Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Co-immunoprecipitation showing interaction between Isl1 and Rnf20 (top). GST pull down assays with recombinant GST protein or GST-Rnf20 fusion protein and recombinant Isl1 protein, showing that the interaction between those two proteins is direct (bottom). (B) Violin plot of Rnf20 expression levels in Isl1+ and NKX2.5+ single cells of E8.25 embryos . Each black dot represents a single cell. FHF, First heart field. SHF, Second heart field. (C) Gross appearance of control ( Isl1 +/+ Rnf20 +/fl ) and Isl1 cre/+ Rnf20 -/fl embryos at E9.5 and E11.5. Scale bars, 2 mm (left), 1 mm (right). (D) Frontal views of E10.5 control and Isl1 cre/+ Rnf20 -/fl hearts, showing short outflow tract (OFT) and a smaller right ventricle (RV). Scale bars, 100 µm. (E) H&E stainings of transverse sections of control and Isl1 cre/+ Rnf20 -/fl embryos at E10.5 showing shortening of the OFT. Scale bars, 200 µm. (F) Histological analysis of control and Rnf20 knockout hearts at E11.5 showing a smaller RV, hypoplastic ventricular wall and abnormal trabeculation. Magnified images of indicated RV regions (square) are shown in the right panel. Scale bars, 200 µm (left), 50 µm (right). (G) RV and left ventricle (LV) wall thickness (top) and trabecular layer thickness (bottom) in control (n=8 for E11.5, n=6 for E12.5) and Isl1 cre/+ Rnf20 -/fl embryos (n=6 for E11.5, n=5 for E12.5) of E11.5 and E12.5 hearts. (H, I) Immunostaining with EdU, anti-MF20 (CMs), anti-IB4 (ECs) and DAPI (nucleus) (H) and quantification of EdU-labeled CMs (EdU+/MF20+ cells) in RV and LV of E12.5 control and Isl1 cre/+ Rnf20 -/fl embryos (n=4) (I), showing a marked reduction in the number of proliferating CMs upon Rnf20 ablation. Scale bars, 100 µm. (J, K) Immunostaining for H2Bub1 catalyzed by Rnf20 and MF20 in RV and LV of E11.5 control and Isl1 cre/+ -Rnf20 -/fl embryos (J) and magnified areas (K). Arrows point to H2Bub1-positive CMs in the LV; asterisks indicate H2Bub1-negative endocardial cells. Scale bars, 50 µm. For this and all other figures, error bars represent mean ±SD. *p<0.05 **p<0.01 ***p<0.001.

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Immunoprecipitation, Recombinant, Expressing, Knock-Out, Immunostaining, Labeling

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Schematic representation of the dissection procedure used for RNA-Seq experiments. RV and OFT of E9.5 and E10.5 control and Isl1 cre/+ Rnf20 -/fl were dissected. (B) Volcano plot showing the distribution of differentially expressed genes in dissected E9.5 OFT and RV of Isl1 cre/+ Rnf20 -/fl versus control OFT and RV (n=4; Log2(FC) ≤ -0.58, ≥0.58; p-value < 0.05). Representative up- (red) and downregulated (blue) genes are indicated. (C) Gene ontology pathway enrichment analysis and representative up- (red) and downregulated (blue) genes in E9.5 Isl1 cre/+ Rnf20 -/fl compared to control hearts. (n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value < 0.05). (D) Gene ontology pathway enrichment analysis and representative up- (red) and downregulated (blue) genes in E10.5 Isl1 cre/+ Rnf20 -/fl compared to control hearts. (E) Heatmap representation of genes involved in extracellular matrix organization and cell differentiation in dissected OFT and RV of Rnf20-deficient and Isl1 haploinsufficient hearts . (F) Circos plot of the significantly changed ligand-receptor interactions at E10.5 upon Rnf20 LOF in Isl1+ cardiovascular precursors. Red arrows represent upregulated ligands, blue arrows represent downregulated ligands and arrow thickness indicates the probability for interaction, i.e. interaction score . (G) Relative mRNA expression levels of genes involved in endothelial-myocardial interactions in ECs isolated from control and Rnf20 KO E10.5 RV and OFT (n= 3). (H) Co-immunoprecipitation with N1ICD and Rnf20, showing that Rnf20 interacts with N1ICD. (I) Overlap between genes differentially expressed in Rnf20 and Rbpj knockout compared to control hearts. (J) Relative expression of genes associated with cardiac jelly protein degradation in control and Isl1 cre/+ Rnf20 -/fl RV and OFT. (K) Images of Alcian blue and nuclear fast red staining of transverse sections of control and Isl1 cre/+ Rnf20 -/fl hearts at E12.5. Magnified images of indicated regions are shown in the right panels. Scale bars, 200 µm (whole heart), 50 µm (magnified regions).

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Dissection, RNA Sequencing Assay, Cell Differentiation, Expressing, Isolation, Immunoprecipitation, Knock-Out, Staining

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Principal component analysis (PCA) of gene expression variation in dissected OFT and RV of E9.5 (n=3) and E10.5 (n=4) control and Isl1 cre/+- -Rnf20 -/fl embryos. (B) Volcano plot showing the distribution of differentially expressed genes in dissected E10.5 OFT and RV of Isl1 cre/+ Rnf20 -/fl versus control OFT and RV (n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value < 0.05). Representative up- (red) and downregulated (blue) genes are indicated. (C) Average ATAC-Seq tag intensities in pharyngeal mesoderm/hearts of wild-type and Isl1-/-embryos at genes downregulated upon Isl1 loss of function in E10.5 (log2FC ≤ -1, p-value < 0.05). (D) Schematic representation of Pol II average profiles and the method used for defining the Pol II pausing index. Pausing index was calculated by the ratio of -50bp to +300bp Pol II signal divided by the Pol II signal within +300bp to +2kb. (E) Pol II pausing index of upregulated, downregulated and non-changed genes in dissected OFT and RV of E10.5 control and Isl1 cre/+- -Rnf20 -/fl embryos, showing that differentially regulated genes are associated with higher elongation rates. Pol II ChIP-Seq data from E14.5 hearts was used (GSE29218) (F) Pol II pausing index of upregulated, downregulated and non-changed cell-cell-signaling genes (GO:0007267).

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Expressing, ChIP-sequencing

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A, B) Gross appearance of control ( Tie2 Cre Rnf20 +/fl ) and Tie2 Cre Rnf20 fl/fl embryos at E10.5 (A) and of dissected hearts (B). (C) Schematic representation of the experimental setup and histological analysis of control and Rnf20 iEC-KO hearts at E12.5 showing a thinner compact layer, less developed trabeculae, disorganized interventricular septum (left) and disorganized endocardial cells (arrows in the right panel). Scale bars, 200 µm. (D) Schematic representation of the experimental setup. For this experiment and all other studies, control ( Cdh5 CreERT2 neg- Rnf20 fl/fl ) and Rnf20 iEC-KO ( Cdh5 CreERT2 pos- Rnf20 fl/fl ) embryos were exposed to tamoxifen from E9.5. (E) H&E staining of representative paraffin sections of E14.5 hearts of control and Rnf20 iEC-KO embryos (left panels), and higher magnification of RVs and LVs (boxed area) showing thinner compact myocardium and trabecular layer (middle and right panels). Abbreviations: Ao, Aorta; LV, left ventricle; RV, right ventricle; IVS, interventricular septum. Star indicates ventricular septal defect (VSD). Scale bars, 200 µm (whole heart), 50 µm (magnified regions). (F) Quantification of the thickness of the compact and trabecular layer as well as the ventricular septum thickness in control and Rnf20 iEC-KO hearts (n=6). (G, H) Immunostaining of E14.5 control (n=5) and Rnf20 iEC-KO (n=4) heart sections with Ser10 phospho-H3 (pH3+) and MF20 antibody (G) and quantification of the percentage of mitotic pH3+ CMs (H). Scale bars, 50 µm. (I) Representative FACS analysis of staining with Vybrant DyeCycle DNA dye for mono- and binucleation in Rnf20 iEC-KO hearts (top panel). The percentage of mononucleated and binucleated CMs (n=6, bottom panel). (J) Representative examples of B- and M-mode echocardiograms of control and Rnf20 iEC-KO embryos at E14.5, showing irregular contractility of the RV. (K) Quantification of the ejection fraction (EF) and fractional shortening (FS) for RV and LV assessed by echocardiography (n=6).

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Staining, Immunostaining

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Experimental regimen to induce Rnf20 ablation (top). First tamoxifen administration in this experiment was conducted at E8.5. Gross appearance of control ( Cdh5 CreERT2 neg-Rnf20 fl/fl ) and Rnf20 iEC-KO ( Cdh5 CreERT2 pos-Rnf20 fl/fl ) embryos (middle panels) and dissected hearts (bottom panels) at E12.5. (B) Experimental regimen to induce Rnf20 ablation (top). First tamoxifen administration for this experiment and all the following experiments was conducted at E9.5. Gross appearance of control ( Cdh5 CreERT2 neg-Rnf20 fl/fl ) and Rnf20 iEC-KO ( Cdh5 CreERT2 pos-Rnf20 fl/fl ) embryos (middle panels) and dissected hearts (bottom panels) at E14.5. (C) Experimental regimen to induce Rnf20 ablation (top). Gross appearance of control ( Cdh5 CreERT2 neg-Rnf20 fl/fl ) and Rnf20 iEC-KO ( Cdh5 CreERT2 pos-Rnf20 fl/fl ) embryos (bottom) at E16.5. (D) Cardiac output assessed by echocardiography of control and Rnf20 iEC-KO hearts (n=6).

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques:

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Schematic representation of the experimental design for the establishment of a co-differentiation system from murine ESC to endocardial cells and cardiomyocytes. (B, C) Representative FACS analysis of direct Nkx2.5-GFP fluorescence and staining for the EC marker CD31 (B) and percentage of Nkx2.5+/CD31+ endocardial cells (n=4) (C). (D) Characterization of simultaneous differentiation of CMs and endocardial cells from ESCs (co-differentiation, top). FACS analysis showing similar percentage of Nkx2.5+/CD31+ endocardial cells and Nkx2.5+/CD31-CMs co-differentiated from ESCs (bottom left) and images of co-differentiated cells (bottom right). (E) Beating rate of CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells at day 9. Beating rate was extracted from video sequences using MYOCYTER. (F) Co-culture of Nkx2.5+/CD31+ endocardial cells and Nkx2.5-/CD31+ hemogenic ECs with human iPSC-derived CMs. Nkx2.5+/CD31+ endocardial cells were sorted from day 7 and differentiated using the protocol represented in panel A, while Nkx2.5-/CD31+ hemogenic ECs were differentiated by the addition of EC differentiation medium at mesoderm stage (protocol adapted for mouse ESCs from . (G) Beating rate of hiPSC-CMs co-cultured either with Nkx2.5+/CD31+ endocardial cells or Nkx2.5-/CD31+ hemogenic ECs for 2 days. Beating rate was extracted from video sequences using MYOCYTER. (H) Relative expression of CM, EC marker genes as well as genes involved in EC-CM crosstalk in CMs differentiated by the direct differentiation protocol of ESCs into CMs or co-differentiated with endocardial cells (top) or ECs differentiated by the direct differentiation protocol of ESCs into ECs or co-differentiated with CMs (bottom). (I) Schematic representation of the methodology used for the generation of control and Rnf20 iEC-KO mouse iPSC lines. (J) Representative FACS analysis of 38 days old CMs (CD31 negative cells in the co-differentiation) stained with Vybrant DyeCycle DNA dye, showing decrease in CMs in G2/M phase upon endothelial Rnf20 ablation. For this experiment as well as the data shown in (K-M), control and Rnf20 iEC-KO iPSCs were co-differentiated in CM and endothelial specific Rnf20 ablation was induced at day 5 by 4-hydroxytamoxifen (4-OHT). (K) Quantification of the percentage of CMs (left) or endocardial cells (right) in G0/G1 and G2/M phase upon EC-specific tamoxifen-mediated Rnf20 ablation (n=4). Cells were differentiated using the co-differentiation protocol and stained with Vybrant DyeCycle DNA dye, and CD31, followed by FACS analysis. (L) Quantification of the percentage of mononucleated and binucleated CMs (day 38) of cells stained with Vybrant DyeCycle DNA dye and subjected to FACS analysis (n=4). (M) Plot of contraction amplitude and speed in spontaneously beating CMs (day 18) from video sequences using MYOCYTER.

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Fluorescence, Staining, Marker, Co-Culture Assay, Derivative Assay, Cell Culture, Expressing

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Schematic representation of the experimental setup for the analysis presented in and . E14.5 control and Rnf20 iEC-KO hearts were used for scRNA-Seq and purified ECs and CMs for RNA-Seq and ATAC-Seq. (B) 10x genomics single-cell RNA-Seq of E14.5 control and Rnf20 iEC-KO . AVC CM, atrioventricular cardiomyocytes (CM); AVcu, atrioventricular cushion cells; Pro. CM, proliferating CM; EndoC, endocardial cells; EndoV, endocardial valve cells; VaMes, valve mesenchyme; SMC, smooth muscle cells; VEC, vascular ECs; EpiC, epicardial cells; MΦ, macrophages. (C) UMAP analysis of CM populations from panel B. Feature plots visualizing Mki67 -expressing proliferative CM. aCM, atrial CM; vCM, ventricular CM. (D) UMAP analysis of CD31+ endothelial and endocardial populations from panel B identifying 7 subclusters. (E) Feature plots of the expression of Mki67 and Col5a2 , showing a decrease in proliferative endocardial cells (circled population) and an increase in Col5a2 expression in subset of endocardial ECs. (F) Violin plots visualizing expression levels of collagens and extracellular matrix proteins in different endothelial and endocardial populations. (G) Gene ontology pathway enrichment analysis and representative genes in upregulated and downregulated genes in Rnf20 iEC-KO ECs compared to ECs from control hearts. n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value < 0.05. (H) Immunostaining of E14.5 control and Rnf20 iEC-KO heart sections with the EndMT marker vimentin (Vim), IB4 for ECs and DAPI for nuclei. Scale bars, 50 µm. (I) Gene ontology pathway enrichment analysis and representative genes in upregulated and downregulated genes in Rnf20 iEC-KO CMs compared to CMs from control hearts. n=3; Log2(FC) ≤ -0.58, ≥0.58; p-value < 0.05.

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Purification, RNA Sequencing Assay, Expressing, Immunostaining, Marker

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Principal component analysis (PCA) of gene expression variation of 1000 most significant genes in ECs and CMs isolated from control embryos (left), showing a clear separation between the different cell populations. PCA of gene expression variation of 1000 most significant genes in ECs (middle) and CMs (right) isolated from control and Rnf20 iEC-KO hearts. (B) Fold-change (FC) of normalized CPM expression of CM marker genes in ECs and CMs isolated from control and Rnf20 iEC-KO hearts. (C) FC of normalized CPM expression of EC marker genes in ECs and CMs isolated from control and Rnf20 iEC-KO hearts. (D) Volcano plots showing the distribution of differentially expressed genes between Cdh5 pos -Rnf20 fl/fl and control ECs (left, brown) and CM (right, red). Representative up- (dark color) and downregulated (light) genes are indicated. n=3; Log2(FC) ≤ -0.58, ≥ 0.58; p-value < 0.05. (E) Gene set enrichment analysis of the pathway cell communication in downregulated genes in EC (top) and CM (bottom) upon endothelial deletion of Rnf20 . (F) Heatmap representing gene expression levels of collagens, protocadherins and factors involved in EndMT in control and Rnf20 iEC-KO ECs. (G) Heatmap representing gene expression levels of genes involved in extracellular matrix organization and cell adhesion and cell division in control and Rnf20 iEC-KO cardiac ECs and HUVECs transfected either with control siRNA or siRNA against Rnf20 . (H) Western blot analysis for RNF20 in HUVECs transfected with siRNA against RNF20 versus control siRNA transfected cells for the analysis in (I, J) . (I, J) Scratch wound healing assay with control and RNF20 knockdown HUVECs (I) and quantification of the migration rate of HUVECs transfected with control siRNA or siRNA against RNF20 (J) . (K) Relative Rnf20 expression levels in sorted ESC-derived CMs cultured alone or together with ESC-derived endocardial ECs, showing increased Rnf20 expression in CMs upon co-culture with ECs.

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Expressing, Isolation, Marker, Transfection, Western Blot, Wound Healing Assay, Migration, Derivative Assay, Cell Culture, Co-Culture Assay

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Volcano plot showing the distribution of differentially accessible chromatin regions between ECs and CMs isolated from control and Rnf20 iEC-KO hearts. n=3; Log2(FC)≤ -0.58, ≥0.58; p-value < 0.05. (B) Gene ontology pathway enrichment analysis of genes showing increased (top) or decreased (bottom) chromatin accessibility in ECs (left panel) and CMs (right panel) isolated from control and Rnf20 iEC-KO hearts. (C, D) Heatmap representation of genes associated to the GO: Tube development showing increased or decreased chromatin accessibility and transcriptional activity (spearman correlation factor r ≥ 0.7) in ECs isolated from control and Rnf20 iEC-KO hearts (C). Examples of genes showing increased chromatin accessibility (D). Genome tracks of ATAC-Seq reads of control and Rnf20 iEC-KO ECs are presented. (E) TOBIAS footprints of chromatin-associated proteins at ATAC-Seq peaks associated to differentially expressed (DE) genes in control and Rnf20 iEC-KO versus control ECs. (F, G) Heatmap representation of genes associated to the GO: Regulation of cell differentiation showing increased or decreased chromatin accessibility and transcriptional activity (spearman correlation factor r ≥ 0.5) in CMs isolated from control and Rnf20 iEC-KO hearts (F). Examples of genes showing decreased (top) or increased (bottom) chromatin accessibility (G). Genome tracks of ATAC-Seq reads of control and Rnf20 iEC-KO CMs are presented. (H) TOBIAS footprints of chromatin-associated proteins at ATAC-Seq peaks associated to differentially expressed (DE) genes in Rnf20 iEC-KO CMs. (I) TOBIAS footprinting analysis on all ATAC peaks associated to upregulated genes (UP) upon Rnf20 LOF in ECs. (J) Venn diagram showing the overlap of genes upregulated in Rnf20 iEC-KO versus control cardiac ECs and in HUVECs overexpressing Sox9 versus control HUVECs. For both datasets: Log2(FC) ≤ -0.58, ≥0.58; p-value < 0.05 (K) Heatmap representing gene expression levels of genes involved in cell adhesion, extracellular matrix organization and regulation of blood pressure in control and Rnf20 iEC-KO cardiac ECs and in control HUVECs and HUVECs overexpressing Sox9. (L, M) Immunostaining for α−actinin and DAPI of CMs co-cultured with HUVECs overexpressing β-gal protein or Sox9 (J) and quantification of binucleated CMs (K). Scale bars, 10 µm.

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Isolation, Activity Assay, Cell Differentiation, Footprinting, Expressing, Immunostaining, Cell Culture

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Principal component analysis (PCA) of genome-wide chromatin accessibility variation in control and Rnf20 iEC-KO ECs (left panel) and CMs (right panel). (B ) Heatmap of 10000 most significantly altered ATAC-Seq peaks between Rnf20 iEC-KO and control ECs (top) and CMs (bottom). (C) Scatter plot showing the spearman correlation between chromatin accessibility and gene expression changes (Log2FC) in Rnf20 iEC-KO vs control ECs for all annotated differentially expressed genes. Pre-filtering of p < 0.05 and Log2(FC) ≤ -0.58 and ≥ 0.58 was applied. Dark brown dots indicate a positive whereas light brown dots indicate a negative correlation. (D) Scatter plot showing the spearman correlation between chromatin accessibility and gene expression changes (Log2(FC)) in Rnf20 iEC-KO vs control CMs for all annotated differentially changed genes. Pre-filtering of p < 0.05 and Log2(FC) ≤ -0.58 and ≥ 0.58 was applied. Dark red dots indicate a positive whereas light red dots indicate a negative correlation. (E) TOBIAS footprints of chromatin-associated proteins at ATAC-Seq peaks on genes that show positive (top) or negative (bottom) correlation between chromatin accessibility with transcriptional activity in ECs, potentially acting as transcriptional activators or repressors, respectively. (F) TOBIAS footprints of chromatin-associated proteins at ATAC-Seq peaks on genes that show positive (top) or negative (bottom) correlation between chromatin accessibility with transcriptional activity in CMs, potentially acting as transcriptional activators or repressors, respectively.

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Genome Wide, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Rnf20 shapes the endothelial control of heart morphogenesis and function

doi: 10.1101/2022.09.16.508288

Figure Lengend Snippet: (A) Bar plot of total numbers of ligand-receptor interactions between ECs and CMs including autocrine signaling in Rnf20 iEC-KO versus control . (B) Circos plot of the significantly changed ligand-receptor interactions between ECs and CMs upon Rnf20 LOF in ECs. Red labeled arrow represent upregulated ligand, blue labeled downregulated ligand and arrow thickness indicates the interaction score . (C) Schematic representation of the experimental design: Lentivirus particles produced by overexpression of lentiviral plasmids from genome-wide libraries in HEK293 cells were used to transduce HUVECs for 2 days. HUVECs overexpressing selected factors were then co-cultured with wild-type P1 rat CM. (D) Percentage of mononucleated and binucleated CMs determined by staining with Vybrant DyeCycle DNA dye and CD31 (to exclude HUVEC cells) and subjected to FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 or 4 days (n=3). (E) Percentage of proliferating EdU-positive CMs determined by EdU-incorporation and FACS analysis. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 2 days. (F) Plot of contraction amplitude and speed in spontaneously beating CMs extracted from video sequences using MYOCYTER. P1 rat CMs were co-cultured with HUVECs overexpressing selected factors for 4 days. (G) Beating rate of rat CMs co-cultured with HUVECs overexpressing selected factors. Beating rate was extracted from video sequences using MYOCYTER. (H) Model of the function of Rnf20 in ECs for proper cardiac morphogenesis and function.

Article Snippet: Upregulated genes were enriched in GO terms linked to cell-cell signaling, regulation of Ca 2+ concentration and heart contraction (Hcn4, Scn10a, Cacna2d2), consistent with the aberrant contractility observed in Rnf20 iEC-KO embryos.

Techniques: Labeling, Produced, Over Expression, Genome Wide, Transduction, Cell Culture, Staining

Journal: eLife

Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

doi: 10.7554/eLife.73524

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-RNF20 (rabbit polyclonal) , Cell Signaling Technology , Cat# 9425S;RRID: AB_2797700 , WB (1:1000).

Techniques: Software

Journal: eLife

Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

doi: 10.7554/eLife.73524

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-RNF20 (rabbit polyclonal) , Cell Signaling Technology , Cat# 9425S;RRID: AB_2797700 , WB (1:1000).

Techniques: Software