Canonical WNT signaling enhances stem cell expression in the developing heart without a corresponding inhibition of cardiogenic differentiation

Abstract

WNT signaling has been shown to influence the development of the heart. Although recent data suggested that canonical WNTs promote the emergence and expansion of cardiac progenitors in the pregastrula embryo, it has long been accepted that once gastrulation begins, canonical WNT signaling needs to be suppressed for cardiac development to proceed. Yet, this latter supposition appears to be odds with the expression of multiple canonical WNTs in the developing heart. The present study examining the effect of ectopic canonical WNT signaling on cardiogenesis in the developing frog was designed to test the hypothesis that heart formation is dependent on the inhibition of canonical WNT activity at the onset of gastrulation. Here we report that cardiac differentiation of explanted precardiac tissue from the dorsal marginal zone was not suppressed by exposure to WNT1 protein, although expression of Tbx5, Tbx20, and Nkx2.5 was selectively reduced. Pharmacological activation of WNT signaling in intact embryos using the GSK3 inhibitor SB415286 did not prevent the formation of an anatomically normal and functionally sound heart, with the only defect observed being lower levels of the cardiac transcription factor Nkx2.5. In both the explant and whole embryo studies, expression of muscle genes and proteins was unaffected by ectopic canonical WNT signaling. In contrast, canonical Wnt signaling upregulated expression of the cardiac stem cell marker c-kit and pluripotency genes Oct25 and Oct60. However, this regulatory stimulation of stem cells did not come at the expense of blocking cardiac progenitors from differentiating.

FIG. 1.

Ectopic activation of canonical WNT signaling in the early blastula. (A) The indicated mRNA was injected into both dorsal blastomeres at the 4-cell stage, and allowed to develop until stage 40 when they were scored for embryonic phenotype and heart formation. Injection of control lacZ RNA did not disrupt development, as normal embryos developed. Approximately 17% and 84% of embryos injected with 300 pg or 1.0 ng of constitutively active β-catenin, respectively, exhibited a ventralized embryonic phenotype without a heart. (B) Representative lacZ-injected embryo, with the arrow showing the position of the contractile heart. (C, D) Examples of β-catenin-injected embryos that displayed a heart-negative phenotype.

FIG. 2.

Canonical WNT activity. (A) Induction of siamois was measured in dorsal marginal zone (DMZ) tissue cultured in the absence or presence of the indicated WNT1 concentration before RNA isolation and polymerase chain reaction (PCR) analysis during day 2 of culture. Highest levels of the WNT target gene siamois were obtained with 20 ng/mL WNT1. **P values between groups was <0.01. *P values between groups was <0.05. (B) Canonical WNT activity of WNT1 protein and the GSK3 inhibitor SB415286 was tested using QCE6 cells transfected with TOPflash-GFP and FOPflash-GFP reporter plasmids. The results are presented as fold-induction over nontreated control, with error bars corresponding to standard error of the mean.

FIG. 3.

Cardiac differentiation of DMZ tissue. DMZ explants were cultured for 4 days in the (A–C) presence or (D) absence of 20 ng/mL WNT1 and immunostained for sarcomeric myosin heavy chain. (A, B) Brightfield and fluorescent images of an individual WNT1-treated explant that displayed sarcomeric myosin immunoreactivity. (C) Another example of an explant incubated with WNT1 exhibiting cardiac tissue that stained positive for sarcomeric myosin. A comparison of the WNT1-treated tissue with (D) nontreated control DMZ demonstrates that ectopic canonical WNT signaling did not alter the pattern of sarcomeric myosin expression within the tissue. (E, F) In contrast, cardiac differentiation of the DMZ tissue was blocked by exposure of DMZ explants to 50 μM LY294002, which inhibits signaling via phosphatidylinositol 3-kinase, as shown for an individual explant imaged by brightfield and fluorescent microscopy. Scale bars=100 μm.

FIG. 4.

Cardiac gene expression in DMZ tissue. DMZ tissue was cultured in the absence or presence of the indicated WNT1 concentration before RNA isolation and PCR analysis of cardiac (A) transcription factors and (B) muscle genes. (A) WNT1 treatment had little impact on of Islet-1 and GATA6 transcription but decreased expression of Tbx5, Tbx20, and Nkx2.5. (B) RNA levels for the cardiac-specific muscle proteins Actc, cMHCα, and cTnI were not significantly affected by WNT1 exposure. *P<0.05; **P<0.01 between WNT1-treated group and control.

FIG. 5.

Ectopic activation of canonical WNT signaling in the early gastrula. This signaling pathway was promoted in the developing frog by treating stage 10.25 embryos with the GSK3 inhibitor SB415286. Types of malformations observed in responses to SB415286 treatment were embryos displaying: (A) mild defects consisting of a bent tail; (B) an anteriorized phenotype that results in a severely truncated tail, but fully developed head; and (C) a compound defect consisting of both a truncated tail and highly deformed head. Despite the differences in the severity of the defects, each of these embryos developed a functional and anatomically normal heart (arrows). Note that the ventral dermis overlaying the developing heart was removed from the latter embryo to better observe the organ's structure. (D) Same embryo as C, showing sarcomeric myosin immunolabeling of the heart (arrow). (E) High magnification of a heart from another SB415286-treated embryo that was incubated to stage 40. The sarcomeric myosin-immunolabeling highlights the developing ventricle (V), left (LA) and right atrium (RA) (F) SB415286-treated embryo that was incubated to stage 46 and exhibited an anatomically normal heart (arrow). (G) Same embryo at higher magnification, with sarcomeric myosin staining showcasing well-formed atrial and ventricular chambers. Scale bars=200 μm.

FIG. 6.

Cardiac gene expression analysis following treatment of the embryo. Cardiac tissue from nontreated or SB415286-treated embryos was isolated and examined for expression of the (A) transcription factors Islet-1, GATA6, Tbx5, Tbx20, and Nkx2.5 and (B) muscle genes Actc, cMHCα, and cTnI by real-time PCR. The only gene whose expression was significantly downregulated by SB415286 exposure was the transcription factor Nkx2.5. **P<0.01 between SB415286-treated group and control.

FIG. 7.

Stem cell marker expression in response to canonical WNT signaling. (A) PCR analysis of DMZ tissue cultured without or in the presence of 20 ng/mL or 50 ng/mL WNT1. Treatment with 20 ng/mL WNT1 promoted an upregulation of both c-kit and oct25 expression. Expression of oct60 in DMZ explants was not affected by WNT1. (B) PCR analysis of cardiac tissue removed from nontreated and SB415286-treated embryos. SB415286 exposure promoted an upregulation of both c-kit and oct60 expression. In contrast, SB415286 did not stimulate expression of oct25. **P<0.01 between treated group and negative control.