The initiation of skeletal myogenesis involves a complex interplay of signalling molecules secreted from the tissues surrounding the somite, including Wnt, Sonic hedgehog, and Bone morphogenetic proteins 4 (BMP4) [1
]. Somites respond to the various signals by activating the expression of transcription factors that specify cells to the skeletal muscle lineage, including Pax3, Meox1 and Gli2 [6
]. Commitment into skeletal myoblasts is dependent on the expression of the myogenic regulatory factors (MRFs), including MyoD, Myf-5, myogenin and myf-6/MRF4/herculin, and is regulated by factors in the dermomyotome [11
P19 cells are pluripotent embryonal carcinoma (EC) cells, derived from mouse embryonic stem (ES) cells, that can differentiate into cardiac and skeletal muscle in a dimethylsulfoxide (DMSO)- and aggregation-dependent manner [13
]. While cells grown in monolayer maintain their stem cell phenotype, the process of cellular aggregation initiates mesoderm induction, shown by the expression of Brachyury T [14
]. Subsequent muscle development proceeds in the presence of DMSO. The order of transcription factors and signalling pathways for myogenesis in P19 cells appear to be similar to those during early embryogenesis. Thus P19 cells are a useful tool for examining in vitro
myogenesis, potentially leading to novel mechanisms relevant to ES stem cell therapy.
Retinoic acid (RA) is a derivative of vitamin A and plays a crucial role in a wide variety of embryonic developmental processes [15
]. In the embryo, the ability of RA to bind its receptors (retinoic acid receptors [RARs]/retinoid × receptors [RXRs]) is precisely controlled by regulating the availability of RA through proteins that synthesize RA, such as retinaldehyde dehydrogenase 2 (RALDH2), and those that metabolize RA, such as Cyp26, and other proteins that transport or bind RA.
Low levels of RA are known to enhance skeletal myogenesis in stem cells and myoblast cell lines [16
]. RA can regulate MRF expression in myoblasts and chick limb [17
], whereas RARs interact and synergize with MRFs [20
]. However, the exact stage(s) at which RA functions to enhance skeletal myogenesis in a stem cell context has not been clearly defined.
Altered RA signalling in vertebrates affects body patterning, generating homeotic transformations and/or segmentation defects [21
]. In Xenopus
embryos, RA signalling regulates segmental patterning by promoting anterior segmental polarity and by positioning segmental boundaries [22
]. In mice, RA coordinates somitogenesis and left-right patterning [23
]. How the effect of RA on the somite impacts on the development of the myotome is not well understood.
In the canonical pathway, Wnt binds to cell surface receptors of the frizzled family, leading to the activation of Dishevelled and stabilization of cytosolic β-catenin [24
]. In a simplified view, β-catenin enters the nucleus, binds the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors, and activates gene expression. Several studies have shown that exogenous Wnt and/or activated β-catenin can replace the dorsal neural tube in the induction of myogenesis in somite explant cultures [25
]. A combination of Wnt and Shh signals regulates the expression levels of both β-catenin and Lef1 in the myotome prior to MyoD expression in the chick [26
]. Further, β-catenin regulates the expression of Pax3 [27
]. In P19 cells, a dominant negative β-catenin inhibits the expression of Pax3, Gli2, Meox1, MyoD, and abrogates myogenesis [8
]. Finally, Wnt was shown to act directly on the Myf5 epaxial enhancer via β-catenin [28
]. Therefore, there is strong evidence that Wnt signalling regulates specification and commitment into the skeletal muscle lineage. How Wnt signalling intersects with RA signalling during myogenesis is unknown.
BMP4 belongs to the TGF-β superfamily of peptide growth factors [29
]. BMPs inhibit myogenesis in myoblast cell lines, limb micromass cultures, and developing somites [3
]. Noggin signals are derived from the notochord and the somite. Noggin signalling is believed to counteract the inhibitory effects of BMP4 on the epaxial somite [32
]. Further experiments using somite explants showed that relative levels of BMP4 and noggin regulated the activity of Pax3 to control the temporal and spatial activation of the MRFs [35
]. Therefore, extensive studies have demonstrated the inhibition of embryonic skeletal myogenesis by BMP. How BMP signalling intersects with RA signalling is unknown.
The role of BMP in cardiomyogenesis has also been extensively studied. In Drosophila
, the BMP homologue decapentaplegic protein (dpp) is secreted from the dorsal ectoderm and maintains tinman expression in the mesoderm [36
]. Similarly, in chick BMP2 or -4 is expressed in tissues adjacent to the precardiac mesoderm and can induce Nkx2-5 and GATA-4 expression [37
]. Conversely, disruption of BMP signalling with noggin or dominant negative receptors can prevent cardiomyogenesis in chick, Xenopus
, ES, P19 and P19CL6 cells [37
]. Therefore, BMP/dpp signalling is essential in controlling cardiomyogenesis.
Studies with embryonic stem and embryonic carcinoma cells have shown that RA inhibits cardiomyogenesis when added at an early stage [16
] and enhances ES cell cardiomyogenesis when added at a late stage of differentiation [46
]. RA can block myocardial gene expression, including XNkx2.5, in Xenopus
] and can alter cardiomyogenesis proliferation and patterning in other model systems [50
]. Mice lacking various combinations of RXRs and RARs have shown that retinoids are required to prevent differentiation and support proliferation of ventricular cardiomyocytes [54
]. RA deficiency in RALDH2 -/- mice alters second heart field formation [56
]. Clearly, RA affects the timing and positioning of cardiomyogenesis at multiple levels and further studies are required to dissect out the role of RA at each step of development.
Here we investigate signalling events leading to the control of stem cell entry into skeletal and cardiac muscle lineages by RA, Wnt, and BMP4. We show that low levels of RA stimulate skeletal myogenesis by accelerating and increasing the expression of Wnt3a, Pax3, Meox1, and MRFs. This early and enhanced activation of skeletal muscle is refractory to inhibitory signals from BMP4 but not from a dominant negative β-catenin. Furthermore, low levels of RA inhibit stem cell differentiation into the cardiac muscle lineage, as shown by the absence of GATA-4 expression. The inhibitory activity of RA on cardiomyogenesis can be abrogated by the presence of BMP4. Therefore, BMP4 and RA function antagonistically to regulate each other's inhibition of entry into skeletal and cardiac muscle lineages, respectively. However, RA functions both upstream and downstream of Wnt signalling through β-catenin.