Endocardial TIE2 orchestrates cardiac trabeculation via sequestering FOXO1 within the cytoplasm and stimulating versican cleavage

Figures

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  Fig. 1.. Endocardial loss of Tie2 resulted in nuclear import of FOXO1, allowing for FOXO1-mediated transcription regulation. (A-D) Control (Tie2+/f and Tie2f/f) and Tie2-cko left ventricular sections at E11.5 were stained with FOXO1 (red) and endomucin (endocardial marker, green) antibodies. (B,D) are the higher magnification images (omitting the green channel) of the boxed areas in A and C showing a diffuse nucleocytoplasmic localization of FOXO1 in control EdCs (arrows, B), but accentuated nuclear localization in the mutant (arrowheads, D). (E) Quantification of EdCs with FOXO1 localized at nucleus (%). Note the mild gradual increase in nuclear import of FOXO1 of wild-type EdCs at E9.5-E13.5 and, by E16.5, the majority of wild-type EdCs demonstrate FOXO1 localized at nucleus. (F,G) Control and Tie2-cko ventricular sections at E10.5 were stained with pAKT (red) and endomucin (green) antibodies, showing reduced phosphorylation of AKT (arrows) in Tie2-cko EdCs compared to the control. Note more intense endomucin staining in Tie2-cko EdCs than in control, although their endocardial network was simplified. DAPI was used to counterstain nuclei in all images. (H) Quantification of pAKT+ EdCs…

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  Fig. 2.. Endocardial loss of Tie2 led to reduced expression of several proliferation promoting genes in EdCs. (A) Control and Tie2-cko left ventricular sections were stained with cyclin D1, cMYC, pYAP and endomucin antibodies. Arrows indicate representative positive staining in EdCs. DAPI was used to counterstain nuclei in all images. (B-D) Quantification of cyclin D1+ EdCs (B), cMYC+ EdCs (C) and pYAP+ EdCs (D). Data represent the mean±s.e.m. Two-way ANOVA in B,C and two-tailed unpaired Student's t-test in D; ***P<0.001. Scale bars: 50 μm.

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  Fig. 3.. Cardiac defects in Tie2-cko mice were phenocopied by endocardial forced activation of FOXO1 and partially rescued by loss of Foxo1. (A) Control (FOXO1CA/+) and FOXO1EC-CA ventricular sections at E11.5 were stained for troponin T (Tnnt2, myocardial marker) and endomucin (endo), showing abnormal trabeculae (arrows) and endocardial network in the mutant heart. (B,C) Quantification of trabeculae density and trabeculae thickness at E11.5. (D) BrdU pulse labeling and co-immunostaining of ventricular sections of control and FOXO1EC-CA embryos at E10.5 with BrdU (red) and ERG (green, EC marker). The cells positive for both BrdU and ERG represent proliferating EdCs (arrows). (E) Quantification of BrdU+/ERG+ cells as a percentage of total ERG+ cells in endocardium. (F) Control (Tie2f/+;Foxo1f/f and Tie2f/f;Foxo1f/f) and Nfatc1Cre;Tie2f/f;Foxo1f/f heart sections at E14.5 were stained with antibodies for troponin T and endomucin. Arrows indicate trabeculae. (G,H) Quantification of trabeculae thickness (G) and thickness of compact wall (H) at E14.5. DAPI was used to counterstain nuclei in all images. Data represent the mean±s.e.m. Two-tailed unpaired Student's t-test; *P<0.05; **P<0.01;…

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  Fig. 4.. Endocardial loss of Vegfr2 led to defective coronary vasculature but normal trabeculation and EdC network, with normal FOXO1 localization. (A-D) Control and Nfatc1Cre;Vegfr2f/f ventricular sections at E13.0 were stained with FOXO1 antibody or co-stained for troponin T (Tnnt2) and endomucin, showing normal nucleocytoplasmic localization of FOXO1 (arrows in A,B), normal trabeculae (arrows in C,D) and endocardial network in both control and mutant hearts, but severely diminished or no coronary vessels present in the ventricular septum (asterisk) of the mutant, compared to normal coronary vessels in the control (arrowheads). (E,F) Control and Nfatc1Cre;Vegfr2f/f ventricular sections at E10.5 were stained for VEGFR2 and ERG, showing only minimal and sporadic VEGFR2 staining (arrowheads) in the mutant endocardium, in contrast to robust staining in the control (arrows). DAPI was used to counterstain nuclei in all images. Scale bars: 50 μm (A,B); 100 μm (C-F).

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  Fig. 5.. Excessive PI3K signaling in EdCs resulted in reduced cardiac trabeculation. (A,B) Heart sections at E11.5 were stained for troponin T and endomucin. (A) Endocardial deletion of Tie2 via the knock-in Nfatc1Cre or the new Bac Nfatc1Cre both led to impaired endocardial network and hyperplastic trabeculae, but the cardiac defects with Nfatc1CreBac were more severe than those with the knock-in Nfatc1Cre. Note increased space between endocardium and myocardium (arrowheads) in both mutant embryos, indicating increased extracellular matrix (ECM) accumulation. (B) Endocardial overexpression of Pik3caH1047R via Nfatc1CreBac led to reduced density of trabeculae in Nfatc1CreBac;Pik3caH1047R ventricles, compared to control littermates. (C) Quantification showing the thickness of the trabecular sheet at E11.5: Tie2-ckoBac (Nfatc1CreBac;Tie2f/f)>Tie2-cko (Nfatc1Cre;Tie2f/f)>Ctrl (control) and H1047R (Nfatc1CreBac;Pik3caH1047R). (D) Alcian Blue and Nuclear Fast Red staining of sagittal paraffin sections of hearts at E11.5, showing normal size of endocardial cushions but reduced trabeculae density in Nfatc1CreBac;Pik3caH1047R hearts, compared to control littermates. (E,F) Quantification of the…

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  Fig. 6.. Identification of versican metabolism as implicated in TIE2 signaling during trabeculation via scRNAseq. (A) UMAP plot of all captured cell populations colored by cluster. (B) Violin plots showing alteration on expression of selected genes in E10.5 wild-type and Tie2-cko ventricular EdCs.

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  Fig. 7.. Endocardial loss of Tie2 and forced activation of FOXO1 resulted in enhanced expression of versican but impaired versican cleavage. (A-D) Immunostaining of heart sections at E12.5 showing that the expression of intact versican (Vcan) was significantly increased, whereas cleaved Vcan (CVcan, DPEAAE) was dramatically reduced in Tie2-cko ventricles, compared to their littermate controls. (E,F) Images of representative immunoblot analysis (E) and comparison of CVcan (F) in whole lysate of the hearts from E12.5 wild-type and Tie2-cko embryos. n=3× two embryos per group. (G-K) Representative immunostaining images and quantitative analysis of E11.5 control and FOXO1EC-CA hearts showing enhanced Vcan expression and reduced CVcan expression in FOXO1EC-CA ventricles, compared to their littermate controls. Arrows show representative positive staining in ventricles; arrowheads show positive staining in AVC cushions. DAPI was used to counterstain nuclei in all images. Data represent the mean±s.e.m. Two-tailed unpaired Student's t-test; *P<0.05; **P<0.01; ****P<0.001. Scale bars: 100 μm.

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  Fig. 8.. Endocardial loss of Tie2 resulted in enhanced expression of ADAMTS1 and TIMP3 in ventricular EdCs. (A-D) Control and Tie2-cko ventricular sections at E13.0 were co-stained for endomucin (green) and ADAMTS1 (red; A,B) or TIMP3 (red; C,D). Arrows show representative positive staining in ventricular EdCs. DAPI was used to counterstain nuclei in all images. (E,F) Quantitative analysis of A-D showing that, compared to their littermate controls, relative staining density of ADAMTS1 was increased in Tie2-cko hearts, although no change was detected on the percentage of ADAMTS1+ EdCs. Relative staining density of TIMP3 and the percentage of TIMP3+ EdCs were, however, both increased in the mutant ventricles. (G) Schematic representation of mouse chromosome 10 region (corresponding to NCBI reference sequence NC_000076.7: base 86136201-86174350) indicating the position of the three putative FOXO1-binding sites (BS). Exonic regions are in blue and the ATG is located in exon 1. The four overlapped insulin response elements (IREs) in the second BS are in red. The primer pairs flanking the putative FOXO1-binding sites (BS1-3) and the pair flanking an intronic region between exon 2 and BS3…

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  Fig. 9.. Proposed TIE2 signaling pathway governing endocardial growth and CM proliferation during trabeculation. In wild-type embryonic hearts, stimulation of TIE2 with ANG1 leads to the activation of PI3K/Akt, which in turn directly phosphorylates FOXO1 at three conserved residues (T24, S256 and S319), promoting its export from the nucleus by binding with the chaperone 14-3-3, and thus resulting in the inhibition of FOXO1-dependent transcription and normal proliferation of EdCs. In Tie2-cko and FOXO1EC-CA embryonic hearts, un-phosphorylated FOXO1 translocates to the nucleus, leading to induction of cell cycle withdrawal and inhibition of EdC proliferation, and increased TIMP3 production in EdCs. The latter impairs Vcan proteolysis by inhibition of ADAMTS1, which results in abnormally high CM proliferation and hyperplastic trabeculation.