A Nodal-to-TGFβ Cascade Exerts Biphasic Control Over CardiopoiesisNovelty and Significance
Rationale: The transforming growth factor-β (TGFβ) family member Nodal promotes cardiogenesis, but the mechanism is unclear despite the relevance of TGFβ family proteins for myocardial remodeling and regeneration.
Objective: To determine the function(s) of TGFβ family members during stem cell cardiogenesis.
Methods and Results: Murine embryonic stem cells were engineered with a constitutively active human type I Nodal receptor (caACVR1b) to mimic activation by Nodal and found to secrete a paracrine signal that promotes cardiogenesis. Transcriptome and gain- and loss-of-function studies identified the factor as TGFβ2. Both Nodal and TGFβ induced early cardiogenic progenitors in embryonic stem cell cultures at day 0 to 2 of differentiation. However, Nodal expression declines by day 4 due to feedback inhibition, whereas TGFβ persists. At later stages (days 4–6), TGFβ suppresses the formation of cardiomyocytes from multipotent Kdr+ progenitors while promoting the differentiation of vascular smooth muscle and endothelial cells.
Conclusions: Nodal induces TGFβ, and both stimulate the formation of multipotent cardiovascular Kdr+ progenitors. TGFβ, however, becomes uniquely responsible for controlling subsequent lineage segregation by stimulating vascular smooth muscle and endothelial lineages and simultaneously blocking cardiomyocyte differentiation.
Embryonic stem cells (ESCs) and induced pluripotent stem cells hold great potential as sources of cardiomyocytes and as models to understand how cardiomyocytes, vascular smooth muscle, and endothelial cells arise from common cardiopoietic progenitors.1 Defining the signals that control cardiopoietic differentiation will be important for numerous applications, including regenerative medicine.
The divergent transforming growth factor-β (TGFβ) family member Nodal is critical for the formation of the heart and other visceral organs. Nodal activates a heteromeric complex of type I [Acvr1b (Alk4) or Acvr1c (Alk7)] and type II (Acvr2a and b) serine/threonine kinase receptors, leading to phosphorylation of Smad2 and Smad3, which then activate target genes.2 Mouse embryos lacking Acvr1b, Smad2, or Nodal, and double knockout of the 2 type II receptors (Acvr2a and Acvr2b) fail to gastrulate or form mesendoderm.3 Genetic deletion of Cripto, an essential Nodal co-receptor in most contexts, is less severe, such that embryos form mesendoderm but are severely deficient in cardiogenic progenitor cells.4,5 The cardiogenesis deficit inherent in Cripto−/− ESCs can be rescued either by incorporation into chimeric (Cripto−/−:wild-type [WT]) embryos4 or by a constitutively active mutant human ACVR1b receptor,6 demonstrating the existence of yet unknown paracrine effectors that propagate the signal from cell to cell.
We used murine ESCs (mESCs) to model cardiogenesis and found that TGFβ2 is induced by Nodal and propagates the cardiogenic signal. The essential nature of TGFβ for cardiogenesis is based on resistance to the feedback inhibitors Lefty1, Lefty2, and Cerberus1 (Cer1) that block Nodal. Consequently, both Nodal and TGFβ induce early cardiogenic progenitors, but Nodal expression declines due to feedback inhibition whereas TGFβ expression persists in Kdr+ cardiopoietic precursors. In this population, TGFβ suppresses cardiomyocyte differentiation while promoting vascular smooth muscle and endothelial cell formation. Thus, a Nodal-to-TGFβ cascade, including feedback inhibition, provides biphasic control over cardiopoietic cell fate.
Protocols and primer sequences are described in the Online Data Supplement.
Cardiogenic Rescue Implicates a Diffusible Factor Downstream of Nodal/Avcr1b
Cripto−/− mESCs are deficient in production of cardiogenic progenitors, exhibiting low Kdr and Mesp1 expression,4,5 (Online Figure IA) and are thus ideal for a cell-mixing study to identify paracrine factors that initiate cardiogenesis downstream of Nodal/Avcr1b (Figure 1A). A constitutively active human ACVR1b receptor (caACVR1b) was stably introduced into Cripto−/− mESCs to activate downstream signaling (Cripto−/−caACVR1b, inducers) (Online Figure IA). Coculture (Figure 1A and 1B) of these cells dramatically restored Kdr and Mesp1 expression in eGFP-labeled Cripto−/− mESCs (responders) (Figure 1C). Coculture also increased the number of Kdr+ progenitors among the responder (eGFP+) population, from 3.34±0.06% to 21.28±1.37% after 5 days (Figure 1D). FACS-isolated GFP+, Kdr+ cells (responders) coexpressed Mesp1 (Figure 1E). By day 9, the induced cells expressed cardiomyocyte markers (Figure 1F) and beat rhythmically (Online Movie I). Residual Cripto−/−caACVR1b cells contaminating the responder population after FACS (0.5%) were insufficient to account for this level of rescue (Online Figure II). Finally, the rescue occurred cell-nonautonomously, since mixtures of eGFP-labeled Cripto−/−caACVR1b inducers with Myh6-mCherry responders revealed clearly distinct patterns of eGFP and mCherry expression (Figure 1G and 1H and Online Movie II).
To test if the induced Kdr+ progenitors autonomously form cardiomyocytes, aggregated responder (Cripto−/−, Myh6-mCherry, eGFP+) and inducer (Cripto−/− caACVR1b) cells were separated by FACS at day 5, reaggregated separately, and cultured for an additional 15 days (Figure 1I). The responder cells expressed Myh6 (Figure 1J) and mCherry (Figure 1K), showing that the paracrine factor(s) initiate cardiogenesis before day 5. Since Cripto−/− cells negligibly respond to Nodal (Online Figure IB), the factor is neither Nodal nor a shed version of Cripto.
TGFβ2 Acts Downstream of Nodal to Induce Cardiogenic Mesoderm
Microarray analysis (not shown) showed that caACVR1b upregulated mRNAs encoding TGFβ1, TGFβ2, TGFβ3, and inhibins. Of these, Tgfb2, Tgfb3, and Inhba were greatly upregulated by caACVR1b transfection in Cripto−/− mESCs (Figure 2A). Since E5.5 to E7.5 mouse embryos express mRNAs encoding Tgfb2 but not Tgfb3 and Inhibins7, TGFβ2 emerged as an attractive candidate for the paracrine factor. Indeed, TGFβ2 treatment from days 0 to 2 gave a dose-dependent induction of genetic markers of mesoderm (Mesp1, Mixl1, Aplnr, and Gsc) and mesoderm derivatives (Myh6, Pecam1, Tagln, Cdh5, and Acta2) and the Myh6-mCherry reporter in Cripto−/− ESCs (Figure 2B and 2C) and even enhanced Mesp1, Kdr, and Myh6 in WT cells (Figure 2D), revealing a functional relationship.
To test if TGFβ is necessary downstream of Nodal/Acvr1b, Cripto−/− responder ESCs were transfected with siRNA against Tgfbr1 before coculture with Cripto−/− caACVR1b ESCs (Figure 2F). Tgfbr1 siRNAs blocked induction of Kdr transcripts (to about 20% of negative control siRNA), establishing TGFβ2 as a paracrine mediator of Nodal signaling.
TGFβ2 Suppresses Cardiomyocyte Differentiation During a Late Stage of Differentiation
The preceding showed that TGFβ2 induces cardiogenic progenitors before day 5. Tgfb2 mRNA, however, continues to rise between days 4 to 8 (Figure 3A), whereas Nodal mRNA declines, suggesting that TGFβ but not Nodal plays a role as Kdr+ progenitors differentiate. To understand the basis for the shift from Nodal to Tgfb2, we examined expression of Lefty1, Lefty2, and Cer1, encoding Nodal inhibitors.3 All 3 became expressed concomitantly with the decline in Nodal levels (Figure 3A), and each was induced by Nodal/TGFβ signaling (Figure 3B and 3C). Moreover, Nodal and TGFβ both induced Nodal (Figure 2A and 3C). The fact that Cer1 and Lefty1 and Lefty2 do not block TGFβ3 probably accounts for the persistence of Tgfb2 after the decline in Nodal. Interestingly, TGFβ2 does not induce Tgfb1 or Tgfb2 and only minimally induced Tgfb3 (Figure 3C), making the cascade inherently self-limiting.
We next asked whether TGFβ influences cardiopoietic differentiation. siRNAs to either Tgfbr1 or Tgfbr2 transfected at day 4 unexpectedly increased expression of Myh6, as well as eGFP driven by the Myh6 promoter (Figure 3D and 3E). At this time, Tgfb2 mRNA predominates in Kdr+ cells (Figure 3F), suggesting autocrine repression of cardiomyocyte differentiation.
To gain further insight into the bimodal function of TGFβ, we treated ESC cultures with SB-431542, a small-molecule inhibitor of Acvr1b/1c and Tgfbr1, at early and late time windows (Figure 3G and 3H). Treatment between 0 to 2 days of culture abolished Mesp1 expression (Figure 3G). Treatment at 4 to 6 days, in contrast, markedly enhanced Myh6 levels in Kdr+ derivatives (Figure 3H). Conversely, recombinant TGFβ2 between days 4 to 6 suppressed Myh6 mRNA as well as Mef2c and Tbx5 protein but increased Pecam1 and Myh11 mRNAs and the level of Pecam1 and Myh11 immunostaining (Figure 3I through 3L). We conclude that a Nodal-to-TGFβ2 cascade enhances production of cardiogenic mesoderm before day 4 and that TGFβ persists to suppress cardiomyocyte differentiation of Kdr+ cells while biasing their differentiation toward endothelial and smooth muscle lineages.
Genetic and stem cell experiments have shown that Nodal acts positively and negatively in cardiogenesis, depending on the developmental stage; however, the identity and function of downstream mediators were unknown.4,6,8,9 Our results define a Nodal-to-TGFβ signaling cascade that exerts positive and negative effects on progenitor induction and cardiomyocyte differentiation, respectively (Figure 4). The biphasic function resembles that of Wnts and BMPs, both of which promote formation of cardiogenic progenitors (eg, Mesp1+, Kdr+) during the period when mesoderm is induced but suppress the subsequent formation of cardiac precursors (eg, Nkx2.5+), and at least BMP acts positively again once Nkx2.5+ progenitors arise.1
Mechanistically, the cascade incorporates auto-induction and inhibition properties that regulate Nodal and TGFβ expression within narrowly delimited developmental times. Nodal is well known for activating its own transcription as well that of its antagonists Lefty1, Lefty 2, and Cer1, yielding an auto-induction cascade that is feedback inhibited. However, TGFβ cannot auto-induce (Figure 2A and 3C) nor is inhibited by Cer1 and Lefty. Consequently, Tgfb2 expression is induced by Nodal and persists after Nodal expression declines.
Considering the possible functions for a time-resolved Nodal-TGFβ cascade led to the finding that TGFβ suppresses cardiomyocyte differentiation while simultaneously enhancing formation of endothelial and smooth muscle lineages (Figure 3E through 3L). The only other factors known to apportion cardiopoietic fate are Wnts, which also suppress cardiomyocyte differentiation at the same developmental stage.1
A specific requirement for TGFβ in cardiac differentiation has implications for understanding congenital heart defects. Genetic deletion of Tgfbr1 in mice causes severe cardiovascular defects,10 and mutation of the latent TGFβ-binding protein 3, which regulates TGFβ bioavailability, impairs differentiation of second heart field cells in zebrafish.11 It will be important to determine if altered TGFβ signaling at the time of cardiac progenitor specification underlies human congenital heart disease, such as the cardiac defects that can present in Loeys-Dietz syndrome caused by mutated TGFBR1 or TGFBR2.
Sources of Funding
This study was supported by the National Institutes of Health (P30AR061303) and California Institute for Regenerative Medicine (RC1-000132).
We thank Yoav Altman, Joseph Russo, and Dr Ed Monosov (SBMRI) for expert assistance.
In June 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.35 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.270272/-/DC1.
Non-standard Abbreviations and Acronyms
- Alk4, activin A receptor, type IB
- Alk7, activin A receptor, type IC
- activin receptor IIA
- activin receptor IIB
- cerberus 1 homolog (Xenopus laevis)
- embryonic stem cells
- kinase insert domain protein receptor
- left-right determination factor 1
- left-right determination factor 2
- murine embryonic stem cells
- mesoderm posterior 1
- myosin, heavy polypeptide 6, cardiac muscle, alpha
- myosin, heavy polypeptide 11, smooth muscle
- platelet/endothelial cell adhesion molecule 1
- transforming growth factorβ
- Alk5, transforming growth factor, beta receptor I
- transforming growth factor, beta receptor IIb
- Received March 28, 2012.
- Revision received July 3, 2012.
- Accepted July 11, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
The divergent TGFβ protein Nodal is well known to play a role in specifying cardiac tissue during early development and is commonly used to generate cardiac cell types, including cardiomyocytes, from pluripotent stem cells.
The cardiogenic activity of Nodal is propagated from cell to cell by unknown paracrine signals, although a shed version of the Nodal co-receptor Cripto has been suggested to be involved.
What New Information Does This Article Contribute?
Nodal induces TGFβ2, and both induce the formation of cardiogenic progenitors in embryonic stem cell cultures.
Nodal expression declines as cardiogenic progenitors form; TGFβ persists and suppresses cardiomyocyte differentiation while simultaneously promoting vascular smooth muscle and endothelial lineages.
TGFβ superfamily members are important for cardiogenesis as well as fibrosis and inflammation associated with myocardial injury. We describe a regulatory cascade that controls the production of TGFβ. TGFβ initially promotes the formation of multipotent cardiac progenitors but subsequently inhibits their differentiation to cardiomyocytes. TGFβ might play a similarly bimodal role in myocardial regeneration.