Temporal and contextual orchestration of cardiac fate by WNT-BMP synergy and threshold

Abstract

Cardiomyogenic development proceeds with a cascade of intricate signalling events that operate in a temporo-spatial fashion to specify cardiac cell fate during early embryogenesis. In fact, conflicting reports exist regarding the role of Wnt/β-catenin signalling during cardiomyogenesis. Here, we describe a dose-dependent and temporal effect of Wnt/β-catenin signalling on in vitro cardiomyogenesis using embryonic stem cells (ESCs) as a model system. We could demonstrate that canonical Wnt activation during early stage of differentiation either through ligand or by GSK3β inhibition helped in maintaining Oct4 and Nanog expressions, and in parallel, it promoted mesoderm and endoderm inductions. In contrast, it led to attenuation in cardiomyogenesis that was reversed by moderate concentration of DKK1, but not soluble Fz8. However, higher DKK1 could also block cardiomyogenesis, suggesting thereby governance of a particular signalling threshold underlying this developmental event. Interestingly, Wnt signalling activation at early stage modulated BMP4 expression in a stage-specific manner. Wnt activation, synchronized with BMP4 and brachyury up-regulation at early stage, correlated well with mesoderm induction. Conversely, Wnt activation led to BMP4 and Wnt5a down-regulation at late stage culminating in cardiomyogenic attenuation. Our findings suggested the existence of precise regulatory machinery with context-dependent role of Wnt for fine tuning mesoderm induction and its derivatives, through establishment of Wnt gradient during ESCs' differentiation. Moreover, contrary to mere activation/inhibition, a specific threshold of Wnt and BMP and their synergy seemed necessary for providing the guiding cues in orchestrating mesoderm induction and subsequent cardiomyogenesis.

Fig 1

Generation and characterizations of βMHC-EYFP clone. (A) Vector construct used to generate stable ESCs clones to demarcate cardiomyocytes expressing βMHC promoter driven EYFP during differentiation. (B) EYFP expression profile during differentiation revealed EYFP expression commencement from d8 onwards in differentiating EBs. (C) Immuno-cytochemical analysis during differentiation and authentication of cardiac-specific EYFP expression. EYFP positive cells showed co-localization with endogenous cardiac markers; α-actinin, Troponin-T and Nkx2–5, thereby specifying those as cardiomyocytes. (D, E) Quantitation of cardiac differentiation from ESCs by counting percentage of EBs beating (D) and beating clusters/EB (E) at the indicated timepoints. n= 5–8: mean ± S.E.M. Scale: 100 μm (B), 50 μm (C).

Fig 2

Authentication of canonical Wnt activation and inhibition of cardiac differentiation upon BIO exposure. (A) Increase in the immunopositivity of β-catenin upon BIO and W3CM treatment (d0–2) in d2 EBs indicated the activation of canonical Wnt/β-catenin signalling. DAPI (blue) was used for nuclear staining (Scale: 20μM). (B, C) Dose-dependent effect of BIO during cardiac differentiation was monitored at d10 by counting percentage EBs beating (B) and beating clusters/EB (C). BIO exposure at 1 uM resulted in attenuation in cardiac differentiation. (D) EBs showed reduced EYFP+ area with BIO exposure during d0–2 time period compared to vehicle control. (E, F) BIO also exerted its effect in a temporal fashion by decreasing the percentage EBs beating (E), and the beating clusters/EB (F) at the indicated time-points compared to control (Ctrl). (G, H) qPCR analysis of cardiac marker expression in EBs at d5 (G; BIO: 10 μM) and d10 (H; BIO: 1 μM) of differentiation, dotted line indicating the control value set as 1. n= 3–4: mean ± S.E.M.

Fig 3

Canonical Wnt signalling and inhibition of cardiac differentiation (d10). (A, B) W3CM inhibited cardiomyogenesis in a temporal fashion as quantified by counting percentage EBs beating (A) and beating clusters/EB (B). (C) In W3CM-treated EBs during d0–2, EYFP expression was less compared to CCM and it was further reduced in EBs with continuous treatment. n= 3: mean ± S.E.M. Scale: 100 μm (C).

Fig 4

Canonical Wnt signalling inhibits cardiac differentiation (d10) in a dose-dependent and temporal fashion. (A, B) rWnt3a (d0–2) blocked cardiac differentiation from ESCs in a dose-dependent manner with 50 ng/ml showing maximum reduction among treatments. (C, D) EBs at early stages and with continuous exposure were more sensitive to rWnt3a (50 ng/ml) block compared to control (Ctrl). (E, F) Flowcytometry quantification revealed reduction in EYFP+ cardiomyocytes following rWnt3a treatment in a concentration-dependent manner (E, d0–2) as well as in a temporal fashion (F), when treated (50 ng/ml) at various time regimens. (G) Live microscopic monitoring of EYFP expressing cardiomyocytes with or without rWnt3a exposure at various time-points during differentiation. Scale: 100 μm. n= 3–6: mean ± S.E.M.

Fig 5

Non-canonical Wnt signalling and cardiac differentiation (d10). (A–E) rWnt5a showed temporal influence on cardiac differentiation. While no difference was noted in percentage EBs beating with rWnt5a exposure during various time-points of differentiation compared to control (A), increase in beating clusters/ EB was evident only with d0–2 exposure (B). (C) EYFP expression in rWnt5a-treated EBs remained unaltered irrespective of the treatment regimen except at d0–2, where it showed an increase in EYFP+ area. (D) Flowcytometry analysis also revealed significant increase in EYFP+ cells following rWnt5a treatment during d0–2 as well as with continuous exposure. (E) Live monitoring of EYFP expressing cardiomyocytes with or without rWnt5a exposure at various time-points during differentiation. (F) W3CM but not CCM could increase the luciferase activity, whereas with non-canonical Wnt5a conditioned medium (W5CM), it was comparable to control (ctrl). Treatment with BIO (1 μM) could also drastically enhance the luciferase activity compared to DMSO control. n= 3–6 mean ± S.E.M. (Scale: 100 μm in E).

Fig 6

Inhibition of Wnt signalling and its influence on cardiomyogenesis (d10) and Wnt-BMP synergy. A–C: Fz8 (200 ng/ml) exposure during d0–2 inhibited cardiac differentiation as determined by counting percentage EBs beating (A), beating clusters/EB (B) and EYFP+ area (C). (D–G) DKK1 exposure (d0–2) influenced the cardiac differentiation in a dose-dependent manner. While, percentage EBs beating remained unchanged (D), variation was noted in the number of beating clusters/EB (E) and in the EYFP+ area (F, G) upon DKK1 treatment. While lower DKK1 (50 ng/ml) treatment could promote cardiomyogenesis, the same at higher concentration (500 ng/ml) abrogated it. (H) Flowcytometry analysis revealed decrease in EYFP+ cardiomyocytes following Fz8 exposure. While DKK1 at 50 ng/ml promoted EYFP+ cardiomyocytes generation, it inhibited the same when used at 500 ng/ml. Exogenous supplementation of BMP4 did not show any appreciable influence on cardiomyogenesis as determined by no difference with respect to EYFP+ cardiomyocytes generation (H), beating clusters/EB (I) and EYFP+ area (J) compared to control. However, blocking endogenous BMP4 with Chordin inhibited cardiomyogenic differentiation from ESCs, the effect of which was nullified by blocking Wnt signalling with DKK1 (50 ng/ml) treatment indicating a possible synergy between Wnt and BMP underlying cardiomyogenic developmental programme. n= 3–6: mean ± S.E.M. (Scale: 100 μm in G).

Fig 7

Wnt-BMP crosstalk and modulation of ESCs' differentiation onset. (A) BIO exposure at d0–2 had a contrasting regulatory influence on BMP4 and Wnt5a expression in d2 and d5 EBs, respectively. Blocking of BMP signalling (d0–2) by using either BMP4 blocking antibody or Noggin in presence of BIO influenced the BMP4 transcript level at d2 indicating a probable interaction between the two. The values have been plotted as fold difference to untreated control (set as 1) at the said time-points. (B) An apparent increase in Oct4 levels and its nuclear localization was seen in W3CM treated EBs (d2). DAPI (blue) was used to mark the nucleus. Scale: 50 μm. (C) BIO treatment (d0–2) increased both Oct4 and Nanog transcripts in d2 EBs. (D, E) Expression profile of Bry (D) and AFP (E) relative to βactin during ESCs differentiation, as analysed by qPCR. (F) Bry expression was up-regulated in d2 EBs upon exposure to either BIO or rWnt3a but not rWnt5a (d0–2). ESCs grown for 6 days (d6) with either BIO or on L-Wnt3a feeders in monolayer culture (mono) could also show increase in Bry expression. (G) BIO did not alter AFP expression in EBs (d2) and also in cells in monolayer culture (d6), whereas Wnt3a and Wnt5a could up-regulate it. n= 3–9: mean ± S.E.M.