YAP1 Drives Lineage Specification and Patterning in hESC-derived Gastruloids
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ABSTRACT:
PROVIDER: PRJNA707055 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13866769_1.fastq.gz | Fastqsanger.gz | |||
| SRR13866769_2.fastq.gz | Fastqsanger.gz | |||
| SRR13866770_1.fastq.gz | Fastqsanger.gz | |||
| SRR13866770_2.fastq.gz | Fastqsanger.gz | |||
| SRR13866771_1.fastq.gz | Fastqsanger.gz |
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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.
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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.
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Project description:The gastrulation process relies on complex interactions between developmental signaling pathways that are not completely understood. Here, we interrogated the contribution of the Hippo signaling effector YAP1 to the formation of the three germ layers by analyzing human embryonic stem cell (hESC)-derived 2D-micropatterned gastruloids. YAP1 knockout gastruloids display a reduced ectoderm layer and enlarged mesoderm and endoderm layers compared with wild type. Furthermore, our epigenome and transcriptome analysis revealed that YAP1 attenuates Nodal signaling by directly repressing the chromatin accessibility and transcription of key genes in the Nodal pathway, including the NODAL and FOXH1 genes. Hence, in the absence of YAP1, hyperactive Nodal signaling retains SMAD2/3 in the nuclei, impeding ectoderm differentiation of hESCs. Thus, our work revealed that YAP1 is a master regulator of Nodal signaling, essential for instructing germ layer fate patterning in human gastruloids.
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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.
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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.
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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
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