Molecular mechanism of symmetry breaking in a 3D model of a human epiblast

ABSTRACTBreaking the anterior-posterior (AP) symmetry in mammals takes place at gastrulation. Much of the signaling network underlying this process has been elucidated in the mouse, however there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro 3D model of a human epiblast whose size, cell polarity, and gene expression are similar to a 10-day human epiblast. A defined dose of bone mor-phogenetic protein 4 (BMP4) spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial to mesenchymal transition. By gene knockouts and live-cell imaging we show that, downstream of BMP4, WNT3 and its inhibitor DKK1 play key roles in this process. Our work demonstrates that a model human epiblast can break axial symmetry despite no asymmetry in the initial signal and in the absence of extraembryonic tissues or maternal cues. Our 3D model opens routes to capturing molecular events underlying axial symmetry breaking phenomena, which have largely been unexplored in model human systems.

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BMP4-induced symmetry breaking in a 3D model of the human epiblast

10.21203/rs.2.9730/v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Mijo Simunovic ◽

Ali H. Brivanlou ◽

Eric D. Siggia

Keyword(s):

Live Cell Imaging ◽

3D Model ◽

Epithelial To Mesenchymal Transition ◽

Cell Imaging ◽

Embryonic Stem ◽

Primitive Streak ◽

Mesenchymal Transition ◽

Pluripotency Markers ◽

Anterior Posterior

Abstract We describe the protocol of generating a 3D stem-cell-based model of the human pre-gastrulation epiblast by culturing human embryonic stem cells in a mix of hydrogel and Matrigel. Much like the epiblast of an in vitro attached day-10 human embryo, this model is an epithelial sphere with a cavity at its center, it is expressing key pluripotency markers, and it displays apico-basal polarity. The 3D colonies can further be differentiated with morphogens and in the case of intermediate concentrations of BMP4, they break the anterior-posterior symmetry characterized by an asymmetric expression of a primitive streak marker and showing signs of epithelial to mesenchymal transition. The protocol described here is suitable for immunofluorescence staining and for live-cell imaging.

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Self-organization of a functional human organizer by combined WNT and NODAL signalling

10.1101/234633 ◽

2017 ◽

Cited By ~ 3

Author(s):

I. Martyn ◽

T.Y. Kanno ◽

A. Ruzo ◽

E.D. Siggia ◽

A.H. Brivanlou

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Embryonic Stem ◽

Primitive Streak ◽

Model Organisms ◽

Detailed Characterization ◽

Mesenchymal Transition ◽

Neural Fate ◽

Secondary Axis ◽

Human Embryology ◽

Emt Markers

In amniotes, the development of the primitive streak (PS) and its accompanying “organizer” define the first stages of gastrulation. Despite detailed characterization in model organisms, the analogous human structures remain a mystery. We have previously shown that when stimulated with BMP4, micropatterned colonies of human embryonic stem cells (hESCs) self-organize to generate early embryonic germ layers1. Here we show that in the same type of colonies WNT signalling is sufficient to induce a PS, and WNT with ACTIVIN is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, epithelial-to-mesenchymal transition (EMT) markers, and expression of the organizer specific transcription factor GSC. Moreover, when grafted into chick embryos, WNT and ACTIVIN treated human cells induce and contribute autonomously to a secondary axis while inducing neural fate in the host. This fulfills the most stringent functional criteria for an organizer, and its discovery represents a major milestone in human embryology.

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FGF signaling mediates definitive endoderm formation by regulating epithelial-to-mesenchymal transition and cell proliferation

The International Journal of Developmental Biology ◽

10.1387/ijdb.190372ql ◽

2020 ◽

Vol 64 (10-11-12) ◽

pp. 471-477

Author(s):

Shengbiao Li ◽

Qingsong Huang ◽

Jianwen Mao ◽

Qiuhong LI

Keyword(s):

Cell Proliferation ◽

Epithelial To Mesenchymal Transition ◽

Embryonic Stem ◽

Selective Inhibitor ◽

Definitive Endoderm ◽

Fgf Signaling ◽

Adhesion Protein ◽

Mesenchymal Transition ◽

Zinc Finger Transcription Factor

FGF signaling pathway is imperative for definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs), which always accompanies an epithelial-to-mesenchymal transition (EMT) process. However, whether there is an association between FGF signaling and the EMT during DE formation in vitro has remained elusive. In the present study, we identify that several FGF family members were significantly activated during the differentiation of hESCs toward DE. Inhibition of FGF signaling by an efficient and selective inhibitor BGJ398 abolishes both the EMT and DE induction by blocking the activation of the zinc-finger transcription factor SNAI1 which is a direct transcriptional repressor of cell adhesion protein CDH1. In addition, cell proliferation is also severely influenced by attenuating the FGF signaling. Collectively, we propose that the FGF signaling promotes the DE formation through mediating the EMT and cell proliferation.

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Endoderm and Hepatic Progenitor Cells Engraft in the Quiescent Liver Concurrent with Intrinsically Activated Epithelial-to-Mesenchymal Transition

Cell Transplantation ◽

10.1177/0963689721993780 ◽

2021 ◽

Vol 30 ◽

pp. 096368972199378

Author(s):

W. Samuel Fagg ◽

Naiyou Liu ◽

Igor Patrikeev ◽

Omar A. Saldarriaga ◽

Massoud Motamedi ◽

...

Keyword(s):

Progenitor Cell ◽

Iron Nanoparticles ◽

Epithelial To Mesenchymal Transition ◽

High Efficiency ◽

Embryonic Stem ◽

Mesenchymal Transition ◽

Cell Engraftment ◽

Resolution Imaging

Stem cell transplantation to the liver is a promising therapeutic strategy for a variety of disorders. Hepatocyte transplantation has short-term efficacy but can be problematic due to portal hypertension, inflammation, and sinusoidal thrombosis. We have previously transplanted small mouse endoderm progenitor (EP) cells to successfully reverse a murine model of hemophilia B, and labeling these cells with iron nanoparticles renders them responsive to magnetic fields, which can be used to enhance engraftment. The mechanisms mediating progenitor cell migration from the sinusoidal space to the hepatocyte compartment are unknown. Here we find human EP and hepatic progenitor (HP) cells can be produced from human embryonic stem cells with high efficiency, and they also readily uptake iron nanoparticles. This provides a simple manner through which one can readily identify transplanted cells in vivo using electron microscopy, shortly after delivery. High resolution imaging shows progenitor cell morphologies consistent with epithelial-to-mesenchymal transition (EMT) mediating invasion into the hepatic parenchyma. This occurs in as little as 3 h, which is considerably faster than observed when hepatocytes are transplanted. We confirmed activated EMT in transplanted cells in vitro, as well as in vivo 24 h after transplantation. We conclude that EMT naturally occurs concurrent with EP and HP cell engraftment, which may mediate the rate, safety, and efficacy of early cell engraftment in the undamaged quiescent liver.

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PI3K/Akt1 signalling specifies foregut precursors by generating regionalized extra-cellular matrix

eLife ◽

10.7554/elife.00806 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 20

Author(s):

S Nahuel Villegas ◽

Michaela Rothová ◽

Martin E Barrios-Llerena ◽

Maria Pulina ◽

Anna-Katerina Hadjantonakis ◽

...

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cell ◽

Central Question ◽

Mesenchymal Transition ◽

Pi3k Activity ◽

Pi3k Signalling ◽

Low Levels

During embryonic development signalling pathways act repeatedly in different contexts to pattern the emerging germ layers. Understanding how these different responses are regulated is a central question for developmental biology. In this study, we used mouse embryonic stem cell (mESC) differentiation to uncover a new mechanism for PI3K signalling that is required for endoderm specification. We found that PI3K signalling promotes the transition from naïve endoderm precursors into committed anterior endoderm. PI3K promoted commitment via an atypical activity that delimited epithelial-to-mesenchymal transition (EMT). Akt1 transduced this activity via modifications to the extracellular matrix (ECM) and appropriate ECM could itself induce anterior endodermal identity in the absence of PI3K signalling. PI3K/Akt1-modified ECM contained low levels of Fibronectin (Fn1) and we found that Fn1 dose was key to specifying anterior endodermal identity in vivo and in vitro. Thus, localized PI3K activity affects ECM composition and ECM in turn patterns the endoderm.

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Recombinant mouse SPARC promotes parietal endoderm differentiation and cardiomyogenesis in embryoid bodies

Biochemistry and Cell Biology ◽

10.1139/o08-141 ◽

2008 ◽

Vol 86 (6) ◽

pp. 487-499 ◽

Cited By ~ 8

Author(s):

Christopher Hrabchak ◽

Maurice Ringuette ◽

Kimberly Woodhouse

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Embryonic Stem ◽

Recombinant Baculovirus ◽

Neuronal Cell ◽

Embryoid Bodies ◽

Marker Genes ◽

Mesenchymal Transition ◽

Enhanced Expression ◽

Parietal Endoderm

In the absence of leukemia inhibitory factor, murine embryonic stem cells cultured in vitro spontaneously aggregate to from three-dimensional embryoid bodies that differentiate to produce hematopoietic, endothelial, muscle, and neuronal cell lineages in a manner recapitulating the events of early embryogenesis. Cardiomyogenesis in embryoid bodies was recently demonstrated to be promoted by PYS-2-derived native SPARC (secreted protein, acidic and rich in cysteine), whose expression is upregulated in parietal endoderm at the onset of the epithelial to mesenchymal transition. Here, we confirm the stimulatory effects of mouse SPARC on cardiomyogenesis using a recombinant baculovirus-produced protein (rmSPARC). Embryoid bodies cultured in the presence of glycosylated rmSPARC, or an unglycosylated peptide spanning the C-terminal EF-hand domain, developed greater numbers of beating cardiomyocytes than did time-matched controls, with enhanced expression of cardiac marker genes including Nkx2.5, Troponin, BMP-2, and MHCα. Histochemical analysis revealed an expansion of the peripheral endoderm, with thicker layers of extracellular matrix (ECM) material observed atop underlying cells. Embryoid bodies treated with SPARC also displayed increased adherence to polystyrene culture dishes, with enhanced expression of ECM mRNAs including collagen IVα3, collagen IVα5, and laminin α1. These results indicate that, in addition to the promotion of cardiomyogenesis, SPARC may also help regulate the molecular composition and organization of ECM secreted by the mesenchymal parietal endoderm.

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Brachyury cooperates with Wnt/β-Catenin signalling to elicit Primitive Streak like behaviour in differentiating mouse ES cells.

10.1101/003871 ◽

2014 ◽

Cited By ~ 1

Author(s):

David Andrew Turner ◽

Pau Rué ◽

Jonathan P Mackenzie ◽

Eleanor Davies ◽

Alfonso Martinez Arias

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Es Cells ◽

Single Cells ◽

Embryonic Stem ◽

Primitive Streak ◽

Mouse Development ◽

Mesenchymal Transition ◽

Mouse Es Cells ◽

Cellular Events ◽

Early Mouse

The formation of the Primitive Streak is the first visible sign of gastrulation, the process by which the three germ layers are formed from a single epithelium during early development. Embryonic Stem Cells (ESCs) provide a good system to understand the molecular and cellular events associated with these processes. Previous work, both in embryos and in culture, has shown how converging signals from both Nodal/TGFβR and Wnt/β-Catenin signalling pathways specify cells to adopt a Primitive Streak like fate and direct them to undertake an epithelial to mesenchymal transition (EMT). However, many of these approaches have relied on genetic analyses without taking into account the temporal progression of events within single cells. In addition, it is still unclear as to what extent events in the embryo are able to be reproduced in culture. Here, we combine flow-cytometry and a quantitative live single-cell imaging approach to demonstrate how the controlled differentiation of mouse ESCs (mESCs) towards a Primitive Streak fate in culture results in cells displaying many of the characteristics observed during early mouse development including transient Brachyury expression, EMT and increased motility. We also find that the EMT initiates the process, and this is both fuelled and terminated by the action of Bra, whose expression is dependent on the EMT and β-Catenin activity. As a consequence of our analysis, we propose that a major output of Brachyury expression is in controlling the velocity of the cells that are transiting out of the Primitive Streak.

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Operative ubiquitin-specific protease 22 deubiquitination confers a more invasive phenotype to cholangiocarcinoma

Cell Death and Disease ◽

10.1038/s41419-021-03940-0 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Yu Tian ◽

Bo Tang ◽

Chengye Wang ◽

Yan Wang ◽

Jiakai Mao ◽

...

Keyword(s):

Tumour Growth ◽

Molecular Mechanisms ◽

Epithelial To Mesenchymal Transition ◽

Sirtuin 1 ◽

Mesenchymal Transition ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Paired Tumour

AbstractOncogenic ubiquitin-specific protease 22 (USP22) is implicated in a variety of tumours; however, evidence of its role and underlying molecular mechanisms in cholangiocarcinoma (CCA) development remains unknown. We collected paired tumour and adjacent non-tumour tissues from 57 intrahepatic CCA (iCCA) patients and evaluated levels of the USP22 gene and protein by qPCR and immunohistochemistry. Both the mRNA and protein were significantly upregulated, correlated with the malignant invasion and worse OS of iCCA. In cell cultures, USP22 overexpression increased CCA cell proliferation and mobility, and induced epithelial-to-mesenchymal transition (EMT). Upon an interaction, USP22 deubiquitinated and stabilized sirtuin-1 (SIRT1), in conjunction with Akt/ERK activation. In implantation xenografts, USP22 overexpression stimulated tumour growth and metastasis to the lungs of mice. Conversely, the knockdown by USP22 shRNA attenuated the tumour growth and invasiveness in vitro and in vivo. Furthermore, SIRT1 overexpression reversed the USP22 functional deficiency, while the knockdown acetylated TGF-β-activated kinase 1 (TAK1) and Akt. Our present study defines USP22 as a poor prognostic predictor in iCCA that cooperates with SIRT1 and facilitates tumour development.

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Plasticity in Neuroblastoma Cell Identity Defines a Noradrenergic-to-Mesenchymal Transition (NMT)

Cancers ◽

10.3390/cancers13122904 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2904

Author(s):

Margot Gautier ◽

Cécile Thirant ◽

Olivier Delattre ◽

Isabelle Janoueix-Lerosey

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Neuroblastoma Cell ◽

Neuroblastoma Cells ◽

Biochemical Properties ◽

Poor Overall Survival ◽

Cell Identity ◽

Mesenchymal Transition ◽

Neuroblastoma Cell Lines

Neuroblastoma, a pediatric cancer of the peripheral sympathetic nervous system, is characterized by an important clinical heterogeneity, and high-risk tumors are associated with a poor overall survival. Neuroblastoma cells may present with diverse morphological and biochemical properties in vitro, and seminal observations suggested that interconversion between two phenotypes called N-type and S-type may occur. In 2017, two main studies provided novel insights into these subtypes through the characterization of the transcriptomic and epigenetic landscapes of a panel of neuroblastoma cell lines. In this review, we focus on the available data that define neuroblastoma cell identity and propose to use the term noradrenergic (NOR) and mesenchymal (MES) to refer to these identities. We also address the question of transdifferentiation between both states and suggest that the plasticity between the NOR identity and the MES identity defines a noradrenergic-to-mesenchymal transition, reminiscent of but different from the well-established epithelial-to-mesenchymal transition.

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