During early heart development at the avian pregastrula stage (stages X–XIV; Eyal-Giladi and Kochav, 1976), cells that contribute to the heart are present in the posterior half of the blastoderm epiblast (Hatada and Stern, 1994). At the onset of gastrulation at stages 2–3 (Hamburger and Hamilton, 1951), the primitive streak begins to develop and prospective heart cells are located within a broad zone in the primitive streak. This broad zone consists of most of the primitive streak, excluding the most anterior and posterior portions (Garcia-Martinez and Schoenwolf, 1993). The anterior region of the primitive streak contains presumptive cells of Hensen's node. At stages 4–4+, Hensen's node develops and prospective heart cells (including cardiomyocyte progenitors) leave the primitive streak, migrate into the mesoderm and spread anterolaterally, resulting in the formation of the anterior lateral-plate mesoderm (Garcia-Martinez and Schoenwolf, 1993; Schultheiss and Lassar, 1999). During gastrulation, right and left anterior endoderms develop from the node itself, and thereafter make contact with the anterior lateral-plate mesoderm (Sellek and Stern, 1991). Developmental biology has shown that the interaction between the anterior lateral-plate mesoderm and its subjacent endoderm is necessary to generate beating tissue (Orts-Llorca, 1963; for reviews, see Schultheiss and Lassar, 1999; Lough and Sugi, 2000; Brand, 2003). After the mesoderm is committed to forming the heart, the pleuro-pericardial coelom, which consists of visceral and somatic coelomic epithelia, develops in the anterior lateral-plate mesoderm. During the subsequent process of embryonic folding, the right and left visceral coelomic epithelia of the precardiac mesoderm migrate toward the ventral midline of the intestinal portal and fuse to each other, resulting in the formation of the primitive heart tube. At this time (stage 9), myofibrillogenesis occurs rapidly and the heart starts to beat spontaneously. Therefore, myofibrillogenesis occurs immediately after the commitment of the myocardial lineage.
During gastrulation, the prospective heart mesoderm becomes committed to the cardiac lineage and thereafter undergoes terminal cardiomyocyte differentiation (Antin et al., 1994). At this time, the anterior lateral endoderm that is subjacent to the precardiac mesoderm expresses BMP2 and BMP4 (Lough et al., 1996; Schultheiss et al., 1997), and fibroblast growth factor (FGF) -8 (Alsan and Schultheiss, 2002). Loss-of-function and gain-of-function experiments indicate that both BMP and FGF signalling is required to regulate cardiac specification, as well as its terminal differentiation. In other words, cooperation between BMP and FGF signalling regulates the expression of early heart marker genes, such as Nkx2.5 and GATA4, as well as proteins of the muscle-specific contractile apparatus (Schultheiss et al., 1995, 1997; Lough et al., 1996; Ladd et al., 1998; Barron et al., 2000; Walters et al., 2001; Alsan and Schultheiss, 2002; Nakajima et al., 2002). Molecules that antagonize signalling by means of the Wnt pathway also have cardiac-inducing properties (Marvin et al., 2001; Schneider and Mercola, 2001; Tzahor and Lassar, 2001). Crescent—a Wnt antagonist that is expressed in the anterior endoderm—is able to induce cardiac-specific gene expression in noncardiogenic tissue in culture (Marvin et al., 2001). Accordingly, in addition to BMP and FGF, inhibition of Wnt signalling is required to promote the formation of the heart in the anterior lateral mesoderm.
During early cardiac myofibrillogenesis observed in cultured anterior lateral mesoderm, the BMP antagonist noggin inhibits the expression of sarcomeric α-actinin, Z-line titin and sarcomeric myosin. It does not, however, inhibit the expression of the initial heart α-actin—smooth muscle α-actin (SMA)—which is first expressed in the anterior lateral mesoderm and then recruited into the initial myofibrils (Sugi and Lough, 1992; Colas et al., 2000; Nakajima et al., 2002). Therefore, there are strong indications that the up-regulation of SMA is activated by a signalling pathway other than that of BMP (Nakajima et al., 2002); this pathway has not yet been elucidated. During the pregastrula stages, a signal(s) from the hypoblast is required to induce cardiac myogenesis in the epiblast. The hypoblast-derived signal(s) acts upstream of the proposed heart-inducing signals provided by the anterior lateral endoderm, and can be mimicked by activin (Yatskievych et al., 1997). Another experiment showed that BMP2 or BMP4 inhibits cardiogenesis before stage 3, suggesting that spatiotemporally restricted BMP antagonism is responsible for cardiomyocyte differentiation in the prestreak stage of avian embryo development (Ladd et al., 1998). However, the signal(s) that regulates the expression of SMA during early cardiogenesis remains unknown.
In the present study, we performed an explantation experiment, using pregastrula chick embryos, to decipher the molecular mechanisms by which the expression of SMA is up-regulated during early cardiogenesis. Our results showed that interaction between tissues of the posterior epiblast and its subjacent hypoblast at early pregastrula stages (stages X–XI) was necessary to initiate the initial heart muscle α-actin, SMA. A similar result was achieved if TGFβ or activin, rather than hypoblast, was added to the cultured posterior epiblast. The results also showed that adding BMP to the cultured blastoderm inhibited the expression of SMA, whereas BMP antagonists, such as chordin, were able to induce the expression of SMA in cultured posterior epiblast that had not been cultivated with its associated hypoblast. In addition, adding lefty-1, a nodal antagonist, to the blastoderm inhibited the expression of SMA, and nodal plus BMP antagonist but not nodal alone induced the strong expression of SMA in cultured posterior epiblast.
Posterolateral Blastoderm Elicits Cardiomyocytes in Culture
First, we attempted to determine the heart-forming region in the pregastrula epiblast from which cardiomyocytes develop at later stages. For this purpose, stage X–XIII blastoderms (epiblast + hypoblast) were obtained and cut into 12 pieces (explants #1 to 6 on right/left half of blastoderm), as shown in Figure 1A. The resulting explants were cultured for 48 hr and stained with a monoclonal antibody specific for sarcomeric α-actinin, an initial cardiomyocyte sarcomeric protein (Nakajima et al., 2002). After 48 hr in culture, explants expressing sarcomeric α-actinin were counted: the results showed that posterior lateral regions (#2, 4, and 6), not including Koller's sickle and the posterior marginal zone, expressed sarcomeric α-actinin (Fig. 1B). By contrast, we could not detect any apparent staining of sarcomeric α-actinin in regions #1, 3, and 5. The incidence of sarcomeric α-actinin expression was higher in explants obtained from stage XII–XIII embryos than stage X–XI embryos, and the incidence was higher in the posterior region (#6) at all stages. The expression of SMA was also significant in regions #2, 4, and 6 (not shown). There was no detectable immunoreactivity of anti-skeletal muscle marker antibody (12/101 antibody, data not shown). In this culture experiment, posterior blastoderm explants generated the contractile apparatus (sarcomere) after 72 hr in incubation (data not shown), at which time cardiomyocytes will have already generated mature myofibrils with a sarcomeric pattern. Mature skeletal muscle, by contrast, does not develop in ovo at 72 hr in incubation (Allen and Pepe, 1965).
We further analyzed whether these regional explants expressed cardiac marker genes, such as Nkx2.5, GATA4, and sarcomeric α-actinin, by using reverse transcriptase polymerase chain reaction (RT-PCR; Fig. 1C) and found that GATA4 and sarcomeric α-actinin were present in regions #2, 4, and 6 but not in anterior medial regions (regions #1, 3, and 5). A large amount of PCR products for Nkx2.5 and GATA4 were detected in regions #4 and 6. A small amount of PCR products for MyoD (skeletal specific transcription factor) was also amplified in the same areas, indicating that regions #4 and 6 contain not only prospective cardiac muscle cells but also skeletal muscle, as described previously (Hatada and Stern, 1994; George-Weinstein et al., 1997). These results indicated that regions #2, 4 and 6, obtained from pregastrula blastoderm, differentiated into cardiomyocytes in culture supplemented with serum-free defined medium, indicating that the posterior lateral region of the pregastrula epiblast contains prospective cardiomyocytes.
Stage X–XI Posterior Epiblast Requires Hypoblast to Express SMA
To decipher the molecular mechanisms that regulate the expression of initial heart α-actin, SMA, we next examined whether an interaction between the tissues of posterior epiblast and its adjacent hypoblast was required for the expression of SMA in cultured posterior blastoderm. In this experiment, we first examined whether or not posterior epiblast alone can express SMA after 48 hr in culture. As shown in Figure 2A, although more than 80% of stage XII–XIII posterior epiblasts alone (region #6) expressed SMA after 48 hr in culture, only 20–30% of explants from stage X–XI posterior epiblasts expressed SMA (P < 0.001). RT-PCR analysis showed that the reduced PCR products of SMA were evident in cultured stage X–XI posterior epiblast (data not shown). We next attempted the heterotypic culture experiment as shown in Figure 2B: region #6 quail posterior epiblast (stage X–XI) was co-cultured with chick posterior hypoblast (stage XI). The resulting cultures were then double-stained with antibodies against SMA and quail nuclear marker protein (QCPN). When the #6 posterior epiblasts from stage X–XI quail embryos were cultured, only 9% (n = 11) of explants expressed SMA after 48 hr in culture. By contrast, when similarly prepared quail #6 epiblast explants were co-cultured with chick posterior hypoblasts from stage XI blastoderm, 75% (n = 12) of quail epiblast explants (possessing QCPN) expressed SMA (Fig. 2B,D; P < 0.01).
We next examined whether stage XI posterior hypoblast could induce the expression of SMA in noncardiogenic anterior epiblast. When stage X–XI quail anterior epiblasts (#1) were co-cultured with stage XI chick posterior hypoblasts, 56% (n = 39) of the noncardiogenic #1 explants, possessing QCPN, expressed SMA after 48 hr in culture. By contrast, only 11% of the control explants (anterior epiblast alone) expressed SMA (n = 46; P < 0.001; Fig. 2C,D). In addition, quail #1 epiblasts of stage XIII embryos that had been co-cultured with stage XI chick posterior hypoblasts were not stimulated to express SMA (Fig. 2D). These results indicated that the posterior epiblasts of stage XII–XIII embryos were already committed to express SMA without co-cultivation of associated hypoblast. By contrast, posterior epiblasts of stage X–XI embryos seemed to require the hypoblast to express SMA. Furthermore, signal(s) from the stage X–XI hypoblast cells induced SMA in early noncardiogenic anterior epiblast (stage X–XI), but not in late anterior epiblast (stage XIII). Therefore, we suggest that short-term interaction between the tissues of the posterior epiblast and its associated hypoblast at an early pregastrula stage is necessary for SMA to be expressed in the developing cardiomyocyte.
TGF-β or Activin Can Induce the Expression of SMA in Cultured Posterior Epiblast
The above experiments indicate that tissue interactions, which were probably regulated by secreted molecules emitted from the hypoblast, were evident in the initial expression of SMA during early cardiogenesis. TGF-β or activin are potent inducers of cardiogenesis in avian posterior epiblast culture (Yatskievych et al., 1997; Ladd et al., 1998). In addition, nodal and activin have similar activities and are potent mesoderm inducers in vertebrates (Schier and Shen, 2000; Schier, 2003). To investigate whether TGF-β, activin or nodal can induce the expression of SMA in cultured early posterior epiblasts of stage X–XI embryos, stage X–XI epiblast explants were cultured in serum-free defined medium with or without recombinant TGF-β3, activin or nodal, and the cultures were stained with antibodies against SMA and sarcomeric α-actinin. As shown in Figure 3, when the explants were cultured in the defined medium alone, only 21% expressed SMA after 48 hr. Conversely, in explants cultured in the medium containing TGF-β3 or activin, 80% or more expressed SMA after 48 hr in culture, and some explants showed a bead-like deposition of sarcomeric α-actinin (Fig. 3). In explants cultured in medium containing nodal or nodal plus CFC2 (nodal cofactor, data not shown), only 27% or less expressed SMA. To investigate whether TGF-β or activin were endogenous signalling molecules responsible for the expression of SMA during cardiogenesis in ovo, we cultured the posterior region of blastoderm (epiblast + hypoblast) in a medium containing either a neutralizing anti–TGF-β antibody mixture (anti-TGF-β1, -2, and -3) or follistatin, a natural inhibitor of activin (Nakamura et al., 1990). In such neutralizing experiments, posterior blastoderm treated with neutralizing anti–TGF-β antibodies or follistatin expressed SMA in a similar manner to that observed in control cultures (data not shown). Results indicated that the signalling pathway that is regulated by TGF-β or activin could induce the expression of SMA in cultured posterior epiblast explants, in a way similar to that seen when posterior epiblast is co-cultured with associated hypoblast. However, neither TGF-β nor activin seem to be endogenous molecules acting on the epiblast to induce the expression of SMA during the earliest process of cardiogenesis in ovo.
BMP4 Inhibits the Expression of SMA in Cultured Posterior Blastoderm
It has been reported that BMP is a potent inducer of cardiac specification and its terminal differentiation after gastrula stages during chick cardiogenesis (Lough et al., 1996; Schultheiss et al., 1997; Walters et al., 2001; Nakajima et al., 2002). We examined the effect of BMP on SMA expression in stage X–XI posterior epiblast (region #6). Posterior epiblasts were cultured in the presence of recombinant BMP4. None of the explants cultured in a medium containing 100 ng/ml of BMP4 expressed SMA (0 of 23) (data not shown, 28% of control epiblast cells express SMA). Moreover, explants that were cultured in the presence of both BMP4 (100 ng/ml) and TGF-β3 (100 ng/ml) did not express SMA (0 of 21). These results indicated that BMP4 inhibited the expression of SMA in cultured epiblast and that this inhibitory effect was not reversed by TGF-β3.
We next examined whether BMP4 could inhibit the expression of SMA in cultured posterior blastoderm (region #6 epiblast + hypoblast), from which cardiomyocytes would later develop. Posterior blastoderm was resected from stage X–XIII embryos and cultured in the medium containing recombinant BMP4. As shown in Figure 4, BMP4 inhibited the expression of SMA in a dose-dependent manner at all pregastrula stages. The explants from stage X–XIII embryos did not express SMA when they were cultured in a medium supplemented with 100 ng/ml of BMP4. RT-PCR analysis showed that the reduced or no PCR products of SMA were detectable when cultured with 100 ng/ml of BMP4 (data not shown). These results indicated that BMP4 inhibited the expression of SMA in cultured posterior epiblast (the presumptive heart-forming region) even if it was cultured with the associated hypoblast or medium containing TGF-β3.
BMP Antagonists Initiate the Expression of SMA in Cultured Stage X Posterior Epiblast
The results mentioned above indicate that BMP4 was able to inhibit the expression of SMA in the developing posterior blastoderm in a dose-dependent manner. Therefore, strong or predominant BMP signals at the pregastrula stage might result in repression of SMA expression in the developing cardiomyocyte. It has been reported that BMP4 is expressed in the epiblast (area pellucida) of the pregastrula embryo and BMP7 in area opaca (Streit et al., 1998; Chapman et al., 2002; Faure et al., 2002). Furthermore, chordin (a natural BMP antagonist), is expressed in the posterior blastoderm and, subsequently, in the anterior half of the primitive streak, from which cardiomyocytes will later develop (Lawson et al., 2001). Therefore, natural BMP antagonists might be important in regulating the expression of SMA in the nascent heart mesoderm from the posterior epiblast (Nakamura et al., 1990; Sasai et al., 1994; Re'em-kalma et al., 1995; Zimmerman et al., 1996). To investigate whether the BMP antagonists were capable of inducing the expression of SMA in cultured posterior epiblast, posterior epiblast explants of stage X embryos were cultured in the presence of BMP antagonists (noggin, follistatin, or chordin). As shown in Figure 5, each BMP antagonist induced the expression of SMA in cultured stage X posterior epiblasts. When the explants were treated with both BMP and chordin, the expression of SMA was similar to that observed in control explants. Sarcomeric α-actinin, the expression of which is induced by endoderm-derived BMP signals, was not found in explants treated with BMP antagonists (data not shown). By contrast, when noncardiogenic anterior epiblasts (#1 explant of stage X–XI embryos) were cultured in a medium containing chordin, the percentage expression of SMA was similar to that in control cultures (n = 26, data not shown). Results indicated that BMP antagonists were able to up-regulate the expression of SMA in the cultured stage X–XI posterior epiblast, in which the expression of SMA was not yet committed. However, the BMP antagonist chordin failed to induce the expression of SMA in noncardiogenic anterior epiblast.
Lefty-1 Inhibits the Expression of SMA in Cultured Posterior Blastoderm
Lefty-1 is a divergent member of the TGF-β family and acts as an antagonist to the nodal signalling pathway (Schier, 2003). To investigate whether lefty could inhibit the expression of SMA in cultured posterior blastoderm of stage X–XI embryos, stage X–XI #6 blastoderm explants (Fig. 1A) were cultured in medium with or without recombinant lefty-1, and cultures were assessed the expression of SMA and sarcomeric α-actinin immunologically. As shown in Figure 6, when the explants were cultured in the defined medium alone, 93% of explants expressed SMA and 40% sarcomeric α-actinin after 48 hr. Conversely, in explants cultured in the medium containing recombinant lefty-1, the expression of SMA and sarcomeric α-actinin was down-regulated in a dose-dependent manner (Fig. 6). RT-PCR showed that the reduced PCR products of SMA were apparent when the explants were cultured in medium containing 1 μg/ml of lefty-1 (data not shown). Results suggested that nodal appears to be one of signalling molecules that are regulating the expression of SMA during early cardiogenesis.
Nodal plus BMP Antagonist Up-Regulates and Enhances the Expression of SMA in Cultured Posterior Epiblast
The results mentioned above indicated that either lefty-1 or BMP was able to inhibit the expression of SMA in cultured posterior blastoderm, and BMP antagonist, but not nodal alone, was able to induce the expression of SMA in cultured preactivated posterior epiblast. Therefore, results suggested that nodal plus BMP antagonist might up-regulate/enhance the expression of SMA in cultured stage X–XI posterior epiblast. To examine whether nodal plus BMP antagonist was capable of enhancing the expression of SMA in cultured posterior epiblast, posterior epiblast explants of stage X–XI embryos were cultured in medium supplemented with recombinant nodal, nodal plus CFC2 (nodal co-factor) or nodal plus BMP antagonist for 48 hr. As shown in Figure 7, when the explants were treated with nodal alone or nodal plus CFC2, there was no detectable staining of SMA in Western blot. Explants treated with follistatin yielded a faint band of SMA (Fig. 7). By contrast, when the explants were treated with follistatin plus nodal, the expression of SMA was up-regulated and gave an intense band of SMA in Western blot (Fig. 7). Explants treated with chordin plus nodal also showed a similar result (data not shown). The expression of SMA induced by nodal plus BMP antagonist was down-regulated by addition of lefty-1 (data not shown).
Posterior and Posterolateral Epiblast Contain Prospective Cardiomyocytes
Several fate-mapping analyses of cardiac progenitor cells have been conducted and have provided evidence that cardiogenic mesoderm is localized to the anterior lateral mesoderm (Rosenquist and DeHaan, 1966; Redkar et al., 2001; Yutzey and Kirby, 2002). However, there is less of a consensus regarding the origin of the heart mesoderm (or cardiogenic cells) in the prestreak-stage blastoderm. Hatada and Stern (1994) mapped the pregastrula epiblast using a vital-dye injection method and reported that the cardiac progenitors originated in the posterior epiblast at stages XII–XIII before gastrulation. This localization of presumptive heart cells in the posterior epiblast is supported by explantation experiments, in which posterior epiblast cells were found to have the potential to generate cardiomyocytes in a serum-free defined medium (Yatskievych et al., 1997; Ladd et al., 1998). Our regionally divided blastoderm (epiblast + hypoblast) culture experiment (Fig. 1) showed that, in addition to the posterior epiblast, the posterior lateral and posterior half of the anterior lateral epiblast also contained cardiogenic cells and that they differentiated into cardiomyocytes expressing Nkx2.5, GATA4, and sarcomeric α-actinin. Accordingly, at the onset of gastrulation, presumptive heart-forming cells in the posterior lateral epiblast migrate to the midline region of the epiblast and form the primitive streak at Hamburger and Hamilton stage 2–3 (early primitive-streak stage; Hamburger and Hamilton, 1951). At the primitive-streak stage, fate-mapping analysis showed that prospective heart cells, including those of the myocardium, endocardium and epicardium, are localized to the rostral half of the primitive streak (Garcia-Martinez and Schoenwolf, 1993). These presumptive heart cells are among the first mesodermal cells to gastrulate through the primitive streak. They eventually migrate to the anterior lateral mesoderm, resulting in the formation of cardiogenic mesoderm (Schoenwolf and Garcia-Martinez, 1995). According to these fate-mapping and regional explantation experiments, the presumptive heart-forming cells at prestreak and primitive-streak stages are clarified in early embryos. However, there is less of a consensus regarding the migration route that is used by heart progenitor cells to migrate from the posterior epiblast to the anterior lateral-plate mesoderm during gastrulation.
Tissue Interaction Between the Epiblast and Hypoblast Is Required to Initiate the Expression of SMA in Cultured Early Posterior Epiblast
SMA, which is expressed initially in the heart-forming mesoderm at mRNA and protein levels by stage 4, is the initial heart α-actin as well as a marker protein of the anterior lateral-plate mesoderm (Ruzicka and Schwartz, 1988; Saint-Jeannet et al., 1992; Sugi and Lough, 1992; Colas et al., 2000; our unpublished observations). In the initial myofibrillogenesis that occurs during chick cardiogenesis, at least two signalling pathways are involved in the expression of cardiac genes: one is an upstream pathway that is responsible for the expression of SMA (Nakajima et al., 2002); the other is BMP signalling, which is involved in the expression of the cardiac transcription factor Nkx2.5, as well as sarcomeric proteins, such as sarcomeric α-actinin, titin, and sarcomeric myosin (Schultheiss et al., 1997; Walters et al., 2001; Nakajima et al., 2002). To decipher the molecular mechanisms that regulate the expression of SMA during initial cardiogenesis, we cultured avian posterior epiblast with or without associated hypoblast and found that only 20–30% of the presumptive heart-forming epiblast explants of stage X–XI embryos expressed SMA. By contrast, more than 80% of epiblast explants from stage XII–XIII embryos expressed SMA without co-cultivation of the associated hypoblast. Furthermore, stage XI posterior hypoblast up-regulated the expression of SMA in noncardiogenic anterior epiblast of stage X–XI embryos. Accordingly, posterior hypoblast can induce, instructively, the expression of SMA during early cardiogenesis. It has been reported that the heart-inducing activity produced by the hypoblast is not replaced by stage 5 anterior endoderm; therefore, the presumptive heart-inducing signals from the hypoblast are different from those produced by heart-forming endoderm (Yatskievych et al., 1997). The hypoblast is established from at least two populations of cells: one component migrates anteriorly from the posterior marginal zone and Koller's sickle, the other component is an ingress from the epiblast at around stage X–XIII (Eyal-Giladi, 1984; Stern, 1990; Bachvarova et al., 1998). Stage X–XI early posterior epiblast explants failed to generate a high incidence of SMA-positive tissue in comparison with those co-cultured with the associated hypoblasts. Therefore, a specific cellular population(s) in the hypoblast, which may originate from Koller's sickle as well as from the posterior marginal zone, might be able to stimulate the expression of SMA in the responding epiblast cells. These results indicated that the interaction between the tissues of the posterior epiblast and the subjacent hypoblast during the early pregastrula stage (stage X–XI) was a prerequisite for the initiation of the expression of SMA in the developing anterior lateral mesoderm and nascent cardiomyocyte. It was suggested that the interactions between the tissues of the two germ layers might contribute to the generation of endoderm cells, which are involved in heart specification and terminal differentiation at late gastrula stages.
Nodal Is likely To Be an Endogenous Molecule Induces SMA Expression During Early Cardiogenesis
Our previous experiments showed that at least two signalling pathways are involved in initial cardiac myofibrillogenesis. One is an unknown pathway, which was regulated by interactions between the tissues of the posterior epiblast and the adjacent hypoblast and is responsible for the expression of SMA (present study). The other is BMP signalling, which is involved in the expression of sarcomeric α-actinin, titin, and sarcomeric myosin (Nakajima et al., 2002). In the present experiment, we showed that exogenously administered TGF-β, activin, BMP antagonist, or nodal plus BMP antagonist induced the expression of SMA in cultured posterior epiblast explants from stage X–XI embryos, in which the expression of SMA was not yet committed. Therefore, TGF-β or activin/nodal signalling(s) seemed to initiate the expression of SMA during early cardiogenesis in vivo. However, neutralizing antibodies against TGF-β1–3, as well as follistatin, did not inhibit the expression of SMA in cultured posterior blastoderm. So, TGF-β or activin were not likely to be endogenous molecules that act on the epiblast cells to initiate heart mesoderm or initial cardiomyocyte development in vivo. By contrast, lefty-1 inhibited the expression of SMA in cultured posterior blastoderm, suggesting that nodal is likely to be an endogenous molecule responsible for the SMA-positive heart mesoderm formation. Nodal belongs to a TGF-β-related signal molecule that appears to be involved in primitive-streak and mesoderm development. In fact, it has been shown that mouse embryos that lack nodal function fail to form a primitive streak and do not undergo mesoderm development (Conlon et al., 1994; Schier and Shen, 2000). Furthermore, nodal and its co-factor, CFC transcripts are found in the posterior region of the chick blastoderm (Schlange et al., 2001; Chapman et al., 2002). Yatskievych et al. (1997) reported that TGF-β or activin can induce the generation of cardiomyocytes in cultured posterior epiblast and that this signalling occurs upstream of the endoderm-derived BMP, which acts on the anterior lateral mesoderm and specifies the cardiomyocyte lineage. The results shown here, as well as the results of other researchers, show that cardiac myogenesis is probably regulated by at least two temporally distinct signals: an early signal that acts on the epiblast (mimicked by activin-like signal) and later endoderm-derived signals, involving BMP, FGF8 and Crescent, that act on the mesoderm (Lough et al., 1996; Schultheiss et al., 1997; Marvin et al., 2000; Alsan and Schultheiss 2002). Our results suggest strongly that nodal is one of the endogenous molecules that are acting on the epiblast to induce the expression of SMA during early cardiogenesis. Further experiments are necessary to elucidate the molecular natures that are responsible for interactions between tissues of the early posterior epiblast and its adjacent hypoblast.
BMP Antagonist Plays an Important Role in the Regulation of SMA Expression
In this study, we have shown that natural BMP antagonists, such as follistatin, noggin, and chordin, induced the expression of SMA in cultured preactivated early posterior epiblast explants of stage X–XI embryos. Another experiment showed that coadministration of nodal and BMP antagonist up-regulated and enhanced the expression of SMA in cultured posterior epiblast in comparison with cultures treated with BMP antagonist. Furthermore, BMP inhibited the expression of SMA at mRNA and protein levels in cultured posterior blastoderm from which cardiomyocytes would later develop. Therefore, strong or predominant BMP signals in the cardiogenic region seemed to inhibit the formation of precardiac mesoderm that expresses SMA.
In Xenopus laevis, BMP counteracts nodal/activin-like signalling; therefore, it could be suggested that the balance between the phospho-SMAD1 and -SMAD2 is important for determining the mesoderm specification (Massague, 2003). In the developing pregastrula and early gastrula embryos, chordin is expressed in the posterior epiblast cells that are just anterior to Koller's sickle. This expression eventually becomes restricted to the anterior primitive-streak region, from which heart mesoderm will later develop (Lawson et al., 2001; Chapman et al., 2002). Chordin is a natural BMP antagonist and was identified in X. laevis animal caps that were treated with activin. When overexpressed, chordin can recapitulate most of the activities of the organizer (Sasai et al., 1994, 1995). Targeting chordin in X. laevis with antisense morpholino oligomers showed that embryos develop with reduced dorsoanterior structures and expanded ventroposterior tissues (Oelgeschlager et al., 2003). In zebrafish embryos, the chordin homologue chordino has been identified in a ventralizing mutant, in which gene expression of the dorsal marker is reduced and dorsoanterior structures are defective (Schulte-Merker et al., 1998). In the mouse, double-homozygous mutants for chordin and noggin show severe defects in the development of the forebrain, indicating that BMP antagonists are required for head development as well as dorsoanterior structures (Bachiller et al., 2000). In addition to the induction of the forebrain, our results indicate that BMP antagonists seem to play an important role in the formation of the anterior lateral-plate mesoderm (heart-forming mesoderm).
In our experiments, hypoblast, activin/nodal signalling or BMP antagonist induced the expression of SMA, which is initially expressed in heart-forming mesoderm and is then subsequently recruited into the early cardiac sarcomere. Recent experiments showed that the hypoblast of the chick pregastrula plays a role in the formation of the anterior–posterior axis, as well as in forebrain induction (Foley et al., 2000; Bertocchini and Stern, 2002; Chapman et al., 2003). It has been reported that patterning of anterior–positional identity and specification of neural identity are separable events—that is, the initial anterior–positional identity is established in the epiblast independently by the hypoblast before neural specification occurs (Chapman et al., 2003). Interestingly, this two-step model of forebrain formation is compatible with early cardiogenesis—that is, a signal(s) from the hypoblast is responsible for the expression of SMA (early lateral-plate mesoderm marker and the initial α-actin of the sarcomere); endoderm-derived signals then regulate heart specification as well as terminal differentiation. Further investigations are necessary to elucidate the definitive molecular nature of the signals responsible for early cardiogenesis.
Stage X–XIII pregastrula blastoderms (Eyal-Giladi and Kochav, 1976; incubation time, 0–3 hr) were collected on ice-cooled phosphate-buffered saline (PBS), and cut into 12 pieces (Fig. 1A) using a sharp tungsten needle. The resulting blastoderm explants (regions #1–6) were explanted onto bovine fibronectin-coated (GIBCO) chamber slides (Nunc) and cultured in a serum-free defined medium (75% DMEM, 25% McCoy's medium, supplemented with 10−7 M dexamethasone and penicillin-streptomycin; Ladd et al., 1998). The posterior blastoderm region (region #6, Fig. 1A) containing two layers—epiblast and hypoblast (but not including Koller's sickle)—was excised carefully to prevent contamination with sickle. It was then separated into epiblast and hypoblast using a thin tungsten needle or an eyebrow hair. The region #6 explant was cultured in the defined medium alone or under various test conditions, in which the medium contained either recombinant activin (donated by Dr. M. Asashima), follistatin (donated by Dr. M. Asashima or purchased from Wako, Tokyo, Japan), TGF-β3, noggin, BMP4, chordin, nodal, CFC2, or lefty-1 (R&D). A heterotypic culture (quail epiblast + chick hypoblast) was performed, as described in Figure 5.
Indirect Immunofluorescence Microscopy
Immunohistochemistry was performed, as described by Nakajima et al. (2002). Cultures were drained of medium and then rinsed with PBS. They were then fixed with 4% paraformaldehyde/PBS for 30 min at room temperature, and again rinsed with PBS. Specimens were blocked for 1 hr with 1% bovine serum albumin/PBS containing 0.1% Triton X-100, and then incubated with primary antibody (or primary antibody mixture) at 4°C overnight. They were then rinsed with PBS and incubated with fluorescein isothiocyanate (FITC) -conjugated or rhodamine isothiocyanate (RITC) -conjugated secondary antibody (or secondary antibody mixture) for 1 hr at room temperature. Samples were observed under a fluorescence microscope using narrow-band mirror units (U-MNIBA and U-MNG; Olympus) and a laser confocal microscope (Zeiss).
The monoclonal antibodies anti-Z-line titin (anti-titin, clone 9D10, IgM; and anti-zeugmatin, clone mab20, IgG2a; Maher et al., 1985; Turnacioglu et al., 1997) and anti-skeletal muscle sarcoplasmic reticulum (clone 12/101, IgG1; Griffin et al., 1987) were obtained from the Developmental Studies Hybridoma Bank (Iowa). The monoclonal antibodies anti-smooth muscle α-actin (clone 1A4, IgG2a; Skalli et al., 1986) and anti-sarcomeric α-actinin (clone EA53, IgG1) were purchased from Sigma. For double immunohistochemistry, we used FITC-conjugated goat anti-mouse IgG2a, RITC-conjugated goat anti-mouse IgG2a, FITC-conjugated goat anti-mouse IgG1, FITC-conjugated goat anti-mouse IgG2b, and RITC-conjugated goat anti-mouse IgM (Southern Biotechnology) as secondary antibodies.
Explants were collected and RNA extracted, as described by Yamagishi et al. (1999). cDNAs were synthesized from 2 μg of total RNA, and PCR was carried out in 10 μl of reaction buffer (0.2 mM dNTPs, 1 mM primers and 0.05 U of Taq DNA polymerase). The sequences of the primers have been described elsewhere—Nkx2.5 (Schultheiss et al., 1995), GATA4 (Schultheiss et al., 1997), MyoD (pair “a”; Lin-Jones and Hauschka, 1996), sarcomeric α-actinin (Nakajima et al., 2002); glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Yamagishi et al., 1999). Amplification of chick SMA transcripts was performed using primers specific for chick SMA (5′-CCACTTACAACAGCATCATG-3′ and 5′-CCAGCCATTACGATGAAAGA-3′). Samples were cycled at 93°C for 30 sec at the annealing temperature (sarcomeric α-actinin, 57°C; Nkx2.5, 57°C; GATA4, 57°C; MyoD, 55°C; GAPDH, 45°C; SMA, 55°C), and at 72°C for 90 sec, with a final extension at 72°C for 10 min. The cycle numbers for the various primers were as follows: sarcomeric α-actinin, 26; Nkx2.5, 30; GATA4, 26; MyoD, 28; SMA, 23; and GAPDH, 28. PCR products were electrophoresed and stained with ethidium bromide.
Explants were homogenized in sample buffer (62.5 mM Tris, 0.1% glycerol, 2% sodium dodecyl sulfate (SDS), and 5% 2-mercaptoethanol, pH 6.8). After heat-denaturation (95°C for 5 min), equal amounts of protein obtained from 15 explants were subjected to SDS-polyacrylamide gel electrophoresis (10% polyacrylamide), and then transferred to Immunobilon-P membrane (Millipore). The membrane was subsequently treated with 5% nonfat dry milk, and was incubated with the primary antibody against SMA or GAPDH for 2 hr at room temperature. After extensive washing, the membrane was incubated with peroxidase-conjugated secondary antibody for 2 hr. Immunoreactive bands were visualized by using ECL detection reagent (Amersham).
The authors thank Dr M. Asashima, University of Tokyo, for donating recombinant activin and follistatin. The monoclonal antibodies used were obtained from the Developmental Studies Hybridoma Bank, which was developed under the auspices of the National Institute of Child Health and Development, and which is maintained by the University of Iowa, Department of Biological Sciences, Iowa City, Iowa. Y.N. was funded by the Ministry of Education, Science and Culture of Japan, The Japan Cardiovascular Research Foundation, The Takeda Science Foundation, and The Terumo Life Science Foundation.