scholarly journals Seamless Genetic Recording of Transiently Activated Mesenchymal Gene Expression in Endothelial Cells During Cardiac Fibrosis

Circulation ◽  
2021 ◽  
Author(s):  
Shaohua Zhang ◽  
Yan Li ◽  
Xiuzhen Huang ◽  
Kuo Liu ◽  
Qing-Dong Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Epithelial To Mesenchymal Transition ◽  
Cardiac Fibrosis ◽  
Marker Gene ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Mesenchymal Transition ◽  
Heart Injury

Background: Cardiac fibrosis is a lethal outcome of excessive formation of myofibroblasts that are scar-forming cells accumulated after heart injury. It has been reported that cardiac endothelial cells (ECs) contribute to a substantial portion of myofibroblasts through EndoMT. Recent lineage tracing studies demonstrate that myofibroblasts are derived from expansion of resident fibroblasts rather than from transdifferentiation of ECs. However, it remains unknown whether ECs can transdifferentiate into myofibroblasts reversibly or EndoMT genes were just transiently activated in ECs during cardiac fibrosis. Methods: By using the dual recombination technology based on Cre-loxP and Dre-rox, we generated a genetic lineage tracing system for tracking EndoMT in cardiac ECs. We used it to examine if there is transiently activated mesenchymal gene expression in ECs during cardiac fibrosis. Activation of the broadly used marker gene in myofibroblasts, αSMA, and the transcription factor that induces epithelial to mesenchymal transition (EMT), Zeb1, was examined. Results: The genetic system enables continuous tracing of transcriptional activity of targeted genes in vivo . Our genetic fate mapping results revealed that a subset of cardiac ECs transiently expressed αSMA and Zeb1 during embryonic valve formation and transdifferentiated into mesenchymal cells through EndoMT. Nonetheless, they did not contribute to myofibroblasts; nor transiently expressed αSMA or Zeb1 after heart injury. Instead, expression of αSMA was activated in resident fibroblasts during cardiac fibrosis. Conclusions: Mesenchymal gene expression is activated in cardiac ECs through EndoMT in the developing heart; but ECs do not transdifferentiate into myofibroblasts, nor transiently express some known mesenchymal genes during homeostasis and fibrosis in the adult heart. Resident fibroblasts that are converted to myofibroblasts by activating mesenchymal gene expression are the major contributors to cardiac fibrosis.

scholarly journals Direct neuronal reprogramming by temporal identity factors

2021 ◽  
Author(s):  
Camille Boudreau-Pinsonneault ◽  
Awais Javed ◽  
Michel Fries ◽  
Pierre Mattar ◽  
Michel Cayouette
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Lineage Tracing ◽  
Developmental Competence ◽  
Rapid Screening ◽  
Mouse Retina ◽  
Genetic Lineage ◽  
Differentiated Cells ◽  
Temporal Identity

Temporal identity factors are sufficient to reprogram developmental competence of neural progenitors, but whether they could also reprogram the identity of fully differentiated cells is unknown. To address this question, we designed a conditional gene expression system combined with genetic lineage tracing that allows rapid screening of potential reprogramming factors in the mouse retina. Using this assay, we report that co-expression of the early temporal identity transcription factor Ikzf1, together with Ikzf4, another Ikaros family member, is sufficient to directly convert adult Muller glial cells into neuron-like cells in vivo, without inducing a proliferative progenitor state. scRNA-seq analysis shows that the reprogrammed cells share some transcriptional signatures with both cone photoreceptors and bipolar cells. Furthermore, we show that co-expression of Ikzf1 and Ikzf4 can reprogram mouse embryonic fibroblasts to induced neurons by remodeling chromatin and promoting a neuronal gene expression program. This work uncovers general neuronal reprogramming properties for temporal identity factors in differentiated cells, opening new opportunities for cell therapy development.

Short-term effect of FSH on gene expression in bovine granulosa cells in vitro

2018 ◽  
Vol 30 (8) ◽  
pp. 1154 ◽  
Author(s):  
Anne-Laure Nivet ◽  
Isabelle Dufort ◽  
Isabelle Gilbert ◽  
Marc-André Sirard
Keyword(s):  
Gene Expression ◽  
Cellular Response ◽  
Epithelial To Mesenchymal Transition ◽  
Short Term ◽  
Mesenchymal Transition ◽  
Vitro Model ◽  
Cell Transcriptome ◽  
Physical Changes

In reproduction, FSH is one of the most important hormones, especially in females, because it controls the number of follicles and the rate of follicular growth. Although several studies have examined the follicular response at the transcriptome level, it is difficult to obtain a clear and complete picture of the genes responding to an increase in FSH in an in vivo context because follicles undergo rapid morphological and physical changes during their growth. To help define the transcriptome downstream response to FSH, an in vitro model was used in the present study to observe the short-term (4 h) cellular response. Gene expression analysis highlighted a set of novel transcripts that had not been reported previously as being part of the FSH response. Moreover, the results of the present study indicate that the epithelial to mesenchymal transition pathway is inhibited by short-term FSH stimuli, maintaining follicles in a growth phase and preventing differentiation. Modulating gene expression in vitro has physiological limitations, but it can help assess the potential downstream response and begin the mapping of the granulosa cell transcriptome in relation to FSH. This information is a key feature to help discriminate between the effects of FSH and LH, or to elucidate the overlapping of insulin-like growth factor 1 and FSH in the granulosa mitogenic response.

scholarly journals Fibroblast State Reversal By MBNL1-Dependent Transcriptome Modification Regulates Cardiac Repair

2021 ◽  
Author(s):  
Darrian Bugg ◽  
Ross Bretherton ◽  
Kylie Beach ◽  
Anna Reese ◽  
Jagadambika Gunaje ◽  
...  
Keyword(s):  
Cardiac Fibrosis ◽  
State Transition ◽  
Rna Binding ◽  
Cardiac Fibroblasts ◽  
Lineage Tracing ◽  
State Transitions ◽  
Genetic Lineage ◽  
Fibrotic Response ◽  
Mesenchymal Transition ◽  
Genetic Lineage Tracing

SUMMARYDynamic fibroblast state transitions are responsible for the heart’s fibrotic response to injury, raising the possibility that tactical control of these transitions could alter maladaptive fibrotic outcomes. Transcriptome maturation by the RNA binding protein Muscleblind Like 1 (MBNL1) has emerged as a potential driver of differentiated cell states. Here genetic lineage tracing of myofibroblasts in the injured heart demonstrated that gains in MBNL1 function corresponded to profibrotic fibroblast states. Similarly, in mice cardiac fibroblast specific MBNL1 overexpression induced a transcriptional myofibroblast profile in healthy cardiac fibroblasts that prevented the fibroproliferative phase of cardiac wound healing. By contrast loss of MBNL1 reverted cardiac fibroblasts to a pro-proliferative epicardial progenitor state that limited cardiac fibrosis following myocardial infarction. This progenitor state transition was associated with an MBNL1-dependent destabilization of the mesenchymal transition gene, Sox9. These findings suggest that MBNL1 regulation of the fibroblast transcriptome drives state transitions underlying cardiac fibrosis and repair.

scholarly journals Nonmuscle myosin IIA and IIB differentially modulate migration and alter gene expression in primary mouse tumorigenic cells

2019 ◽  
Vol 30 (12) ◽  
pp. 1463-1476 ◽  
Author(s):  
Debdatta Halder ◽  
Shekhar Saha ◽  
Raman K. Singh ◽  
Indranil Ghosh ◽  
Ditipriya Mallick ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial To Mesenchymal Transition ◽  
Pcr Analysis ◽  
Nonmuscle Myosin ◽  
Altered Expression ◽  
Mesenchymal Transition ◽  
Expression Of Genes ◽  
Primary Mouse ◽  
Perinuclear Area

Though many cancers are known to show up-regulation of nonmuscle myosin (NM) IIA and IIB, the mechanism by which NMIIs aid in cancer development remains unexplored. Here we demonstrate that tumor-generating, fibroblast-like cells isolated from 3-methylcholanthrene (3MC)-induced murine tumor exhibit distinct phospho-dependent localization of NMIIA and NMIIB at the perinuclear area and tip of the filopodia and affect cell migration differentially. While NMIIA-KD affects protrusion dynamics and increases cell directionality, NMIIB-KD lowers migration speed and increases filopodial branching. Strategically located NMIIs at the perinuclear area colocalize with the linker of nucleoskeleton and cytoskeleton (LINC) protein Nesprin2 and maintain the integrity of the nuclear-actin cap. Interestingly, knockdown of NMIIs results in altered expression of genes involved in epithelial-to-mesenchymal transition, angiogenesis, and cellular senescence. NMIIB-KD cells display down-regulation of Gsc and Serpinb2, which is strikingly similar to Nesprin2-KD cells as assessed by quantitative PCR analysis. Further gene network analysis predicts that NMIIA and NMIIB may act on similar pathways but through different regulators. Concomitantly, knockdown of NMIIA or NMIIB lowers the growth rate and tumor volume of 3MC-induced tumor in vivo. Altogether, these results open a new window to further investigate the effect of LINC-associated perinuclear actomyosin complex on mechanoresponsive gene expression in the growing tumor.

scholarly journals Elevated PTH induces endothelial-to-chondrogenic transition in aortic endothelial cells

2017 ◽  
Vol 312 (3) ◽  
pp. F436-F444 ◽  
Author(s):  
Min Wu ◽  
Jian-Dong Zhang ◽  
Ri-Ning Tang ◽  
Steven D. Crowley ◽  
Hong Liu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
In Vitro Study ◽  
Green Fluorescent Protein Fluorescence ◽  
Lineage Tracing ◽  
Protein Fluorescence ◽  
Mesenchymal Transition

Previous studies have shown that increased parathyroid hormone (PTH) attributable to secondary hyperparathyroidism in chronic kidney disease accelerates the arteriosclerotic fibrosis and calcification. Although the underlying mechanisms remain largely unknown, endothelial cells (ECs) have recently been demonstrated to participate in calcification in part by providing chondrogenic cells via the endothelial-to-mesenchymal transition (EndMT). Therefore, this study aimed to investigate whether elevated PTH could induce endothelial-to-chondrogenic transition in aortic ECs and to determine the possible underlying signaling pathway. We found that treatment of ECs with PTH significantly upregulated the expression of EndMT-related markers. Accordingly, ECs treated with PTH exhibited chondrogenic potential. In vivo, lineage-tracing model-subjected mice with endothelial-specific green fluorescent protein fluorescence to chronic PTH infusion showed a marked increase in the aortic expression of chondrocyte markers, and confocal microscopy revealed the endothelial origin of cells expressing chondrocyte markers in the aorta after PTH infusion. Furthermore, this in vitro study showed that PTH enhanced the nuclear localization of β-catenin in ECs, whereas β-catenin siRNA or DKK1, an inhibitor of β-catenin nuclear translocation, attenuated the upregulation of EndMT-associated and chondrogenic markers induced by PTH. In summary, our study demonstrated that elevated PTH could induce the transition of ECs to chondrogenic cells via EndMT, possibly mediated by the nuclear translocation of β-catenin.

scholarly journals Visualization of stem cell activity in pancreatic cancer expansion by direct lineage tracing with live imaging

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Takahisa Maruno ◽  
Akihisa Fukuda ◽  
Norihiro Goto ◽  
Motoyuki Tsuda ◽  
Kozo Ikuta ◽  
...  
Keyword(s):  
Stem Cell ◽  
Epithelial Mesenchymal Transition ◽  
Cell Activity ◽  
Live Imaging ◽  
Lineage Tracing ◽  
Ductal Adenocarcinoma ◽  
Genetic Lineage ◽  
Mesenchymal Transition ◽  
Stem Cell Activity

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. Although rigorous efforts identified the presence of ‘cancer stem cells (CSCs)’ in PDAC and molecular markers for them, stem cell dynamics in vivo have not been clearly demonstrated. Here we focused on Doublecortin-like kinase 1 (Dclk1), known as a CSC marker of PDAC. Using genetic lineage tracing with a dual-recombinase system and live imaging, we showed that Dclk1+ tumor cells continuously provided progeny cells within pancreatic intraepithelial neoplasia, primary and metastatic PDAC, and PDAC-derived spheroids in vivo and in vitro. Furthermore, genes associated with CSC and epithelial mesenchymal transition were enriched in mouse Dclk1+ and human DCLK1-high PDAC cells. Thus, we provided direct functional evidence for the stem cell activity of Dclk1+ cells in vivo, revealing the essential roles of Dclk1+ cells in expansion of pancreatic neoplasia in all progressive stages.

scholarly journals ets1 associates with KMT5A to participate in high glucose-mediated EndMT via upregulation of PFN2 expression in diabetic nephropathy

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Lihong Lu ◽  
Ziwen Zhong ◽  
Jiahui Gu ◽  
Ke Nan ◽  
Minmin Zhu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Diabetic Nephropathy ◽  
High Glucose ◽  
Epithelial To Mesenchymal Transition ◽  
Umbilical Vein ◽  
Human Subjects ◽  
Histone H4 ◽  
Mesenchymal Transition ◽  
Glomerular Endothelial Cells

Abstract Background Diabetic nephropathy (DN) is currently the leading cause of end-stage renal disease globally. The endothelial-to-mesenchymal transition (EndMT) of glomerular endothelial cells has been reported to play a crucial role in DN. As a specific form of epithelial-to-mesenchymal transition, EndMT and epithelial-to-mesenchymal transition may exhibit mutual modulators. Profilin 2 (PFN2) has been reported to participate in epithelial-to-mesenchymal transition. Moreover, ETS proto-oncogene 1 (ets1) and lysine methyltransferase 5A (KMT5A) have been reported to contribute to high glucose-mediated endothelial injury and epithelial-to-mesenchymal transition. In this study, we hypothesize ets1 associates with KMT5A to modulate PFN2 transcription, thus participating in high glucose-mediated EndMT in glomerular endothelial cells. Methods Immunohistochemistry (IHC) was performed to detect protein levels in the kidney tissues and/or aorta tissues of human subjects and rats. Western blot, qPCR and immunofluorescence were performed using human umbilical vein endothelial cells (HUVECs). Chromatin immunoprecipitation (ChIP) assays and dual luciferase assays were performed to assess transcriptional activity. The difference between the groups was compared by two-tailed unpaired t-tests or one-way ANOVAs. Results Our data indicated that vimentin, αSMA, S100A4 and PFN2 levels were increased, and CD31 levels were reduced in glomerular endothelial cells of DN patients and rats. Our cell experiments showed that high glucose induced EndMT by augmenting PFN2 expression in HUVECs. Moreover, high glucose increased ets1 expression. si-ets1 suppressed high glucose-induced PFN2 levels and EndMT. ets1 overexpression-mediated EndMT was reversed by si-PFN2. Furthermore, ets1 was determined to associate with KMT5A. High glucose attenuated KMT5A levels and histone H4 lysine 20 methylation (H4K20me1), one of the downstream targets of KMT5A. KMT5A upregulation suppressed high glucose-induced PFN2 levels and EndMT. sh-KMT5A-mediated EndMT was counteracted by si-PFN2. Furthermore, H4K20me1 and ets1 occupied the PFN2 promoter region. sh-KMT5A cooperated with ets1 overexpression to activate PFN2 promoter activity. Our in vivo study demonstrated that KMT5A was reduced, while ets1 was augmented, in glomerular endothelial cells of DN patients and rats. Conclusions The present study indicated that ets1 cooperated with KMT5A to transcribe PFN2, thus contributing to hyperglycemia-induced EndMT in the glomerular endothelial cells of DN patients and rats. Trial registration ChiCTR, ChiCTR2000029425. 2020/1/31, http://www.chictr.org.cn/showproj.aspx?proj=48548

scholarly journals Extracellular Vesicles Containing MicroRNA-92a-3p Facilitate Partial Endothelial-Mesenchymal Transition and Angiogenesis in Endothelial Cells

2019 ◽  
Vol 20 (18) ◽  
pp. 4406 ◽  
Author(s):  
Nami O. Yamada ◽  
Kazuki Heishima ◽  
Yukihiro Akao ◽  
Takao Senda
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Epithelial To Mesenchymal Transition ◽  
Ectopic Expression ◽  
Cell Communication ◽  
Related Gene ◽  
Mesenchymal Transition ◽  
Essential Components ◽  
Endothelial To Mesenchymal Transition

Extracellular vesicles (EVs) are nanometer-sized membranous vesicles used for primitive cell-to-cell communication. We previously reported that colon cancer-derived EVs contain abundant miR-92a-3p and have a pro-angiogenic function. We previously identified Dickkopf-3 (Dkk-3) as a direct target of miR-92a-3p; however, the pro-angiogenic function of miR-92a-3p cannot only be attributed to downregulation of Dkk-3. Therefore, the complete molecular mechanism by which miR-92a-3p exerts pro-angiogenic effects is still unclear. Here, we comprehensively analyzed the gene sets affected by ectopic expression of miR-92a-3p in endothelial cells to elucidate processes underlying EV-induced angiogenesis. We found that the ectopic expression of miR-92a-3p upregulated cell cycle- and mitosis-related gene expression and downregulated adhesion-related gene expression in endothelial cells. We also identified a novel target gene of miR-92a-3p, claudin-11. Claudin-11 belongs to the claudin gene family, which encodes essential components expressed at tight junctions (TJs). Disruption of TJs with a concomitant loss of claudin expression is a significant event in the process of epithelial-to-mesenchymal transition. Our findings have unveiled a new EV-mediated mechanism for tumor angiogenesis through the induction of partial endothelial-to-mesenchymal transition in endothelial cells.

scholarly journals Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenic lineages

2021 ◽  
Author(s):  
Stephanie A. Redmond ◽  
Arantxa Cebrian Silla ◽  
Marcos Assis Nascimento ◽  
Benjamin Mansky ◽  
David Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Subventricular Zone ◽  
Adult Mouse ◽  
Marker Gene ◽  
Lineage Tracing ◽  
Molecular Heterogeneity ◽  
Genetic Lineage ◽  
Cell Potential ◽  
Single Nucleus

AbstractThe ventricular-subventricular zone (V-SVZ) is home to the largest neurogenic niche in the adult mouse brain. Previous work has demonstrated that resident stem cells in different locations within the V-SVZ produce different subtypes of new neurons for the olfactory bulb. While great progress has been made in understanding the differences in regional stem cell potential using viral and genetic lineage tracing strategies, the core molecular heterogeneity that underlies these regional differences is largely unknown. Here we present single whole-cell and single nucleus sequencing datasets of microdissected adult mouse V-SVZ, and evidence for the existence of two broad populations of adult neural stem cells. By using spatially resolved microdissections in the single nucleus sequencing dataset as a reference, and mapping marker gene expression in the V-SVZ, we find that these two populations reside in largely non-overlapping domains in either the dorsal or ventral V-SVZ. Furthermore, we identified two subpopulations of newly born neurons that have gene expression consistent with dorsal or ventral origins. Finally, we identify genes expressed by both stem cells and the neurons they generate that specifically mark either the dorsal or ventral adult neurogenic lineage. These datasets, methods and findings will facilitate the study of region-specific regulation of adult neurogenesis.

scholarly journals Identification of an Epithelial-to-Mesenchymal Transition (EMT)-like Programme in t(4;14)-Positive Multiple Myeloma Reveals Novel Targets for Therapeutic Intervention

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 647-647
Author(s):  
Kate Vandyke ◽  
Krzysztof M. Mrozik ◽  
Chee Man Cheong ◽  
Annie W.S. Chow ◽  
Chung H Kok ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Epithelial To Mesenchymal Transition ◽  
Plasma Cells ◽  
Gene Expression Signature ◽  
Newly Diagnosed ◽  
Related Gene ◽  
Tumour Burden ◽  
Mesenchymal Transition

Abstract Introduction: Prognosis for multiple myeloma (MM) patients has improved dramatically since the introduction of novel therapies. However, prognosis in the t(4;14) subgroup, characterized by expression of the histone methyltransferase MMSET, remains relatively poor due to the acquisition of a highly aggressive, motile and invasive phenotype and a generally modest response to therapy. We have previously demonstrated that MMSET promotes an epithelial-to-mesenchymal transition (EMT) in prostate cancer1. While the term EMT is not commonly used to describe MM, we hypothesise that an EMT-like process plays a critical role in t(4;14)­-positive MM disease pathogenesis. In this study, we conducted a comprehensive evaluation of the association between t(4;14) and an EMT-related gene expression signature in MM and identify N-cadherin as a therapeutically-targetable EMT-related gene in t(4;14)-positive MM. Methods and results: Expression of a core EMT-related signature, comprising 169 mesenchymal genes and 49 epithelial genes, was assessed in CD138-selected MM plasma cells from newly-diagnosed MM patients in four independent microarray datasets (E-GEOD-19784 [n = 327 MM patients], E-GEOD-26863 [n = 304], E-MTAB-317 [n = 226] and E-MTAB-363 [n = 155]), accessed through ArrayExpress (EMBL-EBI). In each dataset, gene expression was compared in t(4;14)-positive and t(4;14)-negative patients using linear models for microarray data (LIMMA). Twenty six mesenchymal genes were found to be upregulated in t(4;14)-positive patients across the 4 datasets (p < 0.05, Fisher’s method). A general loss of epithelial genes was not observed, likely due to the haematopoietic origins of MM. Upregulated genes included key EMT drivers (TWIST1, SOX9, TCF4) and genes associated with the cytoskeleton (VIM), adhesion and migration (CDH2, ITGB1, NCAM1) and signalling pathways involved in EMT (BMPR1A, IL6R, TGFB2). The t(4;14)-mediated regulation of EMT-related genes including TWIST1, CDH2, BMPR1A and ITGB1 was confirmed by assessing the effects of MMSET knockdown, knockout and add-back in the t(4;14)-positive human myeloma cell line KMS-11. We have previously demonstrated that N-cadherin (CDH2) expression is elevated in plasma cells from approximately 50% of newly diagnosed multiple myeloma (MM) patients and that elevated serum N-cadherin is associated with poor prognosis2. In order to identify whether N-cadherin is a potential therapeutic target in t(4;14)-positive myeloma, the effects of an N-cadherin peptide inhibitor ADH-1 (Exherin™) or shRNA-mediated N-cadherin knock-down were assessed on MM PC adhesion and proliferation in vivo and in vitro. The role of N-cadherin in MM tumour establishment and intramedullary growth was investigated using the C57BL/KalwRijHsd mouse model of MM. In this model, intravenously injected luciferase-expressing mouse MM PC cells (5TGM1-SFG) home to the bone marrow and initiate systemic MM disease. C57BL/KalwRijHsd mice bearing 5TGM1-SFG N-cadherin knock-down cells had significantly reduced tumour burden, as assessed by bioluminescent imaging and serum paraprotein levels, after 4 weeks compared with mice bearing control 5TGM1-SFG cells. Furthermore, daily intraperitoneal ADH-1 administration (100 mg/kg/day) to 5TGM1-SFG cell-bearing mice significantly significantly decreased tumour burden when administered at the time of tumour inoculation, but had no effect on established tumours. These results suggest that N-cadherin plays a role in extravasation, homing and/or tumour establishment in vivo. Conclusion: This study identified an extensive EMT-like gene expression signature driven by MMSET in t(4;14)-positive MM patients. This provides insight as to how the t(4;14) translocation leads poor prognostic outcomes in up to 20% of MM patients. Furthermore, these studies demonstrate a potential role for N-cadherin in MM tumour dissemination in t(4;14)-positive MM and suggest that N-cadherin represents a novel candidate for therapeutic targeting in these patients. 1Ezponda et al. Oncogene, 2013 2Vandyke et al. Br J Haematol, 2013 Disclosures No relevant conflicts of interest to declare.

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