PRDM16 Is a Compact Myocardium-Enriched Transcription Factor Required to Maintain Compact Myocardial Cardiomyocyte Identity in Left Ventricle

Circulation ◽  
2021 ◽  
Author(s):  
Tongbin Wu ◽  
Zhengyu Liang ◽  
Zengming Zhang ◽  
Canzhao Liu ◽  
Lunfeng Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Rna Sequencing ◽  
Mouse Models ◽  
Molecular Mechanisms ◽  
Left Ventricular ◽  
Ventricular Noncompaction ◽  
Chip Sequencing ◽  
Transcriptional Signature ◽  
Underlying Mechanisms

Background: Left ventricular noncompaction cardiomyopathy (LVNC) was discovered half a century ago as a cardiomyopathy with excessive trabeculation and a thin ventricular wall. In the decades since, numerous studies have demonstrated that LVNC primarily impacts left ventricles (LVs), and is often associated with LV dilation and dysfunction. However, owing in part to the lack of suitable mouse models that faithfully mirror the selective LV vulnerability in patients, mechanisms underlying susceptibility of LV to dilation and dysfunction in LVNC remain unknown. Genetic studies have revealed that deletions and mutations in PRDM16 cause LVNC, but previous conditional Prdm16 knockout mouse models do not mirror the LVNC phenotype in patients, and importantly, the underlying molecular mechanisms by which PRDM16 deficiency causes LVNC are still unclear. Methods: Prdm16 cardiomyocyte (CM)-specific knockout ( Prdm16 cKO ) mice were generated and analyzed for cardiac phenotypes. RNA sequencing and ChIP sequencing were performed to identify direct transcriptional targets of PRDM16 in CMs. Single cell RNA sequencing in combination with Spatial Transcriptomics were employed to determine CM identity at single cell level. Results: CM-specific ablation of Prdm16 in mice caused LV-specific dilation and dysfunction, as well as biventricular noncompaction, which fully recapitulated LVNC in patients. Mechanistically, PRDM16 functioned as a compact myocardium-enriched transcription factor, which activated compact myocardial genes while repressing trabecular myocardial genes in LV compact myocardium. Consequently, Prdm16 cKO LV compact myocardial CMs shifted from their normal transcriptomic identity to a transcriptional signature resembling trabecular myocardial CMs and/or neurons. Chamber-specific transcriptional regulation by PRDM16 was in part due to its cooperation with LV-enriched transcription factors Tbx5 and Hand1. Conclusions: These results demonstrate that disruption of proper specification of compact CM may play a key role in the pathogenesis of LVNC. They also shed light on underlying mechanisms of LV-restricted transcriptional program governing LV chamber growth and maturation, providing a tangible explanation for the susceptibility of LV in a subset of LVNC cardiomyopathies.

P4996The adult heart requires baseline expression of the transcription factor Hand2 to withstand right ventricular pressure overload

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
A M C Koop ◽  
R F Videira ◽  
L Ottaviani ◽  
E M Poels ◽  
K W Van De Kolk ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Right Ventricular ◽  
Rna Sequencing ◽  
Cardiac Remodeling ◽  
Cardiac Dysfunction ◽  
Molecular Mechanisms ◽  
Pressure Overload ◽  
Left Ventricular ◽  
Adult Heart ◽  
Hypertrophic Growth

Abstract Introduction Heart and neural crest derivatives expressed-2 (Hand2) has been identified as an important embryonic basic helix-loop-helix-transcription factor, with different functions in the development of the first and second heart field, from which the left and right ventricle originate, respectively. Our previous work revealed that Hand2, under conditions of left ventricular (LV) pressure overload, is re-expressed in the adult heart and activates a “fetal gene” program contributing to pathological cardiac remodeling. Ablation of cardiac expression of Hand2 resulted in protection to cardiac stress and attenuated maladaptive remodeling. Purpose In this study, we aimed at unraveling the role of Hand2 during cardiac remodeling in response to right ventricular (RV) pressure overload induced by pulmonary artery banding (PAB). Methods Hand2F/F and MCM− Hand2F/F mice were treated with tamoxifen (control and knockout, respectively) and subjected to six weeks of RV pressure overload induced by PAB. Echocardiographic and MRI derived hemodynamic parameters, and molecular remodelling were assessed for experimental groups and compared to sham-operated controls (Fig. 1a). RNA sequencing and gene ontology enrichment analysis were performed to compare the dysregulated genes between the pressure overloaded RV of the control and Hand2 knockout mice. Results After six weeks of increased pressure load (Fig. 1b), levels of Hand2 increased in the control banded animals but, as expected, remained absent in the knockout hearts (Fig. 1c). In contrast to the what was previously observed for the pressure overloaded LV, in the pressure loaded RV, Hand2 depletion resulted in more severe remodelling and dysfunction as reflected by increased hypertrophic growth, increased RV end-diastolic and end-systolic volumes as well as decreased RV ejection fraction (Fig. 1d–g). In addition, RNA sequencing revealed a distinct set of genes that are dysregulated in the pressure-overloaded RV, compared to the previously described pressure-overloaded LV. These include components of the extracellular matrix structure, collagen assembly and organization and several types of collagens. Genes associated with inflammation response, adhesion and muscle organization were also affected in the RV of the Hand2 KO mice (Fig. 1h). Figure 1 Conclusion Cardiac-specific depletion of Hand2 is associated with severe cardiac dysfunction in conditions of RV pressure overload. While inhibiting Hand2 expression can prevent cardiac dysfunction in conditions of LV pressure overload, the same does not hold true for conditions of RV pressure overload. This study highlights the need to better understand the molecular mechanisms driving pathological remodelling of the RV, in contrast to the LV, in order to better diagnose and treat patients with RV or LV failure.

scholarly journals Dissecting the heterogeneity of DENV vaccine-elicited cellular immunity using single-cell RNA sequencing and cellular metabolic profiling

2019 ◽  
Author(s):  
Adam T. Waickman ◽  
Kaitlin Victor ◽  
Tao Li ◽  
Kristin Hatch ◽  
Wiriya Rutvisuttinnunt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
T Cell Immunity ◽  
Functional Markers ◽  
Transcriptional Signature ◽  
Cell Immunity ◽  
Single Cell Rna Sequencing

ABSTRACTGenerating effective and durable T cell immunity is a critical prerequisite for vaccination against dengue virus (DENV) and other viral diseases. Understanding the precise molecular mechanisms of vaccine-elicited T cell immunity remains a critical knowledge gap in vaccinology. In this study, we utilized single-cell RNA sequencing (scRNAseq) and TCR clonotype analysis to demonstrate that a unique transcriptional signature is present in acutely-activated and clonally-expanded T cells that become committed to the memory repertoire. This effector-associated transcriptional signature is dominated by a unique metabolic transcriptional program. Based on this transcriptional signature, we were able to define a set of functional markers that identify the most potent and durable vaccine-reactive memory precursor CD8+ T cells. The transferrin receptor (CD71) other important transporters of amino acids, and direct measurements of glucose and fatty acid uptake, were further validated as early markers of durable T cell memory using conventional flow cytometry. These data suggest that generating durable T cell immunity is a process that is determined by metabolic programming and metabolite availability during the acute phase of the immune response. This study illustrates the power of scRNAseq as an analytical tool to assess the molecular mechanisms of host control and vaccine modality in determining the magnitude, diversity, and persistence of vaccine-elicited cell-mediated immunity.

scholarly journals Single-cell RNA Sequencing Reveals That Injected ADSCs Change the Macrophage Profile in a Murine Model of Pulmonary Fibrosis

2021 ◽  
Author(s):  
Mamatali Rahman ◽  
Zhao-Yan Wang ◽  
Jun-Xiang Li ◽  
Hao-Wei Xu ◽  
Qiong Wu
Keyword(s):  
Pulmonary Fibrosis ◽  
Single Cell ◽  
Rna Sequencing ◽  
Treatment Options ◽  
Mouse Lung ◽  
Therapeutic Effects ◽  
Intratracheal Administration ◽  
Cell Based Therapy ◽  
Single Cell Rna Sequencing ◽  
Underlying Mechanisms

Abstract Background: Idiopathic pulmonary fibrosis (IPF) is a deadly chronic interstitial lung disease with no effective treatment options other than lung transplantation. Allogeneic adipose-derived mesenchymal stem cells (ADSCs) are considered ideal as seed cells for stem cell-based therapy, and some studies illustrated the therapeutic effect of ADSCs on IPF, but the underlying mechanisms remain unclear.Methods: A single intratracheal dose of bleomycin (BLM) was administered to induce pulmonary injury/fibrosis in C57BL/6 mice, after GFP-labeled mouse ADSCs (mADSCs) were implanted intratracheally to explore their potential therapeutic effects in the inflamed/fibrotic lung microenvironment. The mADSCs were then retrieved through fluorescence-activated cell sorting and subjected to single-cell RNA sequencing (scRNA-seq).Results: Our data indicate that the single-dose intratracheal administration of mADSCs could significantly increase the life span of IPF mice by remodeling the extracellular matrix and promoting the polarization of macrophages to an anti-inflammatory phenotype. Conclusions: A single intratracheal injection of mADSCs alleviated BLM-induced pulmonary fibrosis by readjustment of the mouse lung microenvironment, which was reflected in changes of the lung C1QB+, APOE+ and TREM2+ macrophages in the mouse model.

scholarly journals OR16-05 Single-Cell Sequencing Identifies Novel Regulators of Thyrotrope Populations and POU1F1-Independent Thyroid-Stimulating Hormone Expression

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Leonard Cheung ◽  
Alexandre Daly ◽  
Michelle Brinkmeier ◽  
Sally Ann Camper
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Molecular Mechanisms ◽  
Thyroid Stimulating Hormone ◽  
Pars Tuberalis ◽  
Pituitary Cells ◽  
Pars Distalis ◽  
Mouse Development ◽  
Single Cell Sequencing ◽  
Stimulating Hormone

Abstract We implemented single-cell RNA sequencing (scRNAseq) technology as a discovery tool to identify factors enriched in differentiated thyrotropes. Thyroid-stimulating hormone (TSH) is produced in the pars distalis of the anterior pituitary (AP) and primarily acts on the thyroid gland to regulate metabolism through T3/T4. However, TSH is also produced by cells in the pars tuberalis (PT), which is comprised of a thin layer of cells that extends rostrally from the pars distalis along the pituitary stalk to the median eminence in the hypothalamus. TSH produced by PT thyrotropes acts on hypothalamic tanycytes to regulate seasonal reproduction. PT thyrotropes likely descend from rostral tip thyrotropes that arise at e12.5 of mouse development, which transcribe the TSH beta subunit (Tshb) without detectable expression of the transcription factor POU1F1. POU1F1 is required for Tshb transcription in thyrotropes of the adenohypophysis, and it acts synergistically with GATA2 to drive cell fate. The molecular mechanisms driving Tshb expression independently of Pou1f1 in PT thyrotropes are unclear. Thyrotropes are the least abundant endocrine cell-type in the pituitary gland. We used genetic labeling and fluorescence-activated cell sorting (FACS) to enrich for thyrotropes for single-cell sequencing. We performed scRNAseq on 7-day-old GFP-positive pituitary cells from Tshb-Cre; R26-LSL-eYFP and intact whole pituitaries, recovering more than 15,000 cells altogether. We observe two distinct populations of cells expressing Tshb. The larger thyrotrope population has approximately twenty fold higher levels of Tshb and five fold higher Cga transcripts than the smaller population, and they are also distinguished by expression of Pou1f1, TSH-releasing hormone receptor (Trhr), and deiodinase 2 (Dio2), consistent with expectations for AP thyrotropes. The smaller thyrotrope population does not express Pou1f1, but those cells are characterized by expression of TSH receptor (Tshr) and melatonin receptor 1A (Mtnr1a), consistent with expectations for PT thyrotropes. They express mildly increased levels of Eya3 and Six1, although these genes are expressed in other cell-types including AP thyrotropes, stem cells, and gonadotropes. They have two-fold higher levels of Gata2 transcripts and uniquely express the transcription factor Sox14. SOX14 is a SoxB2 family transcription factor that counteracts the transcriptional activity of SoxB1 family members, such as Sox2. In conclusion, our scRNAseq has identified novel markers of PT thyrotropes and unveils novel insights into the similarities and differences in the development and function of pituitary thyrotrope subpopulations.

scholarly journals Single-Cell RNA Sequencing to Disentangle the Blood System

2021 ◽  
Vol 41 (3) ◽  
pp. 1012-1018
Author(s):  
Jean Acosta ◽  
Daniel Ssozi ◽  
Peter van Galen
Keyword(s):  
Blood Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Single Cells ◽  
Cell Types ◽  
Blood System ◽  
Differentiated Cells ◽  
Tissue Organization ◽  
Single Cell Rna Sequencing

The blood system is often represented as a tree-like structure with stem cells that give rise to mature blood cell types through a series of demarcated steps. Although this representation has served as a model of hierarchical tissue organization for decades, single-cell technologies are shedding new light on the abundance of cell type intermediates and the molecular mechanisms that ensure balanced replenishment of differentiated cells. In this Brief Review, we exemplify new insights into blood cell differentiation generated by single-cell RNA sequencing, summarize considerations for the application of this technology, and highlight innovations that are leading the way to understand hematopoiesis at the resolution of single cells. Graphic Abstract: A graphic abstract is available for this article.

scholarly journals Single-Cell RNA Sequencing of Tumor-Infiltrating NK Cells Reveals that Inhibition of Transcription Factor HIF-1α Unleashes NK Cell Activity

Immunity ◽  
2020 ◽  
Vol 52 (6) ◽  
pp. 1075-1087.e8 ◽  
Author(s):  
Jing Ni ◽  
Xi Wang ◽  
Ana Stojanovic ◽  
Qin Zhang ◽  
Marian Wincher ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Nk Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Nk Cell ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Single Cell Rna Sequencing

scholarly journals Review of Single-Cell RNA Sequencing in the Heart

2020 ◽  
Vol 21 (21) ◽  
pp. 8345
Author(s):  
Shintaro Yamada ◽  
Seitaro Nomura
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Clinical Medicine ◽  
Basic Research ◽  
Cellular Level ◽  
Cardiovascular Development ◽  
Biological Mechanisms ◽  
Technological Advances ◽  
Single Cell Rna Sequencing

Single-cell RNA sequencing (scRNA-seq) technology is a powerful, rapidly developing tool for characterizing individual cells and elucidating biological mechanisms at the cellular level. Cardiovascular disease is one of the major causes of death worldwide and its precise pathology remains unclear. scRNA-seq has provided many novel insights into both healthy and pathological hearts. In this review, we summarize the various scRNA-seq platforms and describe the molecular mechanisms of cardiovascular development and disease revealed by scRNA-seq analysis. We then describe the latest technological advances in scRNA-seq. Finally, we discuss how to translate basic research into clinical medicine using scRNA-seq technology.

#71: Single-cell RNA Sequencing Analysis of Zika Virus Infection in Human Stem Cell-Derived Neuroprogenitor Cells and Cerebral Organoids

2021 ◽  
Vol 10 (Supplement_1) ◽  
pp. S14-S14
Author(s):  
K E Ocwieja ◽  
T K Hughes ◽  
J M Antonucci ◽  
A L Richards ◽  
A C Stanton ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Zika Virus ◽  
Molecular Mechanisms ◽  
Sequencing Analysis ◽  
Human Stem Cell ◽  
Zikv Infection ◽  
Single Cell Sequencing ◽  
Single Cell Rna Sequencing

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. It is also unclear why congenital ZIKV disease was not observed prior to the recent epidemics in French Polynesia and the Americas, despite evidence that the Zika virus has actively circulated in parts of Africa and Asia since 1947 and 1966, respectively. Methods Due to advances in stem cell-based technologies, we can now model ZIKV infections of the central nervous system in human stem cell-derived neuroprogenitor cells and cerebral organoids, which recapitulate complex three-dimensional neural architecture. We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize these neural models infected with ZIKV. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, thereby defining transcriptional features of both uninfected and infected cells. Results In neuroprogenitor cells, single-cell sequencing reveals that while uninfected bystander cells strongly upregulate interferon pathway genes, these are largely suppressed in cells infected with ZIKV within the same culture dish. In our organoid model, single-cell sequencing allows us to identify multiple cellular populations, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. In this model of the developing brain, we identify preferred tropisms of ZIKV infection. Our data additionally reveal differences in cell-type frequencies and gene expression within organoids infected by historic and contemporary ZIKV strains from a variety of geographic locations. Conclusions These findings may help explain phenotypic differences attributed to the viruses, including variable propensities to cause microcephaly. Overall, our work provides insight into normal and diseased human brain development and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection.

scholarly journals Single-cell RNA sequencing of Trypanosoma brucei from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens

2020 ◽  
Vol 117 (5) ◽  
pp. 2613-2621 ◽  
Author(s):  
Aurélien Vigneron ◽  
Michelle B. O’Neill ◽  
Brian L. Weiss ◽  
Amy F. Savage ◽  
Olivia C. Campbell ◽  
...  
Keyword(s):  
Single Cell ◽  
Salivary Glands ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Developmental Process ◽  
Surface Glycoprotein ◽  
Mammalian Host ◽  
Transmission Blocking ◽  
African Trypanosomes ◽  
Single Cell Rna Sequencing

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly’s salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.

scholarly journals Neddylation mediates ventricular chamber maturation through repression of Hippo signaling

2018 ◽  
Vol 115 (17) ◽  
pp. E4101-E4110 ◽  
Author(s):  
Jianqiu Zou ◽  
Wenxia Ma ◽  
Jie Li ◽  
Rodney Littlejohn ◽  
Hongyi Zhou ◽  
...  
Keyword(s):  
Heart Failure ◽  
Heart Development ◽  
Molecular Mechanisms ◽  
Heart Defects ◽  
Left Ventricular ◽  
Late Gestation ◽  
Hippo Signaling ◽  
Cardiomyocyte Proliferation ◽  
Ventricular Noncompaction ◽  
Cullin 7

During development, ventricular chamber maturation is a crucial step in the formation of a functionally competent postnatal heart. Defects in this process can lead to left ventricular noncompaction cardiomyopathy and heart failure. However, molecular mechanisms underlying ventricular chamber development remain incompletely understood. Neddylation is a posttranslational modification that attaches ubiquitin-like protein NEDD8 to protein targets via NEDD8-specific E1-E2-E3 enzymes. Here, we report that neddylation is temporally regulated in the heart and plays a key role in cardiac development. Cardiomyocyte-specific knockout of NAE1, a subunit of the E1 neddylation activating enzyme, significantly decreased neddylated proteins in the heart. Mice lacking NAE1 developed myocardial hypoplasia, ventricular noncompaction, and heart failure at late gestation, which led to perinatal lethality. NAE1 deletion resulted in dysregulation of cell cycle-regulatory genes and blockade of cardiomyocyte proliferation in vivo and in vitro, which was accompanied by the accumulation of the Hippo kinases Mst1 and LATS1/2 and the inactivation of the YAP pathway. Furthermore, reactivation of YAP signaling in NAE1-inactivated cardiomyocytes restored cell proliferation, and YAP-deficient hearts displayed a noncompaction phenotype, supporting an important role of Hippo-YAP signaling in NAE1-depleted hearts. Mechanistically, we found that neddylation regulates Mst1 and LATS2 degradation and that Cullin 7, a NEDD8 substrate, acts as the ubiquitin ligase of Mst1 to enable YAP signaling and cardiomyocyte proliferation. Together, these findings demonstrate a role for neddylation in heart development and, more specifically, in the maturation of ventricular chambers and also identify the NEDD8 substrate Cullin 7 as a regulator of Hippo-YAP signaling.

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