scholarly journals EOMES is responsible for WNT memory and can substitute for WNT in mesendoderm specification

2021 ◽  
Author(s):  
Anna Yoney ◽  
Lu Bai ◽  
Ali H. Brivanlou ◽  
Eric D Siggia
Keyword(s):  
Target Genes ◽  
Embryonic Stem ◽  
Model Systems ◽  
Mechanistic Basis ◽  
Vitro Model ◽  
Transcriptional Output ◽  
Accessible Chromatin ◽  
Pluripotency Maintenance

Embryogenesis is guided by a limited set of signaling pathways that are reused at different times and places throughout development. How a context dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro model systems. Our previous work in human embryonic stem cells (hESCs) established that pre-exposure of cells to WNT/β-catenin signaling is sufficient to switch the output of ACTIVIN/SMAD2 signaling from pluripotency maintenance to mesendoderm (ME) differentiation. A body of previous literature has established the role of both pathways in ME differentiation. However, our work demonstrated that the two signals do not need to be present simultaneously and that hESCs have a means to record WNT signals. Here we demonstrate that hESCs have accessible chromatin at SMAD2 binding sites near pluripotency and ME-associated target genes and that WNT priming does not alter SMAD2 binding. Rather our results indicate that stable transcriptional output at ME genes results from WNT-dependent production of an additional SMAD2 co-factor, EOMES. We show that expression of EOMES can replace WNT signaling in ME differentiation, providing a mechanistic basis for WNT-priming and memory in early development.

AK048794 maintains the mouse embryonic stem cell pluripotency by functioning as an miRNA sponge for miR-592

2016 ◽  
Vol 473 (20) ◽  
pp. 3639-3654 ◽  
Author(s):  
Yang Zhou ◽  
Qing-Song Dai ◽  
Shi-Chang Zhu ◽  
Yue-Hua Han ◽  
Hai-Long Han ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Luciferase Reporter ◽  
Stem Cell Pluripotency ◽  
Reporter Assays ◽  
Pluripotency Maintenance ◽  
Embryonic Stem Cell Pluripotency

MiR-592 has been identified as a neural-enriched microRNA, plays an important role in mNPCs differentiation, could induce astrogliogenesis differentiation arrest or/and enhance neurogenesis in vitro. Previous studies showed that long noncoding RNAs (lncRNAs) were involved in the neuronal development and activity. To investigate the role of miR-592 in neurogenesis, we described the expression profile of lncRNAs in miR-592 knockout mouse embryonic stem cells (mESCs) and the corresponding normal mESCs by microarray. By the microarray analysis and luciferase reporter assays, we demonstrated that lncRNA - AK048794, regulated by transcription factor GATA1, functioned as a competing endogenous RNA (ceRNA) for miR-592 and led to the de-repression of its endogenous target FAM91A1, which is involved in mESC pluripotency maintenance. Taken together, these observations imply that AK048794 modulated the expression of multiple genes involved in mESC pluripotency maintenance by acting as a ceRNA for miR-592, which may build up the link between the regulatory miRNA network and mESC pluripotency.

Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Anichavezhi Devendran ◽  
Rasheed Bailey ◽  
Sumanta Kar ◽  
Francesca Stillitano ◽  
Irene Turnbull ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Mononuclear Cells ◽  
Embryonic Stem ◽  
Model Systems ◽  
Patient Specific ◽  
Induced Pluripotent

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

786 ROLE OF LXRALPHA AND ITS LIPOGENIC TARGET GENES IN THE PATHOGENESIS OF HCV-RELATED STEATOSIS IN AN IN VITRO MODEL

2010 ◽  
Vol 52 ◽  
pp. S305-S306
Author(s):  
M.V. García-Mediavilla ◽  
E. Lima ◽  
S. Pisonero-Vaquero ◽  
B. Merino-Antolín ◽  
P.L. Majano ◽  
...  
Keyword(s):  
In Vitro Model ◽  
Target Genes ◽  
Vitro Model

scholarly journals A Targeted Deletion of a Region Upstream from the Jκ Cluster Impairs κ Chain Rearrangement In Cis in Mice and in the 103/bcl2 Cell Line

1999 ◽  
Vol 189 (9) ◽  
pp. 1443-1450 ◽  
Author(s):  
Laurentiu Cocea ◽  
Annie De Smet ◽  
Mahasti Saghatchian ◽  
Simon Fillatreau ◽  
Laurent Ferradini ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Line ◽  
Embryonic Stem ◽  
Similar Reduction ◽  
Targeted Deletion ◽  
Vitro Model ◽  
Germline Transcript ◽  
In Cis

We have shown previously that a mutation of the KI-KII site immediately 5′ to Jκ1 on the mouse immunoglobulin light chain κ locus reduces the rearrangement level in cis, although it does not affect transcription. Here we deleted by homologous recombination in mouse embryonic stem cells a 4-kb DNA fragment, located immediately upstream of the KI-KII element, which contains the promoter of the long germline transcript. Analysis of gene-targeted heterozygous mouse splenic B cells showed a strong decrease in rearrangement for the allele bearing the deletion. When both the KI-KII mutation and the 4-kb deletion were present on the same allele, the overall reduction in rearrangement was stronger than with the 4-kb deletion alone underlying the role of these two elements in the regulation of rearrangement. The same deletion was performed by homologous recombination on one allele of the rearrangement- inducible mouse 103/bcl2-hygroR pre-B cell line, and resulted in a similar reduction in the induction of rearrangement of the mutated allele. This result validates this cell line as an in vitro model for studying the incidence of gene-targeted modifications of the κ locus on the regulation of rearrangement.

scholarly journals NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3666
Author(s):  
Md Imtiaz Khalil ◽  
Ishita Ghosh ◽  
Vibha Singh ◽  
Jing Chen ◽  
Haining Zhu ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Target Genes ◽  
Progressive Failure ◽  
Lncap Cells ◽  
Cycle Arrest ◽  
Castration Resistant ◽  
Damage Response ◽  
Transcriptional Output

Most prostate cancer (PCa) deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa (mCRPC); however, the mechanism and key players leading to this are not fully understood. While studying the role of tousled-like kinase 1 (TLK1) and never in mitosis gene A (NIMA)-related kinase 1 (NEK1) in a DNA damage response (DDR)-mediated cell cycle arrest in LNCaP cells treated with bicalutamide, we uncovered that overexpression of wt-NEK1 resulted in a rapid conversion to androgen-independent (AI) growth, analogous to what has been observed when YAP1 is overexpressed. We now report that overexpression of wt-NEK1 results in accumulation of YAP1, suggesting the existence of a TLK1>NEK1>YAP1 axis that leads to adaptation to AI growth. Further, YAP1 is co-immunoprecipitated with NEK1. Importantly, NEK1 was able to phosphorylate YAP1 on six residues in vitro, which we believe are important for stabilization of the protein, possibly by increasing its interaction with transcriptional partners. In fact, knockout (KO) of NEK1 in NT1 PCa cells resulted in a parallel decrease of YAP1 level and reduced expression of typical YAP-regulated target genes. In terms of cancer potential implications, the expression of NEK1 and YAP1 proteins was found to be increased and correlated in several cancers. These include PCa stages according to Gleason score, head and neck squamous cell carcinoma, and glioblastoma, suggesting that this co-regulation is imparted by increased YAP1 stability when NEK1 is overexpressed or activated by TLK1, and not through transcriptional co-expression. We propose that the TLK1>NEK1>YAP1 axis is a key determinant for cancer progression, particularly during the process of androgen-sensitive to -independent conversion during progression to mCRPC.

scholarly journals The Lysine Methylase SMYD3 Modulates Mesendodermal Commitment during Development

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1233
Author(s):  
Raffaella Fittipaldi ◽  
Pamela Floris ◽  
Valentina Proserpio ◽  
Franco Cotelli ◽  
Monica Beltrame ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Model Systems ◽  
Embryonic Stem Cell Differentiation ◽  
In Vitro Differentiation ◽  
Cancer Models ◽  
Lineage Markers

SMYD3 (SET and MYND domain containing protein 3) is a methylase over-expressed in cancer cells and involved in oncogenesis. While several studies uncovered key functions for SMYD3 in cancer models, the SMYD3 role in physiological conditions has not been fully elucidated yet. Here, we dissect the role of SMYD3 at early stages of development, employing mouse embryonic stem cells (ESCs) and zebrafish as model systems. We report that SMYD3 depletion promotes the induction of the mesodermal pattern during in vitro differentiation of ESCs and is linked to an upregulation of cardiovascular lineage markers at later stages. In vivo, smyd3 knockdown in zebrafish favors the upregulation of mesendodermal markers during zebrafish gastrulation. Overall, our study reveals that SMYD3 modulates levels of mesendodermal markers, both in development and in embryonic stem cell differentiation.

scholarly journals Amnion signals are essential for mesoderm formation in primates

2020 ◽  
Author(s):  
Ran Yang ◽  
Alexander Goedel ◽  
Yu Kang ◽  
Chenyang Si ◽  
Chu Chu ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Embryonic Stem ◽  
Cellular Level ◽  
Model Systems ◽  
Loss Of Function ◽  
Primate Model ◽  
Mesoderm Formation ◽  
Human Primate

AbstractEssential genes for murine embryonic development can demonstrate a disparate phenotype in human cohorts. By generating a transcriptional atlas containing >30,000 cells from postimplantation non-human primate embryos, we discovered that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 resulted in non-human primate embryos which did not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos displayed a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirmed a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development.

scholarly journals Self-organization of human dorsal-ventral forebrain structures by light induced SHH

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Riccardo De Santis ◽  
Fred Etoc ◽  
Edwin A. Rosado-Olivieri ◽  
Ali H. Brivanlou
Keyword(s):  
In Vitro Model ◽  
Light Stimulus ◽  
Embryonic Stem ◽  
The Self ◽  
Model Systems ◽  
Self Organization ◽  
Light Stimulation ◽  
Vitro Model ◽  
Molecular Aspects

AbstractOrganizing centers secrete morphogens that specify the emergence of germ layers and the establishment of the body’s axes during embryogenesis. While traditional experimental embryology tools have been instrumental in dissecting the molecular aspects of organizers in model systems, they are impractical in human in-vitro model systems to dissect the relationships between signaling and fate along embryonic coordinates. To systematically study human embryonic organizer centers, we devised a collection of optogenetic ePiggyBac vectors to express a photoactivatable Cre-loxP recombinase, that allows the systematic induction of organizer structures by shining blue-light on human embryonic stem cells (hESCs). We used a light stimulus to geometrically confine SHH expression in neuralizing hESCs. This led to the self-organization of mediolateral neural patterns. scRNA-seq analysis established that these structures represent the dorsal-ventral forebrain, at the end of the first month of development. Here, we show that morphogen light-stimulation is a scalable tool that induces self-organizing centers.

scholarly journals Amnion signals are essential for mesoderm formation in primates

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ran Yang ◽  
Alexander Goedel ◽  
Yu Kang ◽  
Chenyang Si ◽  
Chu Chu ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Embryonic Stem ◽  
Cellular Level ◽  
Model Systems ◽  
Loss Of Function ◽  
Primate Model ◽  
Mesoderm Formation ◽  
Human Primate

AbstractEmbryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation non-human primate embryos, we uncover that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development.

The aggravating role of the ubiquitin–proteasome system in neurodegenerative disease

2006 ◽  
Vol 34 (5) ◽  
pp. 743-745 ◽  
Author(s):  
C.-C. Hung ◽  
E.J. Davison ◽  
P.A. Robinson ◽  
H.C. Ardley
Keyword(s):  
Ubiquitin Proteasome System ◽  
Model Systems ◽  
Protective Mechanism ◽  
Ubiquitinated Proteins ◽  
Age Related ◽  
Ubiquitin Proteasome ◽  
Vitro Model ◽  
Novel Therapeutic Strategies

Intraneuronal inclusion bodies are key pathological features of most age-related neurodegenerative disorders including Parkinson's disease and Alzheimer's disease. These inclusions are commonly characterized both by the presence of ubiquitinated proteins and the sequestration of components of the UPS (ubiquitin–proteasome system). Unfortunately, as we age, the efficiency of the UPS declines, suggesting that the presence of ubiquitinated proteins and UPS components in inclusions may reflect unsuccessful attempts by the (failing) UPS to remove the aggregating proteins. Whether the physical presence of inclusions causes cell death or, conversely, whether they are non-toxic and their presence reflects a cellular protective mechanism remains highly controversial. Animal and in vitro model systems that allow detailed characterization of the inclusions and their effects on the cell have been developed by us and others. Identification of the mechanisms involved in inclusion formation is already aiding the development of novel therapeutic strategies to prevent or alleviate aggregate-associated neurodegenerative diseases.

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