The mesoderm arises during vertebrate gastrulation as a distinct cell layer situated between the ectoderm and the endoderm (Schoenwolf and Smith, 2000). During and shortly after gastrulation, the mesoderm becomes segregated into distinct precursor cell populations in response to signals from the primitive streak or adjacent cell layers. The heart arises from anterior lateral (AL) mesoderm, and although the gene regulatory pathways governing cardiac myogenesis have not been fully defined, the transition from emerging mesoderm to differentiated cardiac myocytes involves multiple regulatory events (Srivastiva and Olson, 2000).
In chick, cells fated to form the heart arise from anterior portions of the primitive streak just posterior to Hensen's node (Garcia-Martinez and Schoenwolf, 1993). Premyocardial cell specification appears to begin during gastrulation (Antin et al., 1994; Montgomery et al., 1994; Gannon and Bader, 1995; Ladd et al., 1998) and is dramatically enhanced by later signals from AL endoderm (reviewed in Schultheiss and Lassar, 1999; Sugi and Lough, 2000). AL endoderm produces bone morphogenetic protein 2 (BMP2) beginning at Hamburger and Hamilton (HH) stage 5 (Andrée et al., 1998; Schultheiss et al., 1997), and experiments have shown that the BMP inhibitor noggin can block the appearance of differentiated cardiac myocytes if applied before stage 6, at least 8 hr before the onset of myocyte differentiation (Schultheiss et al., 1997; Andrée et al., 1998; Ladd et al., 1998; Schlange et al., 2000). BMP signaling, therefore, plays an important role in early stages of myocardial cell development.
Mechanisms regulating myocardial cell differentiation are less well defined. In chick, cardiac myocyte differentiation begins around stage 8 (González-Sanchez and Bader, 1990; Han et al., 1992) and continues for several days. Differentiation of cardiac myocytes is marked by the transcriptional activation of a large number of genes that code for proteins involved in both structural and metabolic processes. Many of these proteins comprise the contractile myofibril. Although, in skeletal muscle, myofibrillar proteins appear sequentially during myoblast differentiation (Fürst et al., 1989; Lin et al., 1994), in heart muscle, it appears that the major myofibrillar components are expressed almost simultaneously at the onset of myocyte differentiation (Ehler et al., 1999; Rudy et al., 2001). Components of thick and thin filament systems appear to assemble independently and are then integrated to form the contractile myofibril (Ehler et al., 1999; Gregorio and Antin, 2000; Rudy et al., 2001).
Major protein constituents of cardiac thin filaments include actin, tropomyosin, and troponin. The troponin complex, consisting of troponin C (TNC; calcium binding), troponin I (TNI; inhibitory), and troponin T (TNT; tropomyosin binding), confers calcium sensitivity to striated muscle contraction (Tobacman, 1996). Troponin T is a protein of approximately 35 kDa that is responsible for binding of the troponin complex to tropomyosin (Perry, 1998). Chicken cardiac troponin T (cTNT) is initially expressed in both embryonic skeletal and cardiac muscle but is transcriptionally down-regulated in skeletal muscle beginning at midgestation (Cooper and Ordahl, 1985). Although it is generally accepted that components of the contractile myofibril are expressed specifically within differentiated cardiac and/or skeletal muscle cells, expression of some contractile protein genes has been detected in nonmuscle cell types. Expression of chicken slow TNT, for example, has been documented in the liver (Berezowsky and Bag, 1992; Grewal and Bag, 1996), and cTNT transcripts are present in avian limb mesoderm before skeletal muscle cell differentiation (Swiderski and Solursh, 1990). Expression of the mouse cTNT gene is first detected at day 7.5 post coitum in the lateral plate mesoderm before the onset of myocardial cell differentiation (Wang et al., 2001).
While investigating growth factor regulation of cardiac myogenesis in chick, we noticed that cTNT mRNAs were detectable by reverse transcriptase-polymerase chain reaction (RT-PCR) at least 12 hr before the onset of heart muscle cell differentiation, and at least 30 hr before the appearance of the first somitic skeletal muscle cells. We therefore undertook an analysis of cTNT gene expression in early avian embryos. Here we show that cTNT transcripts are initially present throughout much of the mesodermal layer of late gastrula stage embryos. As development progresses, cTNT transcripts become progressively localized to the heart-forming region and, by the early looping stages, are restricted to differentiated cardiac myocytes. cTNT protein, in contrast, is first detected at stage 9 around the onset of cardiac myocyte differentiation. cTNT expression in lateral mesoderm does not require the endoderm and is independent of BMP signaling, even in the heart-forming region. These findings define a BMP independent pathway for the precocious transcription of one component of the troponin complex in cardiac myocytes.
Developmental Expression of cTNT
While investigating signaling interactions regulating cardiac myogenesis, we observed that cTNT mRNAs were detectable in chick embryos during mid- to late gastrula stages, at least 12 hr before the onset of cardiac myocyte differentiation. To compare cTNT expression with other genes expressed during early heart development, RT-PCR analyses were performed by using RNA isolated from whole embryos at HH stages 5, 7, or 11. At stage 5, premyocardial cell specification is under way; at stage 7, a significant proportion of premyocardial cells are determined but have not yet begun to differentiate; whereas at stage 11, a fully formed, beating heart tube is present. As shown in Figure 1, both Nkx 2.5 and cTNT mRNAs were detectable at stage 5. In contrast, mRNAs coding for two genes expressed in differentiated cardiac myocytes, ventricular myosin heavy chain (vMHC) and cTNC, were detectable at stage 11 but not in mRNA samples from stages 5 or 7 embryos.
To investigate the spatial and temporal expression pattern of cTNT expression, whole-mount in situ hybridization analyses were performed on stage 4–12 embryos by using a probe generated from the full-length cTNT cDNA. cTNT mRNAs were first detected at stage 4 in mesoderm on either side of the anterior portion of the primitive streak, and in extraembryonic mesoderm (Fig. 2). By stages 5–6, cTNT expression was observed throughout the lateral and extraembryonic mesoderm, with particularly robust expression within the AL precardiac regions (Fig. 2C). Embryo sections showed that cTNT transcripts were present in the AL region within both the splanchnic and somatic mesoderm (Fig. 2H) and throughout the extraembryonic mesoderm. By stage 9, as cardiac myocyte differentiation commenced, cTNT expression within extraembryonic mesoderm diminished and became more localized to the anterior embryonic heart-forming regions (Fig. 2E,F). cTNT expression continued within mesoderm lateral to the forming heart through stage 10. As the heart tube became fully formed, cTNT expression was progressively more restricted to the myocardium. By stage 12, cTNT mRNAs were present exclusively within differentiated cardiac myocytes (Fig. 2G,I).
To investigate the onset of cTNT protein accumulation, homogenates of stage 4–10 embryos were analyzed by Western blot. As shown in Figure 3, cTNT protein was not detectable until stage 9, concomitant with the onset of cardiac myocyte differentiation.
cTNT Expression Is Independent of Endoderm and BMP Signaling
Chick AL endoderm plays an important role in the activation and maintenance of cardiogenic genes such as Nkx2.5, eHAND, and Mef2C in adjacent mesoderm (Schultheiss and Lassar, 1999; Sugi and Lough, 2000), ultimately leading to cardiac myocyte differentiation and activation of a large number of genes coding for myofibrillar proteins. Removal of endoderm from HH stage 5–6 embryos inhibits expression of Nkx 2.5 and Mef2C (Alsan and Schultheiss, 2002). Two experiments were performed to determine whether cTNT expression requires endoderm-derived signals. First, anterior endoderm was removed from one side of stage 4+ embryos, with the contralateral side serving as an internal control. After 12–24 hr of incubation, embryos were processed for whole-mount in situ hybridization by using the cTNT probe. When embryos in which endoderm had been unilaterally removed were allowed to develop to stage 6, cTNT expression was observed at control levels in lateral mesoderm lacking adjacent endoderm (Fig. 4A). Transverse sections verified that endoderm was absent from the operated side of the embryo (Fig. 4B). When embryos were allowed to develop until stage 10, endoderm removal resulted in cardia bifida (Fig. 4C). On the control side, a beating cTNT-positive heart structure formed in the AL region, whereas on the side lacking endoderm, cTNT was expressed at high levels despite the absence of a three-dimensional heart structure or beating.
To determine whether cTNT expression within the heart field is dependent on BMP2 signaling, aggregates of CHO cells expressing the BMP antagonist noggin were implanted into the anterior region of chick embryos at the late gastrula stage (HH stage 5). Embryos were allowed to develop for 8 hr and then processed for whole-mount in situ hybridization. As previously reported (Schultheiss et al., 1997; Andrée et al, 1998; Schlange et al., 2000), noggin effectively inhibited expression of Nkx 2.5 (Fig. 5C; 5 of 5 embryos showed absence of Nkx 2.5 staining in the region of noggin-expressing cells). In contrast, noggin had no effect on cTNT expression in AL mesoderm of stage 6–7 embryos (Fig. 5A,B; eight of eight embryos showed no inhibition of cTNT mRNA levels by noggin-expressing cells). When noggin-expressing cells were implanted near the anterior midline before stage 7 and the embryos allowed to develop until control embryos reached stage 10, heart formation was inhibited and bilateral cTNT-expressing regions of mesoderm were observed (Fig. 5D). Anterior somite development was also partially inhibited within the vicinity of the noggin-expressing cells (Andrée et al., 1998). Implantation of noggin-expressing cells at stage 7 near the anterior midline resulted in cardia bifida and two beating heart tubes on either side of the midline (Fig. 5E,F). In some cases, relatively normal hearts formed in which cTNT expression was restricted to the myocardium (Fig. 5F). Taken together, these results show that cTNT expression is independent of BMP2 or other signals from the endoderm and that BMP signaling plays a role in the morphogenetic movements required to bring the two heart fields together along the ventral midline.
Muscle cell differentiation involves the coordinate activation of a large number of genes, many of which code for protein constituents of the contractile myofibril. In contrast to skeletal myogenesis in which lineage identity is associated with expression of one or more of the myoD family of basic helix loop helix proteins (Olson, 1993), specification of premyocardial cells involves the coordinate expression of several classes of regulatory proteins, including but likely not limited to members of the Nkx, Gata, and SRF family of transcription factors (Balaguli et al., 1997; Patterson et al., 1998; Charron and Nemer, 1999). In chick, premyocardial cell specification begins during gastrulation and continues through neurula stages (Antin et al., 1994; Montgomery et al., 1994; Gannon and Bader, 1995; Ladd et al., 1998). Expression of Nkx 2.5 and Gatas 4–6 are detectable around stage 5, whereas the transcription factors Mef2C, dHAND, and eHAND are coexpressed in heart-forming cells beginning around stage 8, almost coincident with the onset of myocyte differentiation and expression of genes coding for myofibrillar proteins. It is important to note that these transcriptional regulatory genes are expressed only in anterior mesoderm that is in close proximity to BMP2- producing endoderm, which is restricted to a lateral arc that essentially defines the heart field (Schultheiss et al., 1997; Andrée et al., 1998; Ehrman and Yutzey, 1999). Expression of many differentiation-specific cardiogenic genes, therefore, is directly or indirectly downstream of BMP signaling.
An increasing number of reports, however, indicate that BMP signaling may not be required for all aspects of early cardiogenesis. Although treatment of heart-forming cells from stage 4 embryos with noggin results in loss of Nkx2.5, Gata4, eHAND, Mef2C, and vMHC expression, noggin fails to block vMHC transcription in explants from stage 5–8 embryos and Gata4 expression is actually enhanced by inhibition of BMP signaling (Schlange et al., 2000). Expression of the transcription factor TBX5 (Yamada et al., 2000) and the LEK-related protein CMF-1 (Pabón-Peña et al., 2000) is also independent of BMP signaling.
Here, we show that transcripts encoding cTNT, a protein whose only known function is as part of the troponin complex of myofibrils in cardiac and embryonic skeletal muscles, are present in the chick embryo beginning at stage 4, at least 14 hr and several cell cycles before the onset of cardiac myocyte differentiation. At gastrula stages, cTNT transcripts are present throughout most of the mesodermal cell layer; as the heart tube forms and differentiated myocardial cells appear, transcripts become progressively more restricted to the myocardium. Three lines of evidence indicate that precocious transcription of the cTNT gene throughout the lateral and extraembryonic mesoderm is independent of BMP signaling. First, cTNT is expressed in a pattern that is far broader than BMP2 or BMP4 in the early embryo (Schultheiss et al., 1997; Andrée et al., 1998). Second, removal of endoderm from one side of stage 5 embryos has no effect on cTNT mRNA levels. Third, noggin fails to inhibit cTNT expression, even when noggin-expressing cells are placed within the heart-forming region.
By what mechanism, then, is cTNT transcription up-regulated in the lateral and extraembryonic mesoderm of gastrula stage embryos? Detailed analyses of cTNT promoter function conducted by Ordahl and colleagues have shown that just 99nt of cTNT upstream promoter sequence is sufficient to drive skeletal muscle-specific gene transcription (Mar et al., 1988; Mar and Ordahl, 1988; Mar et al., 1992). This proximal promoter fragment is also weakly active in cardiac muscle cells, although its activity is dramatically increased by an enhancer located between nt-247 and -201 (Mar et al., 1988; Ianello et al., 1991). Two closely spaced MCAT elements within the proximal promoter fragment are required for promoter activity in both muscle cell types. These elements bind TEF proteins, of which RTEF is enriched in striated muscle cells (Farrance et al., 1996). Binding of the ubiquitously expressed poly(ADP-ribose) polymerase (PARP) protein to TEFs and to DNA sequence immediately adjacent to the upstream MCAT element is required for muscle specificity of transcription (Larkin et al., 1996). Specificity of cTNT gene expression, therefore, is thought to arise through the binding of PARP and muscle-enriched forms of TEFs to cis elements and to each other, an interaction that is thought to result in poly(ADP) ribosylation of transcription complex proteins (Butler and Ordahl, 1999). In the context of the early avian embryo, precocious cTNT expression may result from the regional expression of specific TEF isoforms, which interact with ubiquitously expressed PARP. A detailed analysis of TEF isoform expression patterns in embryos has not yet been performed. The cardiac-specific enhancer element located immediately upstream of nt-200 in the cTNT promoter seems unlikely to be involved in the early and widespread cTNT gene expression reported here, because it shows no activity in nonmuscle cells and contains binding sites for cardiogenic regulatory factors that are expressed only within the cardiogenic regions and in differentiated muscle cells (Mar et al., 1988; Iannello et al., 1991).
BMP2 is expressed in the heart-forming region endoderm of chick embryos and plays an important role in the molecular regulation of avian cardiac myocyte development (Schultheiss et al., 1997; Andrée et al., 1998; Ladd et al., 1998). BMP signaling also appears to be required for heart morphogenesis, likely through an autocrine pathway. Removal of anterior endoderm from gastrula stage embryos causes cardia bifida (Fig. 4; Gannon and Bader, 1995), a phenotype that also results from implantation of noggin-expressing cells along the anterior midline (Fig. 5). Similar findings were reported in Xenopus after inhibition of BMP signaling (Walters et al., 2001). Although mice lacking a functional BMP2/4 signaling pathway are uninformative with regard to heart formation due to early embryonic lethality (Mishina et al., 1995; Beppu et al., 2000), Gata4 expression in foregut endoderm is dependent on BMP signaling (Rossi et al., 2001) and Gata4 null mice develop cardia bifida, a phenotype that can be rescued by wild-type endoderm (Narita et al., 1976). Reduction in levels of Gata4–6 also induces cardia bifida in chick, apparently due to defects in the endoderm (Jiang et al., 1998). These findings indicate that BMP signaling regulates cardiac morphogenesis at least partly through autocrine regulation of Gata transcription factors.
Despite the presence of cTNT mRNAs in embryos as early as stage 4, cTNT protein was not detectable by Western blot until stage 9, coincident with the onset of cardiac myocyte differentiation. Expression of cTNT protein, therefore, is under translational control. Swiderski and Solursh (1990) reported that cTNT mRNAs but not protein are also present in developing limb buds approximately 1 day before the onset of limb muscle cell differentiation. For both the late gastrula embryo and the limb bud, cTNT is expressed before the onset of differentiation and in many cells that will not ultimately become cardiac or skeletal muscle cells. As cardiac myocyte and limb muscle cell differentiation commences, however, cTNT expression becomes restricted to cardiac or skeletal myocytes. Similarities between cTNT expression in the late gastrula embryo and in the limb bud suggest that common mechanisms might regulate precocious cTNT gene transcription. It will be interesting to determine whether specific TEF isoforms are selectively expressed within these regions of the embryo.
Whole-Mount In Situ Hybridization
Embryos were collected as described (Yatskievych et al., 1997) and staged according to Hamburger and Hamilton (1951). After fixation in 4% paraformaldehyde in phosphate-buffered saline for 2–24 hr at 4°C, embryos were processed according to Nieto et al. (1996), except that proteinase K treatment was omitted for early gastrula stage embryos. Digoxigenin-labeled antisense cTNT probe was generated according to manufacturer's instructions (Roche) from the full-length cTNT cDNA (accession no. M10013), and hybridizations were carried out at high stringency. BLAST comparisons showed no significant homology of the cTNT sequence with any chicken or mammalian sequence, including other troponins. Some embryos were processed for in situ hybridization by using an Abimed hybridization robot. For histology, stained embryos were post-fixed, dehydrated through an ethanol series, embedded in paraffin, and serially sectioned.
Chick Embryo Culture
Embryos were removed from fertile chick eggs (Gallus domesticus) between HH stage 5 and 8 and cultured as described by New (1955). For some embryos, tungsten needles were used to tease the endoderm away from the mesoderm in the entire anterior region on one side of the embryo. Embryos were then incubated for 12 hr before fixation and processing for in situ hybridization. Aggregates of control or noggin-expressing CHO cells (gift of R. Harland) were prepared by placing 2,000–5,000 cells in hanging drop culture for 3 to 5 days in aMEM plus 5% fetal bovine serum. Resulting aggregates were implanted into the ventral side of the embryo through a small slit cut into the endoderm. Embryos were incubated for 6–24 hr and then processed for whole-mount in situ hybridization.
RNA was isolated from HH stage 5, 7, or 11 whole embryos by using an RNeasy kit (Qiagen, Chatsworth, CA). RNA was treated with 1 U RNAse-free DNAse (Statagene, La Jolla, CA) for 15 min and then re-purified. RT and PCR reactions were performed as previously described (Ladd et al., 1998). Glyceraldehyde-3-phosphate dehydrogenase (51°C), cTNC (56°C), Nkx 2.5 (51°C), and vMHC (56°C) primer sequences and amplification conditions are as described (Schultheiss et al., 1995). cTNT primer sequences were 5′-CAAACTGAGGGACAAAGCCAAG-3′ (forward) and 5′-TGGGGGTGTGGAGATGAGAATC-3′ (reverse), generating a 373-bp PCR product. Cycle number for each primer pair was chosen to fall within linear range of amplification by using positive control RNA samples, and all PCR products were sequenced to confirm their identity.
Western Blot Analysis
Western blots were performed as described by Towbin et al. (1979). Whole embryos between HH stages 3–11 were homogenized directly into gel-loading buffer, boiled for 5 min, electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Membranes were incubated in 5% nonfat dry milk in 1× TTBS buffer (0.1% Tween 20, 100 mM Tris-HCl, pH 7.5, and 0.9% NaCl) at room temperature for 1 hr, washed in 1× TTBS, and then incubated in 1× TTBS/1% BSA plus an affinity purified rabbit immunoglobulin (Ig) G fraction (5 μg/ml final concentration) specific for cTNT (Toyota and Shimada, 1981). After washes and incubation with horseradish peroxidase-conjugated donkey anti-rabbit IgG, peroxidase was visualized by using SuperSignal substrate (Pierce).
We thank Y. Toyota for the gift of the cTNT antibody, and R. Harland for the gift of Noggin-expressing CHO cells. P.B.A. received funding from the NIH.