Early cardiac development: A Wnt beat away
Embryonic cell fate decisions require an integrated symphony of growth factors presented in a specific sequence and in discreet locations within the developing embryo. Because of species-specific organization of cellular topology in utero, growth factor regulation may differ among organisms. Accordingly, a better understanding of species-conserved growth factor requirements necessitates the need for precise modeling of cell differentiation and movement during construction of definitive embryonic tissues. Ideally, this model would allow control of growth factor production to experimentally dissect the relationship between growth factor signal and cell fate decision.
In this issue of PNAS, Ueno et al. (1) compare the role of Wnt signaling during cardiovascular development between mouse embryonic stem cells (ESC) and zebrafish models (1). Transgenic zebrafish expressing Wnt activators (Wnt8) and inhibitor (Dkk-1) under heat-shock control allowed for interrogation of the temporal effects of Wnt signaling in cardiac development. This approach avoided the use of constitutive activation or inhibition that does not mimic the temporal regulation of signaling pathways during development. Their results indicate that canonical Wnt/β-catenin signaling functions in a biphasic manner to enhance cardiac cell fate in both zebrafish embryos and mouse ESCs. A similar role for Wnt/β-catenin signaling was shown recently by Naito et al. (2) using mouse ESCs. Those authors demonstrated that stage-specific Wnt/β-catenin signaling not only prompted cardiac lineage commitment, but inhibited hematopoietic cell fate. These important studies not only emphasize the relevance of tight regulation of Wnt/β-catenin signaling in cell fate choices, but also that such restricted responses in the zebrafish embryo are conserved in the mouse ESC system. Modestly, their observations understate the importance of such signaling in “priming” developmental programs during germ layer maturation.
Embryonic Space and Time: Relevance to Cell Fate
The canonical Wnt signaling pathway is vital for early specification events involving primitive streak formation and generation of the ESC-derived mesoderm (3, 4). It has been shown that mice deficient in Wnt3 or β-catenin fail to develop a primitive streak and lack mesoderm (4, 5). However, the extended role of the canonical pathway in tissues derived from the mesoderm, like that of the cardiovascular systems, remains unclear. Previous studies have shown mixed responses in relation to cardiac emergence to Wnt/β-catenin, displaying either temporal procardiac effects in the Drosophila and P19CL6 mouse embryonic carcinoma cell line (6, 7) or being anticardiogenic in chick and Xenopus models (8, 9). These differences may be attributed to experimental approaches, cross-talk among signaling pathways, or species-specific differences in spatial organization of the mesodermal cells. The approach used by Ueno et al. (1) to understand how and why the cardiac-mesoderm compartment changes its ability to respond to pulses of Wnt/β-catenin signaling has revealed developmental phases important for embryonic cardiac development. In the initial phases of zebrafish development, activation of Wnt/β-catenin signaling was shown to be crucial for mesoderm cells to be specified into the cardiac lineage, whereas later inhibition of the Wnt/β-catenin pathway was key for defining size or maturation status of the heart field. Curiously enough, the overall changes of mesoderm gene expression were minimal in the zebrafish model compared with the mouse ESC system upon activation of the canonical pathway. In the mouse ESC system, both the pan-mesoderm marker, Brachyury, and precardiac marker, Mesp1, were detected earlier and at higher levels during Wnt activation, neither of which was observed in the zebrafish system upon Wnt activation. These differences in expression kinetics and levels may be a function of increased specification of cells to mesoderm compartment seen specifically in mouse embryoid bodies (EBs). It is possible that in the zebrafish model the specification events are more controlled because of the restricted spatial organization required to form the embryo proper. This forced organization, essential in utero, may be lacking in the mouse EB model, thereby providing a more permissive system for Wnt activation to specify cells to mesoderm. The tighter controls of the contributors to the cardiac lineage in the zebrafish may reflect the roles of spatial organization of regions that spur inhibitory signals. Such cross-regulation seems to be reflected as negative-feedback responses in the mouse ES to help maintain signal duration to induce biphasic responses for cell determination. Are the negative-feedback responses a compensatory mechanism that mouse ESC have developed because of “lack of” complex topological organization, or are such Wnt-related mechanisms that regulate the cardiac development normally seen in mouse embryo? Interestingly, negative regulation of Wnt/β-catenin signaling was only important during later stages of cardiac development, when cardiac cells have migrated to anterior regions of the embryo and are undergoing maturation. This observation may represent a required movement from the field of Wnt activation necessary for maturation, in contrast to required Wnt signals during cardiac specification. As shown by Ueno et al. (1), cross-regulation between the canonical and noncanonical Wnt signaling pathways may be necessary for guided differentiation of the cardiac field; however, it has yet to be defined. Characterization of the potency of such Wnt/β-catenin responsive cardiovascular progenitors will require clonal in vitro assays and in vivo adoptive transfer assays to provide compelling evidence for such a paradigm.
Negative regulation of Wnt/β-catenin signaling was only important during later stages of cardiac development.
Implications to the Human
The studies by Ueno et al. (1) and Naito et al. (2) are particularly interesting when comparing human cells and beg the question of whether human ESCs would respond similarly to biphasic patterns of Wnt/β-catenin signaling. Because there are growing concerns regarding the differences in growth factor requirements in human and mouse ESCs (10–15) it is unclear what results may ensue. Interestingly, the similarities in temporal response to Wnt/β-catenin between highly organized zebrafish models and poorly organized in vitro mouse EB models challenge the requirement for organization of cellular structures to govern cell fate decisions. Because Wnt signaling is common to these experimental systems and observations, it is possible that Wnt signaling not only directs differentiation of cells to a specific lineage, but dually orchestrates spatial organization in the mouse EBs to mimic embryonic patterns preexisting and therefore not required in the zebrafish model (16). Determining the specific cellular cues required for human cardiovascular development and regeneration is fundamental to the success of therapies that would benefit from cardiac remodeling. As early trials with VEGF and FGFs have been shown to be ineffective (17), the studies by Ueno et al. (1) and Naito et al. (2) suggest that Wnt signaling may be worthy of exploration as a therapeutic intervention in patients suffering from ischemic insult and worthy of investigation of the role of Wnts in generating mature cardiac cells from human ESC lines.
Acknowledgments
M.B. holds a Canada Research Chair in human stem cell biology. This work was suppor ted by grants from the Canadian Institutes of Health Research and the National Cancer Institute.
Footnotes
- *To whom correspondence should be addressed. E-mail: mbhatia{at}mcmaster.ca
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Author contributions: K.V. and M.B. wrote the paper.
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The authors declare no conflict of interest.
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See companion article on page 9685.
- © 2007 by The National Academy of Sciences of the USA
