The heart is formed through multiple developmental steps which include the determination of the cardiac field in the mesoderm, differentiation of cardiac precursor cells, and maturation of the heart (42
). Many classical embryonic studies have implicated the mechanism of how and where these steps take place in the developing embryos. The vertebrate heart arises from paired mesodermal primordia that migrate to the anterior ventral midline, where they fuse and undergo terminal differentiation (18
). In Xenopus
embryos, the cardiac field is located in the dorsal mesoderm lateral to the Spemann organizer and is specified prior to the end of gastrulation. In this relatively early step of cardiac development, inductive signals from the adjacent deep endoderm and the organizer region play a pivotal role in the determination of the cardiac field (13
). Subsequently, the cardiac primordia always lie in close contact with the endoderm while migrating anterolaterally, and interactions between the endoderm and the overlying mesoderm are thought to be important for the promotion of cardiomyocyte differentiation in cardiac mesodermal cells. In Xenopus
, the presence of the deep dorsoanterior endoderm markedly enhances the heart formation in explants of heart primordia, and the presence of both the endoderm and the organizer is necessary and sufficient to induce beating heart tissue in ventral mesoderm explants (42
). In chicks, the anterior endoderm also induces the differentiation of nonprecardiac mesodermal cells into heart tissue (48
). These observations also suggest that the endoderm-derived signals play a vital role both in the specification of the cardiac field and in the differentiation of determined cardiac precursor cells. However, the precise molecular mechanisms that regulate these inductive events during the formation of the heart are largely unknown at present.
Recent advances in understanding the genetic pathway of heart development have allowed us to use cardiac-restricted transcription factors as early heart-specific markers for dissecting the molecular mechanism of cardiogenesis. Among the several transcription factors implicated in cardiac development, Csx/Nkx-2.5, MEF2C, and GATA-4 have been well characterized in recent years. Csx/Nkx-2.5 is an NK-2 class homeodomain factor that was originally identified as a potential vertebrate homolog of Drosophila tinman
). The tinman
gene is initially expressed in all mesodermal cells, but subsequently its expression domain is restricted to the dorsal part of the mesoderm, and later in development, expression of tinman
is observed only in the dorsal vessel, an insect equivalent for the vertebrate heart (4
). Murine Csx/Nkx-2.5 is also predominantly expressed in the heart and in cardiac progenitor cells from the early developmental stage when two heart primordia are symmetrically situated in the anterior lateral mesoderm. The heart does not form at all in the tinman
mutant of Drosophila
), whereas in Csx/Nkx-2.5 knockout mice, the heart forms, but its development stops at the looping stage (32
). MEF2C belongs to the MEF2 subfamily of MADS-box transcription factors and binds to the AT-rich element in regulatory regions of numerous muscle-specific genes (6
). GATA-4 is a member of the cardiac GATA subfamily, which consists of GATA-4, -5, and -6, and binds to the WGATAR motif in promoter regions of cardiac- or gut-specific genes (9
). MEF2C and GATA-4 are also thought to be involved in the early stage of cardiogenesis. Both of them started to be expressed in the precardiac mesoderm almost simultaneously with Csx/Nkx-2.5. Targeted disruption of MEF2C results in right ventricular dysplasia (30
), and bilateral cardiac primordia fail to fuse in GATA-4−/−
mice because of the ventral folding and fusion defects of the developing embryo (26
). Thus, Drosophila tinman
, vertebrate Csx/Nkx-2.5, MEF2C, and GATA-4 are critical regulators of cardiac development and are useful molecular markers for examining effects of inductive signals from other tissues or germ layers.
In this respect, several experiments were performed by using cardiac-specific transcription factors as cardiac markers to elucidate the molecular mechanism of cardiogenesis and have demonstrated that bone morphogenetic proteins (BMPs) play a vital role in cardiac development. Initially, it was reported that expression of tinman
is restricted to the dorsal part of the mesoderm by the ectodermally expressed decapentaplegic
), a member of the transforming growth factor β (TGF-β) superfamily that is most closely related to vertebrate BMP-2 or BMP-4 (10
). Recently, the ectopic expression of Csx/Nkx-2.5 and GATA-4 was also induced by the implantation of BMP-2-soaked beads in nonprecardiac mesoderms in chicks (49
), suggesting that BMPs play a pivotal role in the induction of vertebrate cardiac development. At present, however, the precise mechanism by which BMPs induce the differentiation of cardiac precursor cells is largely unknown.
In the investigation of the molecular mechanisms of cardiomyocyte differentiation, the in vitro culture system presents a great advantage. P19 embryonal carcinoma cells are undifferentiated stem cells derived from murine teratocarcinoma (34
) and differentiate into a variety of cell types representative of all three germ layers after suspension culture in the presence of several chemical inducers. When exposed to a relatively low concentration of retinoic acid (1 to 10 nM) or dimethyl sulfoxide (DMSO) (0.5 to 1%), some P19 cells differentiate into endodermal and mesodermal cells, including cardiomyocytes (8
). Although the P19 cell line has been widely used as a model system of cardiogenesis in vitro, its utility is limited because of its quite low efficiency of differentiation into cardiomyocytes. Recently, a clonal derivative named P19CL6 was isolated from P19 cells (17
). Unlike P19 cells, this subline efficiently differentiates into beating cardiomyocytes with adherent conditions when treated with 1% DMSO. Since almost all cells differentiate into cardiomyocytes which express cardiac-specific genes, P19CL6 cells are a useful in vitro model to study cardiomyocyte differentiation (17
In the present study, we examined the role of BMPs in the differentiation of cardiomyocytes utilizing the P19CL6 in vitro system. For this purpose, we isolated a permanent P19CL6 cell line named P19CL6noggin that stably overexpresses the BMP antagonist noggin. In contrast to parental P19CL6 cells, P19CL6noggin cells did not differentiate into beating cardiomyocytes and expression of cardiac-specific genes was not induced when treated with DMSO. Overexpression of BMP-2 or addition of BMP protein to the medium restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes, suggesting that BMPs were indispensable for cardiomyocyte differentiation. The failure of P19CL6noggin cells to differentiate into cardiomyocytes was also rescued by overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) superfamily that has been demonstrated to be involved in BMP signaling, whereas overexpression of the dominant negative form of TAK1 inhibited differentiation of parental P19CL6 cells into cardiomyocytes. Simultaneous overexpression of Csx/Nkx-2.5 and GATA-4 also rescued the differentiation defect of P19CL6noggin cells, although overexpression of Csx/Nkx-2.5 or GATA-4 alone did not. These results suggest that the MAPK pathway activated by TAK1 and two cardiac transcription factors, Csx/Nkx-2.5 and GATA-4, mediate BMP-induced cardiomyocyte differentiation.