Wnt and TGF-β signaling are required for the induction of an
            <i>in vitro</i>
            model of primitive streak formation using embryonic stem cells

Gadue, Paul; Huber, Tara L.; Paddison, Patrick J.; Keller, Gordon

doi:10.1073/pnas.0603916103

Cited by 516 publications

(555 citation statements)

References 42 publications

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“…Cells were plated in ActivinA and CH after 25 h of N2B27 + BI treatment to initiate primitive streak‐like differentiation 71, 72, 73, 74. On day 3, BI‐treated cells upregulated Brachyury similar to untreated controls (Fig 5I).…”

Section: Resultsmentioning

confidence: 87%

Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency

et al. 2018

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Mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) signalling is implicated in initiation of embryonic stem (ES) cell differentiation. The pathway is subject to complex feedback regulation. Here, we examined the ERK‐responsive phosphoproteome in ES cells and identified the negative regulator RSK1 as a prominent target. We used CRISPR/Cas9 to create combinatorial mutations in RSK family genes. Genotypes that included homozygous null mutations in Rps6ka1, encoding RSK1, resulted in elevated ERK phosphorylation. These RSK‐depleted ES cells exhibit altered kinetics of transition into differentiation, with accelerated downregulation of naïve pluripotency factors, precocious expression of transitional epiblast markers and early onset of lineage specification. We further show that chemical inhibition of RSK increases ERK phosphorylation and expedites ES cell transition without compromising multilineage potential. These findings demonstrate that the ERK activation profile influences the dynamics of pluripotency progression and highlight the role of signalling feedback in temporal control of cell state transitions.

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Section: Resultsmentioning

confidence: 87%

Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency

et al. 2018

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“…We used the well-established Activin-A-mediated differentiation protocol that efficiently drives ES cells to form primitive streak cells in vitro (9,14) (SI Appendix, Fig. S1A).…”

Section: Resultsmentioning

confidence: 99%

Charting Brachyury-mediated developmental pathways during early mouse embryogenesis

Lolas

Valenzuela

Tjian

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator-Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genomewide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the T c mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and musclefate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination.primitive streak | early development | mesoendoderm differentiation I n the past decade, significant insights have been gained in understanding gene-regulatory programs responsible for embryonic stem (ES) cell pluripotency and self-renewal. However, transcriptional control mechanisms underlying finely balanced lineage-segregation and morphogenetic processes during early mammalian embryogenesis remained elusive. During early mouse gastrulation, Brachyury (T), a classical enhancer-binding transcription factor (TF), has been reported to be required for the proper development of primitive streak, allantois, axial, and posterior mesoderm (reviewed in ref. 1).Specifically, mouse embryos that are homozygous for the Brachyury (T) deletion die at midgestation (2). T −/− mutant epiblast cells are compromised in their ability to migrate away from the primitive streak and, therefore, are unable to undergo the morphogenetic movements carried out by their wild-type (WT) counterparts during gastrulation. In addition to defects in the primitive streak, the notochord is absent in posterior portions of the embryo. Although the anterior portions of T mutant mice contain notochordal precursor-like cells, they fail to undergo normal terminal differentiation (3, 4). The embryonic pattern posterior to the forelimb region of T −/− animals is also disturbed, with somites posterior to the seventh pair absent or abnormal. Although neural folds fuse to form the neural tube, they are severely kinked in the caudal region, and the surface ectoderm tends to form fluid-filled blisters (2, 4, 5). In addition to these well-documented defects, numerous other phenotypic abnormalities have been reported in T −/− and T mutant embryos, including left-right pattern...

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“…This likely reflects the different regulatory factors involved in the formation of separate lineages. Sox1 expression initially requires upregulation of FGF-mediated signals [27,28,30,40]; whereas early hematopoiesis requires signaling via BMP4 and VEGF [41,42]. This demonstrates both how versatile oligosaccharides could be as culture additives, but also highlights the need for careful selection with a thorough knowledge of the specific pathways involved and the GAG structures needed to regulate them.…”

Section: Discussionmentioning

confidence: 99%

Specific Glycosaminoglycans Modulate Neural Specification of Mouse Embryonic Stem Cells

et al. 2011

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Mouse embryonic stem (mES) cells express a low sulfated form of heparan sulfate (HS). HS chains displayed by ES cells and their progeny become more complex and more sulfated during progression from pluripotency to neuroectodermal precursors. Sulfated epitopes are important for recognition and binding of a variety of ligands including members of the fibroblast growth factor (FGF) family. We demonstrated previously that mES cells lacking HS cannot undergo neural specification but this activity can be recovered by adding soluble heparin, a highly sulfated glycosaminoglycan (GAG). Therefore, we hypothesized that soluble GAGs might be used to support neural differentiation of HS competent cells and that the mechanisms underlying this activity might provide useful information about the signaling pathways critical for loss of pluripotency and early lineage commitment. In this study, we demonstrate that specific HS/heparin polysaccharides support formation of Sox1 1 neural progenitor cells from wild-type ES cells. This effect is dependent on sulfation pattern, concentration, and length of saccharide. Using a selective inhibitor of FGF signal transduction, we show that heparin modulates signaling events regulating exit from pluripotency and commitment to primitive ectoderm and subsequently neuroectoderm. Interestingly, we were also able to demonstrate that multiple receptor tyrosine kinases were influenced by HS in this system. This suggests roles for additional factors, possibly in cell proliferation or protection from apoptosis, during the process of neural specification. Therefore, we conclude that soluble GAGs or synthetic mimics could be considered as suitable low-cost factors for addition to ES cell differentiation regimes. STEM CELLS 2011;29:629-640 Disclosure of potential conflicts of interest is found at the end of this article.

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Wnt and TGF-β signaling are required for the induction of an in vitro model of primitive streak formation using embryonic stem cells

Cited by 516 publications

References 42 publications

Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency

Negative feedback via RSK modulates Erk‐dependent progression from naïve pluripotency

Charting Brachyury-mediated developmental pathways during early mouse embryogenesis

Specific Glycosaminoglycans Modulate Neural Specification of Mouse Embryonic Stem Cells

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