Nkx2-5 activity is essential for cardiomyogenesis.

The homeobox transcription factor tinman is essential for heart vessel formation in Drosophila. In contrast, mice lacking the murine homologue Nkx2-5 are defective in cardiac looping but not in cardiac myocyte development. The lack of an essential role for Nkx2-5 in cardiomyogenesis in mammalian systems is most likely the result of genetic redundancy with family members. In this study, we used a dominant negative mutant of Nkx2-5, created by fusing the repressor domain of engrailed 2 to the Nkx2-5 homeodomain, termed Nkx/EnR. Expression of Nkx/EnR inhibited Me(2)SO-induced cardiomyogenesis in P19 cells but not skeletal myogenesis. Nkx/EnR inhibited expression of cardiomyoblast markers, such as GATA-4 and MEF2C, but not of mesoderm markers, such as Brachyury T and Wnt5b, or of skeletal lineage markers, such as MyoD and Mox1. To identify the minimal region of Nkx2-5 that can trigger cardiomyogenesis, we analyzed the activity of various Nkx2-5 deletion mutants. The C-terminal domain was not necessary for the ability of Nkx2-5 to induce cardiomyogenesis and loss of this domain did not enhance myogenesis. Therefore, Nkx2-5 function is essential for commitment of mesoderm into the cardiac muscle lineage, and the N-terminal region, together with the homeodomain, is sufficient for cardiomyogenesis in P19 cells.

There are several families of transcription factors that play a role in cardiomyogenesis, including the Nkx2, GATA, and MEF2 families. In the fruit fly, tinman is expressed in the early mesoderm and the dorsal vessel and is essential for dorsal vessel formation (1,2). A vertebrate homologue of tinman, Nkx2-5 or Csx, is initially present in cardiac progenitors and the pharyngeal endoderm, as early as 7.5 days postcoitum (3). Mice homozygous for a null mutation in Nkx2-5 still form the heart tube and express most cardiac-specific genes (4 -6). The heart tube bends correctly but fails to undergo complete looping, possibly as a result of abnormal muscle growth. The finding that Nkx2-5 is not essential for the formation of cardiomyocytes in mice, but is essential in Drosophila, may be attributed to an overlapping of function between the family of Nkx2-5-related genes in vertebrates (7).
Because characterization of the role of Nkx2-5 in vertebrate early heart formation has been complicated by a potential functional redundancy of the Nkx homologues, single knock-outs have not been completely informative. To circumvent this problem, the genetic redundancy of Nkx and its requirement for cardiac specification and determination were investigated in a novel approach using dominantly acting repressor constructs specific for chosen Nkx homologues (8,9). These dominant repressors were made by fusion of the active repressor domain of the Drosophila engrailed homeodomain protein to the homeodomain of Xenopus Nkx family members (10,11). In this way, co-ectopic expression of the chimeric constructs for both XNkx2-3 and XNkx2-5 in developing Xenopus embryos was shown to be synergistic in completely blocking heart formation and cardiac gene expression such as XNkx2-5, Xenopus cardiac troponin I, and XMLC2. Therefore, these genes are required for the earliest stages of cardiac development in a functionally redundant manner.
Detailed structure/function studies have been performed on Nkx2-5 using transient transfection assays. In the homeodomain region, an amino acid substitution from a conserved asparagine to a glutamine at position 52 in helix 3 blocked DNA binding and reporter gene activation, demonstrating the importance of the homeodomain in binding specificity. Immediately N-terminal to the homeodomain is a putative activation domain, a region of high charge density with overlapping alanine-and proline-rich sequences. This domain appears to be masked by a C-terminal inhibitory region rich in alanine and proline (12)(13)(14). Deletion of the C-terminal region domain causes a 50 -300-fold enhancement of Nkx2-5 function, resulting in activity that is similar in strength to wild type Nkx2-5 in the presence of GATA-4. Consequently it has been proposed that the binding of GATA-4 to the homeodomain changes the protein conformation and relieves the inhibition by the C terminus.
Embryonal carcinoma cells, such as the P19 cell line, are an attractive alternative to the embryo in the identification and analysis of components and events that comprise cardiomyogenesis. P19 cells are capable of differentiating into a variety of cell types representative of all three germ layers. Cellular aggregation in the presence of dimethyl sulfoxide (Me 2 SO) results in the development of cardiac muscle by day 6 of differentiation, whereas the development of skeletal muscle is not observed until day 9 of differentiation. On day 6 of differentiation, markers found in cardiac muscle, such as GATA-4, MEF2C, and cardiac ␣-actin, are observed, whereas skeletal muscle-specific markers, such as MyoD and myogenin, are absent. By day 9, markers for both cardiac and skeletal muscle are present (for review, see Ref. 20). The cardiac myocytes derived from P19 cells display the biochemical and physiological properties that occur during early embryonic development (21)(22)(23). Aggregated P19 cells provide the only environment identified to date that will support the induction of cardiomyogenesis by Nkx2-5, MEF2C, or GATA-4 (24,25).
In the present study, we have used a dominant negative approach to determine whether Nkx2-5 function is essential for cardiomyogenesis in P19 cells. Expression of the dominant negative Nkx2-5 protein completely abolished cardiomyogenesis but not skeletal myogenesis. Furthermore, the N-terminal domain and homeodomain define the minimal region of Nkx2-5 necessary for its function in P19 cells.

EXPERIMENTAL PROCEDURES
Plasmid Constructs-Nkx2-5 full-length (Nkx2-5) cDNA was kindly provided by R. Harvey. The Nkx2-5 deletion mutants were constructed by polymerase chain reaction (PCR) 1 amplification with appropriate primers corresponding to the fragment end points. The cDNA fragment Nkx⌬N was constructed by PCR of the homeodomain and the C-terminal domain (aa 137-319) using the 5Ј-oligonucleotide (oligo) P7 (5Ј-CGC CAC CAT GCG ACG GAA GCC ACG CGT GCT CTT C-3Ј) and the 3Ј-oligo P8 (5Ј-ggatcc CTA CCA GGC ACG GAT GCC GTG CAG C-3Ј). The cDNA fragment NkxHD was constructed by PCR of the homeodomain (aa 137-197) using the 5Ј-oligo P7 and the 3Ј-oligo P6 (5Ј-ggatcc CTA CCG CTG TCG CTT GCA CTT GTA GCG-3Ј). The cDNA fragment Nkx⌬C contained the N-terminal domain and homeodomain of Nkx2-5 (aa  and was PCR-amplified using the 5Ј-oligo P5 (5Ј-C gagctc GCG CTG TGA AAC CTG CGT CGC CAC C-3Ј) and the 3Ј-oligo P6. Each of the deletion products (except those primed with P7) were designed to contain flanking SstI and BamHI (indicated above in lowercase) restriction enzyme sites to facilitate directional cloning into those two sites in the phosphoglycerate kinase (PGK) vector multiple cloning site.
The DNA construct Nkx/EnR contains the 768-bp EN2-Nkx2-5HD fusion cDNA. The 196-aa N-terminal repression domain of the mouse engrailed-2 (EN2) protein was fused to the N terminus of the 60-aa Nkx2-5 homeodomain by PCR of EN2 using the 5Ј-oligo (5Ј-G gagctc GCC ACC ATG GAG GAG AAG GAT TCC-3Ј) and the 3Ј-oligo (5Ј-G ctcgag GGC TTT CTT GAT TTT GGC CCG CTT G-3Ј). Both SstI-and XhoI-flanking sites were engineered into the EN2 product to facilitate in-frame fusion with the homeodomain.
All PCR reactions were performed using Qiagen Taq DNA polymerase using Q-Solution under the following conditions: initial cycle 6 min in duration at 94°C; 25 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C; and a final cycle for 6 min at 72°C. All plasmids were verified by DNA sequencing (Robarts Research Institute Sequencing Facility) using a primer to the PGK promoter and the appropriate antisense primer crossing the putative deletion region.
All expression vectors utilized the pgk-1 promoter to drive the expression of various cDNAs. The construct PGK-lacZ contains the gene encoding ␤-galactosidase, the construct PGK-puro contains the gene encoding puromycin resistance (26), and B17 is a 17-kb BamHI fragment of the pgk-1 promoter (27).
Cell Culture and Transfections-P19 cells were obtained from the American Type Culture Collection (ATCC CRL-1825) and were cultured in ␣-minimum essential medium (Life Technologies, Inc.) supplemented with 5% (v/v) Cosmic Calf Serum (HyClone, Logan, UT), 5% (v/v) fetal bovine serum (CanSera, Rexdale, Ontario, Canada) and 100 g/ml gentamycin (Life Technologies, Inc.). Cells were plated at a density of 1ϫ10 5  ) were generated using either the calcium phosphate coprecipitation method (28) or FuGENE 6 transfection reagent according to the manufacturer's protocol (Roche Molecular Biochemicals). Cells were seeded at 0.75 ϫ 10 6 cells in 5 ml of medium in a 60-mm culture dish 24 h before transfection. For each calcium phosphate cotransfection, a total of 12.5 g of plasmid was used: 1 g of PGK-lacZ, 1 g of PGK-puro, 2.5 g of B17, and 8 g of the desired expression plasmid. After 9 h of transfection, the dishes were washed with phosphate-buffered saline (PBS) and incubated for 24 h in growth medium before harvest. For each FuGENE 6 transfection, a total of 9 g of plasmid was used: 0.5 g of PGK-lacZ, 0.5 g of PGK-puro, 2 g of B17, and 6 g of the desired expression plasmid. For P19 transfections using FuGENE 6 reagent, the optimal reagent (l) to DNA (g) ratio has been determined to be 6:3 for high transfection efficiencies. Transfected cells were harvested in 0.25 M Tris, pH 7.8, and ␤-galactosidase reporter gene assays performed to ensure high transfection efficiencies. The cells were then plated at a density of 2 ϫ 10 6 cells/150-mm culture dish and grown in medium under the selection of puromycin (2 g/ml). Several high expressing clones were selected and transferred to 100-mm culture dishes. Once confluent, 1 ϫ 10 6 cells were plated for a "day zero" harvest 48 h later, and 0.5 ϫ 10 6 cells were plated for differentiation.
P19 Cell Differentiation Experiments-Differentiation was induced as described previously (29) by aggregating 0.5 ϫ 10 6 cells in 60-mm bacterial dishes with or without 0.8% Me 2 SO. The presence of Me 2 SO during the aggregation initiates cardiomyogenesis and cardiac muscle is observed after 6 days of culture, in the appropriate serum (30). Control cell lines aggregated in the absence of Me 2 SO did not undergo cardiomyogenesis. After 4 days of aggregation, cells were plated in 150-mm culture dishes and harvested for RNA on day 6, 2 days later. Cells were also plated onto 0.1% (w/v) gelatin-coated coverslips and fixed for immunofluorescence on day 6.
To determine the hierarchy of gene expression in the Nkx2-5 cardiac pathway, an aggregation time course was carried out in the presence of 0.8% Me 2 SO with two of the P19[Nkx/EnR] cell lines isolated and P19[control] cells.
Northern Blot Analysis-Northern blots were performed as described previously (31). Total RNA was isolated from day 0 and day 6 P19 cell lines and control cells by the lithium chloride/urea extraction method (32). Separation of total RNA (6 g) was performed on a 1% agaroseformaldehyde gel by electrophoresis at 24 V overnight. After RNA was transferred to Hybond-N (Amersham Canada, Ltd., Oakville, Canada) by capillary blotting overnight, it was cross-linked by UV irradiation. Hybridization was performed with DNA probes labeled to over 10 9 cpm/g with [␣-32 P]dCTP using a Multiprime labeling kit (Amersham Canada, Ltd.). The probes were purified on a G-50 Sephadex spin column (Amersham Pharmacia Biotech, Baie d'Urfé, Quebec, Canada), denatured by boiling, and then cooled on ice before use. After the blots were prehybridized for 3-4 h, they were hybridized to the labeled probe for 16 h at 42°C in either NorthernMax (Ambion) or a standard hybridization buffer (5ϫ SSPE, 5ϫ Denhardt's solution, 0.1% SDS, 100 g/ml denatured salmon sperm DNA (Life Technologies, Inc.), and 50% deionized formamide). Blots were subsequently washed for 5ϫ5 min at 42°C in 2ϫ SSC, 0.1% SDS, and for 15 min at 65°C in 0.2ϫ SSC, 0.1% SDS. Hybridization was visualized by autoradiography and with a PhosphorImager SI from Molecular Dynamics.
Immunofluorescence-After a 5-min fixation in methanol at Ϫ20°C, cells were rehydrated in PBS for 15 min at room temperature. The fixed cells were incubated for 1 h at room temperature in the presence of 50 l of a mouse anti-myosin heavy chain (MyHC) monoclonal antibody supernatant, MF20, in a humidified chamber (42). After three 5-min PBS washes, cells were similarly incubated for 1 h at room temperature in the dark in 50 l of PBS with 1 l of goat anti-mouse IgG(HϩL) Cy3-linked antibody (Jackson Immunoresearch Laboratories). After the cells were washed with PBS, coverslips were mounted in a solution of 50% glycerol, 40% PBS, 9.9% paraphenylene diamine, and 0.1% Hoechst stain onto glass slides. Immunofluorescence was visualized with a Zeiss Axioscope microscope; images were captured with a Sony 3CCD color video camera and processed using Northern Eclipse, Adobe Photoshop 5.5 and Canvas 7 (Deneba) software. Immunofluorescence experiments were carried out in duplicate.

Nkx2-5 Function Is Essential for Cardiomyogenesis-Be-
cause Nkx2-5 is sufficient to induce cardiomyogenesis in aggregated P19 cells (25), we investigated whether it is also essential for early cardiomyogenesis by constructing a dominantly acting repressor protein. Previous studies have shown that Nkx2-5 is required for heart formation in Xenopus by using chimeric dominant repressors consisting of the Drosophila engrailed homeoprotein fused to the Nkx2-5 homeodomain (8). To create this dominant negative Nkx2-5 for our study, the active repression domain of mouse engrailed-2 was fused inframe to the DNA-binding homeodomain region of mouse Nkx2-5 ( Fig. 1, panel I) (Fig. 1, panel II, B) but did differentiate into bipolar skeletal myocytes on day 9 (Fig. 1, panel II, D). Therefore, Nkx2-5 function appears essential for cardiomyogenesis but not skeletal myogenesis in P19 cells.
As a control, the engrailed-2 repressor domain was expressed stably in P19 cells, without fusion to any DNA binding domain. P19[EnR] cells differentiated efficiently into both cardiac and skeletal muscle, indicated by the formation of MyHC-positive muscle and the presence of the cardiac and skeletal musclespecific transcripts, respectively (data not shown).
To identify which cardiac-specific transcripts are regulated by Nkx2-5, Northern blots were examined. The expression profiles of two P19[Nkx/EnR] cell lines were compared with a P19[control] cell line (Fig. 2). Total RNA was isolated on days 0 and 6 of differentiation in the presence of Me 2 SO and subjected to Northern blot analysis. High levels of the transfected Nkx/ EnR fusion construct were present on both day 0 and day 6 in P19[Nkx/EnR] cultures ( Fig. 2A, lanes 1-4) but not in P19 control cultures ( Fig. 2A, lanes 5 and 6). The lack of cardiac muscle in P19[Nkx/EnR] cultures in comparison to control cultures was demonstrated by the lack of cardiac ␣-actin expression on day 6 (Fig. 2B, lanes 2 and 4 compared with lane 6).
To confirm the result shown by immunofluorescence experiments that skeletal myogenesis proceeded efficiently in P19[Nkx/EnR] cultures, the expression levels of the myogenic regulatory factors, MyoD and myogenin, were examined by Northern blot analysis (Fig. 3)   tures, but this was not consistent. MyoD and myogenin transcripts were not observed in cultures prior to day 7 (data not shown).
To  (Fig. 4A). The cardiomyoblast marker GATA-4 was expressed by day 2 in P19[control] cells but was not present at any time in P19[Nkx/EnR] cultures (Fig. 4B). The expression of BMP4 was examined because BMP4 plays an integral role in early cardiomyogenesis and tissue that expresses BMP4 is in contact with pre-cardiac cells (43,44). BMP4 expression was greatly reduced in P19[Nkx/EnR] cultures. Although BMP4 was present initially on day 4 in control cells, it was not observed at this time point in P19[Nkx/EnR] cultures (Fig. 4C). BMP4 transcripts were present on day 6 in P19[Nkx/EnR] cultures ϳ8fold lower than in control cells (data not shown).
Several factors are present during early P19 cell differentiation and in the primitive streak of the developing embryo, including Brachyury T, Wnt3a, and Wnt5b (31,45,46). The expression of these genes was relatively unaffected in P19[Nkx/ EnR] cells compared with control cell lines (Fig. 4, D-F, respectively), indicating that mesoderm induction occurred normally. Furthermore, genes involved in commitment to the skeletal muscle lineage were examined. Expression of genes for Mox1 and Pax3 was not lost in P19[Nkx/EnR] cultures compared with control cultures (Fig. 4, G and H, respectively). Therefore, mesoderm induction and skeletal myogenesis were unaffected by the presence of Nkx/EnR.
The N Terminus and the Homeodomain of Nkx2-5 Are Sufficient to Induce Cardiomyogenesis-Previous studies have shown that Nkx2-5 can induce cardiomyogenesis in aggregated P19 cells (25). Furthermore, Nkx2-5 mutants have been analyzed for their ability to transactivate exogenous promoters in transient transfection assays (13)(14)(15)(16). However, a dissection of Nkx2-5 functional domains has not been performed on endogenous promoters. In an effort to identify the minimal region of Nkx2-5 that can activate endogenous cardiac muscle-specific gene expression, we analyzed the functional domains of Nkx2-5 in terms of their activity in P19 cells.
PCR amplification was carried out on full-length Nkx2-5 to delete the N terminus, the C terminus, and both N and C termini, termed Nkx⌬N, Nkx⌬C, and NkxHD, respectively  (Fig. 6B). Aggregation of  cultures in the absence of Me 2 SO also resulted in the differentiation of abundant cardiac muscle (Fig. 6D), confirming previous results by Skerjanc et al. (25). P19[Nkx⌬N] (Fig. 6F), P19[NkxHD] (Fig. 6H), and P19[control] (data not shown) cell lines aggregated in the absence of Me 2 SO did not differentiate into cardiomyocytes on day 6. In contrast, P19[Nkx⌬C] cultures did form heart muscle under identical conditions (Fig. 6J). Therefore, the C terminus is not essential for the ability of Nkx2-5 to induce cardiomyogenesis in P19 cells and the N terminus, along with the homeodomain, constitute the minimal essential region of Nkx2-5.
To compare levels of cardiac-specific transcripts up-regulated by wild-type and mutant Nkx2-5, total RNA was isolated from P19  (Fig. 7A). Endogenous Nkx2-5 from Me 2 SO-or Nkx2-5-induced cardiomyocytes was not present at sufficient levels to   (Fig. 7B, lanes 2-4, 14 -16, and 18, 7C), indicating a requirement for the N terminus and homeodomain of Nkx2-5 for the regulation of MEF2C expression. MyoD transcripts were not induced by the expression of Nkx2-5 or Nkx⌬C, indicating that skeletal muscle was not formed (data not shown).
In contrast to previous observations with exogenous promoter studies (12,14), the Nkx⌬C mutant did not appear to function in an enhanced capacity compared with wild type. Nkx⌬C was not able to induce cardiomyogenesis in the absence of cellular aggregation, both by Northern blot analysis (Fig. 7, B and C, lane 13, Day 0) and by immunofluorescence with an anti-MyHC antibody (data not shown). Thus, the loss of the C-terminal domain did not enhance the ability of Nkx2-5 to function in monolayer cultures. Furthermore, for three cell lines examined in two separate experiments, the extent of myogenesis occurring in P19[Nkx2-5] and P19[Nkx⌬C] cell lines did not differ by more than 10 Ϯ 30%, as measured by densitometry analysis of the level of cardiac ␣-actin expression. In summary, dissecting the Nkx2-5 protein has revealed that the N terminus and homeodomain comprise the minimal region of Nkx2-5 necessary for the initiation of cardiomyogenesis. Furthermore, this minimal region behaved similarly to the wild type Nkx2-5 protein in that it was regulated by the cellular aggregation of P19 cells.

Nkx2-5 Activity Is Essential for Cardiomyogenesis-Ectopic
expression of the Nkx/EnR fusion protein resulted in an abrogation of cardiomyocyte formation but not skeletal myogenesis in aggregated P19 cells, indicating a specific functional requirement for the Nkx2-5 gene product in the cardiac gene program. To begin to dissect the molecular mechanisms that orchestrate cardiac muscle development, we have focused on the hierarchical contributions of several key regulatory factors. We have shown that the inability to form cardiac muscle because of the loss of Nkx2-5 activity is also accompanied by the loss of endogenous expression of cardiomyoblast genes, such as GATA-4 and MEF2C. Furthermore, there was no significant change in the level of genes expressed during mesoderm induction, such as Brachyury T, Wnt5b, and Wnt3a, or in genes expressed in the skeletal muscle pathway, such as Mox1, Pax1, MyoD, and myogenin. Therefore, our results indicate that  Nkx2-5 function is essential for the commitment of mesoderm into cardiomyoblasts, but not into skeletal myocytes. We propose a hierarchy of gene expression controlled by Nkx2-5 that parallels the genetic hierarchy observed in Drosophila (47)(48)(49)(50)(51)(52), suggesting a conservation of pathways in fly and mammalian cardiomyogenesis.
Our approach of using an active dominant-negative Nkx2-5 has yielded a more severe phenotype than results of others from several gene inactivation studies. In mice lacking Nkx2-5, the ventricular specific MLC2V gene, eHAND, and the cardiac ankyrin repeat protein were the only genes shown to have been down-regulated (4 -6). In a more recent study, the expression of 20 candidate genes was examined by in situ hybridization of mice completely null for Nkx2-5, revealing a down-regulation in the expression of the ANF, BNP, MLC2V, N-Myc, Msx2, eHAND, and MEF2C genes in the mutant heart (53). Contrary to our findings, this latter study detected no change in the expression of GATA-4, cardiac ␣-actin, and BMP4 in the mutant mice and no effect on the early formation of cardiomyocytes. It is likely that the many murine homologues of Nkx2-5 have overlapping and compensatory functions, and thus, deletion of any one of these genes may not suffice to completely block cardiomyocyte differentiation. For this reason, dominant negative strategies, as the one used in this study, can bypass potential functional redundancies with other family members and allow for the determination of the role of Nkx2-5.
Several studies employing dominant-negative strategies have taken a similar experimental approach by using the active repressor domain of the engrailed homeoprotein (8,42,54). In studies using early Xenopus blastomeres, ectopic coexpression of dominant-negative engrailed fusions of XNkx2-3 and XNkx2-5 in synergy resulted in the blocking of heart formation, as observed by the lack of a heart tube and complete inhibition of molecular markers including Nkx2-5, cardiac troponin I, and MLC2 (8). A similar phenotype was obtained after injecting XNkx2-3 or XNkx2-5 containing a point mutation in the homeodomain, allowing for their association in protein complexes that can no longer bind DNA (9). The data presented here support these findings and extend them to mammalian cardiomyogenesis. Furthermore, a more detailed examination of molecular markers has shown that, in our system, Nkx2-5 is essential for the earliest stages of commitment of mesoderm into cardiomyoblasts, such as expression of GATA-4.
Taking advantage of the pluripotent nature of P19 cells, it has been possible to determine the specificity of the observed inhibition of cardiomyogenesis. We have been able to show that Nkx/EnR does not affect other pathways of mesodermal differentiation, since the mesodermal markers, Brachyury T, Wnt3a, and Wnt5b, were present at high levels. We tested for potential pleiotropic effects of the dominant-negative Nkx2-5 on skeletal myogenesis and have found that the skeletal differentiation pathway remains unperturbed. The specificity of the Nkx dominant-negative mutant for the cardiac gene program also argues against unforeseen toxicity effects that may arise from expressing some foreign genes to high levels in culture. Furthermore, expression of the engrailed-2 repressor domain without fusion to a DNA binding domain had no effect on the ability of P19 cells to differentiate into cardiac or skeletal muscle.
Our findings suggest that GATA-4 is a target of mouse Nkx2-5. Nkx2-5 synergistically interacts with GATA-4 at cardiac gene promoters and enhancers containing their binding sites in close proximity (13,14,16). Whereas the mouse Nkx2-5 gene is controlled by two enhancers containing GATA-4/5/6 binding sites (55,56), the chicken and mouse GATA6 genes have been shown to be regulated by a Nkx-dependent enhancer (57,58). Together, these studies suggest that Nkx2-5 and GATA proteins may participate in transcriptional regulatory loops in which expression of both factors is reinforced throughout cardiac muscle development. Our data support the presence of this loop and furthermore indicate that a functional Nkx2-5 is essential for its proper formation, resulting in the stable expression of GATA-4 and subsequent cardiomyogenesis. Our results also agree with the findings in Drosophila that show the GATA-4 homologue, Pannier to be genetically downstream of tinman, and the D-MEF2 gene to be down-regulated but not lost (47,52). In addition, loss of GATA-4 function in P19 cells blocked cardiomyogenesis at the cardioblast stage. Nkx2-5 and MEF2C expression was down-regulated but not lost, consistent with a regulatory loop in which Nkx2-5 is upstream of GATA-4 (24).
Our finding of a loss of MEF2C expression in the absence of Nkx2-5 activity is consistent with studies in Drosophila in which D-MEF2 expression is lost in the heart precursors of Drosophila lacking tinman (50), whereas tinman is still expressed in Drosophila lacking D-MEF2 (51). Furthermore, the D-MEF2 gene was shown to be a direct target gene of tinman (48,49), and the level of MEF2C in Nkx2-5 null mice heart is down-regulated by ϳ50% (53). Our results indicate that functional Nkx2-5 is essential for MEF2C expression in mammalian cardiomyogenesis.
Loss of Nkx2-5 activity was shown to result in a downregulation of BMP4 transcript level, and we have previously shown that Nkx2-5 expression can up-regulate BMP4 transcripts in aggregated P19 cells (25). The presence of BMP in the endoderm contacting the heart field promotes cardiomyogenesis by regulating the expression of Nkx2-5 and GATA-4 (43, 44, 59 -64). Our results imply that the formation of cardiomyocytes results in the secretion of a factor that can regulate the expression of BMP4, suggesting that BMP4 and Nkx2-5 function in a complex positive regulatory loop.
The C Terminus Is Not Required for Nkx2-5 Activity-These experiments are the first to analyze the functional domains of Nkx2-5 using endogenous cardiac muscle-specific gene expression. We have shown that deletion of the N terminus completely inhibits cardiomyogenesis in P19 cells, whereas deletion of the C terminus does not significantly perturb Nkx2-5-induced cardiomyocyte formation. Our results do not support previous studies in which the ability of various Nkx2-5 mutants to transactivate exogenous promoters was examined in transient transfection assays (12,14). Transient transfection studies using an Nkx2-5 C-terminal deletion mutant have revealed a 50 -300fold stimulation in transcriptional activity of a luciferase gene carrying multimerized NKE binding sites, depending upon the complexity of the minimal promoter used (14). Such studies using synthetic minimal promoters may omit possible participation of ancillary proteins, owing to the absence of their expression in the cell culture system used, and/or may omit important DNA binding sites, which may be absent in the promoter fragment utilized.
Our results suggest that the previously proposed Nkx2-5 C-terminal inhibition is not involved in the induction of cardiomyogenesis in P19 cells; however, this does not preclude the theory of C-terminal inhibition. It is possible that the C-terminal inhibition or the interaction with putative coactivator(s) may not be the rate-limiting step(s) controlling P19 cell cardiomyogenesis. This scenario would result in similar rates and extents of myogenesis for the Nkx2-5 C-terminal deletion mutant compared with the wild-type protein. Alternatively, it is possible that another factor present in aggregated P19 cells can replace the activation of Nkx2-5 by GATA-4. Although our results do not suggest a functional role for the C terminus of Nkx2-5 in the formation of cardiomyocytes in P19 cells, the precise function of the C-terminal domain in the embryo re-mains to be established, considering that more complex mechanisms are involved in the development of the vertebrate heart.