Developmental Biology

Endocardial TIE1 synergizes with TIE2 to regulate the atrial internal muscular network assembly

Kai Ding
Beibei Xu
Xinhao Yu
Xiwen Jia
Taotao Li
Xin Shen
Junda Li
Xudong Cao
Yahui Liu
Zhen Zhang
Yulong He author has email address

Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, National Clinical Research Center for Hematologic Diseases, State Key Laboratory of Radiation Medicine and Protection, Cam-Su Genomic Resources Center, Suzhou Medical College of Soochow University, Suzhou, China
Pediatric Translational Medicine Institute and Pediatric Congenital Heart Disease Institute, Shanghai Children’s Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Shanghai Collaborative Innovative Center of Intelligent Medical Device and Active Health, Shanghai University of Medicine & Health Sciences, Shanghai, China

https://doi.org/10.7554/eLife.111156.1

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Figures and data

Analysis of TIE1 expression in endocardial cells by the single cell RNA-seq data and its role in cardiac trabeculation during embryogenesis.
A-B. Analysis of the single-cell RNA-seq data of embryonic hearts from E10.5 to E18.5 by Feng et al. Nat Commun 2022 (DOI: 10.1038/s41467-022-35691-7). UMAP visualization of 10 distinct cell clusters in the murine hearts during embryogenesis (E10.5 to E18.5, A). Tie1 and Tek are highly expressed in the endocardial cell population (Npr3⁺) as shown in feature plots. Angpt1 are highly expressed in the atrial cardiomyocytes (B). Black arrows point to the coronary EC population (Fabp4⁺). Red arrows point to the endocardial cell population (Npr3⁺). Blue arrows point to the atrial cardiomyocytes. The quantitative results are shown in Table 1. C-J. Analysis of heart trabeculae of Tie1^tm^1a^/tm^1a and littermate controls by immunostaining for EMCN (endomucin; green), PECAM1 (platelet endothelial cell adhesion molecule 1; red) and DAPI (blue) at E12.5 (C-D) and E14.5 (G-H). Note that Tie1^tm^1a^/tm^1a mice displayed defective trabeculation in the atrium (white arrows) and sparse trabecular structures in the ventricles (white arrows) at E12.5, with more severe defects by E14.5, lacking atrial trabeculae (white arrows) and sparser ventricular trabeculae (white arrows). Asterisks in C and G point to the delayed formation of interventricular septum in Tie1^tm^1a^/tm^1a mice. Arrows point to the trabecula-associated endocardium. E-F. Quantification of the EMCN-positive area in the atrial and ventricular trabeculae at E12.5 (E, LA: Tie1^tm^1a^/tm^1a: 0.63±0.33, n=6; Control: 1.00±0.22, n=8; P=0.054. RA: Tie1^tm^1a^/tm^1a: 0.77±0.26, n=6; Control: 1.00±0.13, n=8; P=0.075. LV: Tie1^tm^1a^/tm^1a: 0.86±0.16, n=6; Control: 1.00±0.046, n=8; P=0.054. RV: Tie1^tm^1a^/tm^1a: 0.91±0.11, n=6; Control: 1.00±0.12, n=8; P=0.13.). Quantification of the ventricular wall thickness (F, LV: Tie1^tm^1a^/tm^1a: 24.87±3.70 µm, n=6; Control: 27.67±4.52 µm, n=8; P=0.41. RV: Tie1^tm^1a^/tm^1a: 26.01±8.42 µm, n=6; Control: 32.84±11.21 µm, n=8; P=0.28.). I-J. Quantification of the EMCN-positive area in the atrial and ventricular trabeculae at E14.5 (I, LA: Tie1^tm^1a^/tm^1a: 0.40±0.16, n=7; Control: 1.00±0.079, n=7; P=0.00060. RA: Tie1^tm^1a^/tm^1a: 0.36±0.17, n=7; Control: 1.00±0.092, n=7; P=0.00060. LV: Tie1^tm^1a^/tm^1a: 0.81±0.22, n=7; Control: 1.00±0.033, n=7; P=0.036. RV: Tie1^tm^1a^/tm^1a: 0.77±0.23, n=7; Control: 1.00±0.040, n=7; P=0.026.). Quantification of the PECAM1-positive area in ventricular walls (J, LV: Tie1^tm^1a^/tm^1a: 0.91±0.15, n=4; Control: 1.00±0.013, n=6; P=0.56. RV: Tie1^tm^1a^/tm^1a: 0.64±0.096, n=4; Control: 1.00±0.024, n=6; P=0.0095.). The quantification data of mutant mice was normalized against that of littermate control mice. In E-J, statistical analysis was performed using the unpaired nonparametric Mann-Whitney U test. Values are represented as means ± SD of at least three independent technical replicates. LA, Left atria, RA, Right atria, LV, Left ventricle, RV, Right ventricle, Scale bar: Scale bar: 400 μm in C and G, 50 μm in D and H.

The transcript levels of Tie1, Tek in atrial and ventricular endocardial cells and Angpt1 in atrial and ventricular cardiomyocytes during the embryonic stage from E10.5-E18.5 (based on the published scRNA-seq data, Feng et al., Nat Commun 2022; DOI: 10.1038/s41467-022-35691-7).
Values are represented as means ± SD and the number referred to cell counts.

Suppression of cardiac trabeculation-related gene expression by the loss of TIE1.
A-B. Analysis of heart trabeculae of Tie1^tm^1a^/tm^1a and littermate controls by immunostaining for EMCN (green), PECAM1 (red) and DAPI (blue) at E17.5. Note that the ventricular trabeculae of Tie1^tm^1a^/tm^1a mice become sparse compared with those of control mice (white arrows). Arrows point to the trabecula-associated endocardium. C-F. Quantification of the EMCN-positive area in the atrial and ventricular trabeculae (C, LA: Tie1^tm^1a^/tm^1a: 0.56±0.16, n=6; Control: 1.00±0.22, n=8; P=0.0043. RA: Tie1^tm^1a^/tm^1a: 0.57±0.33, n=6; Control: 1.00±0.24, n=8; P=0.026. D, LV: Tie1^tm^1a^/tm^1a: 0.86±0.18, n=6; Control: 1.00±0.082, n=8; P=0.054. RV: Tie1^tm^1a^/tm^1a: 0.61±0.18, n=6; Control: 1.00±0.10, n=8; P=0.00070). Quantification of the PECAM1-positive area in ventricular walls (E, LV: Tie1^tm^1a^/tm^1a: 0.66±0.14, n=6; Control: 1.00±0.11, n=8; P=0.00070. RV: Tie1^tm^1a^/tm^1a: 0.55±0.10, n=6; Control: 1.00±0.088, n=8; P=0.00070.). Quantification of the ventricular wall thickness (F, LV: Tie1^tm^1a^/tm^1a: 101.97 ± 22.44 µm, n=6; Control: 174.09 ± 22.84 µm, n=8; P=0.0013. RV: Tie1^tm^1a^/tm^1a: 87.14±26.20 µm, n=6; Control: 137.35±24.06 µm, n=8; P=0.0080.) at E17.5. Except the ventricular wall thickness, the quantification data of mutant mice was normalized against that of littermate control mice. G-L. RNA-seq analysis of whole hearts of Tie1^tm^1a^/tm^1a and littermate controls (E14.5, n=4 per group). G. PCA analysis showed that Tie1^tm^1a^/tm^1a and control groups were divided into two clusters. H. Volcano plot visualization of the differentially expressed genes (DEGs, P-value ≤ 0.05, |Log2Foldchange| > 0). I. Heatmap of significantly downregulated cardiac trabeculation genes (P-value ≤ 0.05). J. GSEA analysis showed the decrease of enrichment in trabeculation, venous development, and endothelial cell migration pathways. K-L. GO analysis revealed that gene related to cardiac chamber morphogenesis and trabeculae development, extracellular matrix, cell adhesion and endothelial migration/differentiation were significantly downregulated (K), while cardiac contraction and apoptosis related genes were significantly upregulated (L). In C-F, statistical analysis was performed using the unpaired nonparametric Mann-Whitney U test. Values are represented as means ± SD of at least three independent technical replicates. LA: Left atria, RA: Right atria, LV: Left ventricle, RV: Right ventricle; Scale bar: 400 μm in A, 50 μm in B; c-c: cell-cell, p-m: plasma membrane in K.

Differential effect of Tie1 deficiency on atrial versus ventricular trabeculation related gene network.
A. Visualization of heart trabeculae structure of Tie1^tm^1a^/tm^1a and littermate controls by whole-mount immunostaining for EMCN (green), PECAM1 (red) and DAPI (blue) at E14.5. Note that Tie1 deletion resulted in the loss of atrial trabecular network with minor defects with ventricular trabecular structures (white arrows). Arrows point to the trabecula-associated endocardium. B-J. RNA-seq analysis of atrium and ventricles of Tie1^tm^1a^/tm^1a and littermate controls (E14.5, n=3 per group). B. PCA analysis results showed clear separation between the atria and ventricles of Tie1^tm^1a^/tm^1a and control mice. C. Differential expression analysis (P-value ≤ 0.05, |log2(Foldchange)| ≥ 0.15) identified over 693 down-regulated and 948 up-regulated atrial genes, while 144 down-regulated and 190 up-regulated genes in ventricles. D-E. Volcano plots of atrial (D) and ventricular (E) differential expressed genes (DEGs). F-G. Heatmap showed significantly downregulated (P-value ≤ 0.05) heart trabeculation genes in atrium (F) and ventricles (G), including Tek, Dll1 and Notch1. H. Quantitation of Tie1 and Tek transcript levels (normalized by Pecam1) in atrium and ventricles of Tie1^tm^1a^/tm^1a and littermate controls (Tie1: Ventricles: 0.72 ± 0.021, n = 5; Atria: 1.00 ± 0.026; P = 0.0079. Tek: Ventricle: 0.55 ± 0.024; Atria: 1.00 ± 0.048; P = 0.0079). I-J. GO enrichment analysis of the DEGs in atrium and ventricles including the heart trabecula morphogenesis, extracellular matrix, cell adhesion and endothelial migration related process down-regulated while genes related to the cardiac contraction, apoptosis process up-regulated. There was a more obvious enrichment detected in the atria. In H, statistical analysis was performed using the unpaired nonparametric Mann-Whitney U test. Values are represented as means ± SD of at least three technical replicates. LA: left atria, RA: right atria, LV: left ventricle, RV: right ventricle; Scale bar: 50 μm in A; c-c: cell-cell, p-m: plasma membrane in I-J.

Impaired atrial trabecular formation after TIE2 insufficiency.
A. Tamoxifen intraperitoneal (i.p.) administration and analysis scheme. B-D. Analysis of heart trabeculae of Tek^iECKO and littermate controls by immunostaining for EMCN (green), PECAM1 (red) and DAPI (blue) 48 hours later (tamoxifen treatment starting at E12.5). E-F. Quantification of the EMCN-positive area in the atrial and ventricular trabeculae (E, LA: Tek^iECKO: 0.60±0.20, n=9; Control: 1.00±0.097, n=8; P=0.00010. RA: Tek^iECKO: 0.64±0.23, n=9; Control: 1.00±0.22, n=8; P=0.0047. LV: Tek^iECKO: 0.85±0.15, n=9; Control: 1.00±0.057, n=8; P=0.020. RV: Tek^iECKO: 0.90±0.16, n=9; Control: 1.00±0.089, n=8; P=0.13.), quantification of the PECAM1-positive area in ventricular walls (F, LV: Tek^iECKO: 0.92±0.25, n=7; Control: 1.00±0.048, n=6; P=0.81. RV: Tek^iECKO: 1.22±0.53, n=7; Control: 1.00±0.13, n=6; P=0.28.) and quantification of the ventricular wall thickness (F, LV: Tek^iECKO: 62.35±11.70 µm, n=9; Control: 62.59±10.26 µm, n=8; P=0.96. RV: Tek^iECKO: 63.87± 9.19 µm, n=9; Control: 58.87±11.02 µm, n=8; P=0.32.) at E14.5. Except the ventricular wall thickness, the quantification data of mutant mice was normalized against that of littermate control mice. G-H. Analysis of heart trabeculae of Tek^iECKO and littermate controls by immunostaining for EMCN (green), PECAM1 (red) and DAPI (blue) 72 hours after the induced gene deletion or later (tamoxifen treatment starting at E12.5). Arrows point to the trabecula-associated endocardium. In E-F, statistical analysis was performed using the unpaired t test. Values are represented as means ± SD of at least three independent technical replicates. LA: left atria, RA: right atria, LV: left ventricle, RV: right ventricle; Scale bar: 500 μm (embryo) and 400 μm (heart) in B, 400 μm in C, 50 μm in D, G, H.

Developmental requirement of TIE1 and TIE2 in atrial and ventricular chamber morphogenesis.
A-D. Analysis of heart trabeculae of Tie1^ΔICD/ΔICD, Tie1^ΔICD/ΔICD;Tek^+/- and littermate controls by immunostaining for EMCN (green), PECAM1 (red) and DAPI (blue) at E10.5. Note that there were no obvious defects observed with the atrial or ventricular structures upon Tie1 deficiency alone at E10.5, while loss of Tie1 combined with the heterozygous deletion of Tek suppressed both atria and ventricle development. White arrows point to the trabecular structures. Quantification of the EMCN-positive area in the ventricular trabeculae at E10.5, and the quantification data of mutant mice was normalized against that of littermate control mice (B, LV: Tie1^ΔICD/ΔICD: 0.86±0.11, n=4; Control: 1.00±0.14, n=5; P=0.29. RV: Tie1^ΔICD/ΔICD: 0.79±0.19, n=4; Control: 1.00±0.15, n=5; P=0.19. D, Tie1^ΔICD/ΔICD;Tek^+/-: 0.64±0.22, n=6; Control: 1.00±0.16, n=7; P=0.0047). E. For the induced gene deletion, tamoxifen intraperitoneal (i.p.) administration (starting from E10.5) and analysis scheme (at E14.5-E16.5). F. The deletion efficiency of TIE1 was examined by the immunostaining of skins from Tie1ICD^iECKO;Tek^+/- and littermate control mice at E16.5 for PECAM1 (green) and TIE1 (red). G-H. Analysis of heart trabeculae of Tie1ICD^iECKO;Tek^+/- mice and littermate controls by immunostaining for EMCN (green), PECAM1 (red) and DAPI (blue) at E14.5. Note that the atrial trabeculae structure was missing, while the ventricular trabeculae became sparse (white arrows). Arrows point to the trabecula-associated endocardium. In B and D, statistical analysis was performed using the unpaired nonparametric Mann-Whitney U test. Values are represented as means ± SD of at least three independent technical replicates. LA: left atria, RA: right atria, LV: left ventricle, RV: right ventricle. Scale bar: 200 μm in A(left) and C(left), 30 μm in A(right) and C (right), 400 μm in G, 200 μm in F and 50 μm in H.

Synergistic role of TIE1 and TIE2 in the atrial trabeculation.
A. Tamoxifen intraperitoneal (i.p.) administration and analysis scheme. B-H. The induced gene deletion by tamoxifen started at E12.5. Analysis of heart trabeculae of Tie1ICD^iECKO;Tek^+/-and littermate controls by immunostaining for EMCN (green), PECAM1 (red) and DAPI (blue) at E14.5 (B-C) and E16.5 (F). Note that the atrial trabeculae were absent and the ventricular trabeculae were slightly sparse in the doubly mutant mice at E14.5 (B-C), and the phenotype became more severe at E16.5 (F). Quantification of the EMCN-positive area in the atrial and ventricular trabeculae (D, E14.5; G, E16.5), the PECAM1-positive area in ventricular walls and the ventricular wall thickness (E, E14.5; H, E16.5). Arrows point to the trabecula-associated endocardium. The quantification data of mutant mice was normalized against that of littermate control mice as shown in Table 2 and Table 3. Arrows in C and F point to the trabecula-associated endocardium. In D, E, G and H, statistical analysis was performed using the unpaired nonparametric Mann-Whitney U test. Values are represented as means ± SD of at least three independent technical replicates. LA: left atria, RA: right atria, LV: left ventricle, RV: right ventricle. Scale bar: 400 μm in B, 50 μm in C and F.

Quantitation of the trabecular area (EMCN⁺), blood vessel density in ventricular wall (PECAM1⁺) and ventricular wall thickness in Tie1ICD^iECKO;Tek^+/- and littermate controls (at E14.5, 48 hours after the induced gene deletion).
Statistical analysis was performed using the unpaired nonparametric Mann-Whitney U test. Values are represented as means ± SD of at least three independent technical replicates.

Quantitation of the trabecular area (EMCN⁺), blood vessel density in ventricular wall (PECAM1⁺) and ventricular wall thickness in Tie1ICD^iECKO;Tek^+/- and littermate controls (at E14.5, 48 hours after the induced gene deletion).
Statistical analysis was performed using the unpaired nonparametric Mann-Whitney U test. Values are represented as means ± SD of at least three independent technical replicates.

Quantitation of the trabecular area (EMCN⁺), coronary vessel density in ventricular wall (PECAM1⁺) and ventricular wall thickness in Tie1ICD^iECKO;Tek^+/- and littermate controls 96 hours after the induced gene deletion (E16.5; tamoxifen treatment from E12.5-E13.5).
Statistical analysis was performed using the unpaired nonparametric Mann-Whitney U test. Values are represented as means ± SD of at least three independent technical replicates.

Abnormal atrial trabecular remodeling after the postnatal deletion of Tie1 combined with one null allele of Tek.
A. Tamoxifen intragastric (i.g.) administration and the analysis scheme. B-H. Analysis of blood vessels in the retinas of Tie1ICD^iECKO (B), Tie1ICD^iECKO;Tek^+/−(E) and control mice by whole-mount immunostaining for PECAM1 (green), DLL4 (red) at P21. Note that hemangioma-like vascular tufts observed in Tie1ICD^iECKO and Tie1ICD^iECKO;Tek^+/−mice. The retinal vascular defects were used to confirm the efficiency of induced gene deletion. Analysis of heart trabeculae in Tie1ICD^iECKO (C), Tie1ICD^iECKO;Tek^+/−(F) and control mice by the immunostaining for EMCN (green), PECAM1 (red) and DAPI (blue) at P21. TIE1 insufficiency alone had no obvious effect on heart size (D, Tie1ICD^iECKO: 0.54±0.021, n=7; Control: 0.54±0.014, n=5; P=0.91.) and atrial trabecular structures (C). The Tie1ICD^iECKO;Tek^+/- mice displayed smaller hearts (G, Tie1ICD^iECKO;Tek^+/-: 0.49±0.086, n=8; Control: 0.59±0.10, n=11; P=0.035.) and abnormal atrial trabecular morphology (F,H). White arrows in H point to the trabecula-associated endocardium in the atria. I. Schematic illustration of TIE1, TIE2 and their synergy in cardiac chamber morphogenesis. TIE1 deficiency, TIE2 or TIE1/TIE2 insufficiency results in a more severe defect with the atrial trabeculation than that of ventricles. Based on the previous findings about the TIE1/TIE2 in venous EC specification ^28,30 and findings from this study, we propose that TIE1 acts in synergy with TIE2 in the specification and remodeling of atrial endocardium, and that there is a differential requirement of TIE receptors in atrial versus ventricular wall morphogenesis. In D and G, statistical analysis was performed using the unpaired nonparametric Mann-Whitney U test (D) and the unpaired t test (G). Values are represented as means ± SD of at least three independent technical replicates. Scale bar: 50 μm in B and E, 1 mm in C and F, 200 μm in H.

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