RA inhibits cardiomyogenesis and enhances entry into the skeletal muscle lineage in P19 cells

Previous studies have shown that RA can inhibit cardiomyogenesis and enhance skeletal myogenesis [16, 47], but the stage at which this occurs and the interaction with other signalling pathways have not been clearly defined. To investigate the mechanisms by which RA modulates myogenesis in P19 cells, various concentrations of RA, in the presence of DMSO, were examined. In agreement with previous results [47], it was found that skeletal myogenesis occurred in the presence of DMSO but not in its absence (summarized in Table 1). Furthermore, 3-30 nM of RA with DMSO was sufficient to block cardiac and enhance skeletal muscle development (data not shown). A time course of P19 cell differentiation was carried out in the presence of DMSO, with and without 30 nM of RA. Cells were fixed on day 9 for immunofluorescence and stained with an anti-myosin heavy chain (MyHC) antibody, MF20, which identifies MyHC in both cardiac and skeletal myocytes. Cardiac and skeletal myocytes can be distinguished by their morphology and time of appearance, with rounded cardiac myocytes appearing by day 6 and elongated, bipolar skeletal myocytes by day 9 [13]. Cells treated with RA did not differentiate into cardiac muscle, evidenced by the absence of rounded cardiac myocytes, expressing MyHC, compared to cells not treated with RA (Figure 1, panels IB and D). In contrast, significantly enhanced levels of bipolar skeletal myocytes were observed in the presence compared to the absence of RA (Figure 1, panels IA and C). Quantification of the number of cardiac and skeletal myocytes after treatment with RA showed that the fourfold increase in skeletal myocytes and eightfold loss of cardiac myocytes were statistically significant (Figure 1, panel II). Therefore, in agreement with others [58], RA inhibited cardiomyogenesis and enhanced skeletal myogenesis in P19 cells.

		Cardiac	Skeletal Markers
Cell line	Conditions	GATA-4	Pax3	Gli2	Meox1	MRFs	Figure
P19	-	-	-	-	-	-	Fig. 7 & Data not shown
P19	+DMSO	+	+	+	+	+	Figs. 1 & 4
P19	+DMSO&RA	-	+++	+	+++	+++	Figs. 1 & 4
P19 [β-cat/EnR]	+DMSO	N.D.	-	-	-	-	Figs. 3 & 4
P19 [β-cat/EnR]	+DMSO&RA	N.D.	+++	+++	-	-	Figs. 3 & 4
P19 [BMP]	-	-	-	-	-	-	Data not shown
P19 [BMP]	+DMSO	+	-	N.D.	-	-	Fig. 6, 7 & 8
P19 [BMP]	+DMSO&RA	+	+++	N.D.	N.D.	+++	Fig. 8
Mouse ES	-	+	-	N.D.	+/-	-	Fig. 2
Mouse ES	+RA	+/-	+++	N.D.	+++	+++	Fig. 2

P19 cell lines indicated on the right were aggregated under the conditions described and the induction of muscle marker gene expression was monitored.

(+++ = high expression; + = normal expression; +/- = partial expression, - = not expressed; N.D. = not determined)

In order to examine the molecular basis of the effects of RA on myogenesis, total RNA was harvested from a time course of cells differentiated with and without RA and subjected to northern blot analysis. Endogenous RALDH2 levels were enhanced on day 2 of the DMSO-induced differentiation in the absence of exogenous RA (Figure 1, panel IIIA), indicating that endogenous RA signalling could be involved at this stage of development [59]. RA-treated cells showed enhanced expression of RALDH2 from days 3-5 (Figure 1, panel IIIA). To determine at what stage cardiomyogenesis was inhibited by RA, expression of the cardiomyoblast gene GATA-4, was examined [60]. Northern blot analysis revealed a lack of induction of GATA-4 transcripts in cells treated with RA (Figure 1, panel IIIB), consistent with the interpretation that under these conditions RA inhibits commitment into the cardiac muscle lineage.

In order to determine at which stage skeletal myogenesis was enhanced, the expression of genes expressed in the primitive streak (Brachyury T and Wnt3a), the dermomyotome (Pax3 and Meox1), and skeletal myoblasts (MyoD and myogenin) was examined. Mesoderm induction occurred in the presence of RA, shown by the expression of Brachyury T (Figure 1, panel IIIC). The levels of Brachyury T appear to be slightly decreased with RA, consistent with the increasing differentiation of mesodermal progenitors. In contrast to the results with GATA-4 expression, Wnt3a (days 4-6), Pax3 (days 2-9), Meox1 (days 3-4), MyoD (days 6-9) and Myogenin (days 6-9) transcripts were upregulated with RA treatment (Figure 1, panels IIID-H), which is consistent with an increase in skeletal myogenesis. Quantitative polymerase chain reaction (Q-PCR) was used to quantify the levels of Pax3, MyoD and Wnt3a transcripts, showing a 9-95-fold upregulation of these factors with RA treatment (Figure 1, panel IV). Hence, RA enhances skeletal myogenesis by upregulating Wnt3a, Pax3 and Meox1 (summarized in Table 1).

Since BMP4 is known to inhibit skeletal and enhance cardiac myogenesis [61], and RA has been shown to inhibit BMP4 expression in the limb forebud [62], we examined the expression of BMP4 transcripts in the presence and absence of RA (Figure 1, panel III- I). The endogenous levels of BMP4 were down-regulated in RA-treated cells relative to the controls from days 3-9. Finally, we examined the expression of the transducer of ErbB2 (Tob1), an intrinsic inhibitor of BMP signalling [63, 64]. Tob1 was upregulated by RA from days 2-9 (Figure 1, panel III-J). Thus, RA may function to enhance skeletal myogenesis and inhibit cardiomyogenesis in part by inhibiting BMP4 expression and/or function.

To identify direct chromatin targets of RAR binding, we used multiple sequence local alignment and visualization http://mulan.dcode.org/ to find RA response elements (RARE) sequences (defined as two tandem repeats of the RGKTCA element in DR1-7 arrangements) located within +/- 100 Kb of the start site for Wnt3a, Pax3 and Meox1. While all three genes contained RARE sequences, only one of the two Meox1 sites (Meox1-2) and one of the two Pax3 sites (Pax3-2) were conserved between mouse and human, located in Mus musculus at -33869 bp and + 114735, respectively (Figure 1, panel VI). Chromatin immunoprecipitation experiments were performed on day 2 of differentiation, using an antibody that recognizes all RARs. We detected a significant 3.6-fold enrichment in chromatin fragments corresponding to the Meox1-2 site, and a significant 2.3-fold enrichment in chromatin fragments corresponding to the Pax3-2 site compared to immunoprecipitation with a rabbit IgG as a negative control (Figure 1, panel V). We also tested several of the non-conserved RAREs identified upstream of Wnt3a. One of these RAREs (Wnt3a-2 at position -36625) showed a significant 1.9-fold enrichment. Several non-conserved RARE sequences in Wnt3a, Meox1, or Pax3 genes were not significantly associated with immunoprecipitated RARs, whereas a known site in RARβ2 was associated, as expected [65]. Thus, RARs bind to conserved elements in the Meox1 and Pax3 genes as well as to one non-conserved element upstream of Wnt3a in a population of differentiating P19 cells, indicating that RA functions both upstream and downstream of Wnt3a signalling.

RA enhances skeletal myogenesis in mES cells

To determine if our results in P19 cells were applicable to mouse embryonic stem (mES) cells, we differentiated mES cells in hanging drops for 2 days and in suspension culture for an additional 5 days, the latter with 0-50 nM RA. After re-plating in tissue culture dishes, cells were harvested on days 7 and 15 for RNA and fixed on day 20 for immunofluorescence. Examination of RNA by RT-PCR indicated increasing transcript levels of Meox1, Pax3, and MyoD in the presence of retinoic acid, peaking at 25 nM (Figure 2, panel I). Quantification of gene expression changes by Q-PCR indicated statistically significant 20-fold increases in Pax3 and Meox1 expression on day 7 and a significant fivefold increase in myogenin on day 15 (Figure 2, panel II). Finally, skeletal myocytes were not observed on day 20 in the absence of RA, as shown by the lack of bipolar cells reacting with MF20 (Figure 2, panel III-B). In the presence of 25 nM RA, MyHC^+ve bipolar skeletal myocytes were visible (Figure 2, panel IIID). Thus, similar to the findings in P19 cells, RA treatment of mES cells results in the enhancement of skeletal muscle progenitor formation, shown by the increase in expression of Pax3, Meox1, MyoD, and myogenin (summarized in Table 1).

RA cannot bypass the inhibition of skeletal myogenesis by a dominant negative β-catenin

Since earlier studies have shown that Wnt signalling, via β-catenin, activates Pax3, Gli2 and Meox1 expression, inducing skeletal myogenesis in P19 cells [8], we were interested in determining how the Wnt signalling pathway intersects with RA signalling. Furthermore, a dominant negative β-catenin, with the transcriptional activation domain replaced by an engrailed repressor domain (β-Cat/EnR), inhibited skeletal myogenesis in P19 cells [8]. This dominant negative approach identifies genes bound by β-catenin and their downstream targets. In order to determine if RA can bypass this inhibition, P19 cells expressing β-Cat/EnR were differentiated in the presence of DMSO, with and without RA and compared to control P19 cells. Cultures were fixed on day 9 and examined by immunofluorescence with MF20. In agreement with previous results [8], the overexpression of β-Cat/EnR resulted in the loss of skeletal myogenesis, shown by the lack of bipolar skeletal myocytes, compared to control cells (Figure 3D versus B). The addition of RA resulted in an increase in the number of skeletal myocytes observed in control cells (Figure 3F versus B), but not in P19 [β-Cat/EnR] cells (Figure 3H). Therefore, RA was not sufficient to circumvent the inhibition of skeletal myogenesis by β-Cat/EnR.

To examine at which time point the RA enhancement of skeletal myogenesis was inhibited by β-Cat/EnR, RNA was harvested on days 0, 5 and 9 from P19 [control] and P19 [β-Cat/EnR] cultures differentiated with increasing amounts of RA. Northern blots showed the expression of exogenous β-Cat/EnR in the P19 [β-Cat/EnR] cells and not in P19 [control] cells (Figure 4, panel IA). Endogenous β-Catenin expression was constitutive, as expected (Figure 4, panel IB). In agreement with Figure 1, MyoD, Meox1 and Pax3 were upregulated with 3 and 10 nM RA in P19 [control] cells, compared to no RA treatment (Figure 4, panels IC - E, lanes 4-7 versus lanes 2-3). Gli2 was upregulated during myogenesis on days 5 and 9, in agreement with previous results [9], but was not further upregulated by RA (Figure 4, panel IF). Interestingly, while MyoD and Meox1 were no longer upregulated by RA in the presence of β-Cat/EnR (Figure 4, panels IC - D, lanes 11-14), Pax3 transcript levels were increased by RA treatment (Figure 4, panel IE, lanes 11-14). Myogenin and Myf-5 were also not upregulated in P19 [β-Cat/EnR] cells treated with RA (data not shown). The enhancement of Gli2 during myogenesis (Figure 4, panel IF, lanes 2-3) was abrogated in the presence of β-Cat/EnR (lanes 9-10), as shown previously [8]. This loss of Gli2 expression was reversed in the presence of RA (Figure 4, panel IF, lanes 11-14). Therefore, the RA enhancement of MyoD and Meox1 was abrogated in the presence of β-Cat/EnR, but not the enhancement of Pax3. Furthermore, the Gli2 expression became responsive to RA in the presence of β-Cat/EnR (summarized in Table 1).

Changes in gene expression for Pax3 and Pax7 were quantified by Q-PCR (Figure 4, panel II). RA treatment resulted in the increased expression of Pax3 and Pax7 in P19 [β-Cat/EnR] cells on days 5 and 9 (Figure 4, panel II). Thus, both Pax3 and Pax7 were upregulated by RA in the presence of β-Cat/EnR.

To further assess the relationship between RA and Wnt, the ability of RA to activate a β-catenin-responsive promoter - TOPflash - which contains 8 high mobility group (HMG) box sites, was examined using luciferase assays and compared to a mutated HMG box promoter, FOPflash [66]. Both 10 and 100 nM RA were sufficient to induce four- to fivefold increases in β-catenin activity in aggregated P19 cells (Figure 4, panel III). Interestingly, the activation by RA required cellular aggregation, since RA did not significantly enhance β-catenin function in monolayer cultures. Furthermore, no evidence was obtained of synergy or inhibition of RARβ on β-catenin activity using a β-catenin-responsive TOPflash reporter (data not shown), in contrast to previous reports of inhibition [67]. Thus RA can activate β-catenin function in DMSO-treated P19 aggregates, likely by enhancing β-catenin translocation to the nucleus.

Induction of Pax3 by RA is not due to enhanced neurogenesis

Since high levels of RA (1 μM) can induce neurogenesis in P19 cells and both Pax3 and Shh signalling via Gli2 can regulate neurogenesis in spinal cord and brain [68–70], the increase in Pax3 mRNA may not reflect skeletal myogenesis but, rather, result from an increase in neurogenesis. To test this hypothesis, we quantified the percentage of cells that differentiated to a neuronal phenotype in P19 [control] and P19 [β-Cat/EnR] cells under cardiac and skeletal myogenic conditions (1%DMSO), skeletal myogenic conditions (1%DMSO+10 nM RA), or neurogenic conditions (1%DMSO+ 1 μM RA) (Figure 5). Differentiation to a post-mitotic neuronal phenotype was identified by labelling of neuron-specific β-III tubulin using anti-Tuj1 antibodies. As expected, 1 μM RA triggered a significant neuronal differentiation compared to DMSO alone (Figure 5, panels I and III). However, neurogenesis with 10 nM RA was variable and was not significantly different compared to DMSO alone (Figure 5, panels I and III). Consistent with previous reports that the dominant-negative inhibition of β-catenin signalling reduces neurogenesis [71], we found that significantly fewer cells differentiated to Tuj1-positive neurons in P19 [β-Cat/EnR] cultures treated with 1 μM RA (Figure 5, panels II and III).

In order to determine whether Pax3/7 proteins were found in neurons, or in neural precursor cells, we used immunofluorescence to co-localize Pax3/7 proteins with neuronal markers (Figure 5, panels IV and V). Anti-Tuj1 antibodies were used to detect immature neurons and anti-double cortin (DCX) antibodies were used to detect committed neuronal precursor cells. DCX protein is expressed as early as 1 day after the initiation of neurogenesis in P19 cells and is the earliest known marker of neurogenesis [72]. To confirm that P19 cells do not express Pax3/7 following neuronal commitment, we quantified the percentage of Pax3/7-positive neuronal precursors and immature neurons in our day 9 cultures (Figure 5, panels IV and V). Under all conditions tested, the overwhelming majority of Pax3/7-positive cells were non-neuronal (Figure 5, panels IV and V). No Pax3/7-positive neurons were detected in P19 [control] cultures under conditions promoting skeletal myogenesis (DMSO + 10 nM RA) (Figure 5, panels IV and V). Taken together, these results provide strong evidence that enhancement of Pax3 and Gli2 expression by low concentrations of RA is indicative of increased skeletal myogenesis and not neurogenesis.

Overexpression of BMP4 blocks skeletal myogenesis in P19 cells

Since RA enhances skeletal myogenesis, while downregulating BMP4 expression, we were interested in examining the interplay between RA and BMP4 in P19 cell myogenesis. In order to examine the effect of BMP4 on skeletal myogenesis in a stem cell context, stable cell lines expressing BMP4 were isolated and termed P19 [BMP4] cells. When aggregated in the absence of DMSO and examined for immunofluorescence with MF20, neither skeletal nor cardiac myogenesis occurred in P19 [BMP4] cells (data not shown). When cells were aggregated in the presence of DMSO, P19 [BMP4] cells differentiated into cardiac muscle (Figure 6, panel IA) but not skeletal muscle (Figure 6, panel IB). Under these conditions, control cells differentiated efficiently into both cardiac (Figure 6, panel IC) and skeletal muscle (Figure 6, panel ID). Furthermore, a similar inhibition of skeletal myogenesis was obtained when parental P19 cells were mixed with P19 [BMP4] cells in various ratios and aggregated in the presence of DMSO (data not shown). This indicated that BMP4 can function extracellularly. By counting the number of MHC+ve cells, skeletal myogenesis was inhibited an average of approximately fivefold in the presence of BMP4 and cardiomyogenesis was enhanced about 1.6-fold (Figure 6, panel II). Thus, overexpression of BMP4 in P19 cells is sufficient to block skeletal muscle and to slightly enhance cardiac muscle development.

BMP4 inhibits skeletal muscle specification

In order to investigate at what point in the pathway BMP4 inhibited myogenesis, the expression patterns of skeletal muscle-specific markers in P19 [BMP4] and P19 [control] cells during DMSO-induced differentiation were compared. Total RNA was harvested on days 0, 6 and 9 for northern blot analysis. P19 [BMP4] cell lines expressed high levels of BMP4 (Figure 7, panel IA), but failed to express early markers of skeletal myogenesis such as Meox1 and Pax3 (Figure 7, panels IB - C) and late markers such as MyoD (Figure 7, panel ID) compared to P19 [control] cells. A 17-fold loss of MyoD transcript levels was detected by Q-PCR in the presence of BMP4 (Figure 8, panel II). These findings suggest that BMP4 inhibited an early stage of skeletal muscle development by preventing proper muscle specification (summarized in Table 1).

To determine if mesoderm induction, the stage prior to muscle specification, was affected by BMP4 expression, a time course of DMSO-induced differentiation was performed. P19 [BMP4] and P19 [control] cells were aggregated in the presence of DMSO and total RNA was harvested during the time course of differentiation for northern blot analysis. P19 [BMP4] and P19 [control] cell lines both expressed the mesoderm markers BrachyuryT and Wnt5b (Figure 7, panels IIA and B). Wnt5b expression levels appeared to be slightly increased. Finally, in order to compare the abilities of RA and BMP4 to enhance β-catenin activity, P19 cells were aggregated with DMSO in the presence and absence of BMP4 and the β-catenin-responsive TOPflash promoter was examined by luciferase assay. BMP4 was able to activate the β-catenin-responsive reporter in aggregated P19 cells treated with DMSO (Figure 7, panel III). Taken together, these results show that BMP4 interfered with skeletal myogenesis at a time point after mesoderm induction but before the specification of skeletal muscle.

Reciprocal regulation of myogenesis by RA and BMP4

Since BMP4 was found to inhibit, and RA to enhance skeletal muscle specification in P19 cells, we wanted to determine if BMP4 could override the enhancement of skeletal myogenesis by RA, and vice versa for cardiomyogenesis. To this end, P19 cells were mixed with P19 [BMP4] or P19 [control] cells and aggregated in the presence of DMSO with or without RA. As expected, P19 cells, mixed with P19 [control] cells and aggregated in the presence of DMSO, differentiated readily into cardiac and skeletal muscle, as seen by positive MyHC staining. As shown in Figures 1 and 4, and quantified in Figure 8, RA inhibited cardiac myogenesis (3.6-fold), but not skeletal myogenesis, and BMP4 inhibited skeletal myogenesis (5.5-fold), but not cardiac myogenesis (Figure 8, panels II and III). However, P19 cells mixed with P19 [BMP4] cells and treated with RA differentiated efficiently into both cardiac and skeletal muscle (Figure 8, panels I-III), indicating that RA and BMP4 could antagonize each other's inhibitory activities.

The results of immunofluorescence were confirmed by RNA analysis. Cultures of P19 cells mixed with P19 [BMP4] cell lines contained high levels of BMP4 transcripts on days 6 and 9 (Figure 8, panel IVA, lanes 2-9). GATA-4 transcripts were present in cultures containing both BMP4 and RA, indicating that BMP4 abrogated the inhibition of cardiomyogenesis by RA (Figure 8, panel IVB, lanes 6-9). MyoD and myosin light chain1/3 (MLC1/3) transcripts were absent in P19 [BMP4], compared to P19 [control] cells (Figure 8, panel IVC - D, lanes 2-5 compared to 1). In contrast, P19 cells treated with BMP4 and RA robustly expressed MyoD and MLC1/3 transcripts (Figure 8, panels IVC and D, lanes 7 and 9). Quantification of MyoD transcript levels under the four conditions by Q-PCR was consistent with the results from counting skeletal myocytes (Figure 8, panel II). Therefore RA enhanced skeletal myogenesis and antagonized the inhibitory actions of BMP4 signalling.

Based on the inability of BMP4 to inhibit skeletal muscle development in the presence of RA, we predicted that the accelerated expression of Pax3 with RA treatment would still be observed in the presence of BMP4. To test this, we examined a time course of P19 cell differentiation in the presence of P19 [BMP4] cells and RA. Total RNA was harvested on days 1-4, 6, and 9 for northern blot analysis. Exogenous BMP4 transcripts were present in P19 [BMP4] cultures (Figure 8, panel VA). Pax3 and MyoD transcripts were detected in cells treated with RA alone in a similar expression pattern compared to cultures containing both RA and BMP4 (Figure 8, panels VB and C, lanes 1-6 compared to 7-12). These results are in contrast to the loss of Pax3 and MyoD expression shown in the presence of BMP4 alone (Figure 7). Therefore, the early enhancement of Pax3 expression by RA treatment still occurs in the presence of BMP4 (summarized in Table 1).

Plasmid constructs

The expression construct phosphoglycerate kinase (PGK)-BMP4 contains a 1.8 Kb EcoRI BMP4 cDNA fragment, containing the complete coding sequence of human BMP4 (Wyeth Pharmaceuticals, MA, USA), driven by the pgk-1 promoter. The empty PGK vector was used as control. The constructs PGK-Puro, B17, and PGK-Lac-Z were previously described [92].

P19 Cell culture and isolation of stable cell lines

P19 embryonal carcinoma cells (American Type Culture Collection, VA, USA) were cultured as previously described [93] in α-minimum essential media supplemented with 5% cosmic calf serum (Hyclone, UT, USA) and 5% fetal bovine serum (Cansera, Rexdale, ON, Canada). P19 [β-cat/EnR] cells were isolated and described previously [8]. For P19 [BMP4] cells, stable cell lines were created using similar protocols. Briefly, P19 cells were transfected with the DNA constructs PGK-BMP4 or the empty PGK vector along with PGK-puro, PGK-LacZ and B17 with the aid of the Fugene 6 transfection kit (Roche Diagnostics Canada, Quebec, Canada) as per manufacturer's instructions. Twenty-four hours after transfection, cells were grown under puromycin selection for 10 days. Individual colonies were analysed for BMP4 expression.

To examine the role of BMP4 in P19 cell differentiation, P19 cells, P19 [BMP4] cells, P19 [control] cells, 1:1 mixtures of P19 and P19 [BMP4] cells, and 1:1 mixture of P19 and P19 [control] cells were aggregated with or without 0.8-1% DMSO for 4 days in petri dishes [94]. Subsequently cells were transferred to tissue culture plates or gelatin coated cover slips and examined for differentiation on days 6 or 9.

To examine the role of RA in P19 cell differentiation, P19 cells, P19 [β-cat/EnR] cells P19 [BMP4] cells, P19 [control] cells, 1:1 mixtures of P19 and P19 [BMP4] cells, and 1:1 mixture of P19 and P19 [control] cells were aggregated with or without 0.8-1% DMSO in the presence of 0-30 nM all-trans retinoic acid. Stocks of retinoic acid were purchased and prepared every 3-4 months. Each new stock was titrated for optimal skeletal myogenesis.

Immunofluorescence

For analysis of muscle-specific markers, cultures were treated as described previously [95] with MF20 [96]. Staining was visualized with a Zeiss Axioskop microscope and photographed with a Sony 3CCD camera. Images were formatted using Adobe Photoshop and Canvas software. Myogenic differentiation was quantified by counting five to 10 fields containing 100-200 cells.

For analysis of neuron-specific markers, cultures were plated on gelatin-coated coverslips and cultures were fixed with methanol at -20°C. Antigenic analysis was performed using mouse-anti-Tuj1 (Research Diagnostics, USA) guinea pig anti-DCX (Chemicon, USA) and Hoechst dye as a nuclear marker. To identify PAX3/7-positive cells, some cultures were also labeled with goat-anti-PAX3/7 (Invitrogen, Canada). Secondary antibodies were AMCA- or FITC-conjugated anti-mouse-IgG, Cy3-conjugated anti-guinea pig-IgG, (Jackson ImmunoResearch Laboratories, USA) and Alexa-488 conjugated anti-goat IgG (Invitrogen, Canada) where appropriate. Immunofluorescence was evaluated using OpenLab Software, version 3.4 (Improvision, MA, USA) on a Leica DMXRA2 microscope equipped for epifluorescence. Antigen-positive cells were counted in three to 10 fields in triplicate samples performed over two independent experiments and data expressed as a percentage of the total cell number (n = 6-10). Counts were performed by two investigators blind as to the treatment conditions of each field.

Northern blot analysis

Cells were harvested for RNA extraction using the Urea/lithium chloride method [97] as previously described [98]. The cDNA fragments used to probe for MLC1/3 and MyoD [47] and for BMP4, GATA4, Pax3, Meox1, Myogenin, BrachyuryT, Wnt5b and 18s have been described previously [10]. A 1.8 Kb Kpn1/Mlu1 full length human cDNA fragment was used to probe for Tob1 (GeneBank Accession Number: BC031406) and a 400 bp HindIII fragment for mouse RALDH2 (GI Number 31982069).

mES cell culture and differentiation

J1 or D3 mES cells were maintained in 15% fetal bovine serum in the presence of leukemia inhibitory factor (LIF). Differentiation was induced by aggregating cells in the absence of LIF. Cells were aggregated in hanging drops containing 800 cells for 2 days and in suspension for a further 5 days. Aggregates were treated with increasing concentrations of retinoic acid from day 2 to day 5 or 7 of differentiation. On day 7 of differentiation, cells were plated on tissue culture plates and allowed to differentiate for a further 13 days. The efficiency of myocyte formation was enhanced by plating at a high cell density. Experiments were performed at least twice with each cell line with similar enhancement of skeletal myogenic precursors.

PCR Analysis

For RT-PCR southern analysis, total RNA was harvested from day 7 mES cells and 0.8 μg was subjected to reverse transcription (Qiagen, Mississauga, Canada). The resultant cDNA was subjected to PCR with primers specific for Pax3, Gata-4 and β-actin [85, 99] and for MyoD and Meox1 [100] described previously. The PCR product was hybridized to DNA probes and visualized by autoradiography.

For quantitative real-time PCR (Q-PCR) expression analysis of mES cells, total RNA was harvested from differentiating cells using the RNeasy Micro kit (Qiagen, Mississauga, Canada). RNA harvested from P19 cells using the urea/lithium chloride extraction was further purified using the RNeasy Micro kit (Qiagen, Mississauga, Canada). The resultant cDNAs were used for Q-PCR, using protocols and primers for Pax3/7, Meox1, MyoD, myogenin and GAPDH as described previously [100]. Primers for β-actin are found in Table 1. mRNA levels were normalized to β-actin or glyceraldehyde-3-phosphate dehydrogenase levels for the corresponding day and subsequently to levels in untreated samples for the corresponding day.

Promoter analysis

P19 cells were transfected with 6 μg of the super8 TOPFlash [101] or FOPFlash reporter plasmids (generous gifts from R Moon), as well as with 2 μg of Renilla Luciferase (Promega, WI, USA), using Fugene Transfection Reagent (Roche Applied Sciences, QC, Canada). Cells were either left in monolayer culture or aggregated in the presence of 1% DMSO, 20 mM LiCl, 1% DMSO with retinoic acid, or 1%DMSO with 50 ng/ml BMP4. Luciferase assays were conducted using the Dual Luciferase Reporter Assay System from Promega, 24 hours after treatment. Luciferase activity was normalized to Renilla activity. Data from samples treated with lithium chloride or RA were normalized to samples treated with DMSO alone.

Chromatin Immunoprecipitation