EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS Zeb2 Regulates Cell Fate at the Exit from Epiblast State in Mouse Embryonic Stem Cells aDepartment of Development and Regeneration, KU a a,b c,d,e,f a b Leuven, Leuven 3000, AGATA STRYJEWSKA, RUBEN DRIES, TIM PIETERS, GRIET VERSTAPPEN, ANDREA CONIDI, a a a b,g Belgium; bDepartment of Cell KATHLEEN CODDENS, ANNICK FRANCIS, LIEVE UMANS, WILFRED F. J. VAN IJCKEN, g d,e h b c,d,e,i Biology, Center for Biomics, GEERT BERX, LEO A. VAN GRUNSVEN, FRANK G. GROSVELD, STEVEN GOOSSENS, c,d,i a,b Erasmus University Medical JODY J. HAIGH, DANNY HUYLEBROECK Center, Rotterdam 3015 CN, c The Netherlands; VIB Key Words. Cell differentiation • DNA-methylation • Embryonic stem cells • Pluripotent stem cells Inflammation Research • Repressors • RNA-sequencing • Transcription factors • Transcriptom Center (IRC), Unit Vascular Cell Biology, dDepartment of Biomedical Molecular Biology, eVIB-IRC, Unit ABSTRACT Molecular and Cellular In human embryonic stem cells (ESCs) the transcription factor Zeb2 regulates neuroectoderm ver- Oncology, Ghent University, sus mesendoderm formation, but it is unclear how Zeb2 affects the global transcriptional regulato- f Ghent 9052, Belgium; Center ry network in these cell-fate decisions. We generated Zeb2 knockout (KO) mouse ESCs, subjected for Medical Genetics, Ghent them as embryoid bodies (EBs) to neural and general differentiation and carried out temporal University Hospital, Ghent h RNA-sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS) analysis in neu- 9000, Belgium; Department ral differentiation. This shows that Zeb2 acts preferentially as a transcriptional repressor associated of Cell Biology, Liver Cell with developmental progression and that Zeb2 KO ESCs can exit from their na€ıve state. However, Biology Lab, Vrije Universiteit most cells in these EBs stall in an early epiblast-like state and are impaired in both neural and mes- Brussel, Jette 1090, Belgium; i endodermal differentiation. Genes involved in pluripotency, epithelial-to-mesenchymal transition ACBD - Blood Cancers and (EMT), and DNA-(de)methylation, including Tet1, are deregulated in the absence of Zeb2. The Stem Cells, Group observed elevated Tet1 levels in the mutant cells and the knowledge of previously mapped Tet1- Mammalian Functional binding sites correlate with loss-of-methylation in neural-stimulating conditions, however, after Genetics, Monash University, the cells initially acquired the correct DNA-methyl marks. Interestingly, cells from such Zeb2 KO EBs Melbourne, VIC, 3004, maintain the ability to re-adapt to 2i 1 LIF conditions even after prolonged differentiation, while Australia 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 A.S. and R.D. are co-first DNA-methylation with irreversible commitment to differentiation. STEM CELLS 2016; 00:000–000 authors. Correspondence: Danny Huylebroeck, Ph.D., Department SIGNIFICANCE STATEMENT of Cell Biology, Erasmus MC The transcription factor Zeb2 is critical for exit from the epiblast state in mouse ESCs and Ee-1040b, Wytemaweg 80, Rotter- dam 3015 CN, The Netherlands. for neural and general differentiation. In addition to its role in EMT it links the pluripotency Telephone: 131-10-7043169; network and DNA-methylation with irreversible commitment to differentiation. Fax: 131-10-7044743; e-mail: [email protected] INTRODUCTION DNA-methylation status has profound Received March 3, 2016; effects on embryonic gene expression. It is accepted for publication Na€ıve mouse embryonic stem cells (mESCs), controlled by DNA (cytosine-5)-methyltransfer- September 12, 2016; first primed epiblast stem cells (EpiSCs), and ases (Dnmt3a/3b/3l) that are highly active in published online in STEM CELLS embryonic germ cells are pluripotent cells that EXPRESS October 14, 2016. ESCs and early embryos and establish new can be used as cell culture models to study methylation patterns and by Dnmt1 that cop- C pluripotent cell states and fate decisions that V AlphaMed Press ies the patterns onto daughter cells [10, 11]. 1066-5099/2016/$30.00/0 occur during embryogenesis [1–6], transitions Active demethylation is orchestrated by Ten- that require changes of the transcriptome and http://dx.doi.org/ eleven translocation methylcytosine dioxyge- 10.1002/stem.2521 methylome. The ground state of self-renewing mESCs can be achieved by simultaneous addi- nases (Tet) [12, 13]. Tet1 levels are high in tion of chemical inhibitors (of MAPK and GSK3 ESCs and decrease upon differentiation, corre- This is an open access lating with exit from pluripotency, and Tet1 article under the terms of the signaling) and LIF (referred to as 2i 1 LIF) [7]. Creative Commons Attribution- When compared to a population of na€ıve steers mesendoderm versus trophectoderm NonCommercial License, which embryonic stem cells (ESCs), ground-state ESCs decisions in preimplantation embryos [14, 15]. permits use, distribution and display higher and more homogeneous expres- Tet1 is also important during somatic reprog- reproduction in any medium, ramming for genome demethylation as well as provided the original work is sion of key pluripotency genes, lower levels of properly cited and is not used differentiation markers and reduced DNA- activation/maintenance of Oct4 and Nanog for commercial purposes. methylation [8, 9]. [16–18]. STEM CELLS 2016;00:00–00 www.StemCells.com VC 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2 Balancing Fate Decisions in Embryonic Stem Cells Zeb2 (Sip1, Zfhx1b) downregulates E-cadherin (Cdh1) and mouse embryonic fibroblasts (mitC-MEFs) in ESC derivation thereby steers epithelial-to-mesenchymal transition (EMT) medium 1 LIF, and allowed to attach, and were re-fed daily. [19], which is relevant to stem cell fate, but also tumorigene- After 5–6 days, the inner cell mass was separated from the sis [20, 21]. Mutations in ZEB2 cause Mowat-Wilson syndrome trophectodermal layer, trypsinized and replated on mitC-MEFs. (MOWS; OMIM#235730), including defects in the central and They were further grown until subconfluency and expanded. peripheral nervous system (CNS, PNS) [22–24]. Many in vivo From these ESCs, Zeb2 KO lines were derived by nucleofection studies confirm the critical roles of Zeb2 in embryogenesis of linearized, blasticidin-selectable (48 hours) pcDNA6-His- and neurodevelopment in particular. Zeb2 KO mice die shortly eGFP:Cre vector to low-passage ESCs using Amaxa A-23 after E8.5 and have multiple defects, including in somitogene- (Lonza, Braine-l’Alleud, BE, www.lonza.com). Five control ESC sis [25], the neural plate and neural crest cells [26]. Cell-type lines and two KO lines, confirmed as such by genotyping specific Zeb2 KO mice develop defects in, for example, the (details available on request), were established. Second, CNS [27–29] and PNS [30–32]. Such studies in embryonic Zeb21/- mice were crossed with R26-iPSC mice that contain a brain revealed cell autonomous, but also non-autonomous RMCE cassette in the ROSA26 (R26) locus [40]. The second Zeb2 actions. In human (h) ESCs, Zeb2 regulates cell fate: R26 allele contained the LacZ reporter [41]. New control and upon Zeb2 knockdown (KD) they commit toward mesendo- RMCE-compatible Zeb2 KO ESC lines (three clones; mixed derm, while Zeb2 overproduction enhances neurogenesis [33]. 129/Bl6 background) were derived using a protocol [42] in ZEB2 is controlled by Nanog, Oct4, and Sox2 in hESCs, but key which pluripotin was replaced with 1 mM PD0325901 and 3 genes downstream of Zeb2 in ESCs, and during early neural mM CHIR99021. To obtain R26_Zeb2 lines, RMCE technology development, remain to be determined, and Zeb2 KO hESCs [43] was used to insert N-terminally Flag epitope (Flag) have not been reported. In order to enter lineage commit- tagged, wild-type Zeb2 cDNA into R26 of Zeb2 KO ESCs. ment, the pluripotency network in ESCs and EpiSCs needs to be distinguished [34, 35]. The list of factors promoting exit from na€ıve or ground state is growing, yet more key players remain to be identified [36–38]. Exit from pluripotency ESC Cultures and Sorting beyond the primed epiblast state requires efficient, irrevers- ESC maintenance: ESCs were maintained feeder-cell free in ible silencing of the transcriptional pluripotency network 2i 1 LIF medium. N2B27 was prepared as described [44]. For (including Oct4 and Nanog silencing, which persist in EpiSCs), 2i 1 LIF medium, 1 lM PD0325901 (Axon, 1408, Axon Med- acquisition and maintenance of DNA-methyl marks, and initia- chem, Groningen, NL, www.axonmedchem.com), 3 lM tion of differentiation. CHIR99021 (Axon, 1386) [7], and 1,000 U LIF/ml (Millipore, Using Zeb2 KO ESCs, we identified Zeb2 as a critical player ESG1107, Merck Millipore, Zwijndrecht, BE, www.merckmilli- for initiating and executing the differentiation programs. Upon pore.com) were added. Directed neural differentiation: On d0, withdrawal of 2i 1 LIF from Zeb2 KO ESC populations, some 3 3 106 ESCs were plated in a 10-cm bacterial petri dish in cells only sometimes commit to differentiation, but instead embryoid body (EB) medium (KO DMEM (Invitrogen, the gross population usually stalls as pluripotent, epiblast-like 10829018, Thermo Fisher Scientific, Merelbeke, BE, www.ther- cells that maintain the ability to re-adapt to 2i 1 LIF even mofisher.com), 15% fetal bovine serum (FBS, Life Technolo- after prolonged exposure