FOXK1 regulates Wnt signalling to promote cardiogenesis

Abstract

Aims: Congenital heart disease (CHD) is the most common genetic birth defect, which has considerable morbidity and mortality. We focused on deciphering key regulators that govern cardiac progenitors and cardiogenesis. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics and is restricted to mesodermal progenitors, somites, and heart. In the present study, we define an essential role for FOXK1 during cardiovascular development.

Methods and results: We used the mouse embryoid body system to differentiate control and Foxk1 KO embryonic stem cells into mesodermal, cardiac progenitor cells and mature cardiac cells. Using flow cytometry, immunohistochemistry, cardiac beating, transcriptional and chromatin immunoprecipitation quantitative polymerase chain reaction assays, bulk RNA sequencing (RNAseq) and assay for transposase-accessible chromatin using sequencing (ATACseq) analyses, FOXK1 was observed to be an important regulator of cardiogenesis. Flow cytometry analyses revealed perturbed cardiogenesis in Foxk1 KO embryoid bodies (EBs). Bulk RNAseq analysis at two developmental stages showed a significant reduction of the cardiac molecular program in Foxk1 KO EBs compared to the control EBs. ATACseq analysis during EB differentiation demonstrated that the chromatin landscape nearby known important regulators of cardiogenesis was significantly relaxed in control EBs compared to Foxk1 KO EBs. Furthermore, we demonstrated that in the absence of FOXK1, cardiac differentiation was markedly impaired by assaying for cardiac Troponin T expression and cardiac contractility. We demonstrate that FOXK1 is an important regulator of cardiogenesis by repressing the Wnt/β-catenin signalling pathway and thereby promoting differentiation.

Conclusion: These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. Mechanistically, FOXK1 represses Wnt signalling to promote the development of cardiac progenitor cells.

Keywords: Forkhead factors; Cardiovascular development; Foxk1; Wnt signalling.

Figure 1

FOXK1 regulates mesodermal progenitor cell development. (A) Schematic of embryoid body in vitro differentiation and cardiac milestones. (B) Foxk1 transcript expression during EB differentiation from Day 2 (D2) to Day 8 (D8). Note that the expression of Foxk1 peaks at Day 5 (D5). (C–E) Representative flow cytometry profile of control and Foxk1 KO EBs at D3, D5, and D7 of mesodermal differentiation protocol with quantitation of the results. Note that throughout differentiation, there is a significant defect in the ability of Foxk1 null EBs to form mesodermal progenitors, particularly cardiac and skeletal myogenic (n = 3, *P < 0.05). Statistical test: Student’s t-test. Data are presented as mean ± SEM.

Figure 2

FOXK1 regulates cardiac developmental transcriptional networks. (A, C) Gene ontology (GO) pathway analysis highlights pathways and development related terms in D3 and D5 control EBs, the x-axis represents the counts of genes in each GO term. The colour scale shows the increased significance of biological processes using the over-representation test with an adjusted P < 0.05. (B, D) The heatmap represents up-regulated and down-regulated in the control EBs vs. the Foxk1 KO EBs at D3 and D5, respectively. The heatmap colour scheme key is provided, with red representing up-regulated and blue representing down-regulated genes.

Figure 3

FOXK1 is an epigenetic regulator of cardiac development. Enriched heatmap of the nucleoATAC data showing more nucleosome free regions (NFR) in control samples as compared to Foxk1 KO samples at D3 and D5 at the Foxk1-binding sites. We divided the FOXK1-binding sites into four regions, showing NFR in both, NFR in control and nucleosome occupied region in Foxk1 KO, NOR in control and NFR in Foxk1 KO, and NFR in both. (C–D) Venn diagram shows the overlap of increased accessibility and up-regulated genes between ATACseq and RNAseq, respectively, in the control group at D3 and D5 over the Foxk1 KO group. (E–F) The heatmap shows commonly expressed transcription factors in both the ATACseq and RNAseq datasets up-regulated and down-regulated in the control EBs over the Foxk1 KO EBs at D3 and D5. The heatmap colour scheme key is provided, with red representing up-regulated and blue representing down-regulated genes.

Figure 4

FOXK1 regulates cardiogenesis in differentiating EBs. (A) Schematic of embryoid bodies during in vitro differentiation with notation of cardiac milestones. (B) Representative flow cytometry profile of control and Foxk1 KO D10 EBs with quantification of the results. Note the significant decrease in cTnT+ cells in the Foxk1 KO group compared to the control (n = 3, *P < 0.05). (C) Immunohistochemical analysis of D10 EBs demonstrates that Foxk1 KO EBs have perturbed cardiogenesis compared to the control group as assayed by cTnT staining. Quantification of the immunohistochemical results demonstrates normal cardiac differentiation in Day 10 EBs in control group that is significantly reduced in the absence of FOXK1 (n = 3, *P < 0.05). (D) Schematic of control and Foxk1 KO EBs, cardiogenic beating assay, and quantification of results. Note the significant decrease in the number of beating EBs at D10 of differentiation in the Foxk1 KO EB group compared to control (n = 3, *P < 0.05). Statistical test: Student’s t-test. Data are presented as mean ± SEM. (E) Pathway analysis highlights GO pathways and development related terms in D10 control EBs, the x-axis represents the counts of genes in each GO term. The colour scale shows the increased significance of the biological processes using the over-representation test with an adjusted P < 0.05. (F) The heatmap shows significantly (adjusted P < 0.05) differentially expressed transcripts up-regulated and down-regulated with a two-fold between control EBs vs. Foxk1 null EBs at D10. Red represents up-regulation of transcripts and blue represents down-regulation of transcripts. (G) The heatmap represents a significant (adjusted P < 1e⁻⁰⁴) change in chromatin accessibility at D10 for control and Foxk1 KO. Red represents an increase in accessibility and blue represents reduced accessibility for the transcription factor.

Figure 5

FOXK1 regulates Wnt signalling to promote cardiogenesis. (A) RNAseq pathway analysis of Wnt signalling in D5 EBs comparing control and Foxk1 KO groups. Wnt signalling is significantly up-regulated in the Foxk1 KO group over the control group, and expression persists (in the absence of Foxk1) during differentiation. (B) Schematic of EB in vitro differentiation with the Wnt signalling inhibitor (IWR1) and cardiac milestones noted. (C) Representative flow cytometry profile of control + IWR1 and Foxk1 KO + IWR1 D10 EBs with quantification of the results. Note that no significant differences were observed between the two groups (n = 3). (D) Schematic of the beating assay of control + IWR1 and Foxk1 KO + IWR1 D10 EBs with quantification of the results. Note that no significant differences were observed between the two groups (n = 3). Statistical test: Student’s t-test. Data presented as mean ± SEM. (E) The principal component analysis (PCA) of RNAseq of EB differentiation at D10 shows samples with similar gene expression cluster together. The second principal components (PC2) show similarities between control and Foxk1 KO + IWR1 (Ct, control; KO, Foxk1 KO; KO + IWR1, Foxk1 KO + IWR1). (F) Venn diagram shows 320 genes up-regulated in both Ct and KO + IWR1 samples when compared to Foxk1 KO samples. (G) Top 10 GO pathways and development terms significantly enriched using the genes commonly up-regulated in control and Foxk1 KO + IWR1 conditions. The x-axis represents the counts of genes in each GO term. The colour scale shows the increased significance of the biological processes using the over-representation test with an adjusted P < 0.05. (H) The heatmap shows top 20 genes from two GO terms (muscle tissue development and striated muscle tissue development) commonly up-regulated in control and Foxk1 KO + IWR1 samples. Red represents up-regulation of genes and blue represents down-regulation of genes.

Figure 6

FOXK1 is a transcriptional repressor of Wnt signalling. (A) Heatmap highlighting the top 20 transcription factors associated with the Wnt signalling pathway enriched in the Foxk1 null D5 EB group from the RNAseq dataset. (B) Wnt6 transcript expression during EB differentiation from D2 to D8 in the presence and absence of FOXK1. Note that the expression of Wnt6 remains high at later stages of differentiation compared to the control group where it is down-regulated. (C) A region upstream of Wnt6 (chr1:74,770,049-74,773,057) that contains a FOXK1 DNA motif, β-catenin-binding site, and decreased chromatin accessibility in the absence of Foxk1 compared to control EBs at D5. (D) qPCR analysis using FOXK1 ChIP demonstrates a significant enrichment at the Wnt6 upstream region compared to the Gapdh control region. (E) β-Catenin transactivates the TOP-flash reporter, which is inhibited by FOXK1. Statistical test: Student’s t-test. Data presented as mean ± SEM.