BRG1 interacts with GLI2 and binds Mef2c gene in a hedgehog signalling dependent manner during in vitro cardiomyogenesis
- Joel Vincent Fair†1,
- Anastassia Voronova†1,
- Neven Bosiljcic1,
- Rashida Rajgara1,
- Alexandre Blais1, 2Email authorView ORCID ID profile and
- Ilona Sylvia Skerjanc1Email author
© The Author(s). 2016
Received: 6 April 2016
Accepted: 28 July 2016
Published: 2 August 2016
The Hedgehog (HH) signalling pathway regulates cardiomyogenesis in vivo and in differentiating P19 embryonal carcinoma (EC) cells, a mouse embryonic stem (mES) cell model. To further assess the transcriptional role of HH signalling during cardiomyogenesis in stem cells, we studied the effects of overexpressing GLI2, a primary transducer of the HH signalling pathway, in mES cells.
Stable GLI2 overexpression resulted in an enhancement of cardiac progenitor-enriched genes, Mef2c, Nkx2-5, and Tbx5 during mES cell differentiation. In contrast, pharmacological blockade of the HH pathway in mES cells resulted in lower expression of these genes. Mass spectrometric analysis identified the chromatin remodelling factor BRG1 as a protein which co-immunoprecipitates with GLI2 in differentiating mES cells. We then determined that BRG1 is recruited to a GLI2-specific Mef2c gene element in a HH signalling-dependent manner during cardiomyogenesis in P19 EC cells, a mES cell model.
Thus, we propose a mechanism where HH/GLI2 regulates the expression of Mef2c by recruiting BRG1 to the Mef2c gene, most probably via chromatin remodelling, to ultimately regulate in vitro cardiomyogenesis.
The embryonic heart is the first organ to develop and is essential for life. Perturbations in heart development can lead to congenital heart disease, which is the most common birth defect in the Western world . During heart development, cardiac precursors populate the first and the second heart fields (FHF and SHF), which establish the cardiac crescent by embryonic day (E) 7.5 [2, 3]. In the cardiac crescent a transcriptional network regulates cardiac progenitor differentiation in a spatiotemporal manner through a crosstalk of inductive signals from surrounding tissues, including hedgehog (HH) [4, 5]. These signals lead to the expression of cardiac progenitor-enriched transcription factors, including NK2 homeobox 5 protein (NKX2-5), myocyte-specific enhancer factor 2C (MEF2C), T-box protein 5 (TBX5), and GATA-binding protein 4 (GATA-4), which are essential for efficient heart looping and morphogenesis [5–10].
When one of the three mammalian HH ligands - Indian (IHH), desert (DHH), or sonic hedgehog (SHH) - binds to and inhibits the transmembrane protein, patched 1 (PTCH1) [11, 12], smoothened (SMO) initiates the transition of glioma-associated (GLI) proteins into the nucleus for modulation of HH target gene expression . GLI2 is a primary transducer and activator of the response to HH signalling . GLI1 is a transcriptional activator but its expression is dependent on GLI2 and/or GLI3 . In the absence of HH ligand, PTCH1 inhibits SMO activity, which triggers phosphorylation of the GLI proteins and results in their proteolytic cleavage and/or degradation . While phosphorylation of GLI3 mostly yields a partially cleaved GLI3 transcriptional repressor (GLI3R), which represses the expression of HH target genes, the phosphorylation of GLI2 mainly leads to its complete proteasomal degradation .
Shh -/- mice have altered heart looping  and a single outflow tract . Mice with tissue specific knockout of Shh -/- in the SHF, using Nkx2-5-Cre, display outflow tract septation defects . Shh -/- /Ihh -/- or Smo -/- embryos have a delayed Nkx2-5 expression and heart tube formation . In accordance, Ptch1 -/- embryos, which have the negative regulation of HH signalling removed, express higher levels of Nkx2-5 in the cardiac crescent . Gli2 -/- Gli3 +/- embryos have persistent truncus arteriosus (PTA) and a single outflow tract [19, 20]. SMO agonist (SAG), which enhances HH signalling, increases embryonic chick cardiac progenitor cell proliferation in vivo and in vitro . In zebrafish, treatment with SAG or Shh enhances the number of cardiomyocytes in the developing cardiac chambers [22, 23], whereas treatment with the HH signalling inhibitor, cyclopamine, reduces Nkx2-5 and Myhc expression as well as cardiomyocyte proliferation [22, 23]. Together these studies demonstrate that functional HH signalling is important for regulating the number of cardiac progenitor cells and heart development in vivo.
D. melanogaster embryos lacking a single Mef2 gene do not exhibit any muscle development . In mammals, there are four MEF2 members, MEF2A-D . Expression of a dominant-negative fusion protein of MEF2C with an engrailed repression domain (EnR) under the regulation of an Nkx2-5 enhancer (Nkx2-5-MEF2C/EnR), which mediates the repression of all MEF2 target genes, leads to severely disrupted cardiomyogenesis in mice . Mef2c -/- mice or mice with myocardium-specific knockout of Mef2c, through either Myhc6-Cre or Mlc2v-Cre, fail to undergo heart looping morphogenesis, as well as correct development of the right ventricle and outflow tract [8, 9]. Thus, MEF2 factors are important for early heart development.
Differentiating mouse embryonic stem (mES) cells share a similar hierarchical set of gene expression patterns observed during cardiomyogenesis in vivo . The mesoderm marker, Brachyury, and the precardiac mesoderm marker, Mesp1, are expressed by days 3 and 4 of differentiation, respectively ; cardiac progenitor genes Nkx2-5, Gata-4, Tbx5, and Mef2c are expressed by day 6 [27–29]; and both alpha and beta isoforms of MyHC proteins (MyHC6/α-MyHC and MyHC7/β-MyHC, respectively) are expressed in mES cell-derived cardiomyocytes . Although mES cells serve as a useful in vitro model system for studying molecular regulation of cardiomyogenesis, the roles of HH signalling during mES cardiomyogenesis have yet to be assessed.
The role of HH signalling and MEF2 factors during cardiomyogenesis in vitro has been studied in P19 embryonal carcinoma (EC) cells, a mES cell model system [31–33]. P19 cells originate from a mouse teratoma, are pluripotent, give rise to tissues in chimeric mice, and can be induced to differentiate into cardiomyocytes when treated with dimethylsulphoxide (DMSO) [34–36]. In P19 cells, overexpression of MEF2C, SHH, or GLI2 is sufficient to induce and enhance cardiomyogenesis through the upregulation of cardiac progenitor factors like Nkx2-5 and Gata-4 [31, 33]. In agreement, P19 cells treated with cyclopamine show delayed cardiomyogenesis , whereas expression of a dominant-negative GLI/EnR or Nkx2-5-MEF2C/EnR results in reduced cardiomyogenesis and Nkx2-5, Tbx5, Gata-4, and Myhc6 expression . GLI2 and MEF2C can directly bind to each other’s gene regulatory elements in P19 cells undergoing cardiomyogenesis, form a protein complex, and synergistically activate an Nkx2-5 promoter . Therefore, HH signalling and MEF2C may regulate cardiomyogenesis through a common pathway.
Chromatin remodelling factors modulate chromatin density, which affects the ability of transcription factors to regulate gene expression [37, 38]. The Brahma-associated factors (BAF) belong to the switch/sucrose non-fermentable (SWI/SNF) group of complexes and mediate nucleosome shifting on chromatin in an ATP-dependent manner . When the ATPase BAF subunit, Brahma-related gene 1 (BRG1/SMARCA4) is globally knocked out, embryos do not survive past the peri-implantation stage . Embryos with a conditional mutation of Brg1 in cardiac progenitor cells, using Nkx2-5-Cre, have irregular ventricle morphology and die by E10.5 . Therefore, BRG1 is important during heart development.
GLI3 and GLI1 proteins interact with BRG1 in the developing or postnatal brain, respectively . Furthermore, BRG1 is required for both HH target gene repression and activation in mouse embryonic fibroblasts (MEFs), most probably though an interaction with GLI3R and GLI1, respectively , and is recruited to at least some HH target genes in a HH signalling-dependent manner . Although GLI2 and BRG1 co-immunoprecipitate in MEFs, the importance of this interaction has yet to be tested .
Given the role of HH signalling and BAF subunits during cardiomyogenesis [18, 31–33, 41], the requirement of BRG1 for HH target gene activation, and BRG1’s ability to interact with GLI proteins , we hypothesized that GLI2 and BRG1 may function together to regulate cardiomyogenesis in vitro. Here we show that: 1) activation or suppression of HH signalling during mES cell cardiomyogenesis regulates cardiac progenitor transcripts Mef2c, Nkx2-5, and Tbx5; 2) GLI2 co-immunoprecipitates with BRG1 in differentiating mES cells; and 3) BRG1 is recruited to the GLI2 target gene, Mef2c, in a HH signalling-dependent manner in P19 cells undergoing cardiomyogenesis.
mES cell culture
D3 mES cells (ATCC, #CRL-1934) were cultured with 10 % fetal bovine serum (FBS, Wisent Inc.) and leukemia inhibitory factor (LIF, Millipore). D3 cells were stably transfected with the empty pcDNA3.1+ vector or the pcDNA3.1+ vector expressing Flag-Gli2 S662A , a full-length complementary DNA of mouse Gli2 driven by the CMV promoter, in frame with the Flag epitope and containing the serine to alanine mutation at position 662 which prevents phosphorylation and proteasomal degradation . Stable clones were termed mES[Ctrl] and mES[GLI2], respectively. Transfected colonies were selected with 0.8 mg/ml Geneticin (GIBCO) and screened for the highest FLAG-GLI2S662A transcription and protein expression levels by quantitative PCR (qPCR) and western blot analysis, respectively (Additional file 1: Figure S1 and data not shown). To induce differentiation, mES cells were divided into 20 μl hanging drops at 800 cells/drop and allowed to form aggregates for two days without LIF, as described in . For immunoprecipitation studies, mES cells were divided into 20 μl hanging drops at 8000 cells/drop to allow for sufficient starting material. The resulting embryoid bodies (EBs) were pooled and left in suspension for three days. Then the EBs were transferred to tissue culture-treated (TC) plates (Corning) or 0.1 % gelatin-coated (Fisher Scientific) coverslips and cultured until day 7, 10, or 15 for the analysis of cardiomyogenesis, neurogenesis, or skeletal myogenesis, respectively. Medium was changed every two days. For HH inhibition, KAAD-cyclopamine (Toronto Research Chemicals) or the methanol vehicle, were added from day 3 to day 7 at a final concentration of 3 μM where it was added every second day with medium change. KAAD-cyclopamine- and methanol-treated EBs were plated on 0.1 % gelatin-coated coverslips and were cultured until day 7.
P19 EC cell culture
Parental P19 EC cells (ATCC, #CRL-1825) or P19 EC cells transfected with either pcDNA3-GLI2 or an empty vector control as described in [33, 45], termed P19[GLI2] and P19[Ctrl], respectively, were cultured and differentiated as per . Briefly, the P19 EC cell differentiation was initiated by plating 5x104 cells/ml with 1 % v/v DMSO (Sigma-Aldrich) in non-adherent dishes. After 4 days in suspension, the newly aggregated EBs were transferred to TC plates or 0.1 % gelatin-coated (Fisher Scientific) coverslips without DMSO for an additional 2 days of the 6-day protocol. Throughout the differentiation process the EBs were fed fresh medium with or without DMSO at least every two days. To inhibit HH signalling in P19 EC cells, differentiating P19 EC cultures were treated with 5 μM KAAD-cyclopamine, as previously described , or with methanol vehicle alone throughout the entire 6-day protocol.
To detect FLAG-GLI2S662A in the stable cell lines, total protein extracts on days 2-5 of differentiation were prepared by lysing cells in radioimmunoprecipitation (RIPA) buffer [50 mM Tris pH7.5, 150 mM NaCL, 0.2 % NP-40, 2 mM EDTA, 1X PIC (Roche), 0.5 mM PMSF (Sigma-Aldrich)]. The extracts were spun down at 17,000 x g for 30 min and the lysates were collected. 300 μg of clarified total protein lysates was submitted to immunoprecipitation with 20 μl of FLAG-beads (FLAG-IP), as per Sigma-Aldrich’s protocol. Bound proteins were eluted by boiling the beads in SDS-page sample buffer for 10 min.
For mass-spectrometric analysis, FLAG-IP was performed using 5 mg of nuclear protein extracts from day 3 differentiating mES[GLI2] and mES[Ctrl] cells as described above except that the beads with bound proteins were washed in wash buffer containing 300 mM NaCl.
For co-immunoprecipitation assay, 2 mg of total protein lysate from day 3 differentiating mES[Ctrl] and mES[Gli2] cell lines, prepared as described above, was pre-cleared for 1 h using rec-Protein-G-Sepharose beads (Invitrogen) and then were split equally and subjected to immunoprecipitation with either mouse IgG agarose beads as a negative control (Sigma-Aldrich) or FLAG beads (Sigma-Aldrich). The immunoprecipitation was performed as above except low salt wash buffer was used (100 mM NaCl, 50 mM Tris pH7.5) and the IP lasted for 2 h.
The resulting IP eluates and total protein (input) samples were separated using a 4–12 % denaturing polyacrylamide gel (NuPAGE, Invitrogen) with MOPS running buffer according to the manufacturer’s protocol. The resolved proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad), blocked using non-fat dry milk (Carnation) reconstituted with Tris-buffered saline and Tween 20 (TBST), and incubated with GLI2- (kind gift from C.C. Hui ), Brg1- (Millipore), FLAG- (Sigma-Aldrich), or α-tubulin-specific (DM1A, Sigma-Aldrich) antibodies. Membranes were stripped with re-blot plus mild stripping buffer (Millipore) between each primary antibody incubation. The signal was detected using horseradish peroxidase (HRP)-conjugated secondary anti-mouse (Cell Signalling) or anti-rabbit (Santa Cruz) antibodies, followed by a chemiluminescence reaction using Pierce ECL substrate (Fisher Scientific).
Densitometry was performed on the GLI2-specific bands with the ImageJ program (National Institutes of Health, USA) . The densities of the GLI2 protein in the FLAG-IP samples were normalized to the loading control, α-tubulin, and were presented as a percentage of the highest band density.
Mass spectrometric analysis
Eluates from mES[Ctrl] and mES[Gli2] FLAG-IP, prepared as described above, were resolved on a 4–12 % denaturing polyacrylamide gel (NuPAGE, Invitrogen) and silver-stained. The staining identified a unique band profile in the 170-250 kDa range of the mES[GLI2] sample compared to the mES[Ctrl] sample (data not shown). Resolved proteins from both cell lines, within the 170-250 kDa range, were extracted from the gel using in-gel digestion, according to . The extracted samples were submitted to liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Thermo LTQ Orbitrap XL hybrid mass spectrometer with a nanospray ion source. The MS/MS ion spectra were matched against the SwissProt database (version 2013_05) using the MASCOT software (Matrix Science, UK) with a peptide mass tolerance of 10 ppm and a fragment mass tolerance of 0.6 Da .
Quantitative PCR (qPCR) analysis
Sequences of primers used for real time qPCR analyses
Day 7 differentiated mES cells were fixed in −20 °C MeOH (Fisher), incubated 1:1 with monoclonal MF20 antibody supernatant  in phosphate buffered saline (PBS) and Cy3-conjugated goat anti-mouse IgG (Jackson Immuno Research) 1:100 in PBS to detect pan-MyHC expression. Coverslips were mounted in 50 parts PBS, 50 parts glycerol (Fisher), and 1 part Hoechst 33258 dye for staining nuclei. Indirect immunofluorescence of MyHC was visualized using a Leica DMI6000B inverted fluorescent microscope (Leica Microsystems GmbH) and captured with a Hamamatsu Orca AG camera (Hamamatsu Photonics). Pictures were processed with the Volocity 4.3.2 software (Perkin Elmer). Cells were counted based on the number of nuclei and identified as MyHC+ve or MyHC-ve using the Volocity imaging program with automated cell-identification parameters as described in ref. . To measure the intensity of MHC signal, the outline of at least 10 MyHC+ve individual cells in 10 random fields of view per each cell line and experiment was drawn using the software ImageJ. The intensity of the MHC staining in each outlined cell was measured, together with several background measurements. The corrected intensity values = integrated density - (cell area x mean intensity reading for the background) was calculated as in McCloy et al . To assess proliferation, day 7 cells were treated with 10 μM EdU for 1 h prior to fixing with 3.7 % formaldehyde and then continuing with the EdU staining protocol (Invitrogen).
Chromatin immunoprecipitation (ChIP) assays
Sequences of primers used for ChIP-qPCR analyses
Chr 5:142,906,954 - 142,907,148
Chr 15: 70,644,478 - 70,644,564
Mef2c site A
Chr 13: 83,417,148 - 83,417,378
Mef2c site B
Chr 13: 83,450,400 - 83,450,695
Mef2c site C
Chr 13: 83,517,957 - 83,518,157
Mef2c site D
Chr 13: 83,524,812 - 83,524,937
Mef2c site E
Chr 13: 83,595,419 - 83,595,594
Mef2c site F
Chr 13: 83,633,148 - 83,633,305
Mef2c site G
Chr 13: 83,660,831 - 83,661,075
Mef2c site H
Chr 13: 83,664,180 - 83,664,382
Mef2c site I
Chr 13: 83,739,543 - 83,739,715
Chromatin from KAAD-cyclopamine- or MeOH-treated P19 EC cells was prepared as above with the following adapted fixing and sonication conditions. Cells from 4 plates of day 4 P19 EC EBs - treated with KAAD-cyclopamine or MeOH - were fixed with 1.5 mM Ethylene glycol bis[succinimidylsuccinate] (EGS) (Thermo Scientific) for 30 min alone, then with 1 % formaldehyde (Sigma-Aldrich) for an additional 30 min. Chromatin was sonicated in 1 mL, 12x12 AFA tubes (Covaris) for 30 min with an S220 Focused-ultrasonicator (Covaris) as per manufacturer’s recommended operating conditions for a target 200-700 bp fragment range. The IP of these samples was done using 20 μg of chromatin. The qPCR analyses were performed, as above, but with 1/50th of each eluted sample, per reaction.
BRG1 genome-wide ChIP-sequencing peaks described in undifferentiated mES cells  were screened for overlapping conserved GLI consensus binding motifs, which were identified with the Multiple Sequence Local Alignment and Visualization tool (Mulan) as described in . The nearest genes within 50 kb of these overlapping sites in the mouse genome (mm9 genome assembly) were categorized by gene ontology (GO) biological process analysis using the Genomic Regions Enrichment of Annotations Tool (GREAT) bioinformatics system as described in . The entire set of BRG1 target genes in mES cells  was used as background for assessing the enrichment of these identified genes.
Overexpression of FLAG-GLI2S662A results in increased levels of GLI2 protein during mES cell differentiation
Since GLI2 protein is prone to degradation when phosphorylated , we chose to stably transfect mES cells with a Flag-Gli2 S662A , a vector used to express a stabilized version of GLI2 . qPCR showed that over the course of differentiation, transgene expression declines in all four clones analyzed (Additional file 1: Figure S1), possibly reflecting post-transcriptional down-regulation of the Gli2 mRNA or transcript. We note that the Gli2 S662A mRNA that we expressed contains the entire 3’ untranslated region of the gene and as such could be subjected to microRNA-mediated downregulation [61, 62]. A similar downregulation was observed when we expressed a different transcription factor in pluripotent cells . Importantly, the expression of Gli2 S662A mRNA persisted in all clones, albeit at lower levels throughout differentiation (Additional file 1: Figure S1). For subsequent experiments, we selected clone #3, with the highest and most stable levels of Gli2 mRNA during differentiation (Additional file 1: Figure S1). From here on, this clone is referred to as the mES[GLI2] cell line, while control mES cells transfected with only a Flag peptide tag-containing vector (clone Flag#1) are referred to as the mES[Ctrl] cell line.
Comparable results were also observed at the transcriptional level. Flag-Gli2 S662A transcripts were only expressed in the mES[GLI2] cells and were at the highest level in the undifferentiated (day 0) mES[GLI2] cells (Fig. 1b, panel Flag-Gli2 S662A ), and this was mirrored by total Gli2 transcript levels (Fig. 1b and Additional file 1: Figure S1). Such a trend has been noted in other published work on differentiating mES cells . Thus, the stability of the exogenous GLI2S662A protein, which remains higher during the differentiation of mES[GLI2] cells compared to controls, is a key element in our model system.
Overexpression of GLI2 results in enhanced cardiac progenitor gene expression
During differentiation, both the mES[Ctrl] and mES[GLI2] cell lines showed a similar transition through the mesoderm stage of differentiation, as no significant difference was detected for the levels of Brachyury transcripts by day 4 between these two cell lines (Fig. 2, panel Brachyury). Mesp1 levels were significantly upregulated in the mES[GLI2] cultures as compared to control cultures on day 4 of differentiation, however, its level of expression was minor as compared to day 6 values and was not significantly higher than day 0 basal levels (Fig. 2, panel Mesp1). On day 6, no significant difference between mES[Ctrl] and mES[GLI2] cell lines was detected for the Mesp1 expression. Based on this analysis, the mesoderm and precardiac mesoderm stages of cardiomyogenesis during mES cell differentiation did not appear to be significantly regulated by GLI2 overexpression. These results are supported by previous reports, which showed that modulation of HH signalling in mouse P19 EC cells did not significantly regulate mesoderm induction [31–33].
The cardiac progenitor transcripts Nkx2-5, Mef2c, and Tbx5 were significantly upregulated in the mES[GLI2] cultures on day 6 of differentiation as compared to mES[Ctrl] cells (Fig. 2; panels Nkx2-5, Mef2c, and Tbx5). Interestingly, overexpression of GLI2 had no effect on day 6 Gata-4 transcript levels (Fig. 2, panel Gata-4). Therefore, overexpression of GLI2 during mES cell differentiation resulted in enhanced transcription levels of select cardiac progenitor genes. Our data supports and extends previous publications, where overexpression of GLI2 was shown to induce and enhance cardiac progenitor gene expression in P19 EC cells [31, 33].
We tested whether cells of the skeletal muscle and neuronal lineages were affected, since GLI2 and HH have been shown to regulate these processes [14, 65, 66]. However, we did not detect any significant increase in the mRNA levels of Pax3 (Additional file 2: Figures S2A), a gene involved in skeletal myogenesis and downstream of GLI2 [45, 53, 67, 68], or in MyHC-positive skeletal myocytes (data not shown as none of the cultures contained any skeletal myocytes after 10 days of differentiation). A significant increase in Ascl1, a gene involved in neural progenitor differentiation that is downstream of GLI2 and HH signalling [67, 69–71], was detected (Additional file 2: Figure S2B). However, none of the cultures contained NF68-positive neurons, even after 10 days of differentiation (negative data not shown).
Overexpression of GLI2 does not result in an increased number of cardiomyocytes
Although overexpression of GLI2 during mES cell differentiation also did not significantly affect the level of Myh6 or Myh7 transcripts on day 7 (Fig. 3c), we observed a notable increase in MyHC signal intensity in cardiac myocytes in mES[GLI2] cultures (Fig. 3d). This suggests that although the number of cardiomyocytes formed is unchanged, the presence of exogenous GLI2 is associated with a trend towards higher MyHC protein expression.
To test if overexpression of GLI2 had any effect on cell proliferation or apoptosis, we first analyzed EdU-positive cells. When cultures were analyzed for the presence of EdU-positive cells on day 7, there was no difference between mES[Ctrl] and mES[Gli2] cell lines (Additional file 3: Figure S3A). Of note, we also did not observe any differences in RNA yields between mES[Ctrl] and mES[Gli2] cultures throughout the 7-day differentiation protocol (Additional file 3: Figure S3B). This supports that overexpression of GLI2 did not have any overt effects on cell proliferation. Lastly, we analyzed cells with fragmented nuclei as a classical hallmark of apoptosis. Notably, there was no difference in the proportion of apoptotic cells in mES[Gli2] cultures when compared to mES[Ctrl] (Additional file 3: Figure S3C). Thus, overexpression of GLI2 did not have any effect on cell proliferation or survival in differentiating mES cells.
Antagonizing the HH pathway leads to lower cardiac progenitor gene expression
GLI2 interacts with BRG1 during mES cell differentiation
The observed co-immunoprecipitation of GLI2 and BRG1 during mES cell differentiation led to our hypothesis that GLI2 could recruit BRG1 to regulatory regions of GLI2 target genes to modulate their expression. Given the results and the known roles of BRG1 and GLI2 in heart development [19, 20, 41], we were interested in knowing if GLI2 and BRG1 could co-regulate key cardiac progenitor genes implicated in cardiomyogenesis. Mef2c, which is important for proper mammalian heart development [8, 75], was enhanced in day 6 differentiating mES[GLI2] cells compared to mES[Ctrl] cells (Fig. 2) and was recently identified as a direct target of GLI2 during P19 EC cell cardiomyogenesis . Thus, we set out to determine if BRG1 associates with GLI2-bound Mef2c regulatory elements in a HH-dependent manner. Since mES cells spontaneously differentiate into lineages of all three germ layers  and BRG1 is expressed in many mammalian cells and tissues , it is difficult to answer this question in the context of endogenous mES cell cardiomyogenesis. In contrast, P19 EC cells predominantly differentiate into cardiomyocytes by day 6 of differentiation, along with skeletal myogenic progenitors and fibroblast-like cells when treated with DMSO [35, 77, 78]. Moreover, P19 EC differentiation is very similar to mES myogenic differentiation [33, 44, 79, 80]. For these reasons, we used P19 cells to determine the role of BRG1-GLI2 protein complex during myogenic differentiation.
BRG1 associates with Mef2c regulatory elements in HH signalling dependent manner
A selection of proteins that associate with the Mef2c gene
Location (mm10: Chr 13)
Source of Protein
83.504,012 - 83,504,138
83,504,087 - 83,504,115
83,504,111 - 83,504,139
83,504,111 - 83,504,139
83,523,287 - 83,523,306
83,523,351 - 83,523,374
83,523,369 - 83,523,395
83,523,405 - 83,523,430
83,565,883 - 83,565,913
83,565,913 - 83,565,943
83,565,947 - 83,565,977
83,582,754 - 83,582,777
83,582,754 - 83,582,777
83,582,752 - 83,582,791
83,582,752 - 83,582,791
83,582,752 - 83,582,791
Results from the anti-BRG1 ChIP showed an association of BRG1 with Mef2c sites C, D, and G-I in the P19[Ctrl] cells as compared to ChIP with a non-specific rabbit serum control (Fig. 5c). Notably, although there was a similar trend in the P19[GLI2] cells, we did not observe statistical significance for BRG1 association on sites D and G-I when compared to non-specific rabbit serum (Fig. 5c). Mef2c site A, which lacked GLI2 association under the same experimental conditions in a previous study , showed no significant association with BRG1 in either cell line (Fig. 5c). However, the association of BRG1 with the Mef2c site C was significantly (p < 0.05) higher in P19[GLI2] cells as compared to P19[Ctrl] cells (Fig. 5c). This effect was not due to altered BRG1 expression in P19[GLI2] cells as there was no significant change in Brg1 expression in P19[GLI2] cells, as compared to P19[Ctrl] cells, on day 4 of differentiation, when the ChIP assay was performed (Additional file 4: Figure S4B).
Mef2c site C is of significant interest as it is located proximally to the ISL-1-dependent SHF enhancer region  (Fig. 5b, SHF1), it is upstream of the start site of a transcript expressed in the developing heart , and it is also the closest GLI2-binding site to a region previously shown to associate with BRG1 [33, 84] (Fig. 5b, light blue circle). Thus, as overexpression of GLI2 resulted in increased association of BRG1 with Mef2c site C, we chose this site to further investigate the dependence of BRG1 association on endogenous HH signalling during P19 EC cell cardiomyogenesis. KAAD-cyclopamine treatment of P19 EC cells resulted in a 71.7 ± 17.0 % and 60.5 ± 25.0 % decrease in Gli1 expression on days 4 and 6 of P19 EC cell differentiation, respectively, without significantly affecting the day 4 or 6 Brg1 transcript levels, compared to treatment with a vehicle control (Fig. 5d). Similar cyclopamine-mediated reductions of Gli1 expression have been seen before in P19, P19CL6, and mES cells [32, 85, 86]. By day 6, differentiating P19 EC cells treated with KAAD-cyclopamine expressed significantly lower Mef2c (45.9 ± 16.7 %), Gata-4 (74.8 ± 12.2 %), and Tbx5 (65.4 ± 5.9 %) transcript levels, compared to differentiating cells treated with the vehicle control (Fig. 5e; panels Mef2c, Gata-4, and Tbx5). These lower levels of cardiac progenitor-enriched transcripts were expected as cyclopamine-treated P19 EC cells have repressed Gata-4 expression  and cyclopamine-treated P19CL6 EC cells show lower levels of Gata-4 and Nkx2-5 transcripts  and we showed that a similar treatment in mES cells has a comparable effect (Fig. 4). Neither the level of contractile protein transcripts in the day 6 differentiated cells (Fig. 5e, panels Myhc6 and Myhc7) nor the number of MyHC+ve cardiomyocytes (data not shown) significantly differed between KAAD-cyclopamine and vehicle-treated P19 EC cells. This is supported by previous reports, where differentiating P19 EC cells treated with cyclopamine did not show any difference in the number of MyHC+ve cardiomyocytes . Thus, inhibition of HH signalling downregulated, but did not abolish, normal expression of cardiac progenitor specific transcripts and, overall, did not repress the formation of cardiomyocytes, in agreement with .
To test if inhibition of HH signalling attenuated the ability of BRG1 to associate with Mef2c site C, we performed an anti-BRG1 ChIP in day 4 differentiating P19 EC cells treated with vehicle or KAAD-cyclopamine (Fig. 5f). The association of BRG1 with Mef2c site C in P19 EC cells was significantly reduced upon HH inhibition (Fig. 5f). Importantly, KAAD-cyclopamine treatment had no significant effect on BRG1’s association with β-actin a known target of BRG1 in mES cells . A gene desert locus was used as a negative control and showed no significant association with BRG1. Therefore, HH signalling is required, at least to some extent, for the efficient association of BRG1 to the GLI2-specific Mef2c gene element C.
HH signalling is important for cardiac progenitor gene expression during mES cell differentiation
Using a stabilized GLI2 mutant, we have shown that GLI2 enhances early expression of cardiac progenitor transcripts such as Mef2c, Nkx2-5, and Tbx5 during mES cell cardiomyogenesis (Fig. 2). In agreement, when HH signalling in mES cells is blocked with cyclopamine, this leads to a great reduction in the expression of these genes (Fig. 4). This supports and expands previous reports in P19 EC cells [32, 33].
The effect of cardiac progenitor gene upregulation by exogenous Gli2 is probably not due to an increase in mesodermal transcript expression as GLI2 overexpression does not have any significant effect on the levels of Brachyury or Mesp1 on days 4 and 6, respectively (Fig. 2). Notably, the small upregulation of Mesp1 transcript levels on day 4 in mES[GLI2] cells is minor in comparison with day 6 values. This is in agreement with previous studies, which demonstrate that there is no significant HH-dependent effect on mesoderm transcripts in P19 EC cells [31–33].
The enhanced expression of Nkx2-5 in mES[GLI2] cells correlates with the HH-mediated expression of Nkx2-5 in vivo [4, 18] and in P19 EC cells [31–33]. The upregulation of Tbx5 transcript levels in differentiating mES[GLI2] cells (Fig. 2) follows the same pattern of Tbx5 upregulation seen in differentiating P19[GLI2] cells . When HH signalling is inhibited in P19 EC or mES cells by KAAD-cyclopamine, Tbx5 expression is reduced (Figs. 4 and 5e), similarly to P19 EC cells overexpressing dominant-negative GLI/EnR . Interestingly, Tbx5 is expressed in SHF cells that receive HH signals in vivo, and a TBX5-HH network is required for atrial septation . Although the regulation of Tbx5 expression by the HH pathway is yet to be determined in vivo, based on results from previous reports and our observations, Tbx5 expression is regulated by HH signalling in vitro, during P19 EC and mES cell differentiation.
In contrast to Mef2c, Nkx2-5, and Tbx5 upregulation in mES[GLI2] cells, Gata-4 expression is not affected when GLI2 is overexpressed (Fig. 2). Importantly, Gata-4 transcript levels were downregulated in mES cells treated with cyclopamine (Fig. 4), similarly to P19 EC cells (Fig. 5e and ref. ). This suggests that while exogenous Gli2 cannot enhance Gata-4 expression in differentiating mES cells, repression of endogenous HH signalling is sufficient to reduce its expression. It is possible that Gata-4 induction is more efficient with other Gli family members, that Gata-4 responds to Hh signaling with a different kinetic, or that it responds at a different threshold of Hh-Gli activity, compared to the other cardiac transcription factor genes. Previous studies have shown that Gata-4 expression might be independently regulated from Mef2c, Nkx2-5, and/or Tbx5 expression [84, 88, 89]. Removal of the adjacent endoderm from the side of a developing avian heart, or a conditional knockout of Baf250a in the SHF of the developing murine heart results in decreased Nkx2-5 and Mef2c expression while Gata-4 levels remain unaffected [84, 88]. In mES cells, overexpression of the cardiac progenitor gene regulator, Yin Yang 1 (YY1) enhances Nkx2-5 and Tbx5 expression with no apparent effect on Gata-4 expression . Therefore, our observation of Mef2c, Nkx2-5, and Tbx5 upregulation in mES[GLI2] cells with no observable effect on day 6 Gata-4 expression is in line with previous studies and could provide insight into a Gli2-dependent and independent regulation of mES cell cardiomyogenesis.
In summary, the activation or suppression of the HH signaling in mES cells yields a concerted up- or down-regulation of multiple cardiac progenitor transcription factor genes. Since our experiments were carried out using total cell populations, we cannot rule out the possibility that this occurred in a disorganized manner, with individual cells upregulating only one of the multiple cardiac progenitor specific genes we saw induced at the population level. Previous work by our and other labs, as well as this work, demonstrate that the induction patterns of Nkx2-5, Tbx5, MEF2C and Gata-4 in differentiating mES and P19 cells mimic each other, ultimately culminating in maximal transcription on the same days of differentiation, just before or on the onset of the expression of structural cardiac muscle genes (Fig. 2, and [27–29, 32, 33, 45]). Moreover, simultaneous expression of Gata-4 and Tbx5 is detected in hES-derived cardiovascular progenitors and cardiomyocytes, but not in smooth muscle or endothelial cells . These reports suggest that the expression of multiple signature cardiac muscle progenitor genes represents an induction of cardiac muscle progenitors and differentiated cardiomyocytes. Therefore, we deem unlikely the possibility of illegitimate expression of the overall signature cardiac progenitor specific genes in other cell types. Nevertheless, a transcriptome analysis at a single-cell level or after enrichment using cell surface markers specific for cardiac progenitors would help to directly address this question by providing an estimate of cell heterogeneity in the cultures.
Modulation of HH signalling does not alter the number of mES cardiomyocytes
Although modulation of HH signalling through Gli2 overexpression or application of cyclopamine regulates Nkx2-5, Mef2c and Tbx5 cardiac progenitor gene expression in mES cells (Figs. 2 and 4), this does not alter the percentage of cardiomyocytes (Figs. 3 and 4). One explanation might lie in Gata-4 expression. Namely, Gata-4 appears to be a limiting factor, in a transcription factor cocktail, to induce MyHC expression in mES cells . Therefore, the lack of Gata-4 enhancement by exogenous GLI2 may offer an explanation for the lack of cardiomyocyte number increase in mES[GLI2] cultures. HH signalling gain-of-function experiments in P19 cells showed both an induction and enhancement of cardiomyogenesis [31, 33]. P19 cells may be more susceptible to differentiate into cardiomyocytes as they are more prone to differentiate into cells of the mesodermal lineage . In contrast, mES cells differentiating through EBs in the absence of any other external stimuli give rise to cell types from all three germ layers [76, 92, 93] and thus may not have as many mesoderm cells that are susceptible to HH-mediated regulation. It is possible that GLI2 overexpression affects the differentiation of other cell types as HH signalling regulates many developmental programs in vivo [14, 65, 66], resulting in a lower percentage of cardiomyocytes in mES[GLI2] cultures. Another possibility is that there may be other additional factors, mechanisms and signalling pathways in mES cells that regulate the transition of pre-cardiac mesoderm to cardiomyocyte progenitor cell when compared to P19 EC cells. Lastly, HH signalling may enhance mechanisms in the neighbouring lineages that in turn negatively regulate cardiac differentiation in mES cells.
GLI2 immunoprecipitates with BRG1 during mES cardiomyogenesis and active HH signalling regulates BRG1 association with Mef2c gene
To determine the mechanism of Gli2 during in vitro cardiomyogenesis, we used both mES(Gli2) and P19(Gli2) differentiating cells. The co-immunoprecipitation of GLI2 and BRG1 in differentiating mES cells (Fig. 5a) is in agreement with previous observations of HA-tagged GLI2 immunoprecipitation with BRG1 in NIH 3 T3 cells . To investigate the significance of this interaction, we tested the hypothesis that GLI2 and BRG1 co-regulate cardiomyogenesis in vitro by modulating the expression of GLI2 target genes. In this light, we focused on Mef2c gene, a direct target of GLI2 during P19 EC cell cardiomyogenesis . Increased Mef2c transcript levels in mES[GLI2] (Fig. 2) support and extend these results. Moreover, BRG1 associates with the first exon in the Mef2c gene , where other proteins, including MyoD, are known to bind , but which lacks GLI consensus binding site (Fig. 5b).
The anti-BRG1 ChIP analysis from day 4 differentiating P19 EC control cells demonstrates that BRG1 associates with Mef2c sites C, D, and G-I (Fig. 5c), which GLI2 has been shown to bind under similar conditions . These results support and extend a previous report  by showing that BRG1 can associate with GLI2-specific Mef2c sites C and D, which are closer to the ISL-1-dependent and NKX2-5/FOXH1-dependent SHF enhancer regions than sites previously identified (Fig. 5b) . Of these two sites, Brg1 is more efficiently recruited to Mef2c site C in P19 cells overexpressing Gli2. Moreover, when HH signalling in P19 cells is blocked, there is a reduction of Brg1 association with this site. Thus, our findings indicate that BRG1 is recruited to at least one Mef2c gene regulatory element in a HH- and GLI2-dependent manner.
Our hypothesis that Gli2 and Brg1 may share common chromatin targets is also supported by the identification of additional 1892 putative GLI- and BRG1-target genes through an in silico analysis (Additional file 5: Table S1), where BRG1 genome-wide ChIP-sequencing peaks from  were screened for conserved GLI consensus binding motifs. While we did not identify Mef2c as a common putative target, most probably due to the stringent conditions of the analysis, we did identify previously reported shared GLI- and BRG1-target genes, Gli1 and Ptch1 , amongst the theoretical targets, validating this bioinformatic assay (Additional file 5: Table S1).
Selected gene ontology biological processes significantly enriched among genes within 50 kb of a BRG1- associating site and GLI consensus binding motifa
Regulation of gene expression
Atf2, Meis1, Ncoa2, Ncor1, Smad2
Actn1, Ctnna1, Ctnnd1, Mapt, Myo1e
Angpt1, Fgf15, Fgf18, Pdgfa
Creb1, Dhh, Mef2d, Ptch1, Rara, Smo
Nervous system development
Bdnf, Neurod4, Nkx2-2, Pax6
Pattern specification process
Hoxa2, Hoxa9, Hoxd3, Yy1
Atoh1, Otx1, Otx2, Gata2
Foxh1, Notch1, Tbx20, Ttn, Wnt5a
Regulation of cell cycle
Cdc26, Cdk4, Cdk6, E2f1, E2f2
Bmp2, Gli1, Gli2
Epha2, Fat1, Lama5, Lamc1, Tubb2b
Hdac3, Mll3, Myst2, Smarca2, Smarcd1
Taken together, our results indicate that GLI2 and BRG1 co-operate to regulate cardiomyogenesis in vitro. We propose a mechanism where HH signaling, through activated GLI2, recruits BRG1 along with other potential co-factors to the Mef2c gene site C to promote Mef2c expression (Fig. 6b). Inhibition of HH signaling results in reduced BRG1 association at Mef2c site C and reduced Mef2c expression (Fig. 6c). Since BRG1 association correlates with chromatin accessibility , we predict that GLI2 might recruit BRG1 to increase chromatin accessibility on Mef2c and other potential yet unidentified target genes to regulate in vitro cardiomyogenesis.
ChIP, chromatin immunoprecipitation; DHH, desert hedgehog; DMSO, dimethylsulphoxide; EC, embryonal carcinoma; EGS, ethylene glycol bis[succinimidylsuccinate]; EnR, engrailed repression domain; FHF, first heart field; HH, hedgehog; HRP, horseradish peroxidase; IHH, Indian hedgehog; LIF, leukemia inhibitory factor; MEF, mouse embryonic fibroblast; mES, mouse embryonic stem; MyHC, myosin heavy chain; PTA, persistent truncus arteriosus; PVDF, polyvinylidene fluoride; RIPA, radioimmunoprecipitation; SAG, smoothened agonist; SHF, secondary heart field; SHH, sonic hedgehog; SWI/SNF, switch/sucrose non-fermentable; TBST, tris-buffered saline and Tween 20
The authors would like to thank Dr. Spiegelman for providing them with the Flag-Gli2 S662A expression vector and Dr. Imane Chakroun for her technical support.
This work was supported by grants MOP-53277 (to I.S.S.) and MOP-119458 (to A.B.) from the Canadian Institutes of Health Research (http://www.cihr-irsc.gc.ca/e/193.html). J.V.F. was supported by an Ontario Graduate Scholarship and a Canadian Institutes of Health Research Master’s Award. A.V. was supported by an Ontario Graduate Scholarship and a doctoral research award from the Heart and Stroke Foundation of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
All supporting data are presented in the main manuscript and supplementary data file.
Conceived and designed the experiments: JVF AV ISS. Performed the experiments: JVF AV NB ISS RR. Analyzed the data: JVF AV ISS RR. Contributed reagents/materials/analysis tools: JVF AV NB AB ISS. Wrote the manuscript: JVF AV AB. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Bruneau BG. The developmental genetics of congenital heart disease. Nature. 2008;451:943–8.View ArticlePubMedGoogle Scholar
- Buckingham M, Meilhac S, Zaffran S. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet. 2005;6:826–35.View ArticlePubMedGoogle Scholar
- Harvey RP. Patterning the vertebrate heart. Nat Rev Genet. 2002;3(7):544–56.View ArticlePubMedGoogle Scholar
- Xin M, Olson EN, Bassel-Duby R. Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair. Nat Rev Mol Cell Biol. 2013;14:529–41.View ArticlePubMedPubMed CentralGoogle Scholar
- Vincent SD, Buckingham ME. How to make a heart: the origin and regulation of cardiac progenitor cells. Curr Top Dev Biol. 2010;90:1–41.View ArticlePubMedGoogle Scholar
- Lyons I, Parsons LM, Hartley L, Li R, Andrews JE, Robb L, Harvey RP. Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5. Genes Dev. 1995;9:1654–66.View ArticlePubMedGoogle Scholar
- Maitra M, Schluterman MK, Nichols HA, Richardson JA, Lo CW, Srivastava D, Garg V. Interaction of Gata4 and Gata6 with Tbx5 is critical for normal cardiac development. Dev Biol. 2009;326:368–77.View ArticlePubMedGoogle Scholar
- Lin Q, Schwarz J, Bucana C, Olson EN. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science. 1997;276(5317):1404–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Vong LH, Ragusa MJ, Schwarz JJ. Generation of conditional Mef2cloxP/loxP mice for temporal- and tissue-specific analyses. Genesis. 2005;43(1):43–8.View ArticlePubMedGoogle Scholar
- Kuo CT, Morrisey EE, Anandappa R, Sigrist K, Lu MM, Parmacek MS, Soudais C, Leiden JM. GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes Dev. 1997;11:1048–60.View ArticlePubMedGoogle Scholar
- Marigo V, Davey RA, Zuo Y, Cunningham JM, Tabin CJ. Biochemical evidence that patched is the Hedgehog receptor. Nature. 1996;384(6605):176–9.View ArticlePubMedGoogle Scholar
- Stone DM, Hynes M, Armanini M, Swanson TA, Gu Q, Johnson RL, Scott MP, Pennica D, Goddard A, Phillips H, Noll M, Hooper JE, de Sauvage F, Rosenthal A. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature. 1996;384(6605):129–34.View ArticlePubMedGoogle Scholar
- Hui CC, Angers S. Gli proteins in development and disease. Annu Rev Cell Dev Biol. 2011;27:513–37.View ArticlePubMedGoogle Scholar
- Briscoe J, Thérond PP. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat Rev Mol Cell Biol. 2013;14(7):416–29.View ArticlePubMedGoogle Scholar
- Tsukui T, Capdevila J, Tamura K, Ruiz-Lozano P, Rodriguez-Esteban C, Yonei-Tamura S, Magallon J, Chandraratna RA, Chien K, Blumberg B, Evans RM, Belmonte JC. Multiple left-right asymmetry defects in Shh(-/-) mutant mice unveil a convergence of the shh and retinoic acid pathways in the control of Lefty-1. Proc Natl Acad Sci U S A. 1999;96:11376–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Washington Smoak I, Byrd NA, Abu-Issa R, Goddeeris MM, Anderson R, Morris J, Yamamura K, Klingensmith J, Meyers EN. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev Biol. 2005;283(2):357–72.View ArticlePubMedGoogle Scholar
- Goddeeris MM, Schwartz R, Klingensmith J, Meyers EN. Independent requirements for Hedgehog signaling by both the anterior heart field and neural crest cells for outflow tract development. Development. 2007;134(8):1593–604.View ArticlePubMedGoogle Scholar
- Zhang XM, Ramalho-Santos M, McMahon AP. Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R symmetry by the mouse node. Cell. 2001;106:781–92.View ArticlePubMedGoogle Scholar
- Kim PC, Mo R, Hui Cc C. Murine models of VACTERL syndrome: Role of sonic hedgehog signaling pathway. J Pediatr Surg. 2001;36(2):381–4.View ArticlePubMedGoogle Scholar
- Kim J, Kim P, Hui CC. The VACTERL association: lessons from the Sonic hedgehog pathway. Clin Genet. 2001;59(5):306–15.View ArticlePubMedGoogle Scholar
- Dyer LA, Makadia FA, Scott A, Pegram K, Hutson MR, Kirby ML. BMP signaling modulates hedgehog-induced secondary heart field proliferation. Dev Biol. 2010;348(2):167–76.View ArticlePubMedPubMed CentralGoogle Scholar
- Thomas NA, Koudijs M, van Eeden FJ, Joyner AL, Yelon D. Hedgehog signaling plays a cell-autonomous role in maximizing cardiac developmental potential. Development. 2008;135:3789–99.View ArticlePubMedPubMed CentralGoogle Scholar
- Choi W, Gemberling M, Wang J, Holdway JE, Shen M, Karlstrom RO, Poss KD. In vivo monitoring of cardiomyocyte proliferation to identify chemical modifiers of heart regeneration. Development. 2013;140:660–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Lilly B, Zhao B, Ranganayakulu G, Paterson BM, Schulz RA, Olson EN. Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila. Science. 1995;267(5198):688–93.View ArticlePubMedGoogle Scholar
- Potthoff MJ, Olson EN. MEF2: a central regulator of diverse developmental programs. Development. 2007;134:4131–40.View ArticlePubMedGoogle Scholar
- Karamboulas C, Dakubo GD, Liu J, De Repentigny Y, Yutzey K, Wallace VA, Kothary R, Skerjanc IS. Disruption of MEF2 activity in cardiomyoblasts inhibits cardiomyogenesis. J Cell Sci. 2006;119(Pt 20):4315–21.View ArticlePubMedGoogle Scholar
- Bondue A, Lapouge G, Paulissen C, Semeraro C, Iacovino M, Kyba M, Blanpain C. Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specification. Cell Stem Cell. 2008;3:69–84.View ArticlePubMedGoogle Scholar
- Christoforou N, Miller RA, Hill CM, Jie CC, McCallion AS, Gearhart JD. Mouse ES cell-derived cardiac precursor cells are multipotent and facilitate identification of novel cardiac genes. J Clin Invest. 2008;118(3):894–903.PubMedPubMed CentralGoogle Scholar
- Wu J, Kubota J, Hirayama J, Nagai Y, Nishina S, Yokoi T, Asaoka Y, Seo J, Shimizu N, Kajiho H, Watanabe T, Azuma N, Katada T, Nishina H. p38 Mitogen-activated protein kinase controls a switch between cardiomyocyte and neuronal commitment of murine embryonic stem cells by activating myocyte enhancer factor 2C-dependent bone morphogenetic protein 2 transcription. Stem Cells Dev. 2010;19(11):1723–34.View ArticlePubMedGoogle Scholar
- Metzger JM, Lin WI, Johnston RA, Westfall MV, Samuelson LC. Myosin heavy chain expression in contracting myocytes isolated during embryonic stem cell cardiogenesis. Circ Res. 1995;76:710–9.View ArticlePubMedGoogle Scholar
- Gianakopoulos PJ, Skerjanc IS. Hedgehog signaling induces cardiomyogenesis in P19 cells. J Biol Chem. 2005;280:21022–8.View ArticlePubMedGoogle Scholar
- Gianakopoulos PJ, Skerjanc IS. Cross talk between hedgehog and bone morphogenetic proteins occurs during cardiomyogenesis in P19 cells. In Vitro Cell Dev Biol Anim. 2009;45:566–72.View ArticlePubMedGoogle Scholar
- Voronova A, Al Madhoun A, Fischer A, Shelton M, Karamboulas C, Skerjanc IS. Gli2 and MEF2C activate each other’s expression and function synergistically during cardiomyogenesis in vitro. Nucleic Acids Res. 2012;40(8):3329–47.View ArticlePubMedGoogle Scholar
- McBurney MW, Rogers BJ. Isolation of male embryonal carcinoma cells and their chromosome replication patterns. Dev Biol. 1982;89:503–8.View ArticlePubMedGoogle Scholar
- McBurney MW. P19 embryonal carcinoma cells. Int J Dev Biol. 1993;37(1):135–40.PubMedGoogle Scholar
- van der Heyden MA, Defize LH. Twenty one years of P19 cells: what an embryonal carcinoma cell line taught us about cardiomyocyte differentiation. Cardiovasc Res. 2003;58:292–302.View ArticlePubMedGoogle Scholar
- de la Serna IL, Ohkawa Y, Imbalzano AN. Chromatin remodelling in mammalian differentiation: lessons from ATP-dependent remodellers. Nat Rev Genet. 2006;7:461–73.View ArticlePubMedGoogle Scholar
- Paranjape SM, Kamakaka RT, Kadonaga JT. Role of chromatin structure in the regulation of transcription by RNA polymerase II. Annu Rev Biochem. 1994;63:265–97.View ArticlePubMedGoogle Scholar
- Ho L, Crabtree GR. Chromatin remodelling during development. Nature. 2010;463:474–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Bultman S, Gebuhr T, Yee D, La Mantia C, Nicholson J, Gilliam A, Randazzo F, Metzger D, Chambon P, Crabtree G, Magnuson T. A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Mol Cell. 2000;6:1287–95.View ArticlePubMedGoogle Scholar
- Takeuchi JK, Lou X, Alexander JM, Sugizaki H, Delgado-Olguín P, Holloway AK, Mori AD, Wylie JN, Munson C, Zhu Y, Zhou Y, Yeh R, Henkelman RM, Harvey RP, Metzger D, Chambon P, Stainier DYR, Pollard KS, Scott IC, Bruneau BG. Chromatin remodelling complex dosage modulates transcription factor function in heart development. Nat Commun. 2011;2:187.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhan X, Shi X, Zhang Z, Chen Y, Wu JI. Dual role of Brg chromatin remodeling factor in Sonic hedgehog signaling during neural development. Proc Natl Acad Sci U S A. 2011;108:12758–63.View ArticlePubMedPubMed CentralGoogle Scholar
- Bhatia N, Thiyagarajan S, Elcheva I, Saleem M, Dlugosz A, Mukhtar H, Spiegelman VS. Gli2 is targeted for ubiquitination and degradation by beta-TrCP ubiquitin ligase. J Biol Chem. 2006;281:19320–6.View ArticlePubMedGoogle Scholar
- Ryan T, Shelton M, Lambert JP, Malecova B, Boisvenue S, Ruel M, Figeys D, Puri PL, Skerjanc IS. Myosin phosphatase modulates the cardiac cell fate by regulating the subcellular localization of Nkx2.5 in a Wnt/Rho-associated protein kinase-dependent pathway. Circ Res. 2013;112(2):257–66.View ArticlePubMedGoogle Scholar
- Petropoulos H, Gianakopoulos PJ, Ridgeway AG, Skerjanc IS. Disruption of Meox or Gli activity ablates skeletal myogenesis in P19 cells. J Biol Chem. 2004;279:23874–81.View ArticlePubMedGoogle Scholar
- Rudnicki MA, McBurney MW. Cell culture methods and induction of differentiation of embryonal carcinoma cell lines. In: Robertson EJ, editor. Teratocarcinomas and embryonic stem cells: a practical approach. Oxford: IRL Press; 1987.Google Scholar
- Hu MC, Mo R, Bhella S, Wilson CW, Chuang PT, Hui CC, Rosenblum ND. GLI3-dependent transcriptional repression of Gli1, Gli2 and kidney patterning genes disrupts renal morphogenesis. Development. 2006;133:569–78.View ArticlePubMedGoogle Scholar
- Abràmoff MD, Magalhães PJ, Ram SJ. Image processing with imageJ. Biophotonics Int. 2004;11:36–42.Google Scholar
- Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc. 2006;1(6):2856–60.View ArticlePubMedGoogle Scholar
- Eng JK, Searle BC, Clauser KR, Tabb DL. A face in the crowd: recognizing peptides through database search. Mol Cell Proteomics. 2011;10(11):R111.009522.View ArticlePubMedPubMed CentralGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–8.View ArticlePubMedGoogle Scholar
- Gianakopoulos PJ, Mehta V, Voronova A, Cao Y, Yao Z, Coutu J, Wang X, Waddington MS, Tapscott SJ, Skerjanc IS. MyoD directly up-regulates premyogenic mesoderm factors during induction of skeletal myogenesis in stem cells. J Biol Chem. 2011;286:2517–25.View ArticlePubMedGoogle Scholar
- Voronova A, Coyne E, Al Madhoun A, Fair JV, Bosiljcic N, St-Louis C, Li G, Thurig S, Wallace V, Wiper-Bergeron N, Skerjanc IS. Hedgehog signaling regulates MyoD expression and activity. J Biol Chem. 2013;288:4389–404.View ArticlePubMedGoogle Scholar
- Bader D, Masaki T, Fischman DA. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J Cell Biol. 1982;95:763–70.View ArticlePubMedGoogle Scholar
- McCloy RA, Rogers S, Caldon CE, Lorca T, Castro A, Burgess A. Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle. 2014;13(9):1400–12.View ArticlePubMedPubMed CentralGoogle Scholar
- Savage J, Conley AJ, Blais A, Skerjanc IS. SOX15 and SOX7 differentially regulate the myogenic program in P19 cells. Stem Cells. 2009;27:1231–43.View ArticlePubMedGoogle Scholar
- Ho L, Jothi R, Ronan JL, Cui K, Zhao K, Crabtree GR. An embryonic stem cell chromatin remodeling complex, esBAF, is an essential component of the core pluripotency transcriptional network. Proc Natl Acad Sci U S A. 2009;106:5187–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Ovcharenko I, Loots GG, Giardine BM, Hou M, Ma J, Hardison RC, Stubbs L, Miller W. Mulan: multiple-sequence local alignment and visualization for studying function and evolution. Genome Res. 2005;15(1):184–94.View ArticlePubMedPubMed CentralGoogle Scholar
- McLean CY, Bristor D, Hiller M, Clarke SL, Schaar BT, Lowe CB, Wenger AM, Bejerano G. GREAT improves functional interpretation of cis-regulatory regions. Nat Biotechnol. 2010;28(5):495–501.View ArticlePubMedPubMed CentralGoogle Scholar
- Pan Y, Bai CB, Joyner AL, Wang B. Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation. Mol Cell Biol. 2006;26(9):3365–77.View ArticlePubMedPubMed CentralGoogle Scholar
- Sun Z, Zhang T, Hong H, Liu Q, Zhang H. miR-202 suppresses proliferation and induces apoptosis of osteosarcoma cells by downregulating Gli2. Mol Cell Biochem. 2014;397(1-2):277–83.View ArticlePubMedGoogle Scholar
- Jiang Z, Cushing L, Ai X, Lu J. miR-326 is downstream of Sonic hedgehog signaling and regulates the expression of Gli2 and smoothened. Am J Respir Cell Mol Biol. 2014;51(2):273–83.PubMedPubMed CentralGoogle Scholar
- Savage J, Voronova A, Mehta V, Sendi-Mukasa F, Skerjanc IS. Canonical Wnt signaling regulates Foxc1/2 expression in P19 cells. Differentiation. 2010;79(1):31–40.View ArticlePubMedGoogle Scholar
- Agren M, Kogerman P, Kleman MI, Wessling M, Toftgard R. Expression of the PTCH1 tumor suppressor gene is regulated by alternative promoters and a single functional Gli-binding site. Gene. 2004;330:101–14.View ArticlePubMedGoogle Scholar
- Jiang J, Hui CC. Hedgehog signaling in development and cancer. Dev Cell. 2008;15(6):801–12.View ArticlePubMedGoogle Scholar
- Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001;15(23):3059–87.View ArticlePubMedGoogle Scholar
- Voronova A, Fischer A, Ryan T, Al Madhoun A, Skerjanc IS. Ascl1/Mash1 is a novel target of Gli2 during Gli2-induced neurogenesis in P19 EC cells. PLoS One. 2011;6(4):e19174.View ArticlePubMedPubMed CentralGoogle Scholar
- Ridgeway AG, Skerjanc IS. Pax3 is essential for skeletal myogenesis and the expression of Six1 and Eya2. J Biol Chem. 2001;276(22):19033–9.View ArticlePubMedGoogle Scholar
- Guillemot L, Levy A, Raymondjean M, Rothhut B. Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT1A cells. J Biol Chem. 2001;13:13.Google Scholar
- Pattyn A, Guillemot F, Brunet JF. Delays in neuronal differentiation in Mash1/Ascl1 mutants. Dev Biol. 2006;295(1):67–75.View ArticlePubMedGoogle Scholar
- Wang L, Zhang ZG, Gregg SR, Zhang RL, Jiao Z, LeTourneau Y, Liu X, Feng Y, Gerwien J, Torup L, Leist M, Noguchi CT, Chen ZY, Chopp M. The Sonic hedgehog pathway mediates carbamylated erythropoietin-enhanced proliferation and differentiation of adult neural progenitor cells. J Biol Chem. 2007;282(44):32462–70.View ArticlePubMedGoogle Scholar
- Hidaka K, Lee JK, Kim HS, Ihm CH, Iio A, Ogawa M, Nishikawa S, Kodama I, Morisaki T. Chamber-specific differentiation of Nkx2.5-positive cardiac precursor cells from murine embryonic stem cells. FASEB J. 2003;17(6):740–2.PubMedGoogle Scholar
- Taipale J, Chen JK, Cooper MK, Wang BL, Mann RK, Milenkovic L, Scott MP, Beachy PA. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature. 2000;406(6799):1005–9.View ArticlePubMedGoogle Scholar
- Goodrich LV, Milenkovic L, Higgins KM, Scott MP. Altered neural cell fates and medulloblastoma in mouse patched mutants. Science. 1997;277(5329):1109–13.View ArticlePubMedGoogle Scholar
- Barnes RM, Harris IS, Jaehnig EJ, Sauls K, Sinha T, Rojas A, Schachterle W, McCulley DJ, Norris RA, Black BL. MEF2C regulates outflow tract alignment and transcriptional control of Tdgf1. Development. 2016;143(5):774-9.Google Scholar
- Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 1988;336(6200):684–7.View ArticlePubMedGoogle Scholar
- Rudnicki MA, Jackowski G, Saggin L, McBurney MW. Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells. Dev Biol. 1990;138(2):348–58.View ArticlePubMedGoogle Scholar
- Rudnicki MA, Reuhl KR, McBurney MW. Cell lines with developmental potential restricted to mesodermal lineages isolated from differentiating cultures of pluripotential P19 embryonal carcinoma cells. Development. 1989;107(2):361–72.PubMedGoogle Scholar
- Kennedy KA, Porter T, Mehta V, Ryan SD, Price F, Peshdary V, Karamboulas C, Savage J, Drysdale TA, Li SC, Bennett SA, Skerjanc IS. Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative beta-catenin. BMC Biol. 2009;7:67.View ArticlePubMedPubMed CentralGoogle Scholar
- Al Madhoun AS, Mehta V, Li G, Figeys D, Wiper-Bergeron N, Skerjanc IS. Skeletal myosin light chain kinase regulates skeletal myogenesis by phosphorylation of MEF2C. EMBO J. 2011;30(12):2477–89.View ArticlePubMedPubMed CentralGoogle Scholar
- Machida Y, Murai K, Miyake K, Iijima S. Expression of chromatin remodeling factors during neural differentiation. J Biochem. 2001;129(1):43–9.View ArticlePubMedGoogle Scholar
- Dodou E, Verzi MP, Anderson JP, Xu SM, Black BL. Mef2c is a direct transcriptional target of ISL1 and GATA factors in the anterior heart field during mouse embryonic development. Development. 2004;131:3931–42.View ArticlePubMedGoogle Scholar
- Black BL, Cripps RM. Myocyte Enhancer Factor 2 Transcription Factors in Heart Development and Disease. In: Rosenthal N, Harvey RP, editors. Heart Development and Regeneration, vol. 2. Oxford: Academic; 2010. p. 673–99.View ArticleGoogle Scholar
- Lei I, Gao X, Sham MH, Wang Z. SWI/SNF protein component BAF250a regulates cardiac progenitor cell differentiation by modulating chromatin accessibility during second heart field development. J Biol Chem. 2012;287(29):24255–62.View ArticlePubMedPubMed CentralGoogle Scholar
- Clement CA, Kristensen SG, Mollgard K, Pazour GJ, Yoder BK, Larsen LA, Christensen ST. The primary cilium coordinates early cardiogenesis and hedgehog signaling in cardiomyocyte differentiation. J Cell Sci. 2009;122(Pt 17):3070–82.View ArticlePubMedPubMed CentralGoogle Scholar
- Zheng G, Tao Y, Yu W, Schwartz RJ. Brief report: SRF-dependent MiR-210 silences the sonic hedgehog signaling during cardiopoesis. Stem Cells. 2013;31(10):2279–85.View ArticlePubMedGoogle Scholar
- Xie L, Hoffmann AD, Burnicka-Turek O, Friedland-Little JM, Zhang K, Moskowitz IP. Tbx5-hedgehog molecular networks are essential in the second heart field for atrial septation. Dev Cell. 2012;23:280–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Alsan BH, Schultheiss TM. Regulation of avian cardiogenesis by Fgf8 signaling. Development. 2002;129(8):1935–43.PubMedGoogle Scholar
- Gregoire S, Karra R, Passer D, Deutsch M, Krane M, Feistritzer R, Sturzu A, Domian I, Saga Y, Wu SM. Essential and unexpected role of Yin Yang 1 to promote mesodermal cardiac differentiation. Circ Res. 2013;112:900–10.View ArticlePubMedPubMed CentralGoogle Scholar
- Li Y, Lin B, Yang L. Comparative transcriptomic analysis of multiple cardiovascular fates from embryonic stem cells predicts novel regulators in human cardiogenesis. Sci Rep. 2015;5:9758.View ArticlePubMedPubMed CentralGoogle Scholar
- Bai F, Ho Lim C, Jia J, Santostefano K, Simmons C, Kasahara H, Wu W, Terada N, Jin S. Directed differentiation of embryonic stem cells into cardiomyocytes by bacterial injection of defined transcription factors. Sci Rep. 2015;5:15014.View ArticlePubMedPubMed CentralGoogle Scholar
- Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154–6.View ArticlePubMedGoogle Scholar
- Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981;78:7634–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang DZ, Valdez MR, McAnally J, Richardson J, Olson EN. The Mef2c gene is a direct transcriptional target of myogenic bHLH and MEF2 proteins during skeletal muscle development. Development. 2001;128:4623–33.PubMedGoogle Scholar
- Hui CC, Slusarski D, Platt KA, Holmgren R, Joyner AL. Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm- and mesoderm-derived tissues suggests multiple roles during postimplantation development. Dev Biol. 1994;162:402–13.View ArticlePubMedGoogle Scholar
- Agarwal P, Verzi MP, Nguyen T, Hu J, Ehlers ML, McCulley DJ, Xu S, Dodou E, Anderson JP, Wei ML, Black BL. The MADS box transcription factor MEF2C regulates melanocyte development and is a direct transcriptional target and partner of SOX10. Development. 2011;138:2555–65.View ArticlePubMedPubMed CentralGoogle Scholar
- De Val S, Chi NC, Meadows SM, Minovitsky S, Anderson JP, Harris IS, Ehlers ML, Agarwal P, Visel A, Xu S, Pennacchio L, Dubchak I, Krieg P, Stainier DYR, Black BL. Combinatorial regulation of endothelial gene expression by ets and forkhead transcription factors. Cell. 2008;135:1053–64.View ArticlePubMedPubMed CentralGoogle Scholar
- De Val S, Anderson JP, Heidt AB, Khiem D, Xu S, Black BL. Mef2c is activated directly by Ets transcription factors through an evolutionarily conserved endothelial cell-specific enhancer. Dev Biol. 2004;275:424–34.View ArticlePubMedGoogle Scholar
- Dodou E, Xu S, Black BL. mef2c is activated directly by myogenic basic helix-loop-helix proteins during skeletal muscle development in vivo. Mech Dev. 2003;120:1021–32.View ArticlePubMedGoogle Scholar
- Hao H, Tummala P, Guzman E, Mali RS, Gregorski J, Swaroop A, Mitton KP. The transcription factor neural retina leucine zipper (NRL) controls photoreceptor-specific expression of myocyte enhancer factor Mef2c from an alternative promoter. J Biol Chem. 2011;286:34893–902.View ArticlePubMedPubMed CentralGoogle Scholar
- von Both I, Silvestri C, Erdemir T, Lickert H, Walls JR, Henkelman RM, Rossant J, Harvey RP, Attisano L, Wrana JL. Foxh1 is essential for development of the anterior heart field. Dev Cell. 2004;7:331–45.View ArticleGoogle Scholar