Opposing roles of TCF7/LEF1 and TCF7L2 in cyclin D2 and Bmp4 expression and cardiomyocyte cell cycle control during late heart development

Abstract

Bone morphogenetic protein (BMP) and Wnt pathways regulate cell proliferation and differentiation, but how these two pathways interact and mediate their nuclear actions in the heart, especially during late cardiac development, remains poorly defined. T-cell factor (TCF) and lymphoid enhancer factor (LEF) family transcriptional factors, including Lef1, Tcf7, Tcf7l1, and Tcf7l2, are important nuclear mediators of canonical Wnt/β-catenin signaling throughout cardiac development. We reveal that these TCF/LEF family members direct heart maturation through distinct temporal and spatial control. TCF7 and LEF1 decrease while TCF7L1 and TCF7L2 remain relatively stable during heart development. LEF1 is mainly expressed in mesenchymal cells in valvular regions. TCF7 and TCF7L1 are detected in the nucleus of mesothelial and endothelial cells, but not in cardiomyocytes or mesenchymal cells. Tcf7l2 is the primary TCF/LEF family member in cardiomyocytes and undergoes alternative splicing during heart development. A TCF7L2 intensity gradient opposite to that of β-catenin and cardiomyocyte proliferative activity is present in fetal hearts. Wnt activation by cardiac deletion of APC, a negative Wnt regulator, dramatically increases Cyclin D2 and Bmp4 expression. BMP signal transducing transcription factors, the mothers against decapentaplegic homologs (SMADs) are increasingly phosphorylated upon Wnt activation. LEF1/TCF7 displaces TCF7L2 and cooperates with pSMAD 1/5/8 in the regulatory elements of Cyclin D2 and Bmp4 promoters to promote β-catenin recruitment and transcriptional activation. Finally, we demonstrate that TCF7L2 is a transcriptional suppressor of Cyclin D2 and Bmp 4 in a cardiac cell line by overexpression and knockdown experiments.

Figure 1.. TCF/LEFs expression in mouse hearts at E13.5 and E17.5.

A, Cardiac mRNA expressions of Tcf/Lef family at E13.5 are represented as mean delta Ct (ΔCt) values normalized by Gapdh. B, The log2 fold changes of Tcf/Lef mRNA level at E17.5 versus E13.5 in murine hearts. C, A heat map shows fragments per kilobase of transcript per million mapped reads (FPKM) for Tcf/Lef genes in murine hearts from E13.5 to postnatal day 21. D-G, Expression of Lef1, Tcf7, Tcf7l1 and Tcf7l2 in mouse hearts at E13.5 by immunohistochemistry. Lef1 shows clear and strong nuclear staining in cells of atrioventricular (AV) cushions and valves (D3). Scattered CMs showing moderate to weak nuclear staining for Lef1 are also observed in the compact (D1) and trabecular (D2) layers. Moderate nuclear staining of Tcf7l2 is mainly observed in CMs of the trabecular layer (E2) and cells of AV cushions and valves (E3). CMs in the compact layer barely or weakly stain positive for Tcf7l2 (E1). Nuclear Tcf7 and Tcf7l1 are not detected in CMs of both compact and trabecular layers (F-G) at E13.5 by immunohistochemistry. Epicardial (arrows) and endothelial cells (triangles) lining the outer and inner cardiac surfaces respectively are positive for Tcf7 and Tcf7l1. Scale bar=200μm. Data represent mean ± SD. N=4 independent experiments.

Figure 2.. Tcf7l2 gradient across the ventricular wall of normal E13.5 embryos.

A, An illustration (A) shows how nuclear Tcf7l2 intensity was measured from different areas of ventricle walls, including the compact layer (zone I), outer trabecular layer (zone II) and inner trabecular layer (zone III). Region of interests (ROI) are semi-automatically outlined by image J based on Actinin positive areas. Nuclei of CMs within ROIs are semi-automatically selected in different zones according to the DAPI staining. B and C, Bar graphs demonstrate average nuclear Tcf7l2 intensity (B) and the percentage of Tcf7l2 positive CMs (D) in different zones across ventricular walls of E13.5. D, Nuclear Tcf7l2 signal is gradually increased from the outer compact layer to the inner trabecular layer forming an intensity gradient across the ventricle wall. Boxed region (d) is magnified and showed with double staining for Tcf7l2 and Actinin. Tcf7l2 positive cells from zone I, II and III are mainly Actinin positive CMs. Endothelial cells are negative for Tcf7l2 (triangles). Dotted lines show visceral pericardium and bar=50μm. Data represent mean ± SD. N=4 independent experiments.

Figure 3.. Alteration of β-catenin partners by Apc deletion.

A, A bar graph shows the ratio of Tcf/Lef mRNA expression levels of hearts between Apc knockout (KO) animal and their wild type (WT) siblings at E13.5 and E17.5 by q-PCR. B, Upregulation Lef1 and Tcf7, but downregulation of long form of Tcf7l2 (~75 kDa) upon cardiac Apc deletion at E17.5 by western blot. Short form of Tcf7l2 (~54 kDa) is not changed. pSmad1/5/8 is also increased by ablation of Apc. Uncropped images of Western blots are displayed in Fig. S6. C, Band intensities are quantified by ImageJ and normalized to Histone 3. D and E, Tcf7 was not detected in WT (D), but was induced in Apc KO mice and colocalized with nuclear β-Cat in CMs (E) at E17.5. F and G, Tomato hetero hearts revealed efficient conversion of membranous red (not shown well due to weaker signal than that of Lef1) to green fluorescence by αMHC-Cre. Lef1 was not detected in WT (F), but was dramatically elevated in Apc KO mice (G) at E17.5. Scale bars=50μm. Data represent mean ± SD. N=4 independent experiments.

Figure 4.. Upregulation of Bmp-Smad1/5/8 signals in hearts by Apc deletion at E13.5 and E17.5.

A and B, pSmad1/5/8 is upregulated in the cardiomyocytes (arrows) of Apc knockout animals (B) compared to their wild type siblings (A) by immunofluorescent labeling. Non-cardiomyocytes (asterisks) are also positive for pSmad1/5/8, but no obvious changes are observed between wild type and Apc knockout hearts. C, Apc deletion greatly increases Bmp4 mRNA level in E13.5 hearts, but has no effect on mRNA levels of Bmp2, Bmp7 and Bmp10. D and E, Bmp4 in situ hybridization of E13.5 wild-type (D) and Apc deleted (E) hearts that were processed and imaged in parallel, showing an upregulation of Bmp4 mRNA level in the Apc knockout ventricles. Dotted lines indicate visceral pericardium. F, At E17.5, cardiac Bmp4 mRNA remains consistently at high levels in Apc knockout mice. Scale bars=50μm. Data represent mean ± SD. N=3-4 independent experiments.

Figure 5.. Wnt nuclear effectors are recruited to the regulatory element of Cyclin D2 and Bmp4 promoters.

A, Amplicon positions and putative TCF/LEF and SMAD binding sites relative to transcription start site (TSS). B, ChIP shows that the recruitments of β-catenin, Smad4, pSmad1/5/8, Lef1 and Tcf7 to the TCF/LEF and SMAD binding sites in the Cyclin D2 and Bmp4 promoters are increased, while Tcf7l2 is removed from these regions in Apc cKO compared to WT hearts at E17.5. These changes are accompanied by an increase in histone H4 acetylation (Acetyl H4), indicating transcriptional activation. Inputs are 2% of total mount chromatin. C, A diagram shows Wnt target gene regulation. In WT mice, Tcf7l2 interacts with SMAD family members to suppress gene transcription. Upon Apc deletion, Tcf7l2 is removed from TCF/LEF binding sites and replaced with activating forms of TCF/LEF family members, Tcf7 and Lef1 to promote the recruitment of pSmad1/5/8 and β-catenin as well as histone H4 acetylation. D, Semiquantitative analysis of the relative enrichment of proteins bound to the Tcf7l2 or SMAD consensus site. The intensity of each band in the gel images was measured using the ImageJ program and normalized by input. Each bar represents the mean ± SD of 4 independent experiments.

Figure 6.. Tcf7l2 levels control the expression of Lef1, Tcf7, Cyclin D2, and Bmp4.

The mRNA levels of HL-1 CMs transfected with wild type long-form Tcf7l2 (Ade WT), Tcf7l2 shRNA (sh RNA), and control vector (pGPIZ) are quantified and compared with non-transfected group (Control) by real time RT-PCR 72 hours after transfection. Tcf7l2 shRNA significantly increases transcript levels of Lef1, Tcf7, Cyclin D2, and Bmp4. The mRNA levels of Bmp4 and Cyclin D2 are downregulated by Tcf7l2 overexpression. Data are presented as mean±SD from 4 independent experiments.