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 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 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.

Prolonged atrazine exposure beginning in utero and adult uterine morphology in mice

2021 ◽  
pp. 1-10
Author(s):  
Meaghan J. Griffiths ◽  
Amy L. Winship ◽  
Jessica M. Stringer ◽  
Elyse O. Swindells ◽  
Alesia P. Harper ◽  
...  
Keyword(s):  
Drinking Water ◽  
Oestrogen Receptor ◽  
Endometrial Hyperplasia ◽  
Epithelial To Mesenchymal Transition ◽  
Cell Metabolism ◽  
In Utero ◽  
Receptor Expression ◽  
Mesenchymal Transition ◽  
Pregnant Mice ◽  
Emt Markers

Abstract Through drinking water, humans are commonly exposed to atrazine, a herbicide that acts as an endocrine and metabolic disruptor. It interferes with steroidogenesis, including promoting oestrogen production and altering cell metabolism. However, its precise impact on uterine development remains unknown. This study aimed to determine the effect of prolonged atrazine exposure on the uterus. Pregnant mice (n = 5/group) received 5 mg/kg body weight/day atrazine or DMSO in drinking water from gestational day 9.5 until weaning. Offspring continued to be exposed until 3 or 6 months of age (n = 5–9/group), when uteri were collected for morphological and molecular analyses and steroid quantification. Endometrial hyperplasia and leiomyoma were evident in the uteri of atrazine-exposed mice. Uterine oestrogen concentration, oestrogen receptor expression, and localisation were similar between groups, at both ages (P > 0.1). The expression and localisation of key epithelial-to-mesenchymal transition (EMT) genes and proteins, critical for tumourigenesis, remained unchanged between treatments, at both ages (P > 0.1). Hence, oestrogen-mediated changes to established EMT markers do not appear to underlie abnormal uterine morphology evident in atrazine exposure mice. This is the first report of abnormal uterine morphology following prolonged atrazine exposure starting in utero, it is likely that the abnormalities identified would negatively affect female fertility, although mechanisms remain unknown and require further study.

scholarly journals DPYSL3 modulates mitosis, migration, and epithelial-to-mesenchymal transition in claudin-low breast cancer

2018 ◽  
Vol 115 (51) ◽  
pp. E11978-E11987 ◽  
Author(s):  
Ryoichi Matsunuma ◽  
Doug W. Chan ◽  
Beom-Jun Kim ◽  
Purba Singh ◽  
Airi Han ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Negative Feedback ◽  
Epithelial To Mesenchymal Transition ◽  
Triple Negative ◽  
Breast Cancers ◽  
Mesenchymal Transition ◽  
Multinucleated Cells ◽  
P21 Activated Kinase ◽  
Emt Markers ◽  
Mitotic Defect

A Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomic analysis prioritized dihydropyrimidinase-like-3 (DPYSL3) as a multilevel (RNA/protein/phosphoprotein) expression outlier specific to the claudin-low (CLOW) subset of triple-negative breast cancers. A PubMed informatics tool indicated a paucity of data in the context of breast cancer, which further prioritized DPYSL3 for study. DPYSL3 knockdown in DPYSL3-positive (DPYSL3+) CLOW cell lines demonstrated reduced proliferation, yet enhanced motility and increased expression of epithelial-to-mesenchymal transition (EMT) markers, suggesting that DPYSL3 is a multifunctional signaling modulator. Slower proliferation in DPYSL3-negative (DPYSL3−) CLOW cells was associated with accumulation of multinucleated cells, indicating a mitotic defect that was associated with a collapse of the vimentin microfilament network and increased vimentin phosphorylation. DPYSL3 also suppressed the expression of EMT regulators SNAIL and TWIST and opposed p21 activated kinase 2 (PAK2)-dependent migration. However, these EMT regulators in turn induce DPYSL3 expression, suggesting that DPYSL3 participates in negative feedback on EMT. In conclusion, DPYSL3 expression identifies CLOW tumors that will be sensitive to approaches that promote vimentin phosphorylation during mitosis and inhibitors of PAK signaling during migration and EMT.

Dexamethasone-dependent versus -independent markers of epithelial to mesenchymal transition in primary hepatocytes

2010 ◽  
Vol 391 (1) ◽  
Author(s):  
Patricio Godoy ◽  
Sumathi Lakkapamu ◽  
Markus Schug ◽  
Alexander Bauer ◽  
Joanna D. Stewart ◽  
...  
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Culture Media ◽  
Three Dimensional ◽  
Morphological Features ◽  
Collagen Gels ◽  
Mesenchymal Transition ◽  
Subsequent Step ◽  
Twist 1 ◽  
Emt Markers

Abstract Recently, epithelial to mesenchymal transition (EMT) has been shown to represent a feature of dedifferentiating hepatocytes in vitro. Three-dimensional soft collagen gels can antagonize but not completely abolish this effect. Hormonal additives to culture media are known to maintain differentiated hepatocyte functions. Therefore, we studied whether insulin and dexamethasone antagonize EMT in cultured hepatocytes. Both hormones antagonized but not completely abolished certain morphological features of EMT. Dexamethasone antagonized acquisition of fibroblastoid shape, whereas insulin favored bile canaliculi formation. In a subsequent step, we analyzed expression of a battery of EMT-related genes. Of all markers tested, vimentin and snail-1 correlated best with morphological features of EMT. Interestingly, dexamethasone reduced expression levels of both vimentin and snail-1, whereas the influence of insulin was less pronounced. An important result of this study is that 12 out of 17 analyzed EMT markers were transcriptionally influenced by dexamethasone (vimentin, snail-1, snail-2, HNF4α, Twist-1, ZEB2, fibronectin, occludin, MMP14, claudin-1, cytokeratin-8, and cytokeratin-18), whereas the remaining factors seemed to be less dependent on dexamethasone. In conclusion, EMT markers in hepatocytes can be classified as dexamethasone-dependent versus -independent.

scholarly journals Mouse primitive streak forms in situ by initiation of epithelial to mesenchymal transition without migration of a cell population

2011 ◽  
Vol 241 (2) ◽  
pp. 270-283 ◽  
Author(s):  
Margot Williams ◽  
Carol Burdsal ◽  
Ammasi Periasamy ◽  
Mark Lewandoski ◽  
Ann Sutherland
Keyword(s):  
Cell Population ◽  
Epithelial To Mesenchymal Transition ◽  
Primitive Streak ◽  
Mesenchymal Transition ◽  
A Cell

A Novel Biomimetic Model for Studying Mechanics of Embryonic Morphogenesis and Differentiation

2010 ◽  
Author(s):  
Julia A. Henkels ◽  
Evan A. Zamir
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Primitive Streak ◽  
Mitotic Cell ◽  
Embryonic Cells ◽  
Mesenchymal Transition ◽  
Genetics Research ◽  
Undifferentiated Cells ◽  
Organizing Center ◽  
Important Time ◽  
Anterior Posterior

Before the explosion of genetics research in the last century, embryonic development was largely studied from a mechanical perspective. Paired with genetic advances in understanding developmental signaling pathways and induction mechanisms, an important goal for understanding morphogenesis is to discover how the genome codes for changes in the mechanical movements of the embryonic cells. After formation of the zygote, a phase of rapid mitotic cell division is followed by epithelialization resulting in a cohesive sheet of cells termed the epiblast. During the next major phase of triploblastic development called gastrulation, a group of undifferentiated cells in the epiblast moves collectively to the embryonic midline and eventually gives rise to the three primary germ layers: endoderm, mesoderm, and ectoderm. At the primitive streak—the “organizing center” in amniotes (reptiles, birds, and mammals) which delineates anterior-posterior polarity—prospective endodermal and mesodermal precursors undergo epithelial-to-mesenchymal transition (EMT), internalization, and eventually organogenesis. “It is not birth, marriage, or death, but gastrulation which is truly the most important time in your life” (Lewis Wolpert, 1986).

scholarly journals Putative oncogene Brachyury (T) is essential to specify cell fate but dispensable for notochord progenitor proliferation and EMT

2016 ◽  
Vol 113 (14) ◽  
pp. 3820-3825 ◽  
Author(s):  
Jianjian Zhu ◽  
Kin Ming Kwan ◽  
Susan Mackem
Keyword(s):  
Cell Fate ◽  
Epithelial Mesenchymal Transition ◽  
Primitive Streak ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Mesenchymal Transition ◽  
Neural Fate ◽  
Notochord Cell ◽  
Genetic Lineage Tracing ◽  
And Function

The transcription factor Brachyury (T) gene is expressed throughout primary mesoderm (primitive streak and notochord) during early embryonic development and has been strongly implicated in the genesis of chordoma, a sarcoma of notochord cell origin. Additionally, T expression has been found in and proposed to play a role in promoting epithelial–mesenchymal transition (EMT) in various other types of human tumors. However, the role of T in normal mammalian notochord development and function is still not well-understood. We have generated an inducible knockdown model to efficiently and selectively deplete T from notochord in mouse embryos. In combination with genetic lineage tracing, we show that T function is essential for maintaining notochord cell fate and function. Progenitors adopt predominantly a neural fate in the absence of T, consistent with an origin from a common chordoneural progenitor. However, T function is dispensable for progenitor cell survival, proliferation, and EMT, which has implications for the therapeutic targeting of T in chordoma and other cancers.

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