YAP1 Drives Lineage Specification and Patterning in hESC-derived Gastruloids
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PROVIDER: PRJNA707055 | ENA |
REPOSITORIES: ENA
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Project description:The gastrulation process is controlled by the interplay between morphogenetic signals from BMP, WNT and NODAL pathways. Increasing evidences support an emerging role of the Hippo-YAP signaling in the cell-fate decisions that guide lineage specification in mouse and human Embryonic Stem Cells (hESCs). However, the contribution of YAP to the process of gastrulation in hESCs remains unknown. Here, we show that YAP1 regulates the specification, size and patterning of the three-germ layers. Using hESC-derived 2D-micropatterned gastruloids and directed differentiation approaches we show that YAP maintains a semi-active NODAL signaling during gastrulation essential to regulate the exit of pluripotency. In absence of YAP1, a hyperactive NODAL signaling retains SMAD2.3 in the nuclei, impeding the exit of pluripotency and the acquisition of the ectodermal gene program. Accordingly, the inhibition of NODAL signaling is sufficient to rescue the gastrulation-defective phenotype of the YAP1 KO hESCs. Our work revealed that Hippo-YAP1 signaling is an important component of the developmental network that coordinate hESC pluripotency and gastrulation.
2022-01-14 | GSE168377 | GEO
Project description:Patterning and growth are fundamental features of embryonic development that must be tightly coordinated during morphogenesis. While metabolism is known to control cell growth, how it impacts patterning and links to morphogenesis is poorly understood. To understand how metabolism impacts early mesoderm specification during gastrulation, we used in vitro mouse embryonic stem (ES) cell-derived gastruloids, due to ease of metabolic manipulations and high-throughput nature. Gastruloids showed mosaic expression of glucose transporters co-expressing with the mesodermal marker T/Bra. To understand the significance of cellular glucose uptake in development, we used the glucose metabolism inhibitor 2-deoxy-D-glucose (2-DG). 2-DG blocked the expression of T/Bra and abolishes axial elongation in gastruloids. Surprisingly, removing glucose completely from the medium did not phenocopy 2-DG treatment despite a significant decline in glycolytic intermediates occurring under both conditions. As 2-DG can also act as a competitive inhibitor of mannose in protein glycosylation, we added mannose together with 2-DG and found that it could rescue the mesoderm specification. We corroborated these results in vivomouse embryos where supplementing mannose rescued the 2-DG mediated phenotype of mesoderm specification and proximo-distal elongation of the primitive streak. We further showed that blocking production and intracellular recycling of mannose abrogated mesoderm specification. At molecular level, proteomics analysis revealed that mannose reversed glycosylation of the Wnt pathway regulator, Secreted Frizzled Receptor, Frzb, expressed in the primitive streak of the mouse embryo. Our study showed how mannose linked metabolism to glycosylation of a developmental pathway component, crucial in patterning of mesoderm and morphogenesis of gastruloids.
2024-01-12 | MSV000093849 | MassIVE
Project description:Patterning and growth are fundamental features of embryonic development that must be tightly coordinated during morphogenesis. As metabolism can control cell growth while also providing mechanistic links to developmental signalling pathways, it is ideally placed to enable this coordination. To understand how metabolism impacts early mesoderm specification, we used mouse embryonic stem (ES) cell-derived gastruloids, as these enable temporal control over metabolic manipulations and can be generated in large quantities. Gastruloids show mosaic expression of two glucose transporters,Slc2a1andSlc2a3both of which co-express with the expression of both the mesodermal markerT/Braand the neural markerSox2. To understand the significance of cellular glucose uptake in development, we used the glucose metabolism inhibitor 2-deoxy-D-glucose (2-DG). 2-DG specifically blocks the expression ofT/Brawithout affecting the expression ofSox2and abolishes axial elongation in gastruloids. Surprisingly, removing glucose completely from the medium did not phenocopy 2-DG treatment despite a significant decline in glycolytic intermediates occurring under both conditions. As 2-DG can also act as a competitive inhibitor of mannose, we added mannose together with 2-DG and found that it could rescue the mesoderm specification. Together, our results show that while mannose is crucial for mesoderm specification, the glycolytic pathway is dispensable at early stages ofT/Braexpression in gastruloids.
2024-03-11 | GSE236887 | GEO
Project description:The Wnt3a/?-catenin and Activin/Smad2,3 signaling pathways synergize to induce endodermal differentiation of human embryonic stem cells, however the mechanism is not well-understood. Using ChIP-seq and GRO-seq analyses, we report here that hESC enhancers, including Wnt3a/LEF-1 sites, hold enhancer RNAPII complexes (eRNAPII) containing high levels of Ser5P and low Ser7P. In Wnt3a signaling, ?-catenin recruits cohesin to the LEF-1:eRNAPII sites to induce enhancer-promoter looping and activate transcription of mesoendodermal (ME) genes. However, paused Ser5P-RNAPII complexes accumulate at these genes, indicating that elongation remains limiting. Subsequent Activin/Smad2,3 signaling increases P-TEFb occupancy, CTD-Ser7P, and productive elongation at ME genes. Additionally, ME genes, including EOMES and MIXL1, are repressed by the Hippo regulator, Yap1, an essential pluripotency factor. GRO-seq experiments indicate that Yap1 blocks nascent transcription and controls NELF occupancy on ME genes. Thus, Wnt3a/?-catenin and Activin/Smad2,3 pathways up-regulate transcription initiation and elongation, respectively, to overcome Yap1 repression during early hESC differentiation ChIP-seq and GROseq experiments in H1 hESCs. Cells were treated with Wnt3a (200ng/ml), Activin A (100ng/ml) or Wnt3a+Activin A (W200ng/ml+A100ng/ml) for 4h (ChIP-seq) or 6h (GRO-seq). GRO-seq in YAP depleted cells were carried out following transfection with control or YAP siRNAs . After 48h transfection, cells were left untreated or treated with Wnt3a+Activin (W200ng/ml+A100ng/ml) for additional 6h.
2015-05-04 | E-GEOD-64758 | biostudies-arrayexpress
Project description:Gastruloids are three-dimensional aggregates of embryonic stem cells (ESCs) that display key features of mammalian post-implantation development, including germ layer specification and axial organization. Gastruloids have mostly been characterized with microscopy-based approaches, limiting the number of genes that can be explored. It is therefore unclear to what extent gene expression in gastruloids reflects in vivo embryonic expression. Using both single-cell RNA-seq (scRNA-seq) and spatial transcriptomics we systematically compared cell types and spatial expression patterns between mouse gastruloids and mouse embryos.
2019-12-05 | GSE123187 | GEO
Project description:The Wnt3a/β-catenin and Activin/Smad2,3 signaling pathways synergize to induce endodermal differentiation of human embryonic stem cells, however the mechanism is not well-understood. Using ChIP-seq and GRO-seq analyses, we report here that hESC enhancers, including Wnt3a/LEF-1 sites, hold enhancer RNAPII complexes (eRNAPII) containing high levels of Ser5P and low Ser7P. In Wnt3a signaling, β-catenin recruits cohesin to the LEF-1:eRNAPII sites to induce enhancer-promoter looping and activate transcription of mesoendodermal (ME) genes. However, paused Ser5P-RNAPII complexes accumulate at these genes, indicating that elongation remains limiting. Subsequent Activin/Smad2,3 signaling increases P-TEFb occupancy, CTD-Ser7P, and productive elongation at ME genes. Additionally, ME genes, including EOMES and MIXL1, are repressed by the Hippo regulator, Yap1, an essential pluripotency factor. GRO-seq experiments indicate that Yap1 blocks nascent transcription and controls NELF occupancy on ME genes. Thus, Wnt3a/β-catenin and Activin/Smad2,3 pathways up-regulate transcription initiation and elongation, respectively, to overcome Yap1 repression during early hESC differentiation
2015-05-04 | GSE64758 | GEO
Project description:Specifying the primitive streak (PS) guides stem cell differentiation in vitro, however much remains to be learned about the transcription networks that direct anterior and posterior PS cells (APS and PPS, respectively) to differentiate to distinct mesendodermal subpopulations. Here, we show that APS genes are predominantly induced in YAP1-/- hESCs in response to ACTIVIN. This finding establishes the Hippo effector YAP1 as a master regulator of PS specification, functioning to repress ACTIVIN-regulated APS genes in hESCs. Moreover, transient exposure of wild-type hESCs to dasatinib, a potent C-SRC/YAP1 inhibitor, enables differentiation to APS-derived endoderm and cardiac mesoderm in response to ACTIVIN. Importantly, these cells can differentiate efficiently to normal beating cardiomyocytes without the cytoskeletal defect seen in YAP1-/- hESC-derived cardiomyocytes. Overall, we uncovered an induction mechanism to generate APS cells using a cocktail of ACTIVIN and YAP1i molecules that holds practical implications for hESC and iPSC differentiation into distinct mesendodermal lineages.
2018-11-14 | GSE121877 | GEO
Project description:The cellular microenvironment shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and benefit from hypoxic culture in vitro. Yet, how different components of the hypoxia response impact stem cell transcriptional networks and lineage choices remains unclear. Here we investigated the effect of acute and prolonged hypoxia on stem cell states and differentiation efficiencies of embryonic and extraembryonic cells. We show that prolonged hypoxia enhances differentiation of embryonic stem (ES) cells towards the mesendoderm lineage by transcriptionally priming cells with a primitive streak signature including Wnt3 and T expression. Exposure to hypoxia in ES culture or during formation of gastrulation-mimicking organoids (gastruloids) moderates T expression and enhances structural complexity. Hypoxic gastruloids generated without exogenous Wnt induction can spontaneously elongate and self-organize. Direct gene regulation by Hif1a, combined with DNA demethylation and metabolic rewiring modulate the transcriptional response and phenotypic outcome. Our findings highlight the influence of the microenvironment on stem cell function and provide a rationale supportive of applying physiological conditions in synthetic embryo models.
2022-09-05 | PXD026641 | Pride
Project description:Single cell transcriptomic study (using 10x Genomics v3 kits) of gastruloids, aggregates of mESCs, at different developmental timepoints (24h, 48h and 72h post-aggregation). mESCs, corresponding to the 0h timepoint, were also sequenced. The aim of this study was to delineate the cell state transition dynamics underlying anteroposterior symmetry breaking and germ layer formation in gastruloids. Gastruloids used in this study were generated from Bra::GFP reporter mESCs (Fehling et al., 2003).
2023-12-04 | E-MTAB-12749 | biostudies-arrayexpress
Project description:Capturing cardiogenesis in gastruloids
| PRJNA667329 | ENA