Project description:The Wnt/β-catenin signalling pathway is a key regulator of embryonic stem cell self-renewal and differentiation. Constitutive activation of this pathway has been shown to significantly increase mouse embryonic stem cell (mESC) self-renewal and pluripotency marker expression. In this study, we generated a novel β-catenin knock-out model in mESCs by using CRISPR/Cas9 technology to delete putatively functional N-terminally truncated isoforms observed in previous knock-out models. While we showed that aberrant N-terminally truncated isoforms are not functional in mESCS, we observed that canonical Wnt signalling is not active in mESCs, as β-catenin ablation does not alter mESC transcriptional profile in LIF-enriched culture conditions; on the other hand, Wnt signalling activation represses mESC spontaneous differentiation. We also showed that transcriptionally silent β-catenin (ΔC) isoforms can rescue β-catenin knock-out self-renewal defects in mESCs, cooperating with TCF1 and LEF1 in the inhibition of mESC spontaneous differentiation in a Gsk3 dependent manner.
Project description:expression profile of conditional knock out of beta-catenin by K19-CRE at E7.5. Tested a wild type with two alleles of beta-catenin, a heterzyote with one deleted allele and the conditional null in the domain on cytokeratin 19 driven CRE expression
Project description:expression profile of conditional knock out of beta-catenin by K19-CRE at E7.5. Tested a wild type with two alleles of beta-catenin, a heterzyote with one deleted allele and the conditional null in the domain on cytokeratin 19 driven CRE expression Keywords: other
Project description:Canonical Wnt and Nodal signaling are both required for induction of the primitive streak (PS), which guides organization of the early embryo. The Wnt effector β-catenin is thought to function in these early lineage specification decisions via transcriptional activation of Nodal signaling. Here, we demonstrate a broader role for β-catenin in PS formation by analyzing its genome-wide binding in a human embryonic stem cell model of PS induction. β-catenin occupies regulatory regions in numerous PS and neural crest genes, and direct interactions between β-catenin and the Nodal effectors SMAD2/3 are required at these regions for PS gene activation. Furthermore, OCT4 binding in proximity to these sites is likewise required for PS induction, suggesting a collaborative interaction between β-catenin and OCT4. Induction of neural crest genes by β-catenin is repressed by SMAD2/3, ensuring proper lineage specification. This study provides mechanistic insight into how Wnt signaling controls early cell lineage decisions. Examination of β-catenin binding in hESC incubated in media control (RPMI), media containing CHIR or CHIR+SB for 6h and analyzed by ChIP-sequencing
Project description:Activation of Wnt/β-catenin signaling is pivotal to the formation of definitive endoderm (DE) lineage during early embryonic development, but its regulatory mechanism was not yet fully understood. In this study, we generated new CTNNB1-/- human ES cells (hESC) through CRISPR-based insertional gene disruption approach and performed systematic rescue using various truncated or mutant β-catenin to study the Wnt/β-catenin-centered cellular mechanisms underlying DE differentiation from hESC. We used RNA-seq to examine the transcriptional information to understand the functions of β-catenin.
Project description:Heat shock transcription factor 1(HSF1) is an important transcription factor which regulates the expression of a wide array of genes including heat shock proteins and oncogenes. Here, we report that HSF1 as a target of WNT/β-catenin signaling, regulates parts of target genes of WNT/β-catenin signaling. To explore the biological relevance of HSF1 activation to WNT/β-catenin signaling, we profiled gene expression of wild type mouse embryonic fibroblasts (WT MEF) and HSF1 knock out MEF (HSF1 KO MEF) before and after lithium chloride (LiCl) treatment which was a potent GSK3β inhibitor and increased the expression of β-catenin.
Project description:We generated pluripotent stem cells (Mel1 hESC containing a GFP reporter driven by the endogenous insulin promoter) with a functional knock out of PTPN2 by CRISPR/Cas9 genome editing. KO or WT control stem cells were differentiated into beta-like cells (sBC), sorted for GFP, and prepared for deep sequencing.
Project description:β-catenin plays a vital role in various biological processes, such as body axis determination and cell differentiation, during embryonic development in metazoans. These β-catenin functions are thought to be exerted through complexes formed with various types of proteins. Although β-catenin complex proteins have been identified in several bilaterian models, little is known about the structural and functional properties of β-catenin complexes in the early metazoan evolutionary phases. In this study, we performed a comparative analysis of β-catenin sequences in nonbilaterian lineages that branched off early in metazoan evolution. We aslo carried out a transphyletic function experiments of β-catenin from non-bilaterian metazoans using developing Xenopus embryos, which included secondary axis induction in embryos and proteomic analysis of the β-catenin protein complex. Comparative functional analysis of nonbilaterian β-catenins also demonstrated sequence characteristics important for β-catenin function, and the deep origin and evolutionary conservation of the cadherin-catenin complex. Proteins coimmunoprecipitated with β-catenin included several proteins conserved across metazoans. These data provide a new insight into the conserved repertoire of β-catenin complexes.