Role Of β-catenin In Controlling Tri-lineage Specification From Human Embryonic Stem Cells

As an essential event during gastrulation,the signaling,transcriptional and epigenetic regulatory mechanisms in controlling the ectoderm,mesoderm and endoderm cell fate specification from pluripotent stem cells within the epiblast of early human embryos remain largely unknown due to the ethical limitation and the inaccessibility of human embryos.Human embryonic stem cells(h ESCs)are directly derived from epiblast of a human embryo,which hold unlimited self-renewal ability and the potential to differentiate into all three germ layers in vitro under proper culture conditions.h ESCs therefore represent an unprecedented cell type to simulate the process of human tri-lineage specification.Previous studies based on animal models have revealed that loss-of-function of classical Wnt/β-catenin signaling leads to early embryonic lethality,while ectopic activation of classical Wnt/β-catenin signaling results in the formation of the secondary body axis,reinforcing an essential role of Wnt/β-catenin signaling in regulating gastrulation and tri-lineage specification in animals.In vitro studies also found that canonical Wnt signaling was required for the formation of primitive streak and endoderm lineages in h ESCs.However,the exact functions and molecular regulatory mechanisms of Wnt/β-catenin signaling in pluripotency maintenance and tri-lineage specification remain to be fully elucidated.In this study,we focus on the role of Wnt/β-catenin signaling in h ESCs selfrenewal and three germ layer differentiation,via knock out(KO)of β-catenin(CTNNB1),the core downstream effector of classical Wnt signaling,by CRISPR/Cas9-mediated double sg RNA knockout strategy(Paired-KO),inducible overexpression of constitutively active(CA)β-catenin,in vivo teratoma formation assay and in vitro embryoid body(EB)spontaneous differentiation.We also apply a series of RNA-seq and Ch IP-seq analyses to investigate the molecular mechanisms underlying Wnt/β-catenin signaling orchestrating tri-lineage specification from h ESCs.Our results revealed that the maintenance and self-renewal of h ESCs were independent of the activation of canonical Wnt signaling.The h ESCs with β-catenin KO maintained regular clonal morphology and underwent normal self-renewal.β-catenin KO h ESCs expressed high levels of pluripotency genes,including OCT4,SOX2 and NANOG.In comparison with the wild type counterpart,β-catenin KO h ESCs did not show obvious changes regarding cell cycle and apoptosis.RNA-seq experiments also showed that the wild type and β-catenin KO h ESCs had almost similar gene expression patterns,further strengthening the conclusion that Wnt/β-catenin signaling pathway is not necessarily required for pluripotency maintenance in h ESCs.Intriguingly,transient activation of Wnt/β-catenin signaling was necessary for h ESCs to exit the pluripotent state and initiate three germ layer differentiation,while sustained Wnt/β-catenin signaling activation promoted mesendoderm commitment.Compared with wild-type controls,β-catenin KO h ESCs could be long-term maintained in the absence of ectopic supplement of FGF2,suggesting that β-catenin KO h ESCs are prone to self-renewal rather than tri-lineage differentiation.Inoculating β-catenin KO h ESCs subcutaneously into immunodeficient mice failed to form teratomas,further indicating an indispensable role of β-catenin in three germ layer specification from h ESCs.In vitro EB formation assay also confirmed that the differentiation of three germ layers from h ESCs was largely blocked upon β-catenin KO.Rescue experiments revealed that short-term restoring of Wnt/β-catenin signaling by transient inducing CAβ-catenin expression endowed h ESCs with the ability to differentiate into all three germ layers,whereas long-term induction of CA β-catenin expression resulted in prominent mesendoderm specification by sacrificing the ectoderm cell fate.These results highlight a conserved role of Wnt/β-catenin signaling in determining mesendoderm cell fate in humans.However,our study also reveal an overlooked role of Wnt/β-catenin signaling as a “gate-keeper” of tri-lineage entry in human before it implement a role in mesendoderm differentiation.To understand the molecular basis of Wnt/β-catenin signaling in controlling trilineage entry,we performed Ch IP-seq analyses to map the binding sites of β-catenin and surrounding histone modifications in h ESCs.β-catenin was enriched in the promoters and introns of a plethora of early ectodermal,mesodermal and endodermal genes.Upon β-catenin KO,the levels of H3K27me3 in these early ectodermal,mesodermal and endodermal genes,but not in those pluripotent or other types of genes,were specifically and significantly increased.These results demonstrate that during the pluripotency maintenance,β-catenin selectively binds to the promoter regions of early tri-lineage genes and maintains them low levels of H3K27me3 modification to license the early tri-lineage genes a “poised” state for transcription activation once the three germ layer differentiation process initiates.In conclusion,our current study reveals a novel role of Wnt/β-catenin signaling in controlling tri-lineage entry of h ESCs by maintaining tri-lineage genes low levels of H3K27me3 modification.Our study provides a new model of tri-lineage specification in humans and a means to regulate h ESCs maintenance and differentiation.