scholarly journals EZH2 is required for parathyroid and thymic development through differentiation of the third pharyngeal pouch endoderm

2021 ◽  
pp. dmm.046789
Author(s):  
Cinzia Caprio ◽  
Gabriella Lania ◽  
Marchesa Bilio ◽  
Rosa Ferrentino ◽  
Li Chen ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Mouse Embryos ◽  
Pharyngeal Pouch ◽  
Expression Domain ◽  
Polycomb Repressive Complex 2 ◽  
Thymic Development ◽  
Conditional Deletion ◽  
Pharyngeal Apparatus

The Ezh2 gene encodes a histone methyltransferase of the Polycomb Repressive Complex 2 that methylates histone H3 lysine 27. In this work we asked whether EZH2 has a role in the development of the pharyngeal apparatus and whether it regulates the expression of the Tbx1 gene, which encodes a key transcription factor required in pharyngeal development. To these ends, we performed genetic in vivo experiments with mouse embryos and we used mouse embryonic stem cell (ESC)-based protocols to probe endoderm and cardiogenic mesoderm differentiation. Results showed that EZH2 occupies the Tbx1 gene locus in mouse embryos, and that suppression of EZH2 was associated with reduced expression of Tbx1 in differentiated mESCs. Conditional deletion of Ezh2 in the Tbx1 expression domain, which includes the pharyngeal endoderm, did not cause cardiac defects but revealed that the gene has an important role in the morphogenesis of the 3rd pharyngeal pouch (PP). We found that in conditionally deleted embryos the 3rd PP was hypoplastic, had reduced expression of Tbx1, lacked the expression of Gcm2, a gene that marks the parathyroid domain, but expressed FoxN1, a gene marking the thymic domain. Consistently, the parathyroids did not develop, and the thymus was hypoplastic. Thus, Ezh2 is required for parathyroid and thymic development, probably through a function in the pouch endoderm. This discovery also provides a novel interpretational key for the finding of Ezh2 activating mutations in hyperparathyroidism and parathyroid cancer.

scholarly journals Bidirectional Wnt signaling between endoderm and mesoderm confer tracheal identity in mouse and human

2019 ◽  
Author(s):  
Keishi Kishimoto ◽  
Kana T. Furukawa ◽  
Agustin Luz Madrigal ◽  
Akira Yamaoka ◽  
Chisa Matsuoka ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Wnt Signaling ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Bmp Signaling ◽  
Lateral Plate ◽  
Tracheal Tissue ◽  
Canonical Wnt ◽  
Human And Mouse

AbstractThe periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. Loss of β-catenin in fetal mouse mesoderm caused loss of Tbx4+ tracheal mesoderm and tracheal cartilage agenesis. The Tbx4 expression relied on endodermal Wnt activity and its downstream Wnt ligand but independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Repopulating in vivo model, activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generated tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation was required along with Wnt to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.

scholarly journals Insight into Nephrocan Function in Mouse Endoderm Patterning

2019 ◽  
Vol 21 (1) ◽  
pp. 8
Author(s):  
Martina Addeo ◽  
Silvia Buonaiuto ◽  
Ilaria Guerriero ◽  
Elena Amendola ◽  
Feliciano Visconte ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Specific Expression ◽  
Knock Out ◽  
New Variant ◽  
Liver And Pancreas ◽  
Regenerative Therapies

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5–11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn−/− mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

scholarly journals Cripto promotes A–P axis specification independently of its stimulatory effect on Nodal autoinduction

2008 ◽  
Vol 180 (3) ◽  
pp. 597-605 ◽  
Author(s):  
Daniela D'Andrea ◽  
Giovanna L. Liguori ◽  
J. Ann Le Good ◽  
Enza Lonardo ◽  
Olov Andersson ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Embryonic Stem ◽  
Cellular Transformation ◽  
Axis Formation ◽  
Receptor Complex ◽  
Expression Domain ◽  
Vertebrate Embryo ◽  
Axis Specification ◽  
Anterior Posterior

The EGF-CFC gene cripto governs anterior–posterior (A–P) axis specification in the vertebrate embryo. Existing models suggest that Cripto facilitates binding of Nodal to an ActRII–activin-like kinase (ALK) 4 receptor complex. Cripto also has a crucial function in cellular transformation that is independent of Nodal and ALK4. However, how ALK4-independent Cripto pathways function in vivo has remained unclear. We have generated cripto mutants carrying the amino acid substitution F78A, which blocks the Nodal–ALK4–Smad2 signaling both in embryonic stem cells and cell-based assays. In criptoF78A/F78A mouse embryos, Nodal fails to expand its own expression domain and that of cripto, indicating that F78 is essential in vivo to stimulate Smad-dependent Nodal autoinduction. In sharp contrast to cripto-null mutants, criptoF78A/F78A embryos establish an A–P axis and initiate gastrulation movements. Our findings provide in vivo evidence that Cripto is required in the Nodal–Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A–P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal–ALK4–Smad2 signaling pathway.

Abstract 78: Pnmt+ "Primer" Cells Give Rise to Cardiac Myocytes and Neurons in the Mammalian Heart: Development of New Experimental Tools for Cardiac Regenerative Medicine Applications

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Namita M Varudkar ◽  
Jixiang Xia ◽  
Ibrahim Abukenda ◽  
Karl Pfeifer ◽  
Steven Ebert
Keyword(s):  
Stem Cell ◽  
Regenerative Medicine ◽  
Neural Crest ◽  
Heart Development ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Stem Cell Differentiation ◽  
Left Ventricular

Phenylethanolamine n-methyltransferase (Pnmt) catalyzes the conversion of norepinephrine to epinephrine, and thus serves as a marker for adrenergic cells. We employed a combination of immunofluorescent histochemical staining and genetic fate-mapping strategies to show that two separate Pnmt+ cell populations contribute to heart development. Intrinsic cardiac adrenergic (ICA) cells originate from the primary heart field, and contribute to pacemaking, conduction, and working (contractile) myocardium. A second population of cardiac Pnmt+ cells is derived from migrating neural crest. These neural crest adrenergic (NCA) cells appear to contribute to cardiac neurons. By adulthood, most of the Pnmt+ cells show a distinctively left-sided orientation in the heart, with nearly 90% of them being found in the left atrium and ventricle. Surprisingly large swaths of ventricular muscle are derived from Pnmt+ primer cells. Since this region of the heart is highly vulnerable to coronary artery disease and often sustains varying degrees of damage following myocardial infarction, we hypothesize that directed stem cell differentiation into Pnmt+ primer cells could serve as a valuable resource for repair and/or regeneration of left ventricular myocardium for heart disease patients. To test this hypothesis, we have generated stable recombinant mouse embryonic stem cell (mESC) lines that express various fluorescent marker proteins under the control of the endogenous Pnmt gene regulatory network. These cells can be rapidly expanded in culture, sorted, and used for transplantation studies in animal models to determine their therapeutic effectiveness. The cells can be induced along cardiogenic or neurogenic pathways in vitro, and the resulting Pnmt+ cells from each population can then be collected and tested in vivo. To achieve this goal, we have knocked-in a nuclear-localized enhanced green fluorescent protein into the Pnmt locus to create Pnmt-nEGFP recombinant mESCs and mice. We show that nEGFP expression is specifically expressed in Pnmt+ cells in vitro and in vivo. This strategy allows us to identify and isolate Pnmt+ cells to evaluate their effectiveness for cardiac regenerative medicine applications. .

Ultrastructural comparison of developing mouse embryonic stem cell- and in vivo-derived cardiomyocytes

2006 ◽  
Vol 30 (10) ◽  
pp. 800-807 ◽  
Author(s):  
Hossein Baharvand ◽  
Abbas Piryaei ◽  
Razieh Rohani ◽  
Adeleh Taei ◽  
Mohammad Hassan Heidari ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell

scholarly journals RBBP4 modulates gene activity through acetylation and methylation of histone H3 lysine 27

2021 ◽  
Author(s):  
Weipeng Mu ◽  
Noel S Murcia ◽  
Keriayn N. Smith ◽  
Debashish U Menon ◽  
Della Yee ◽  
...  
Keyword(s):  
Histone H3 ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Regulatory Elements ◽  
Chromatin Binding ◽  
Disease Treatment ◽  
Polycomb Repressive Complex 2 ◽  
H3k27 Methylation ◽  
Transcriptional Changes ◽  
Stem Cell Lines

AbstractRBBP4 is a core subunit of polycomb repressive complex 2 (PRC2) and HDAC1/2-containing complexes, which are responsible for histone H3 lysine 27 (H3K27) methylation and deacetylation respectively. However, the mechanisms by which RBBP4 modulates the functions of these complexes remain largely unknown. We generated viable mouse embryonic stem cell lines with RBBP4 mutations that disturbed methylation and acetylation of H3K27 on target chromatin and found that RBBP4 is required for PRC2 assembly and H3K27me3 establishment on target chromatin. Moreover, in the absence of EED and SUZ12, RBBP4 maintained chromatin binding on PRC2 loci, suggesting that the pre-existence of RBBP4 on nucleosomes serves to recruit PRC2 to restore H3K27me3 on newly synthesized histones. As such, disruption of RBBP4 function led to dramatic changes in transcriptional profiles. In spite of the PRC2 association, we found that transcriptional changes were more closely tied to the deregulation of H3K27ac rather than H3K27me3 where increased levels of H3K27ac were found on numerous cis-regulatory elements, especially putative enhancers. These data suggest that RBBP4 controls acetylation levels by adjusting the activity of HDAC complexes. As histone methylation and acetylation have been implicated in cancer and neural disease, RBBP4 could serve as a potential target for disease treatment.

Physiological rationale for responsiveness of mouse embryonic stem cells to gp130 cytokines

Development ◽  
2001 ◽  
Vol 128 (12) ◽  
pp. 2333-2339 ◽  
Author(s):  
Jennifer Nichols ◽  
Ian Chambers ◽  
Tetsuya Taga ◽  
Austin Smith
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Preimplantation Mouse ◽  
In The Wild ◽  
Lactating Females ◽  
Hoechst Staining

Embryonic stem cells are established directly from the pluripotent epiblast of the preimplantation mouse embryo. Their derivation and propagation are dependent upon cytokine-stimulated activation of gp130 signal transduction. Embryonic stem cells maintain a close resemblance to epiblast in developmental potency and gene expression profile. The presumption of equivalence between embryonic stem cells and epiblast is challenged, however, by the finding that early embryogenesis can proceed in the absence of gp130. To explore this issue further, we have examined the capacity of gp130 mutant embryos to accommodate perturbation of normal developmental progression. Mouse embryos arrest at the late blastocyst stage when implantation is prevented. This process of diapause occurs naturally in lactating females or can be induced experimentally by removal of the ovaries. We report that gp130−/− embryos survive unimplanted in the uterus after ovariectomy but, in contrast to wild-type or heterozygous embryos, are subsequently unable to resume development. Inner cell masses explanted from gp130−/− delayed blastocysts produce only parietal endoderm, a derivative of the hypoblast. Intact mutant embryos show an absence of epiblast cells, and Hoechst staining and TUNEL analysis reveal a preceding increased incidence of cell death. These findings establish that gp130 signalling is essential for the prolonged maintenance of epiblast in vivo, which is commonly required of mouse embryos in the wild. We propose that the responsiveness of embryonic stem cells to gp130 signalling has its origin in this adaptive physiological function.

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

eLife ◽  
2013 ◽  
Vol 2 ◽  
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.

scholarly journals Normal Embryonic and Germ Cell Development in Mice Lacking α1,3-Fucosyltransferase IX (Fut9) Which Show Disappearance of Stage-Specific Embryonic Antigen 1

2004 ◽  
Vol 24 (10) ◽  
pp. 4221-4228 ◽  
Author(s):  
Takashi Kudo ◽  
Mika Kaneko ◽  
Hiroko Iwasaki ◽  
Akira Togayachi ◽  
Shoko Nishihara ◽  
...  
Keyword(s):  
Germ Cells ◽  
Primordial Germ Cells ◽  
Embryonic Stem ◽  
Mouse Embryos ◽  
Lewis X ◽  
Strong Activity ◽  
Embryonic Antigen ◽  
Stage Specific Embryonic Antigen

ABSTRACT Stage-specific embryonic antigen 1 (SSEA-1), an antigenic epitope defined as a Lewis x carbohydrate structure, is expressed during the 8-cell to blastocyst stages in mouse embryos and in primordial germ cells, undifferentiated embryonic stem cells, and embryonic carcinoma cells. For many years, SSEA-1 has been implicated in the development of mouse embryos as a functional carbohydrate epitope in cell-to-cell interaction during morula compaction. In a previous study, α1,3-fucosyltransferase IX (Fut9) exhibited very strong activity for the synthesis of Lewis x compared to other α1,3-fucosyltransferases in an in vitro substrate specificity assay. Fut4 and Fut9 transcripts were expressed in mouse embryos. The Fut9 transcript was detected in embryonic-day-13.5 gonads containing primordial germ cells, but the Fut4 transcript was not. In order to identify the role of SSEA-1 and determine the key enzyme for SSEA-1 synthesis in vivo, we have generated Fut9-deficient (Fut9−/−) mice. Fut9−/− mice develop normally, with no gross phenotypic abnormalities, and are fertile. Immunohistochemical analysis revealed an absence of SSEA-1 expression in early embryos and primordial germ cells of Fut9−/− mice. Therefore, we conclude that expression of the SSEA-1 epitope in the developing mouse embryo is not essential for embryogenesis in vivo.

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