Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors

Abstract

Heart disease is the leading cause of mortality and morbidity in the western world. The heart has little regenerative capacity after damage, leading to much interest in understanding the factors required to produce new cardiac myocytes. Despite a robust understanding of the molecular networks regulating cardiac differentiation, no single transcription factor or combination of factors has been shown to activate the cardiac gene program de novo in mammalian cells or tissues. Here we define the minimal requirements for transdifferentiation of mouse mesoderm to cardiac myocytes. We show that two cardiac transcription factors, Gata4 and Tbx5, and a cardiac-specific subunit of BAF chromatin-remodelling complexes, Baf60c (also called Smarcd3), can direct ectopic differentiation of mouse mesoderm into beating cardiomyocytes, including the normally non-cardiogenic posterior mesoderm and the extraembryonic mesoderm of the amnion. Gata4 with Baf60c initiated ectopic cardiac gene expression. Addition of Tbx5 allowed differentiation into contracting cardiomyocytes and repression of non-cardiac mesodermal genes. Baf60c was essential for the ectopic cardiogenic activity of Gata4 and Tbx5, partly by permitting binding of Gata4 to cardiac genes, indicating a novel instructive role for BAF complexes in tissue-specific regulation. The combined function of these factors establishes a robust mechanism for controlling cellular differentiation, and may allow reprogramming of new cardiomyocytes for regenerative purposes.

Figure 1

Ectopic induction of cardiac differentiation in mouse embryos. a, Actc in situ hybridization (top) shows endogenous cardiac crescent at E8.0 and ectopic cardiac gene expression (red arrowheads) in embryos transfected with indicated expression constructs. Bottom row shows EGFP signal. b, Ectopic expression of Myl2, α-TM, and Actc in consecutive sections of an embryo transfected with Tbx5/Nkx2-5/Gata4+Baf60c. Top left panel: EGFP signal and plane of section. h: heart; hf: headfold. c, Ectopic α-TM expression (red) is restricted to EGFP+ cells (green). DAPI shows nuclei (blue). d, Percentage of embryos with ectopic Actc-positive foci and beating tissue.

Figure 2

Induction of cardiac differentiation in embryonic and extraembryonic mesoderm. a, Transfected cells were observed in endoderm and mesoderm (EGFP), but induction of cTnT was mainly restricted to mesoderm. b, cTnT was not induced in extraembryonic endoderm. c, Induction of cTnT in mouse amnion by Gata4/Tbx5+Baf60c. Left panel: low-magnification view of a transfected embryo; arrowheads show ectopic cTnT in the amnion. Right panels: close-up of the amnion, showing transfected cells (EGFP), cTnT expression, and merge of both. Arrowheads indicate transfected cTnT+ cells. amn: amnion; em: embryo; ys: yolk sac.

Figure 3

Minimal requirements for cardiac gene activation in mouse embryos. a, Expression of Actc in embryos transfected with pairwise combinations of transcription factors+Baf60c. Only Gata4+Baf60c induced ectopic Actc (red arrowheads). b, Expression of Nkx2-5; Gata4+Baf60c induced ectopic Nkx2-5 (arrowheads). c, Percentage of Actc-positive and beating embryos for each combination. d, Specificity of cardiogenic factors; percentage of embryos in which Actc was induced is shown.

Figure 4

Mechanism for induction of cardiac differentiation. a, Strategy for isolating and analyzing transfected cells. b, RT-PCR of several cardiac markers in RNA isolated from transfected EGFP-positive mouse embryonic cells obtained by FACS. c, Chromatin immunoprecipitation (ChiP) shows that GATA4 and Brg1 bind Tnnt2 and Nppa only in the presence of Baf60c. Br: brain, He: heart, IgG: nonspecific immunoserum. d, Model for action of Baf60c. e, Minimal transcriptional network for the ectopic induction of cardiac differentiation. Made using Biotapestry software.