultured in nearly identical conditions, with the only difference being that canonical WNT signalling is activated in ESC via GSK3 inhibition and inhibited in RSC with IWP2 (see Methods). In naïve ESCs, canonical ß-catenin signalling exploits TCF3 to support pluripotency, by associating with these regulatory regions and counteracting TCF3-mediated repression (Athanasouli et al, 2023;Watanabe and Dai, 2011). Yet canonical WNT signalling also induces PrE differentiation in ESC and can do so in virtually identical ESC media with one key difference, the removal of the FGF block (Anderson et al, 2017).…”