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

2014 ◽  
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.

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

2018 ◽  
Author(s):  
Mijo Simunovic ◽  
Jakob J. Metzger ◽  
Fred Etoc ◽  
Anna Yoney ◽  
Albert Ruzo ◽  
...  
Keyword(s):  
Symmetry Breaking ◽  
Axial Symmetry ◽  
3D Model ◽  
Epithelial To Mesenchymal Transition ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Molecular Evidence ◽  
Axis Formation ◽  
Mesenchymal Transition

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.

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

2019 ◽  
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.

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

2017 ◽  
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.

scholarly journals An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

2013 ◽  
Author(s):  
David A Turner ◽  
Jamie Trott ◽  
Penelope Hayward ◽  
Pau Rué ◽  
Alfonso Martinez Arias
Keyword(s):  
Cell Fate ◽  
Neural Development ◽  
Es Cells ◽  
Initial Period ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Wnt Signalling ◽  
Dependent Manner ◽  
Cell Fate Decisions ◽  
Mouse Es Cells

Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a ?race for fates? in which the neuroectodermal fate has an advantage.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

Strain-specific transgene methylation occurs early in mouse development and can be recapitulated in embryonic stem cells

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2853-2859 ◽  
Author(s):  
A. Weng ◽  
T. Magnuson ◽  
U. Storb
Keyword(s):  
De Novo ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Mouse Development ◽  
Partial Methylation ◽  
Distal Chromosome ◽  
Before And After ◽  
Endogenous Genes ◽  
De Novo Methylation

A murine transgene, HRD, is methylated only when carried in certain inbred strain backgrounds. A locus on distal chromosome 4, Ssm1 (strain-specific modifier), controls this phenomenon. In order to characterize the activity of Ssm1, we have investigated developmental acquisition of methylation over the transgene. Analysis of postimplantation embryos revealed that strain-specific methylation is initiated prior to embryonic day (E) 6.5. Strain-specific transgene methylation is all-or-none in pattern and occurs exclusively in the primitive ectoderm lineage. A strain-independent pattern of partial methylation occurs in the primitive endoderm and trophectoderm lineages. To examine earlier stages, embryonic stem (ES) cells were derived from E3.5 blastocysts and examined for transgene methylation before and after differentiation. Though the transgene had already acquired some methylation in undifferentiated ES cells, differentiation induced further, de novo methylation in a strain-dependent manner. Analysis of methylation in ES cultures suggests that the transgene and endogenous genes (such as immunoglobulin genes) are synchronously methylated during early development. These results are interpreted in the context of a model in which Ssm1-like modifier genes produce alterations in chromatin structure during and/or shortly after implantation, thereby marking target loci for de novo methylation with the rest of the genome during gastrulation.

Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats

2002 ◽  
Vol 92 (1) ◽  
pp. 288-296 ◽  
Author(s):  
Jiang-Yong Min ◽  
Yinke Yang ◽  
Kimber L. Converso ◽  
Lixin Liu ◽  
Qin Huang ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Cell Transplantation ◽  
Troponin I ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Control Group ◽  
Positive Staining ◽  
Es Cell ◽  
Intramyocardial Injection

Massive loss of cardiac myocytes after myocardial infarction (MI) is a common cause of heart failure. The present study was designed to investigate the improvement of cardiac function in MI rats after embryonic stem (ES) cell transplantation. MI in rats was induced by ligation of the left anterior descending coronary artery. Cultured ES cells used for cell transplantation were transfected with the marker green fluorescent protein (GFP). Animals in the treated group received intramyocardial injection of ES cells in injured myocardium. Compared with the MI control group injected with an equivalent volume of the cell-free medium, cardiac function in ES cell-implanted MI animals was significantly improved 6 wk after cell transplantation. The characteristic phenotype of engrafted ES cells was identified in implanted myocardium by strong positive staining to sarcomeric α-actin, cardiac α-myosin heavy chain, and troponin I. GFP-positive cells in myocardium sectioned from MI hearts confirmed the survival and differentiation of engrafted cells. In addition, single cells isolated from cell-transplanted MI hearts showed rod-shaped GFP-positive myocytes with typical striations. The present data demonstrate that ES cell transplantation is a feasible and novel approach to improve ventricular function in infarcted failing hearts.

Human embryonic stem cells

2000 ◽  
Vol 113 (1) ◽  
pp. 5-10 ◽  
Author(s):  
M.F. Pera ◽  
B. Reubinoff ◽  
A. Trounson
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Carcinoma Cells ◽  
Mouse Es Cells ◽  
Undifferentiated State ◽  
Human Es Cells

Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.

flk-1, an flt-related receptor tyrosine kinase is an early marker for endothelial cell precursors

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 489-498 ◽  
Author(s):  
T.P. Yamaguchi ◽  
D.J. Dumont ◽  
R.A. Conlon ◽  
M.L. Breitman ◽  
J. Rossant
Keyword(s):  
Endothelial Cell ◽  
Tyrosine Kinase ◽  
Blood Vessels ◽  
Tyrosine Kinases ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Morphological Evidence ◽  
Mouse Development ◽  
Early Marker

We have used RT-PCR to screen pluripotent murine embryonic stem cells to identify receptor tyrosine kinases (RTKs) potentially involved in the determination or differentiation of cell lineages during early mouse development. Fourteen different tyrosine kinase sequences were identified. The expression patterns of four RTKs have been examined and all are expressed in the mouse embryo during, or shortly after, gastrulation. We report here the detailed expression pattern of one such RTK, the flt-related gene flk-1. In situ hybridization analysis of the late primitive streak stage embryo revealed that flk-1 was expressed in the proximal-lateral embryonic mesoderm; tissue fated to become heart. By headfold stages, staining was confined to the endocardial cells of the heart primordia as well as to the blood islands of the visceral yolk sac and the developing allantois. Patchy, speckled staining was detected in the endothelium of all the major embryonic and extraembryonic blood vessels as they formed. During early organogenesis, expression was detected in the blood vessels of highly vascularized tissues such as the brain, liver, lungs and placenta. Since flk-1 was expressed in early mesodermal cells prior to any morphological evidence for endothelial cell differentiation (vasculogenesis), as well as in cells that form blood vessels from preexisting ones (angiogenesis), it appears to be a very early marker of endothelial cell precursors. We have previously reported that another novel RTK, designated tek, was expressed in differentiating endothelial cells. We show here that flk-1 transcripts are expressed one full embryonic day earlier than the first tek transcripts. The expression of these two RTKs appear to correlate with the specification and early differentiation of the endothelial cell lineage respectively, and therefore may play important roles in the establishment of this lineage.

The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 877-886 ◽  
Author(s):  
V. Wilson ◽  
L. Manson ◽  
W.C. Skarnes ◽  
R.S. Beddington
Keyword(s):  
T Cells ◽  
Neural Tube ◽  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Distal Portion ◽  
Adhesion Properties ◽  
Cell Clones ◽  
Posterior Neuropore ◽  
Pass Through

The T (Brachyury) deletion in mouse is responsible for defective primitive streak and notochord morphogenesis, leading to a failure of the axis to elongate properly posterior to the forelimb bud. T/T embryonic stem (ES) cells colonise wild-type embryos, but in chimeras at 10.5 days post coitum (dpc) onwards they are found predominantly in the distal tail, while trunk paraxial and lateral mesoderm are deficient in T/T cells (Wilson, V., Rashbass, P. and Beddington, R. S. P. (1992) Development 117, 1321–1331). To determine the origin of this abnormal tissue distribution, we have isolated T/T and control T/+ ES cell clones which express lacZ constitutively using a gene trap strategy. Visualisation of T/T cell distribution in chimeric embryos throughout gastrulation up to 10.5 dpc shows that a progressive buildup of T/T cells in the primitive streak during gastrulation leads to their incorporation into the tailbud. These observations make it likely that one role of the T gene product is to act during gastrulation to alter cell surface (probably adhesion) properties as cells pass through the primitive streak. As the chimeric tail elongates at 10.5 dpc, abnormal morphology in the most distal portion becomes apparent. Comparison of T expression in the developing tailbud with the sites of accumulation of T/T cells in chimeras shows that T/T cells collect in sites where T would normally be expressed. T expression becomes internalised in the tailbud following posterior neuropore closure while, in abnormal chimeric tails, T/T cells remain on the surface of the distal tail. We conclude that prevention of posterior neuropore closure by the wedge of T/T cells remaining in the primitive streak after gastrulation is one source of the abnormal tail phenotypes observed. Accumulation of T/T cells in the node and anterior streak during gastrulation results in the preferential incorporation of T/T cells into the ventral portion of the neural tube and axial mesoderm. The latter forms compact blocks which are often fused with the ventral neural tube, reminiscent of the notochordal defects seen in intact mutants. Such fusions may be attributed to cell-autonomous changes in cell adhesion, possibly related to those observed at earlier stages in the primitive streak.

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