Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells.

Agata Stryjewska,Wilfred F J Van Ijcken,Frank G Grosveld,Leo A Van Grunsven,Steven Goossens,Kathleen Coddens,Annick Francis,Geert Berx,Jody J Haigh,Tim Pieters,Ruben Dries,Andrea Conidi,Lieve Umans,Griet Verstappen,Danny Huylebroeck

doi:10.1002/stem.2521

Abstract

In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm versus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulatory network in these cell‐fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal RNA‐sequencing (RNA‐seq) and reduced representation bisulfite sequencing (RRBS) analysis in neural differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated with developmental progression and that Zeb2 KO ESCs can exit from their naïve state. However, most cells in these EBs stall in an early epiblast‐like state and are impaired in both neural and mesendodermal differentiation. Genes involved in pluripotency, epithelial‐to‐mesenchymal transition (EMT), and DNA‐(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1‐binding sites correlate with loss‐of‐methylation in neural‐stimulating conditions, however, after the cells initially acquired the correct DNA‐methyl marks. Interestingly, cells from such Zeb2 KO EBs maintain the ability to re‐adapt to 2i + LIF conditions even after prolonged differentiation, while knockdown of Tet1 partially rescues their impaired differentiation. Hence, in addition to its role in EMT, Zeb2 is critical in ESCs for exit from the epiblast state, and links the pluripotency network and DNA‐methylation with irreversible commitment to differentiation. Stem Cells 2017;35:611–625

Highlights

The transcription factor Zeb2 is critical for exit from the epiblast state in mouse embryonic stem cells (ESCs) and for neural and general differentiation
Na€ıve mouse embryonic stem cells, primed epiblast stem cells (EpiSCs), and embryonic germ cells are pluripotent cells that can be used as cell culture models to study pluripotent cell states and fate decisions that occur during embryogenesis [1,2,3,4,5,6], transitions that require changes of the transcriptome and methylome
To gain insight into what processes might be involved in the establishment of the early differences between control mESCs (Ctrl) and Zeb2 KO cells, we identified the top positive and negative www.StemCells.com VC 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press genes that contribute to principal component 2 (PC2, which separates the lineage trajectories of Ctrl and Zeb2 KO samples on the Principal Component Analysis (PCA) plot) and performed gene ontology (GO) analysis using Gorilla software [52]

Summary

Introduction

Na€ıve mouse embryonic stem cells (mESCs), primed epiblast stem cells (EpiSCs), and embryonic germ cells are pluripotent cells that can be used as cell culture models to study pluripotent cell states and fate decisions that occur during embryogenesis [1,2,3,4,5,6], transitions that require changes of the transcriptome and methylome. When compared to a population of na€ıve embryonic stem cells (ESCs), ground-state ESCs display higher and more homogeneous expression of key pluripotency genes, lower levels of differentiation markers and reduced DNAmethylation [8, 9]. DNA-methylation status has profound effects on embryonic gene expression. It is controlled by DNA (cytosine-5)-methyltransferases (Dnmt3a/3b/3l) that are highly active in ESCs and early embryos and establish new methylation patterns and by Dnmt that copies the patterns onto daughter cells [10, 11]. Tet levels are high in ESCs and decrease upon differentiation, correlating with exit from pluripotency, and Tet steers mesendoderm versus trophectoderm decisions in preimplantation embryos [14, 15]. Tet is important during somatic reprogramming for genome demethylation as well as activation/maintenance of Oct and Nanog [16,17,18]

Methods

Results

Conclusion