Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells

Abstract

Understanding pathways controlling cardiac development may offer insights that are useful for stem cell-based cardiac repair. Developmental studies indicate that the Wnt/beta-catenin pathway negatively regulates cardiac differentiation, whereas studies with pluripotent embryonal carcinoma cells suggest that this pathway promotes cardiogenesis. This apparent contradiction led us to hypothesize that Wnt/beta-catenin signaling acts biphasically, either promoting or inhibiting cardiogenesis depending on timing. We used inducible promoters to activate or repress Wnt/beta-catenin signaling in zebrafish embryos at different times of development. We found that Wnt/beta-catenin signaling before gastrulation promotes cardiac differentiation, whereas signaling during gastrulation inhibits heart formation. Early treatment of differentiating mouse embryonic stem (ES) cells with Wnt-3A stimulates mesoderm induction, activates a feedback loop that subsequently represses the Wnt pathway, and increases cardiac differentiation. Conversely, late activation of beta-catenin signaling reduces cardiac differentiation in ES cells. Finally, constitutive overexpression of the beta-catenin-independent ligand Wnt-11 increases cardiogenesis in differentiating mouse ES cells. Thus, Wnt/beta-catenin signaling promotes cardiac differentiation at early developmental stages and inhibits it later. Control of this pathway may promote derivation of cardiomyocytes for basic research and cell therapy applications.

Fig. 1.

Wnt/β-catenin signaling promotes zebrafish heart specification at pregastrula stages but inhibits heart formation at gastrula stages. WT, hsWnt8, or hsDkk1 transgenic embryos were heat-shocked for 30 min starting at the indicated times. (A) The embryos were fixed at 13 hpf (eight-somite stage) and processed for nkx2.5 in situ hybridization. The embryos are shown in dorsal view (anterior left); n > 12 for each experimental point. The experiment was repeated twice each for heat shock at 3.5, 6, and 9 hpf. The combined percentages of affected embryos from these three experiments were as follows: Wnt8, 3.5 hpf, up: 86% (n = 43); Wnt8, 6 hpf, up: 36% (n = 59); Wnt8, 9 hpf, down: 100% (n = 28); Dkk1, 3.5 hpf, down: 100% (n = 52); Dkk1, 6 hpf, up: 73% (n = 40); and Dkk1, 9 hpf, up: 18% (n = 61). (B) The embryos were fixed at 18 hpf (18-somite stage) and processed for in situ hybridization with cardiac myosin light chain 2 (cmlc2). Close-up views of cmlc2 expression are shown (anterior left). Wnt8 overexpression at 3.5 hpf results in expansion of cmlc2 expression (24 of 29 embryos) but represses cmlc2 expression when activated at 6 hpf (15 of 19) or 9 hpf (13 of 14). Conversely, Dkk1 overexpression at 3.5 hpf weakly represses cmlc2 (7 of 20) but increases cmlc2 expression when activated at 6 hpf (11 of 12) or 9 hpf (8 of 19).

Fig. 2.

Effect of manipulating Wnt/β-catenin signaling on mesoderm induction and mesoderm patterning. WT, hsWnt8, or hsDkk1 transgenic embryos were heat-shocked at 3.5 hpf for 30 min, fixed at 7 hpf, and processed for in situ hybridization with the marker genes indicated in the figure. (A) Wnt8 overexpression is not sufficient to expand expression of the pan-mesodermal marker ntl, but Dkk1 overexpression results in decreased ntl expression (44 of 44 embryos). Wnt8 overexpression reduces expression of the dorsal marker flh (13 of 20) and expands the ventrolateral markers tbx6 (13 of 18) and eve1 (30 of 31). Conversely, Dkk1 overexpression expands flh (32 of 34) and suppresses tbx6 (15 of 15) and eve1 (30 of 30) expression. (B and C) Wnt/β-catenin signaling at pregastrula and gastrula stages suppresses anterior lateral mesoderm and expands posterior lateral mesoderm. WT, hsWnt8, or hsDkk1 transgenic embryos were heat-shocked for 30 min at the times indicated in the figure, fixed at 13 hpf (eight-somite stage), and processed for in situ hybridization. (B) pu.1 expression in anterior lateral mesoderm (myeloid precursors) is repressed by Wnt8 overexpression at 3.5 hpf (13 of 13 embryos), 6 hpf (16 of 16), and 9 hpf (17 of 17), whereas Dkk1 overexpression results in expanded and ectopic pu.1 expression (3.5 hpf, 19 of 20; 6 hpf, 22 of 22; 9 hpf, 10 of 13). Note that the anterior pole of the embryos is shown to demonstrate the ectopic pu.1 expression in hsDkk1 embryos, which, in other views, would not have been visible. (C) pax2.1 expression in pronephric mesoderm (arrows) is expanded by Wnt8 overexpression at 3.5 hpf (11 of 17 embryos), 6 hpf (26 of 42), or 9 hpf (10 of 10), whereas Dkk1 overexpression results in severe shortening of posterior pax2.1-expressing mesodermal regions (3.5 hpf, 29 of 29; 6 hpf, 21 of 21; 9 hpf, 18 of 18). The posterior half of flat-mounted embryos (anterior left) is shown.

Fig. 3.

Biphasic effect of Wnt/β-catenin signaling on cardiac differentiation in mouse ES cells. (A) Addition of Wnt-3A from day 2 to day 5 dramatically accelerated the onset of beating activity in EBs. ∗∗, P < 0.01 compared with control (CTL). Representative of three independent experiments, each performed in triplicate. (B) α-MHC expression at day 11 was examined by quantitative RT-PCR, normalized to GAPDH, and expressed as a ratio to control EBs. Wnt-3A treatment caused a 22-fold increase in α-MHC expression. ∗∗, P < 0.01 compared with control. Representative of three independent experiments, each performed in triplicate. (C) Analysis of sarcomeric MHC by flow cytometry demonstrates that early treatment with Wnt-3A enhances cardiomyocyte content at days 13 and 21 by 10- and 3-fold, respectively. (D and E) Analysis of beating activity (D) and α-MHC expression at day 13 (E) showed that the glycogen synthase kinase-3β inhibitor BIO has a biphasic role on cardiogenesis, enhancing cardiogenesis when given early and inhibiting cardiogenesis when given late. Note that in this experiment, control cultures exhibited a “catch-up” phase of accelerated differentiation that slightly surpassed that of day 18 cultures receiving early BIO treatment. A similar catch-up phase was occasionally observed by using recombinant Wnt-3A, although in most experiments, both early and late cardiogenesis was increased.

Fig. 4.

Effects of Wnt-3A treatment on expression of markers for mesoderm, the Wnt pathway, and cardiomyocyte differentiation. Differentiating EBs received Wnt-3A from day 2 to day 5. Triplicate cultures were harvested for RNA isolation at the indicated times and analyzed by quantitative RT-PCR for the indicated transcripts. Values were normalized to GAPDH, and control (CTL) values were arbitrarily set to 1.0 on day 3. ∗, P < 0.05 vs. control; ∗∗, P < 0.01 vs. control. (A and B) Wnt-3A accelerated expression of the pan-mesoderm marker Brachyury T by 3 days, indicating enhanced mesoderm induction. Wnt-3A markedly up-regulated expression of the precardiac mesoderm marker Mesp1, peaking at day 5 of differentiation. (C–E) Wnt-3A suppressed expression of Wnt-3A and Wnt-1 while inducing expression of Dkk-1, an inhibitor of Wnt/β-catenin signaling. This demonstrates a negative feedback loop for canonical Wnt signaling in differentiating ES cells. (F) Expression of Wnt-11 was up-regulated at multiple time points by treatment with Wnt-3A. Note the substantial increase in Wnt-11 in untreated control EBs over time (although delayed relative to Wnt-3A-treated). (G and H) Expression of the cardiac homeodomain transcription factor Nkx2.5 and contractile protein α-MHC was markedly increased by Wnt-3A by day 9.

Fig. 5.

Overexpression of Wnt-11 enhances late cardiac differentiation in mouse ES cells. Mouse R1 ES cells were lentivirally transduced with a constitutively expressing human Wnt-11 construct plus EGFP or a GFP-only virus (control); the cells were then differentiated as EBs. (A) Although both groups initiated beating activity at the same time, overexpression of Wnt-11 significantly increased the extent of beating at days 14 and 21 (n = 3 per time point). ∗∗, P < 0.01 compared with control. These data are representative of six independent experiments, each performed in triplicate. In two of the six experiments, poor overall cardiac differentiation was noted (<10% of control EBs beating at 21 days), and Wnt-11 treatment did not improve differentiation in these cases. (B) α-MHC expression in EBs overexpressing human Wnt-11 was increased at day 21 (n = 3). ∗∗, P < 0.01 compared with control EBs expressing only EGFP. Data represent three experiments, each performed in triplicate.