BACKGROUND
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  <cit> .
during heart development, cardiac precursors populate the first and the second heart fields , which establish the cardiac crescent by embryonic day   <dig>   <cit> .
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   <cit> .
these signals lead to the expression of cardiac progenitor-enriched transcription factors, including nk <dig> homeobox  <dig> protein , myocyte-specific enhancer factor 2c , t-box protein  <dig> , and gata-binding protein  <dig> , which are essential for efficient heart looping and morphogenesis .
when one of the three mammalian hh ligands - indian , desert , or sonic hedgehog  - binds to and inhibits the transmembrane protein, patched  <dig>   <cit> , smoothened  initiates the transition of glioma-associated  proteins into the nucleus for modulation of hh target gene expression  <cit> .
gli <dig> is a primary transducer and activator of the response to hh signalling  <cit> .
gli <dig> is a transcriptional activator but its expression is dependent on gli <dig> and/or gli <dig>  <cit> .
in the absence of hh ligand, ptch <dig> inhibits smo activity, which triggers phosphorylation of the gli proteins and results in their proteolytic cleavage and/or degradation  <cit> .
while phosphorylation of gli <dig> mostly yields a partially cleaved gli <dig> transcriptional repressor , which represses the expression of hh target genes, the phosphorylation of gli <dig> mainly leads to its complete proteasomal degradation  <cit> .
shh-/- mice have altered heart looping  <cit>  and a single outflow tract  <cit> .
mice with tissue specific knockout of shh-/- in the shf, using nkx2-5-cre, display outflow tract septation defects  <cit> .
shh-/-/ihh-/- or smo-/- embryos have a delayed nkx2- <dig> expression and heart tube formation  <cit> .
in accordance, ptch1-/- embryos, which have the negative regulation of hh signalling removed, express higher levels of nkx2- <dig> in the cardiac crescent  <cit> .
gli2-/-gli3+/- embryos have persistent truncus arteriosus  and a single outflow tract  <cit> .
smo agonist , which enhances hh signalling, increases embryonic chick cardiac progenitor cell proliferation in vivo and in vitro  <cit> .
in zebrafish, treatment with sag or shh enhances the number of cardiomyocytes in the developing cardiac chambers  <cit> , whereas treatment with the hh signalling inhibitor, cyclopamine, reduces nkx2- <dig> and myhc expression as well as cardiomyocyte proliferation  <cit> .
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 mef <dig> gene do not exhibit any muscle development  <cit> .
in mammals, there are four mef <dig> members, mef2a-d  <cit> .
expression of a dominant-negative fusion protein of mef2c with an engrailed repression domain  under the regulation of an nkx2- <dig> enhancer , which mediates the repression of all mef <dig> target genes, leads to severely disrupted cardiomyogenesis in mice  <cit> .
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  <cit> .
thus, mef <dig> factors are important for early heart development.
differentiating mouse embryonic stem  cells share a similar hierarchical set of gene expression patterns observed during cardiomyogenesis in vivo  <cit> .
the mesoderm marker, brachyury, and the precardiac mesoderm marker, mesp <dig>  are expressed by days  <dig> and  <dig> of differentiation, respectively  <cit> ; cardiac progenitor genes nkx2- <dig>  gata- <dig>  tbx <dig>  and mef2c are expressed by day  <dig> ; and both alpha and beta isoforms of myhc proteins  are expressed in mes cell-derived cardiomyocytes  <cit> .
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 mef <dig> factors during cardiomyogenesis in vitro has been studied in p <dig> embryonal carcinoma  cells, a mes cell model system .
p <dig> 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  .
in p <dig> cells, overexpression of mef2c, shh, or gli <dig> is sufficient to induce and enhance cardiomyogenesis through the upregulation of cardiac progenitor factors like nkx2- <dig> and gata- <dig>  <cit> .
in agreement, p <dig> cells treated with cyclopamine show delayed cardiomyogenesis  <cit> , whereas expression of a dominant-negative gli/enr or nkx2-5-mef2c/enr results in reduced cardiomyogenesis and nkx2- <dig>  tbx <dig>  gata- <dig>  and myhc <dig> expression  <cit> .
gli <dig> and mef2c can directly bind to each other’s gene regulatory elements in p <dig> cells undergoing cardiomyogenesis, form a protein complex, and synergistically activate an nkx2- <dig> promoter  <cit> .
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  <cit> .
the brahma-associated factors  belong to the switch/sucrose non-fermentable  group of complexes and mediate nucleosome shifting on chromatin in an atp-dependent manner  <cit> .
when the atpase baf subunit, brahma-related gene  <dig>  is globally knocked out, embryos do not survive past the peri-implantation stage  <cit> .
embryos with a conditional mutation of brg <dig> in cardiac progenitor cells, using nkx2-5-cre, have irregular ventricle morphology and die by e <dig>   <cit> .
therefore, brg <dig> is important during heart development.
gli <dig> and gli <dig> proteins interact with brg <dig> in the developing or postnatal brain, respectively  <cit> .
furthermore, brg <dig> is required for both hh target gene repression and activation in mouse embryonic fibroblasts , most probably though an interaction with gli3r and gli <dig>  respectively  <cit> , and is recruited to at least some hh target genes in a hh signalling-dependent manner  <cit> .
although gli <dig> and brg <dig> co-immunoprecipitate in mefs, the importance of this interaction has yet to be tested  <cit> .
given the role of hh signalling and baf subunits during cardiomyogenesis , the requirement of brg <dig> for hh target gene activation, and brg1’s ability to interact with gli proteins  <cit> , we hypothesized that gli <dig> and brg <dig> 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- <dig>  and tbx5; 2) gli <dig> co-immunoprecipitates with brg <dig> in differentiating mes cells; and 3) brg <dig> is recruited to the gli <dig> target gene, mef2c, in a hh signalling-dependent manner in p <dig> cells undergoing cardiomyogenesis.
methods
mes cell culture
d <dig> mes cells  were cultured with 10 % fetal bovine serum  and leukemia inhibitory factor .
d <dig> cells were stably transfected with the empty pcdna <dig> + vector or the pcdna <dig> + vector expressing flag-gli2s662a, a full-length complementary dna of mouse gli <dig> driven by the cmv promoter, in frame with the flag epitope and containing the serine to alanine mutation at position  <dig> which prevents phosphorylation and proteasomal degradation  <cit> .
stable clones were termed mes and mes, respectively.
transfected colonies were selected with  <dig>  mg/ml geneticin  and screened for the highest flag-gli2s662a transcription and protein expression levels by quantitative pcr  and western blot analysis, respectively .
to induce differentiation, mes cells were divided into 20 μl hanging drops at  <dig> cells/drop and allowed to form aggregates for two days without lif, as described in  <cit> .
for immunoprecipitation studies, mes cells were divided into 20 μl hanging drops at  <dig> cells/drop to allow for sufficient starting material.
the resulting embryoid bodies  were pooled and left in suspension for three days.
then the ebs were transferred to tissue culture-treated  plates  or  <dig>  % gelatin-coated  coverslips and cultured until day  <dig>   <dig>  or  <dig> for the analysis of cardiomyogenesis, neurogenesis, or skeletal myogenesis, respectively.
medium was changed every two days.
for hh inhibition, kaad-cyclopamine  or the methanol vehicle, were added from day  <dig> to day  <dig> 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  <dig>  % gelatin-coated coverslips and were cultured until day  <dig>
p <dig> ec cell culture
parental p <dig> ec cells  or p <dig> ec cells transfected with either pcdna3-gli <dig> or an empty vector control as described in  <cit> , termed p <dig> and p <dig>  respectively, were cultured and differentiated as per  <cit> .
briefly, the p <dig> ec cell differentiation was initiated by plating 5x <dig> cells/ml with 1 % v/v dmso  in non-adherent dishes.
after 4 days in suspension, the newly aggregated ebs were transferred to tc plates or  <dig>  % gelatin-coated  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 p <dig> ec cells, differentiating p <dig> ec cultures were treated with 5 μm kaad-cyclopamine, as previously described  <cit> , or with methanol vehicle alone throughout the entire 6-day protocol.
immunoprecipitation assays
to detect flag-gli2s662a in the stable cell lines, total protein extracts on days 2- <dig> of differentiation were prepared by lysing cells in radioimmunoprecipitation  buffer .
the extracts were spun down at  <dig>  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 , 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  <dig> differentiating mes and mes 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  <dig> differentiating mes and mes cell lines, prepared as described above, was pre-cleared for 1 h using rec-protein-g-sepharose beads  and then were split equally and subjected to immunoprecipitation with either mouse igg agarose beads as a negative control  or flag beads .
the immunoprecipitation was performed as above except low salt wash buffer was used  and the ip lasted for 2 h.
immunoblot analysis
the resulting ip eluates and total protein  samples were separated using a 4–12 % denaturing polyacrylamide gel  with mops running buffer according to the manufacturer’s protocol.
the resolved proteins were transferred to a polyvinylidene fluoride  membrane , blocked using non-fat dry milk  reconstituted with tris-buffered saline and tween  <dig> , and incubated with gli2- , brg1- , flag- , or α-tubulin-specific  antibodies.
membranes were stripped with re-blot plus mild stripping buffer  between each primary antibody incubation.
the signal was detected using horseradish peroxidase -conjugated secondary anti-mouse  or anti-rabbit  antibodies, followed by a chemiluminescence reaction using pierce ecl substrate .
densitometry was performed on the gli2-specific bands with the imagej program   <cit> .
the densities of the gli <dig> 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 and mes flag-ip, prepared as described above, were resolved on a 4–12 % denaturing polyacrylamide gel  and silver-stained.
the staining identified a unique band profile in the 170-250 kda range of the mes sample compared to the mes sample .
resolved proteins from both cell lines, within the 170-250 kda range, were extracted from the gel using in-gel digestion, according to  <cit> .
the extracted samples were submitted to liquid chromatography-tandem mass spectrometry  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  using the mascot software  with a peptide mass tolerance of 10 ppm and a fragment mass tolerance of  <dig>  da  <cit> .
quantitative pcr  analysis
total rna from mes and p <dig> cells was isolated using rneasy micro kit  and e.z.n.a.
total rna kit  according to manufacturers’ instructions.
cdna was generated using at least 500 ng of total rna using the quantitect reverse transcription kit as per manufacturer’s protocol .
a negative control  was prepared alongside each experiment for every cell line to control against genomic dna contamination.
for each qpcr reaction, 1/40th of the resultant cdna reaction product, a final concentration of  <dig> nm transcript-specific primers and either a gotaq qpcr master mix kit  or a kapa sybr® fast qpcr kit  were used to detect transcripts of interest in a given sample with an eppendorf realplex <dig> mastercycler.
the primer sequences are listed in table  <dig>  threshold amplification cycles  values were determined for each sample, and normalized to the β-actin control using the 2-ΔΔct method  <cit> .
the relative fold changes were presented as a percentage of the highest transcriptional expression for each respective gene , as described in  <cit> .
all error bars represent ± standard error of the mean  from three or more independent biological replicates.
all statistical analyses were done using student’s t-tests.table  <dig> sequences of primers used for real time qpcr analyses
β-actin
ascl1
brachyury
brg1
flag-gli2
s662a
gata-4
gli1
gli2
mef2c
mesp1
myhc6
myhc7
nkx2-5
pax3
ptch1
tbx5
immunofluorescence
day  <dig> differentiated mes cells were fixed in −20 °c meoh , incubated 1: <dig> with monoclonal mf <dig> antibody supernatant  <cit>  in phosphate buffered saline  and cy3-conjugated goat anti-mouse igg  1: <dig> in pbs to detect pan-myhc expression.
coverslips were mounted in  <dig> parts pbs,  <dig> parts glycerol , and  <dig> part hoechst  <dig> dye for staining nuclei.
indirect immunofluorescence of myhc was visualized using a leica dmi6000b inverted fluorescent microscope  and captured with a hamamatsu orca ag camera .
pictures were processed with the volocity  <dig> .
<dig> software .
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.
<cit> .
to measure the intensity of mhc signal, the outline of at least  <dig> myhc+ve individual cells in  <dig> 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 -  was calculated as in mccloy et al  <cit> .
to assess proliferation, day  <dig> cells were treated with 10 μm edu for 1 h prior to fixing with  <dig>  % formaldehyde and then continuing with the edu staining protocol .
chromatin immunoprecipitation  assays
chip assays were performed as previously described  <cit>  using 25 μg of chromatin from day  <dig> differentiating p <dig> or p <dig> cells and 2 μl of anti-snf2β/brg <dig>  antibodies or normal rabbit serum .
briefly, ebs from  <dig> plates  were fixed with 1 % formaldehyde  for 60 min before isolating and shearing chromatin as per  <cit> .
the chromatin-antibody complexes were captured with bsa-blocked rec-protein g-sepharose 4b conjugated beads .
eluted complexes were treated with rnase a  and proteinase k , to remove contaminating rna and proteins, respectively.
the dna was purified using a qiaquick pcr purification kit .
to detect eluted dna fragments, qpcr analyses were performed using 1/40th of each eluted sample, per reaction, with sequence-specific primers listed in table  <dig>  as mentioned above.
for every qpcr reaction, a standard curve was generated to analyze the enrichment of brg1- or igg-associated genomic elements from the input sample.table  <dig> sequences of primers used for chip-qpcr analyses
β-actin
mef2c site c
mef2c site d
mef2c site e
mef2c site f
mef2c site g
mef2c site h
mef2c site i
chr chromosome
chromatin from kaad-cyclopamine- or meoh-treated p <dig> ec cells was prepared as above with the following adapted fixing and sonication conditions.
cells from  <dig> plates of day  <dig> p <dig> ec ebs - treated with kaad-cyclopamine or meoh - were fixed with  <dig>  mm ethylene glycol bis   for 30 min alone, then with 1 % formaldehyde  for an additional 30 min.
chromatin was sonicated in 1 ml, 12x <dig> afa tubes  for 30 min with an s <dig> focused-ultrasonicator  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.
bioinformatics analysis
brg <dig> genome-wide chip-sequencing peaks described in undifferentiated mes cells  <cit>  were screened for overlapping conserved gli consensus binding motifs, which were identified with the multiple sequence local alignment and visualization tool  as described in  <cit> .
the nearest genes within 50 kb of these overlapping sites in the mouse genome  were categorized by gene ontology  biological process analysis using the genomic regions enrichment of annotations tool  bioinformatics system as described in  <cit> .
the entire set of brg <dig> target genes in mes cells  <cit>  was used as background for assessing the enrichment of these identified genes.
RESULTS
overexpression of flag-gli2s662a results in increased levels of gli <dig> protein during mes cell differentiation
since gli <dig> protein is prone to degradation when phosphorylated  <cit> , we chose to stably transfect mes cells with a flag-gli2s662a, a vector used to express a stabilized version of gli <dig>  <cit> .
qpcr showed that over the course of differentiation, transgene expression declines in all four clones analyzed , possibly reflecting post-transcriptional down-regulation of the gli <dig> mrna or transcript.
we note that the gli2s662a mrna that we expressed contains the entire 3’ untranslated region of the gene and as such could be subjected to microrna-mediated downregulation  <cit> .
a similar downregulation was observed when we expressed a different transcription factor in pluripotent cells  <cit> .
importantly, the expression of gli2s662a mrna persisted in all clones, albeit at lower levels throughout differentiation .
for subsequent experiments, we selected clone # <dig>  with the highest and most stable levels of gli <dig> mrna during differentiation .
from here on, this clone is referred to as the mes cell line, while control mes cells transfected with only a flag peptide tag-containing vector  are referred to as the mes cell line.
mes and mes cell lysates were then tested for the presence of exogenous flag-gli2s662a protein by immunoprecipitation with anti-flag beads  and sequential western blot analysis with gli <dig> antibodies .
we observed a significant enrichment of total gli <dig> protein levels in mes cells when compared to mes on days 2- <dig> of differentiation .
we found ~8-fold and ~13-fold enrichment of total gli <dig> protein level in mes cells when compared to mes on days  <dig> and  <dig> of differentiation, respectively , and over-expression persisted on days  <dig> and  <dig>  when no gli <dig> protein was detectable in the mes cell line at this exposure level.
overall, these results show that the gli <dig> protein was overexpressed in mes cells during early stages of mes cell differentiation compared to the control.fig.
<dig> overexpression of gli <dig> protein in mes cultures is maintained during differentiation.
a total protein extracts from corresponding differentiating cells were analyzed using immunoblot with gli <dig> antibodies, with or without prior flag-ip.
α-tubulin was used as a loading control.
relative band densities are listed below blots.
the size of the upper band in the total protein blots ~180 kda, is consistent with other studies  <cit>  and equivalent to the single gli <dig> band that was pulled down by flag-ip.
the non-specific binding seen below the gli <dig> band is typical of this antibody  <cit> .
b transcription levels of the indicated genes in differentiating mes  and mes  cells were quantified using qpcr.
expression levels were normalized to β-actin, calibrated to day  <dig> mes culture expression levels, and presented as a percentage of the highest expression level recorded, per gene.
error bars represent +/- sem.
the number of biological replicates analyzed  is indicated beside each graph.
n =  <dig> for total gli <dig> days  <dig> and  <dig>  one-tailed student’s t-tests were used for statistical analyses.
grey lines represent paired t-tests; black lines represent unpaired t-tests;  p <  <dig>
comparable results were also observed at the transcriptional level.
flag-gli2s662a transcripts were only expressed in the mes cells and were at the highest level in the undifferentiated  mes cells , and this was mirrored by total gli <dig> transcript levels .
such a trend has been noted in other published work on differentiating mes cells  <cit> .
thus, the stability of the exogenous gli2s662a protein, which remains higher during the differentiation of mes cells compared to controls, is a key element in our model system.
overexpression of gli <dig> results in enhanced cardiac progenitor gene expression
to assess the efficacy of the gli <dig> overexpression, we monitored the expression of the gli <dig> target gene, ptch <dig>  <cit> .
overexpression of gli <dig> resulted in a significant  <dig>  ±  <dig> -fold increase of ptch <dig> transcripts in undifferentiated mes cells and a significant  <dig>  ±  <dig> -fold increase in day  <dig> differentiating mes cells compared to control cells .
therefore, the overexpression of gli <dig> in mes cells increased gli <dig> target gene expression.fig.
<dig> overexpression of gli <dig> in mes cells enhances transcript expression of a gli <dig> target and of cardiac progenitor-enriched genes.
qpcr analysis of mrna expression of indicated genes known to be active in hh-responsive cells, mesoderm/precardiac mesoderm cells, and cardiac progenitor cells, in mes  and mes  cultures on the days noted in the figure.
expression levels were normalized to β-actin, calibrated to day  <dig> mes culture expression levels, and presented as a percentage of the highest expression level recorded, per gene.
error bars represent +/- sem.
the number of biological replicates analyzed  is indicated on each graph.
one-tailed student’s t-tests were used for statistical analyses.
grey lines represent paired t-tests; black lines represent unpaired t-tests;  p <  <dig>
during differentiation, both the mes and mes 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  <dig> between these two cell lines .
mesp <dig> levels were significantly upregulated in the mes cultures as compared to control cultures on day  <dig> of differentiation, however, its level of expression was minor as compared to day  <dig> values and was not significantly higher than day  <dig> basal levels .
on day  <dig>  no significant difference between mes and mes cell lines was detected for the mesp <dig> 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 gli <dig> overexpression.
these results are supported by previous reports, which showed that modulation of hh signalling in mouse p <dig> ec cells did not significantly regulate mesoderm induction .
the cardiac progenitor transcripts nkx2- <dig>  mef2c, and tbx <dig> were significantly upregulated in the mes cultures on day  <dig> of differentiation as compared to mes cells .
interestingly, overexpression of gli <dig> had no effect on day  <dig> gata- <dig> transcript levels .
therefore, overexpression of gli <dig> during mes cell differentiation resulted in enhanced transcription levels of select cardiac progenitor genes.
our data supports and extends previous publications, where overexpression of gli <dig> was shown to induce and enhance cardiac progenitor gene expression in p <dig> ec cells  <cit> .
we tested whether cells of the skeletal muscle and neuronal lineages were affected, since gli <dig> and hh have been shown to regulate these processes  <cit> .
however, we did not detect any significant increase in the mrna levels of pax <dig> , a gene involved in skeletal myogenesis and downstream of gli <dig>  <cit> , or in myhc-positive skeletal myocytes .
a significant increase in ascl <dig>  a gene involved in neural progenitor differentiation that is downstream of gli <dig> and hh signalling , was detected .
however, none of the cultures contained nf68-positive neurons, even after 10 days of differentiation .
overexpression of gli <dig> does not result in an increased number of cardiomyocytes
to determine if the enhancement of select cardiac progenitor transcripts led to an overall enhancement of cardiomyocytes in mes cultures, we counted myhc+ve cells in day  <dig> mes and mes cultures .
on average,  <dig>  ±  <dig>  % and  <dig>  ±  <dig>  % of the total cells counted were myhc+ve in the mes and mes cultures, respectively .
similar percentages have been shown before by analysis of myhc, mlc2v and tropomyosin in day  <dig> or  <dig> differentiating mes cells, differentiated using the hanging drop method  <cit> .
although there was a trend towards an increase in cardiomyocytes in the mes cultures compared to the control, the variability between the samples across three biological replicates  and another four additional replicates  resulted in no significant difference in the percentage of myhc+ve cells on day  <dig> fig.
<dig> overexpression of gli <dig> does not significantly upregulate the number of myhc+ve cells.
a myhc+ve cells  were visualized and  counted in corresponding day  <dig> differentiating cells by indirect immunofluorescence.
hoechst  was used to visualize nuclei.
representative images of the cardiomyocyte-enriched areas on the periphery of an eb are shown.
scale bar represents 100 μm.
at least  <dig> nuclei were counted across  <dig> random fields of view, per biological replicate; n =  <dig>  c
myhc6/ <dig> expression levels on the days indicated in mes  and mes  cells were normalized to β-actin, calibrated to day  <dig> mes culture expression levels, and presented as a percentage of the highest expression level recorded, per gene.
error bars represent +/- sem; n =  <dig>  one-tailed student’s t-tests were used for statistical analyses.
grey lines represent paired t-tests; black lines represent unpaired t-tests;  p <  <dig> .
d myhc+ve cells from  were analyzed for myhc signal intensity using the imagej program.
at least  <dig> cells were analyzed across  <dig> random fields of view per biological replicate; n = 2
although overexpression of gli <dig> during mes cell differentiation also did not significantly affect the level of myh <dig> or myh <dig> transcripts on day  <dig> , we observed a notable increase in myhc signal intensity in cardiac myocytes in mes cultures .
this suggests that although the number of cardiomyocytes formed is unchanged, the presence of exogenous gli <dig> is associated with a trend towards higher myhc protein expression.
to test if overexpression of gli <dig> 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  <dig>  there was no difference between mes and mes cell lines .
of note, we also did not observe any differences in rna yields between mes and mes cultures throughout the 7-day differentiation protocol .
this supports that overexpression of gli <dig> 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 cultures when compared to mes .
thus, overexpression of gli <dig> 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
a previous report demonstrated active hh signalling during mes differentiation  <cit> .
our results showing induced levels of ptch <dig> expression, a readout of active hh signalling, during mes differentiation , are in line with this observation.
thus, following the results from gain-of-function experiments, we proceeded to investigate the involvement of gli <dig> through loss-of-function experiments.
for this, we utilized a synthetic derivative of cyclopamine, kaad-cyclopamine, which is approximately 10- <dig> times more potent than cyclopamine without being more toxic  <cit>  and has previously been shown to successfully inhibit hh signalling in a variety of cells, such as p <dig> ec cells, c3h10t1/ <dig> fibroblasts, and adult skeletal muscle satellite cells  <cit> .
hh inhibition was monitored by the downregulation of ptch <dig> mrna, a marker of active hh signalling  <cit> .
kaad-cyclopamine treatment resulted in a 69 ± 6 % decrease in ptch <dig> expression on day  <dig> of mes cell differentiation, as compared to vehicle-treated cells .
hh inhibition also resulted in comparable decreases in expression of nkx2- <dig>  gata- <dig>  tbx <dig> and mef2c .
we also noted a significant, albeit more modest, decrease in myh <dig> expression .
by immunofluorescence, we noted a slight, not statistically significant decrease in the number of myosin heavy chain-positive cells .
to rule out that the observed effect on myhc cardiac myocytes was not due to aberrant cell proliferation or apoptosis, we first tested the rna yields from identical plates of vehicle and cyclopamine treated cultures.
throughout 7-day differentiation protocol, there was no difference in the amount of rna harvested from vehicle-control and cyclopamine treated cultures , suggesting that there was no overt effect on cell proliferation in the presence of cyclopamine.
to test for aberrant apoptosis, cells with fragmented nuclei were analyzed .
these controls rule out the possibility that cell density or survival differences elicited by cyclopamine treatment would have caused the phenotype we observed.
together, our results on hh activation  and inhibition  support the notion that hh signaling is important for cardiac progenitor gene expression, similarly to previous reports in p <dig> cells  <cit> .fig.
<dig> hh signalling blockade prevents the induction of gli <dig> target and of cardiac progenitor-enriched genes.
a qpcr analysis of mrna expression of ptch <dig> and indicated cardiac progenitor-enriched genes in day  <dig> mes cells  or in cultures kept in differentiation condition for 7 days, in the presence of kaad-cyclopamine  or vehicle .
error bars represent +/- sem; n =  <dig>  p <  <dig>  by one-tailed paired t-test.
b immunofluorescence detection of myosin heavy chain  in similar day  <dig> cultures treated with vehicle or kaad-cyclopamine.
dna was counterstained with hoechst .
c myhc+ve cells from  were counted.
error bars represent +/- sem; n =  <dig> biological replicates
gli <dig> interacts with brg <dig> during mes cell differentiation
we further sought to elucidate the molecular mechanisms behind the enhanced mef2c, nkx2- <dig>  and tbx <dig> transcription levels on day  <dig> of mes cell differentiation by gli <dig> overexpression.
to identify potential gli2-interacting proteins, we performed a mass spectrometric and western blot analysis of the flag-ip on mes and mes cell nuclear extracts from day  <dig> differentiating cells.
brg <dig>  an active member of the baf chromatin-remodelling complex was identified and had a higher mascot score in the mes sample than the mes sample, in the mass spectrometry assay .
a sequential western blot assay with anti-gli <dig> antibodies confirmed immunoprecipitation of gli <dig> protein in mes, but not in mes cells .
when the same blot was re-probed with anti-brg <dig> antibodies, co-immunoprecipitation of brg <dig> with flag-gli2s662a was observed .
the co-ip brg <dig> signal in the mes cells was not merely due to modulated levels of brg <dig> expression, as both mes and mes samples showed no significant difference in brg <dig> mrna transcript levels during the first four days of differentiation .
our results showing co-precipitation of brg <dig> with gli <dig> are similar to those reported by others using nih 3 t <dig> cells, where brg <dig> was found to interact with ha-tagged gli proteins, including gli <dig>  <cit> .fig.
<dig> overexpression of gli <dig> recruits brg <dig> to a gli2-specific mef2c gene element.
a anti-flag immunoprecipitation in nuclear extracts from day  <dig> mes or mes cells.
immunoprecipitated proteins were probed by western blot using anti-brg <dig> or anti-gli <dig> antibodies.
b a schematic representation of the mef2c gene and its major sites of interest.
the scale bar represents coordinates of the mef2c gene in the mouse genome .
this schematic was constructed using the ucsc genome browser , transfac, and data from previous publications .
gli-specific mef2c sites a-i are marked with beige circles.
gli <dig> has been shown to bind only sites b-i .
other associating proteins, including brg <dig> and myod, are depicted with coloured circles as outlined in the legend.
a detailed description of the associating protein sites can be found in table  <dig>  chr: chromosome.
shf i: isl-1-dependent shf enhancer.
shf ii: nkx2-5/foxh1-dependent shf enhancer.
c anti-brg <dig> chip was performed on day  <dig> differentiating p <dig> and p <dig> cultures with sequential qpcr analyses of gli-specific mef2c sites a-i, depicted in .
one-tailed student’s t-tests were used for the chip statistical analyses; n =  <dig>  all error bars represent +/- sem.
all grey lines represent paired t-tests; all black lines represent unpaired t-tests;  p <  <dig> .
d
gli <dig> and brg <dig> mrna expression levels were assessed by qpcr in differentiating p <dig> ec cultures, treated with meoh vehicle or kaad-cyclopamine.
these levels were normalized to β-actin and presented as a fold-change over meoh-treated culture expression levels from the same day.
e the effect of kaad-cyclopamine  or vehicle  treatment on the expression level of gli <dig> and indicated cardiomyogenesis-specific genes was assessed using qpcr analysis.
expression levels were normalized to β-actin, calibrated to day  <dig> untreated culture expression levels, and presented as a percentage of the highest expression level recorded, per gene.
f anti-brg <dig> chip was performed on day  <dig> differentiating p <dig> ec cultures that were treated with either meoh vehicle  or kaad-cyclopamine .
each chip was followed by qpcr analyses on a gene desert region , β-actin , and mef2c site c from panel b. all error bars represent +/- sem; n =  <dig>  two-tailed student’s t-tests were used for statistical analyses.
all grey lines represent paired t-tests; all black lines represent unpaired t-tests;  p <  <dig>
the observed co-immunoprecipitation of gli <dig> and brg <dig> during mes cell differentiation led to our hypothesis that gli <dig> could recruit brg <dig> to regulatory regions of gli <dig> target genes to modulate their expression.
given the results and the known roles of brg <dig> and gli <dig> in heart development  <cit> , we were interested in knowing if gli <dig> and brg <dig> could co-regulate key cardiac progenitor genes implicated in cardiomyogenesis.
mef2c, which is important for proper mammalian heart development  <cit> , was enhanced in day  <dig> differentiating mes cells compared to mes cells  and was recently identified as a direct target of gli <dig> during p <dig> ec cell cardiomyogenesis  <cit> .
thus, we set out to determine if brg <dig> associates with gli2-bound mef2c regulatory elements in a hh-dependent manner.
since mes cells spontaneously differentiate into lineages of all three germ layers  <cit>  and brg <dig> is expressed in many mammalian cells and tissues  <cit> , it is difficult to answer this question in the context of endogenous mes cell cardiomyogenesis.
in contrast, p <dig> ec cells predominantly differentiate into cardiomyocytes by day  <dig> of differentiation, along with skeletal myogenic progenitors and fibroblast-like cells when treated with dmso  <cit> .
moreover, p <dig> ec differentiation is very similar to mes myogenic differentiation  <cit> .
for these reasons, we used p <dig> cells to determine the role of brg1-gli <dig> protein complex during myogenic differentiation.
brg <dig> associates with mef2c regulatory elements in hh signalling dependent manner
in line with what has been shown previously  <cit> , we determined that brg <dig> is expressed throughout the differentiation of mes and p <dig> cells, and that gli <dig> over-expression does not drastically affect its expression in either .
our lab has previously shown that gli <dig> associates with eight of the nine conserved gli consensus binding motifs found in the mef2c gene on day  <dig> of p <dig> cell differentiation    <cit> .
given that brg <dig> immunoprecipitates with gli <dig>  we performed an anti-brg <dig> chip on chromatin from day  <dig> differentiating p <dig> and p <dig> cells, to determine if brg <dig> associates with gli2-specific mef2c genomic elements during differentiation, and also if gli <dig> overexpression can modulate this potential association.
notably, p <dig> cells exhibited enhanced cardiomyogenesis as measured by an increased percentage of cardiomyocytes and higher expression levels of cardiac progenitor genes on day  <dig> of differentiation, in agreement with previous reports .table  <dig> a selection of proteins that associate with the mef2c gene
chr chromosome
auppercase nucleotides in the target sequence represent nonconsensus binding domains
ball binding site information for proteins, except brg <dig> and sox <dig>  were compiled with information gathered through transfac
results from the anti-brg <dig> chip showed an association of brg <dig> with mef2c sites c, d, and g-i in the p <dig> cells as compared to chip with a non-specific rabbit serum control .
notably, although there was a similar trend in the p <dig> cells, we did not observe statistical significance for brg <dig> association on sites d and g-i when compared to non-specific rabbit serum .
mef2c site a, which lacked gli <dig> association under the same experimental conditions in a previous study  <cit> , showed no significant association with brg <dig> in either cell line .
however, the association of brg <dig> with the mef2c site c was significantly  higher in p <dig> cells as compared to p <dig> cells .
this effect was not due to altered brg <dig> expression in p <dig> cells as there was no significant change in brg <dig> expression in p <dig> cells, as compared to p <dig> cells, on day  <dig> of differentiation, when the chip assay was performed .
mef2c site c is of significant interest as it is located proximally to the isl-1-dependent shf enhancer region  <cit>  , it is upstream of the start site of a transcript expressed in the developing heart  <cit> , and it is also the closest gli2-binding site to a region previously shown to associate with brg <dig>  <cit>  .
thus, as overexpression of gli <dig> resulted in increased association of brg <dig> with mef2c site c, we chose this site to further investigate the dependence of brg <dig> association on endogenous hh signalling during p <dig> ec cell cardiomyogenesis.
kaad-cyclopamine treatment of p <dig> ec cells resulted in a  <dig>  ±  <dig>  % and  <dig>  ±  <dig>  % decrease in gli <dig> expression on days  <dig> and  <dig> of p <dig> ec cell differentiation, respectively, without significantly affecting the day  <dig> or  <dig> brg <dig> transcript levels, compared to treatment with a vehicle control .
similar cyclopamine-mediated reductions of gli <dig> expression have been seen before in p <dig>  p19cl <dig>  and mes cells  <cit> .
by day  <dig>  differentiating p <dig> ec cells treated with kaad-cyclopamine expressed significantly lower mef2c , gata- <dig> , and tbx <dig>  transcript levels, compared to differentiating cells treated with the vehicle control .
these lower levels of cardiac progenitor-enriched transcripts were expected as cyclopamine-treated p <dig> ec cells have repressed gata- <dig> expression  <cit>  and cyclopamine-treated p19cl <dig> ec cells show lower levels of gata- <dig> and nkx2- <dig> transcripts  <cit>  and we showed that a similar treatment in mes cells has a comparable effect .
neither the level of contractile protein transcripts in the day  <dig> differentiated cells  nor the number of myhc+ve cardiomyocytes  significantly differed between kaad-cyclopamine and vehicle-treated p <dig> ec cells.
this is supported by previous reports, where differentiating p <dig> ec cells treated with cyclopamine did not show any difference in the number of myhc+ve cardiomyocytes  <cit> .
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  <cit> .
to test if inhibition of hh signalling attenuated the ability of brg <dig> to associate with mef2c site c, we performed an anti-brg <dig> chip in day  <dig> differentiating p <dig> ec cells treated with vehicle or kaad-cyclopamine .
the association of brg <dig> with mef2c site c in p <dig> ec cells was significantly reduced upon hh inhibition .
importantly, kaad-cyclopamine treatment had no significant effect on brg1’s association with β-actin a known target of brg <dig> in mes cells  <cit> .
a gene desert locus was used as a negative control and showed no significant association with brg <dig>  therefore, hh signalling is required, at least to some extent, for the efficient association of brg <dig> to the gli2-specific mef2c gene element c.
discussion
in this study we have shown that 1) gli <dig> enhances the expression of cardiac progenitor-enriched genes while blocking hh signalling through cyclopamine decreases the expression of cardiac progenitor-enriched genes; 2) gli <dig> immunoprecipitates with brg <dig> during mes cell cardiomyogenesis; and 3) brg <dig> is recruited to a gli2-specific mef2c gene element in a hh-dependent manner.
thus, we propose a model, where hh/gli <dig> regulates the expression of mef2c via the recruitment of brg <dig> to the mef2c gene element upstream of the shf elements to ultimately regulate cardiomyogenesis in stem cells .fig.
<dig> a model summarizing the role of the hh signalling pathway and its primary transducer, gli <dig>  during mes cell cardiomyogenesis.
a in this study, gli <dig> expression was observed to positively regulate cardiac progenitor-enriched genes in mes cells .
the solid black arrows indicate hh/gli2’s proposed direct regulation.
the hollow green arrow marks the transition that is enhanced by gli <dig>  b gli2-regulated enrichment of cardiac progenitor transcripts may be explained in part by the ability of the transcriptional activator form of gli <dig>   - mediated via a functional hh signalling pathway  - to enrich brg <dig> association at gli2-specific mef2c site c. this enrichment site is proximal to known shf enhancer regions .
other cofactors , including the remaining baf complex members and other unidentified shf-related transcription factors  may support this gli2-mediated association.
c when kaad-cyclopamine inhibits hh signalling, gli <dig> is likely degraded, which leads to a reduction of brg <dig> association on mef2c site c. although we have not observed the formation of gli2r in our system , it is possible that gli <dig> may be processed into gli2r  when hh signalling is blocked  <cit> .
also, gli3r  may contribute to the repression of mef2c expression.
the downstream binding of either gli2r or gli3r to mef2c has yet to be assessed
hh signalling is important for cardiac progenitor gene expression during mes cell differentiation
using a stabilized gli <dig> mutant, we have shown that gli <dig> enhances early expression of cardiac progenitor transcripts such as mef2c, nkx2- <dig>  and tbx <dig> during mes cell cardiomyogenesis .
in agreement, when hh signalling in mes cells is blocked with cyclopamine, this leads to a great reduction in the expression of these genes .
this supports and expands previous reports in p <dig> ec cells  <cit> .
the effect of cardiac progenitor gene upregulation by exogenous gli <dig> is probably not due to an increase in mesodermal transcript expression as gli <dig> overexpression does not have any significant effect on the levels of brachyury or mesp <dig> on days  <dig> and  <dig>  respectively .
notably, the small upregulation of mesp <dig> transcript levels on day  <dig> in mes cells is minor in comparison with day  <dig> values.
this is in agreement with previous studies, which demonstrate that there is no significant hh-dependent effect on mesoderm transcripts in p <dig> ec cells .
the enhanced expression of nkx2- <dig> in mes cells correlates with the hh-mediated expression of nkx2- <dig> in vivo  <cit>  and in p <dig> ec cells .
the upregulation of tbx <dig> transcript levels in differentiating mes cells  follows the same pattern of tbx <dig> upregulation seen in differentiating p <dig> cells  <cit> .
when hh signalling is inhibited in p <dig> ec or mes cells by kaad-cyclopamine, tbx <dig> expression is reduced , similarly to p <dig> ec cells overexpressing dominant-negative gli/enr  <cit> .
interestingly, tbx <dig> is expressed in shf cells that receive hh signals in vivo, and a tbx5-hh network is required for atrial septation  <cit> .
although the regulation of tbx <dig> expression by the hh pathway is yet to be determined in vivo, based on results from previous reports and our observations, tbx <dig> expression is regulated by hh signalling in vitro, during p <dig> ec and mes cell differentiation.
in contrast to mef2c, nkx2- <dig>  and tbx <dig> upregulation in mes cells, gata- <dig> expression is not affected when gli <dig> is overexpressed .
importantly, gata- <dig> transcript levels were downregulated in mes cells treated with cyclopamine , similarly to p <dig> ec cells .
this suggests that while exogenous gli <dig> cannot enhance gata- <dig> expression in differentiating mes cells, repression of endogenous hh signalling is sufficient to reduce its expression.
it is possible that gata- <dig> induction is more efficient with other gli family members, that gata- <dig> 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- <dig> expression might be independently regulated from mef2c, nkx2- <dig>  and/or tbx <dig> expression  <cit> .
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- <dig> and mef2c expression while gata- <dig> levels remain unaffected  <cit> .
in mes cells, overexpression of the cardiac progenitor gene regulator, yin yang  <dig>  enhances nkx2- <dig> and tbx <dig> expression with no apparent effect on gata- <dig> expression  <cit> .
therefore, our observation of mef2c, nkx2- <dig>  and tbx <dig> upregulation in mes cells with no observable effect on day  <dig> gata- <dig> 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- <dig>  tbx <dig>  mef2c and gata- <dig> in differentiating mes and p <dig> 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 .
moreover, simultaneous expression of gata- <dig> and tbx <dig> is detected in hes-derived cardiovascular progenitors and cardiomyocytes, but not in smooth muscle or endothelial cells  <cit> .
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 gli <dig> overexpression or application of cyclopamine regulates nkx2- <dig>  mef2c and tbx <dig> cardiac progenitor gene expression in mes cells , this does not alter the percentage of cardiomyocytes .
one explanation might lie in gata- <dig> expression.
namely, gata- <dig> appears to be a limiting factor, in a transcription factor cocktail, to induce myhc expression in mes cells  <cit> .
therefore, the lack of gata- <dig> enhancement by exogenous gli <dig> may offer an explanation for the lack of cardiomyocyte number increase in mes cultures.
hh signalling gain-of-function experiments in p <dig> cells showed both an induction and enhancement of cardiomyogenesis  <cit> .
p <dig> cells may be more susceptible to differentiate into cardiomyocytes as they are more prone to differentiate into cells of the mesodermal lineage  <cit> .
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  <cit>  and thus may not have as many mesoderm cells that are susceptible to hh-mediated regulation.
it is possible that gli <dig> overexpression affects the differentiation of other cell types as hh signalling regulates many developmental programs in vivo  <cit> , resulting in a lower percentage of cardiomyocytes in mes 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 p <dig> ec cells.
lastly, hh signalling may enhance mechanisms in the neighbouring lineages that in turn negatively regulate cardiac differentiation in mes cells.
gli <dig> immunoprecipitates with brg <dig> during mes cardiomyogenesis and active hh signalling regulates brg <dig> association with mef2c gene
to determine the mechanism of gli <dig> during in vitro cardiomyogenesis, we used both mes and p <dig> differentiating cells.
the co-immunoprecipitation of gli <dig> and brg <dig> in differentiating mes cells  is in agreement with previous observations of ha-tagged gli <dig> immunoprecipitation with brg <dig> in nih 3 t <dig> cells  <cit> .
to investigate the significance of this interaction, we tested the hypothesis that gli <dig> and brg <dig> co-regulate cardiomyogenesis in vitro by modulating the expression of gli <dig> target genes.
in this light, we focused on mef2c gene, a direct target of gli <dig> during p <dig> ec cell cardiomyogenesis  <cit> .
increased mef2c transcript levels in mes  support and extend these results.
moreover, brg <dig> associates with the first exon in the mef2c gene  <cit> , where other proteins, including myod, are known to bind  <cit> , but which lacks gli consensus binding site .
the anti-brg <dig> chip analysis from day  <dig> differentiating p <dig> ec control cells demonstrates that brg <dig> associates with mef2c sites c, d, and g-i , which gli <dig> has been shown to bind under similar conditions  <cit> .
these results support and extend a previous report  <cit>  by showing that brg <dig> 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   <cit> .
of these two sites, brg <dig> is more efficiently recruited to mef2c site c in p <dig> cells overexpressing gli <dig>  moreover, when hh signalling in p <dig> cells is blocked, there is a reduction of brg <dig> association with this site.
thus, our findings indicate that brg <dig> is recruited to at least one mef2c gene regulatory element in a hh- and gli2-dependent manner.
our hypothesis that gli <dig> and brg <dig> may share common chromatin targets is also supported by the identification of additional  <dig> putative gli- and brg1-target genes through an in silico analysis , where brg <dig> genome-wide chip-sequencing peaks from  <cit>  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, gli <dig> and ptch <dig>  <cit> , amongst the theoretical targets, validating this bioinformatic assay .
a gene ontology analysis identified a broad spectrum of biological processes that are significantly enriched for these genes, such as regulation of gene expression, cell differentiation and system development .
as gli <dig> and brg <dig> are ubiquitously expressed proteins  <cit> , it is possible that they might co-regulate a variety of cellular and developmental programs.
notably, we identified nervous system development, where gli <dig> and brg <dig> are known to interact  <cit> , and heart development .
foxh <dig>  tbx <dig>  notch <dig>  and wnt5a are amongst the potential targets in the heart development category.
foxh <dig> and tbx <dig> are expressed in the shf field, while notch and wnt signalling pathways are known to regulate heart development  <cit> .
thus, gli <dig> and brg <dig> may co-regulate cardiac genes that have yet to be identified.
moreover, we predict that this co-regulation may be common to other developmental programs, which is supported by the identification of additional hh-dependent go categories, such as osteoblast differentiation, nervous system development and cell cycle regulation  <cit> .table  <dig> selected gene ontology biological processes significantly enriched among genes within 50 kb of a brg1- associating site and gli consensus binding motifa
atf <dig>  meis <dig>  ncoa <dig>  ncor <dig>  smad2
actn <dig>  ctnna <dig>  ctnnd <dig>  mapt, myo1e
angpt <dig>  fgf <dig>  fgf <dig>  pdgfa
creb <dig>  dhh, mef2d, ptch <dig>  rara, smo
bdnf, neurod <dig>  nkx2- <dig>  pax6
hoxa <dig>  hoxa <dig>  hoxd <dig>  yy1
atoh <dig>  otx <dig>  otx <dig>  gata2
foxh <dig>  notch <dig>  tbx <dig>  ttn, wnt5a
cdc <dig>  cdk <dig>  cdk <dig>  e2f <dig>  e2f2
bmp <dig>  gli <dig>  gli2
epha <dig>  fat <dig>  lama <dig>  lamc <dig>  tubb2b
hdac <dig>  mll <dig>  myst <dig>  smarca <dig>  smarcd1
aa complete list can be found in additional file 5: table s1
CONCLUSIONS
taken together, our results indicate that gli <dig> and brg <dig> co-operate to regulate cardiomyogenesis in vitro.
we propose a mechanism where hh signaling, through activated gli <dig>  recruits brg <dig> along with other potential co-factors to the mef2c gene site c to promote mef2c expression .
inhibition of hh signaling results in reduced brg <dig> association at mef2c site c and reduced mef2c expression .
since brg <dig> association correlates with chromatin accessibility  <cit> , we predict that gli <dig> might recruit brg <dig> to increase chromatin accessibility on mef2c and other potential yet unidentified target genes to regulate in vitro cardiomyogenesis.
abbreviations
chip, chromatin immunoprecipitation; dhh, desert hedgehog; dmso, dimethylsulphoxide; ec, embryonal carcinoma; egs, ethylene glycol bis; 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
additional files
additional file 1: figure s <dig>  mrna expression levels of the gli2s662a-flag transgene in mesc clones stably transfected with a gli <dig> expression plasmid or with the empty vector.
total rna was extracted from cells at various time points during differentiation.
expression values are relative to those at day  <dig> with the clone “mesc#1” and are normalized to β-actin.
for each clone, n =  <dig>
additional file 2: figure s <dig>  gli <dig> may regulate early neurogenesis during mes cell differentiation.
total rna was isolated from differentiating mes and mes cultures on days indicated and analyzed using qpcr for the expression of  pax <dig> and  ascl <dig>  expression levels were normalized to β-actin, calibrated to day  <dig> mes culture expression levels, and presented as a percentage of the highest expression level recorded, per gene.
error bars represent +/- sem; n =  <dig>  one-tailed student’s t-tests were used for statistical analyses.
grey lines represent paired t-tests; black lines represent unpaired t-tests;  p <  <dig> .
additional file 3: figure s <dig>  modulation of hh signalling does not affect cell proliferation or survival.
day  <dig> cells were treated with edu for 1 h prior to staining.
at least  <dig> cells per sample, across  <dig> fields of view, were counted.
error bars represent +/- sem.
student t-tests were used for statistical analyses.
n =  <dig>   the yields of total rna extracted from mes and mes are given as a proxy for cell numbers in the cultures.
for each time point, n is between  <dig> and  <dig>  and error bars represent +/- sem.
the proportion of cells with highly condensed, apoptotic nuclei after hoechst staining at day  <dig> is given.
at least  <dig> cells per condition per replicate were counted.
n =  <dig>   yields of rna in mes cultures treated with methanol vehicle or kaad-cyclopamine.
for each time point, n =  <dig>  and error bars represent +/- sem.
the proportion of cells with highly condensed, apoptotic nuclei is given for vehicle- and kaad-cyclopamine-treated mes cultures at day  <dig>  n =  <dig>  and at least  <dig> cells per replicate and condition were counted.
additional file 4: figure s <dig>  expression of brg <dig> in mes and p <dig> cells overexpressing gli <dig>  qpcr analysis of brg <dig> mrna expression levels in  mes  and mes  cells, or  p <dig>  and p <dig>  cells.
for  expression levels were normalized to β-actin, calibrated to day  <dig> mes or p <dig> culture expression levels, and presented as a percentage of the highest expression level recorded, per gene.
two-tailed student’s t-tests were used for the mrna statistical analyses; n =  <dig>
additional file 5: table s <dig>  a complete list of gene ontology biological processes significantly enriched among genes within 50 kb of a brg1-associating site and gli consensus binding motif.

 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, which establish the cardiac crescent by embryonic day. 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. these signals lead to the expression of cardiac progenitor-enriched transcription factors, including nk homeobox protein, myocyte-specific enhancer factor 2c, t-box protein, and gata-binding protein, which are essential for efficient heart looping and morphogenesis. when one of the three mammalian hh ligands - indian, desert, or sonic hedgehog - binds to and inhibits the transmembrane protein, patched, smoothened initiates the transition of glioma-associated proteins into the nucleus for modulation of hh target gene expression. gli is a primary transducer and activator of the response to hh signalling. gli is a transcriptional activator but its expression is dependent on gli and/or gli. in the absence of hh ligand, ptch inhibits smo activity, which triggers phosphorylation of the gli proteins and results in their proteolytic cleavage and/or degradation. while phosphorylation of gli mostly yields a partially cleaved gli transcriptional repressor, which represses the expression of hh target genes, the phosphorylation of gli 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- expression and heart tube formation. in accordance, ptch1-/- embryos, which have the negative regulation of hh signalling removed, express higher levels of nkx2- in the cardiac crescent. gli2-/-gli3+/- embryos have persistent truncus arteriosus and a single outflow tract. smo agonist, 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, whereas treatment with the hh signalling inhibitor, cyclopamine, reduces nkx2- and myhc expression as well as cardiomyocyte proliferation. 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 mef gene do not exhibit any muscle development. in mammals, there are four mef members, mef2a-d. expression of a dominant-negative fusion protein of mef2c with an engrailed repression domain under the regulation of an nkx2- enhancer, which mediates the repression of all mef 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. thus, mef factors are important for early heart development. differentiating mouse embryonic stem cells share a similar hierarchical set of gene expression patterns observed during cardiomyogenesis in vivo. the mesoderm marker, brachyury, and the precardiac mesoderm marker, mesp are expressed by days and of differentiation, respectively; cardiac progenitor genes nkx2- gata- tbx and mef2c are expressed by day; and both alpha and beta isoforms of myhc proteins 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 mef factors during cardiomyogenesis in vitro has been studied in p embryonal carcinoma cells, a mes cell model system. p 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. in p cells, overexpression of mef2c, shh, or gli is sufficient to induce and enhance cardiomyogenesis through the upregulation of cardiac progenitor factors like nkx2- and gata-. in agreement, p 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- tbx gata- and myhc expression. gli and mef2c can directly bind to each other’s gene regulatory elements in p cells undergoing cardiomyogenesis, form a protein complex, and synergistically activate an nkx2- 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. the brahma-associated factors belong to the switch/sucrose non-fermentable group of complexes and mediate nucleosome shifting on chromatin in an atp-dependent manner. when the atpase baf subunit, brahma-related gene is globally knocked out, embryos do not survive past the peri-implantation stage. embryos with a conditional mutation of brg in cardiac progenitor cells, using nkx2-5-cre, have irregular ventricle morphology and die by e. therefore, brg is important during heart development. gli and gli proteins interact with brg in the developing or postnatal brain, respectively. furthermore, brg is required for both hh target gene repression and activation in mouse embryonic fibroblasts, most probably though an interaction with gli3r and gli respectively, and is recruited to at least some hh target genes in a hh signalling-dependent manner. although gli and brg 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, the requirement of brg for hh target gene activation, and brg1’s ability to interact with gli proteins, we hypothesized that gli and brg 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- and tbx5; 2) gli co-immunoprecipitates with brg in differentiating mes cells; and 3) brg is recruited to the gli target gene, mef2c, in a hh signalling-dependent manner in p cells undergoing cardiomyogenesis. methods mes cell culture d mes cells were cultured with 10 % fetal bovine serum and leukemia inhibitory factor. d cells were stably transfected with the empty pcdna + vector or the pcdna + vector expressing flag-gli2s662a, a full-length complementary dna of mouse gli driven by the cmv promoter, in frame with the flag epitope and containing the serine to alanine mutation at position which prevents phosphorylation and proteasomal degradation. stable clones were termed mes and mes, respectively. transfected colonies were selected with  mg/ml geneticin and screened for the highest flag-gli2s662a transcription and protein expression levels by quantitative pcr and western blot analysis, respectively. to induce differentiation, mes cells were divided into 20 μl hanging drops at 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 cells/drop to allow for sufficient starting material. the resulting embryoid bodies were pooled and left in suspension for three days. then the ebs were transferred to tissue culture-treated plates or  % gelatin-coated coverslips and cultured until day or for the analysis of cardiomyogenesis, neurogenesis, or skeletal myogenesis, respectively. medium was changed every two days. for hh inhibition, kaad-cyclopamine or the methanol vehicle, were added from day to day 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  % gelatin-coated coverslips and were cultured until day p ec cell culture parental p ec cells or p ec cells transfected with either pcdna3-gli or an empty vector control as described in, termed p and p respectively, were cultured and differentiated as per. briefly, the p ec cell differentiation was initiated by plating 5x cells/ml with 1 % v/v dmso in non-adherent dishes. after 4 days in suspension, the newly aggregated ebs were transferred to tc plates or  % gelatin-coated 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 p ec cells, differentiating p ec cultures were treated with 5 μm kaad-cyclopamine, as previously described, or with methanol vehicle alone throughout the entire 6-day protocol. immunoprecipitation assays to detect flag-gli2s662a in the stable cell lines, total protein extracts on days 2- of differentiation were prepared by lysing cells in radioimmunoprecipitation buffer. the extracts were spun down at 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, 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 differentiating mes and mes 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 differentiating mes and mes cell lines, prepared as described above, was pre-cleared for 1 h using rec-protein-g-sepharose beads and then were split equally and subjected to immunoprecipitation with either mouse igg agarose beads as a negative control or flag beads. the immunoprecipitation was performed as above except low salt wash buffer was used and the ip lasted for 2 h. immunoblot analysis the resulting ip eluates and total protein samples were separated using a 4–12 % denaturing polyacrylamide gel with mops running buffer according to the manufacturer’s protocol. the resolved proteins were transferred to a polyvinylidene fluoride membrane, blocked using non-fat dry milk reconstituted with tris-buffered saline and tween, and incubated with gli2-, brg1-, flag-, or α-tubulin-specific antibodies. membranes were stripped with re-blot plus mild stripping buffer between each primary antibody incubation. the signal was detected using horseradish peroxidase -conjugated secondary anti-mouse or anti-rabbit antibodies, followed by a chemiluminescence reaction using pierce ecl substrate. densitometry was performed on the gli2-specific bands with the imagej program. the densities of the gli 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 and mes flag-ip, prepared as described above, were resolved on a 4–12 % denaturing polyacrylamide gel and silver-stained. the staining identified a unique band profile in the 170-250 kda range of the mes sample compared to the mes sample. 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 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 using the mascot software with a peptide mass tolerance of 10 ppm and a fragment mass tolerance of  da. quantitative pcr analysis total rna from mes and p cells was isolated using rneasy micro kit and e.z.n.a. total rna kit according to manufacturers’ instructions. cdna was generated using at least 500 ng of total rna using the quantitect reverse transcription kit as per manufacturer’s protocol. a negative control was prepared alongside each experiment for every cell line to control against genomic dna contamination. for each qpcr reaction, 1/40th of the resultant cdna reaction product, a final concentration of nm transcript-specific primers and either a gotaq qpcr master mix kit or a kapa sybr® fast qpcr kit were used to detect transcripts of interest in a given sample with an eppendorf realplex mastercycler. the primer sequences are listed in table  threshold amplification cycles values were determined for each sample, and normalized to the β-actin control using the 2-ΔΔct method. the relative fold changes were presented as a percentage of the highest transcriptional expression for each respective gene, as described in. all error bars represent ± standard error of the mean from three or more independent biological replicates. all statistical analyses were done using student’s t-tests.table sequences of primers used for real time qpcr analyses β-actin ascl1 brachyury brg1 flag-gli2 s662a gata-4 gli1 gli2 mef2c mesp1 myhc6 myhc7 nkx2-5 pax3 ptch1 tbx5 immunofluorescence day differentiated mes cells were fixed in −20 °c meoh, incubated 1: with monoclonal mf antibody supernatant in phosphate buffered saline and cy3-conjugated goat anti-mouse igg 1: in pbs to detect pan-myhc expression. coverslips were mounted in parts pbs, parts glycerol, and part hoechst dye for staining nuclei. indirect immunofluorescence of myhc was visualized using a leica dmi6000b inverted fluorescent microscope and captured with a hamamatsu orca ag camera. pictures were processed with the volocity. software. 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 myhc+ve individual cells in 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 - was calculated as in mccloy et al. to assess proliferation, day cells were treated with 10 μm edu for 1 h prior to fixing with  % formaldehyde and then continuing with the edu staining protocol. chromatin immunoprecipitation assays chip assays were performed as previously described using 25 μg of chromatin from day differentiating p or p cells and 2 μl of anti-snf2β/brg antibodies or normal rabbit serum. briefly, ebs from plates were fixed with 1 % formaldehyde for 60 min before isolating and shearing chromatin as per. the chromatin-antibody complexes were captured with bsa-blocked rec-protein g-sepharose 4b conjugated beads. eluted complexes were treated with rnase a and proteinase k, to remove contaminating rna and proteins, respectively. the dna was purified using a qiaquick pcr purification kit. to detect eluted dna fragments, qpcr analyses were performed using 1/40th of each eluted sample, per reaction, with sequence-specific primers listed in table  as mentioned above. for every qpcr reaction, a standard curve was generated to analyze the enrichment of brg1- or igg-associated genomic elements from the input sample.table sequences of primers used for chip-qpcr analyses β-actin mef2c site c mef2c site d mef2c site e mef2c site f mef2c site g mef2c site h mef2c site i chr chromosome chromatin from kaad-cyclopamine- or meoh-treated p ec cells was prepared as above with the following adapted fixing and sonication conditions. cells from plates of day p ec ebs - treated with kaad-cyclopamine or meoh - were fixed with  mm ethylene glycol bis for 30 min alone, then with 1 % formaldehyde for an additional 30 min. chromatin was sonicated in 1 ml, 12x afa tubes for 30 min with an s focused-ultrasonicator 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. bioinformatics analysis brg 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 as described in. the nearest genes within 50 kb of these overlapping sites in the mouse genome were categorized by gene ontology biological process analysis using the genomic regions enrichment of annotations tool bioinformatics system as described in. the entire set of brg 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 gli protein during mes cell differentiation since gli protein is prone to degradation when phosphorylated, we chose to stably transfect mes cells with a flag-gli2s662a, a vector used to express a stabilized version of gli. qpcr showed that over the course of differentiation, transgene expression declines in all four clones analyzed, possibly reflecting post-transcriptional down-regulation of the gli mrna or transcript. we note that the gli2s662a mrna that we expressed contains the entire 3’ untranslated region of the gene and as such could be subjected to microrna-mediated downregulation. a similar downregulation was observed when we expressed a different transcription factor in pluripotent cells. importantly, the expression of gli2s662a mrna persisted in all clones, albeit at lower levels throughout differentiation. for subsequent experiments, we selected clone # with the highest and most stable levels of gli mrna during differentiation. from here on, this clone is referred to as the mes cell line, while control mes cells transfected with only a flag peptide tag-containing vector are referred to as the mes cell line. mes and mes cell lysates were then tested for the presence of exogenous flag-gli2s662a protein by immunoprecipitation with anti-flag beads and sequential western blot analysis with gli antibodies. we observed a significant enrichment of total gli protein levels in mes cells when compared to mes on days 2- of differentiation. we found ~8-fold and ~13-fold enrichment of total gli protein level in mes cells when compared to mes on days and of differentiation, respectively, and over-expression persisted on days and when no gli protein was detectable in the mes cell line at this exposure level. overall, these results show that the gli protein was overexpressed in mes cells during early stages of mes cell differentiation compared to the control.fig. overexpression of gli protein in mes cultures is maintained during differentiation. a total protein extracts from corresponding differentiating cells were analyzed using immunoblot with gli antibodies, with or without prior flag-ip. α-tubulin was used as a loading control. relative band densities are listed below blots. the size of the upper band in the total protein blots ~180 kda, is consistent with other studies and equivalent to the single gli band that was pulled down by flag-ip. the non-specific binding seen below the gli band is typical of this antibody. b transcription levels of the indicated genes in differentiating mes and mes cells were quantified using qpcr. expression levels were normalized to β-actin, calibrated to day mes culture expression levels, and presented as a percentage of the highest expression level recorded, per gene. error bars represent +/- sem. the number of biological replicates analyzed is indicated beside each graph. n =  for total gli days and one-tailed student’s t-tests were used for statistical analyses. grey lines represent paired t-tests; black lines represent unpaired t-tests; p <  comparable results were also observed at the transcriptional level. flag-gli2s662a transcripts were only expressed in the mes cells and were at the highest level in the undifferentiated mes cells, and this was mirrored by total gli transcript levels. 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 cells compared to controls, is a key element in our model system. overexpression of gli results in enhanced cardiac progenitor gene expression to assess the efficacy of the gli overexpression, we monitored the expression of the gli target gene, ptch. overexpression of gli resulted in a significant  ±  -fold increase of ptch transcripts in undifferentiated mes cells and a significant  ±  -fold increase in day differentiating mes cells compared to control cells. therefore, the overexpression of gli in mes cells increased gli target gene expression.fig. overexpression of gli in mes cells enhances transcript expression of a gli target and of cardiac progenitor-enriched genes. qpcr analysis of mrna expression of indicated genes known to be active in hh-responsive cells, mesoderm/precardiac mesoderm cells, and cardiac progenitor cells, in mes and mes cultures on the days noted in the figure. expression levels were normalized to β-actin, calibrated to day mes culture expression levels, and presented as a percentage of the highest expression level recorded, per gene. error bars represent +/- sem. the number of biological replicates analyzed is indicated on each graph. one-tailed student’s t-tests were used for statistical analyses. grey lines represent paired t-tests; black lines represent unpaired t-tests; p <  during differentiation, both the mes and mes 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 between these two cell lines. mesp levels were significantly upregulated in the mes cultures as compared to control cultures on day of differentiation, however, its level of expression was minor as compared to day values and was not significantly higher than day basal levels. on day no significant difference between mes and mes cell lines was detected for the mesp 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 gli overexpression. these results are supported by previous reports, which showed that modulation of hh signalling in mouse p ec cells did not significantly regulate mesoderm induction. the cardiac progenitor transcripts nkx2- mef2c, and tbx were significantly upregulated in the mes cultures on day of differentiation as compared to mes cells. interestingly, overexpression of gli had no effect on day gata- transcript levels. therefore, overexpression of gli during mes cell differentiation resulted in enhanced transcription levels of select cardiac progenitor genes. our data supports and extends previous publications, where overexpression of gli was shown to induce and enhance cardiac progenitor gene expression in p ec cells. we tested whether cells of the skeletal muscle and neuronal lineages were affected, since gli and hh have been shown to regulate these processes. however, we did not detect any significant increase in the mrna levels of pax, a gene involved in skeletal myogenesis and downstream of gli, or in myhc-positive skeletal myocytes. a significant increase in ascl a gene involved in neural progenitor differentiation that is downstream of gli and hh signalling, was detected. however, none of the cultures contained nf68-positive neurons, even after 10 days of differentiation. overexpression of gli does not result in an increased number of cardiomyocytes to determine if the enhancement of select cardiac progenitor transcripts led to an overall enhancement of cardiomyocytes in mes cultures, we counted myhc+ve cells in day mes and mes cultures. on average,  ±   % and  ±   % of the total cells counted were myhc+ve in the mes and mes cultures, respectively. similar percentages have been shown before by analysis of myhc, mlc2v and tropomyosin in day or differentiating mes cells, differentiated using the hanging drop method. although there was a trend towards an increase in cardiomyocytes in the mes cultures compared to the control, the variability between the samples across three biological replicates and another four additional replicates resulted in no significant difference in the percentage of myhc+ve cells on day fig. overexpression of gli does not significantly upregulate the number of myhc+ve cells. a myhc+ve cells were visualized and counted in corresponding day differentiating cells by indirect immunofluorescence. hoechst was used to visualize nuclei. representative images of the cardiomyocyte-enriched areas on the periphery of an eb are shown. scale bar represents 100 μm. at least nuclei were counted across random fields of view, per biological replicate; n =  c myhc6/ expression levels on the days indicated in mes and mes cells were normalized to β-actin, calibrated to day mes culture expression levels, and presented as a percentage of the highest expression level recorded, per gene. error bars represent +/- sem; n =  one-tailed student’s t-tests were used for statistical analyses. grey lines represent paired t-tests; black lines represent unpaired t-tests; p < . d myhc+ve cells from were analyzed for myhc signal intensity using the imagej program. at least cells were analyzed across random fields of view per biological replicate; n = 2 although overexpression of gli during mes cell differentiation also did not significantly affect the level of myh or myh transcripts on day, we observed a notable increase in myhc signal intensity in cardiac myocytes in mes cultures. this suggests that although the number of cardiomyocytes formed is unchanged, the presence of exogenous gli is associated with a trend towards higher myhc protein expression. to test if overexpression of gli 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 there was no difference between mes and mes cell lines. of note, we also did not observe any differences in rna yields between mes and mes cultures throughout the 7-day differentiation protocol. this supports that overexpression of gli 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 cultures when compared to mes. thus, overexpression of gli 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 a previous report demonstrated active hh signalling during mes differentiation. our results showing induced levels of ptch expression, a readout of active hh signalling, during mes differentiation, are in line with this observation. thus, following the results from gain-of-function experiments, we proceeded to investigate the involvement of gli through loss-of-function experiments. for this, we utilized a synthetic derivative of cyclopamine, kaad-cyclopamine, which is approximately 10- times more potent than cyclopamine without being more toxic and has previously been shown to successfully inhibit hh signalling in a variety of cells, such as p ec cells, c3h10t1/ fibroblasts, and adult skeletal muscle satellite cells. hh inhibition was monitored by the downregulation of ptch mrna, a marker of active hh signalling. kaad-cyclopamine treatment resulted in a 69 ± 6 % decrease in ptch expression on day of mes cell differentiation, as compared to vehicle-treated cells. hh inhibition also resulted in comparable decreases in expression of nkx2- gata- tbx and mef2c. we also noted a significant, albeit more modest, decrease in myh expression. by immunofluorescence, we noted a slight, not statistically significant decrease in the number of myosin heavy chain-positive cells. to rule out that the observed effect on myhc cardiac myocytes was not due to aberrant cell proliferation or apoptosis, we first tested the rna yields from identical plates of vehicle and cyclopamine treated cultures. throughout 7-day differentiation protocol, there was no difference in the amount of rna harvested from vehicle-control and cyclopamine treated cultures, suggesting that there was no overt effect on cell proliferation in the presence of cyclopamine. to test for aberrant apoptosis, cells with fragmented nuclei were analyzed. these controls rule out the possibility that cell density or survival differences elicited by cyclopamine treatment would have caused the phenotype we observed. together, our results on hh activation and inhibition support the notion that hh signaling is important for cardiac progenitor gene expression, similarly to previous reports in p cells.fig. hh signalling blockade prevents the induction of gli target and of cardiac progenitor-enriched genes. a qpcr analysis of mrna expression of ptch and indicated cardiac progenitor-enriched genes in day mes cells or in cultures kept in differentiation condition for 7 days, in the presence of kaad-cyclopamine or vehicle. error bars represent +/- sem; n =  p <  by one-tailed paired t-test. b immunofluorescence detection of myosin heavy chain in similar day cultures treated with vehicle or kaad-cyclopamine. dna was counterstained with hoechst. c myhc+ve cells from were counted. error bars represent +/- sem; n =  biological replicates gli interacts with brg during mes cell differentiation we further sought to elucidate the molecular mechanisms behind the enhanced mef2c, nkx2- and tbx transcription levels on day of mes cell differentiation by gli overexpression. to identify potential gli2-interacting proteins, we performed a mass spectrometric and western blot analysis of the flag-ip on mes and mes cell nuclear extracts from day differentiating cells. brg an active member of the baf chromatin-remodelling complex was identified and had a higher mascot score in the mes sample than the mes sample, in the mass spectrometry assay. a sequential western blot assay with anti-gli antibodies confirmed immunoprecipitation of gli protein in mes, but not in mes cells. when the same blot was re-probed with anti-brg antibodies, co-immunoprecipitation of brg with flag-gli2s662a was observed. the co-ip brg signal in the mes cells was not merely due to modulated levels of brg expression, as both mes and mes samples showed no significant difference in brg mrna transcript levels during the first four days of differentiation. our results showing co-precipitation of brg with gli are similar to those reported by others using nih 3 t cells, where brg was found to interact with ha-tagged gli proteins, including gli.fig. overexpression of gli recruits brg to a gli2-specific mef2c gene element. a anti-flag immunoprecipitation in nuclear extracts from day mes or mes cells. immunoprecipitated proteins were probed by western blot using anti-brg or anti-gli antibodies. b a schematic representation of the mef2c gene and its major sites of interest. the scale bar represents coordinates of the mef2c gene in the mouse genome. this schematic was constructed using the ucsc genome browser, transfac, and data from previous publications. gli-specific mef2c sites a-i are marked with beige circles. gli has been shown to bind only sites b-i. other associating proteins, including brg and myod, are depicted with coloured circles as outlined in the legend. a detailed description of the associating protein sites can be found in table  chr: chromosome. shf i: isl-1-dependent shf enhancer. shf ii: nkx2-5/foxh1-dependent shf enhancer. c anti-brg chip was performed on day differentiating p and p cultures with sequential qpcr analyses of gli-specific mef2c sites a-i, depicted in. one-tailed student’s t-tests were used for the chip statistical analyses; n =  all error bars represent +/- sem. all grey lines represent paired t-tests; all black lines represent unpaired t-tests; p < . d gli and brg mrna expression levels were assessed by qpcr in differentiating p ec cultures, treated with meoh vehicle or kaad-cyclopamine. these levels were normalized to β-actin and presented as a fold-change over meoh-treated culture expression levels from the same day. e the effect of kaad-cyclopamine or vehicle treatment on the expression level of gli and indicated cardiomyogenesis-specific genes was assessed using qpcr analysis. expression levels were normalized to β-actin, calibrated to day untreated culture expression levels, and presented as a percentage of the highest expression level recorded, per gene. f anti-brg chip was performed on day differentiating p ec cultures that were treated with either meoh vehicle or kaad-cyclopamine. each chip was followed by qpcr analyses on a gene desert region, β-actin, and mef2c site c from panel b. all error bars represent +/- sem; n =  two-tailed student’s t-tests were used for statistical analyses. all grey lines represent paired t-tests; all black lines represent unpaired t-tests; p <  the observed co-immunoprecipitation of gli and brg during mes cell differentiation led to our hypothesis that gli could recruit brg to regulatory regions of gli target genes to modulate their expression. given the results and the known roles of brg and gli in heart development, we were interested in knowing if gli and brg could co-regulate key cardiac progenitor genes implicated in cardiomyogenesis. mef2c, which is important for proper mammalian heart development, was enhanced in day differentiating mes cells compared to mes cells and was recently identified as a direct target of gli during p ec cell cardiomyogenesis. thus, we set out to determine if brg 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 brg 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, p ec cells predominantly differentiate into cardiomyocytes by day of differentiation, along with skeletal myogenic progenitors and fibroblast-like cells when treated with dmso. moreover, p ec differentiation is very similar to mes myogenic differentiation. for these reasons, we used p cells to determine the role of brg1-gli protein complex during myogenic differentiation. brg associates with mef2c regulatory elements in hh signalling dependent manner in line with what has been shown previously, we determined that brg is expressed throughout the differentiation of mes and p cells, and that gli over-expression does not drastically affect its expression in either. our lab has previously shown that gli associates with eight of the nine conserved gli consensus binding motifs found in the mef2c gene on day of p cell differentiation. given that brg immunoprecipitates with gli we performed an anti-brg chip on chromatin from day differentiating p and p cells, to determine if brg associates with gli2-specific mef2c genomic elements during differentiation, and also if gli overexpression can modulate this potential association. notably, p cells exhibited enhanced cardiomyogenesis as measured by an increased percentage of cardiomyocytes and higher expression levels of cardiac progenitor genes on day of differentiation, in agreement with previous reports.table a selection of proteins that associate with the mef2c gene chr chromosome auppercase nucleotides in the target sequence represent nonconsensus binding domains ball binding site information for proteins, except brg and sox were compiled with information gathered through transfac results from the anti-brg chip showed an association of brg with mef2c sites c, d, and g-i in the p cells as compared to chip with a non-specific rabbit serum control. notably, although there was a similar trend in the p cells, we did not observe statistical significance for brg association on sites d and g-i when compared to non-specific rabbit serum. mef2c site a, which lacked gli association under the same experimental conditions in a previous study, showed no significant association with brg in either cell line. however, the association of brg with the mef2c site c was significantly higher in p cells as compared to p cells. this effect was not due to altered brg expression in p cells as there was no significant change in brg expression in p cells, as compared to p cells, on day of differentiation, when the chip assay was performed. mef2c site c is of significant interest as it is located proximally to the isl-1-dependent shf enhancer region, 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 brg. thus, as overexpression of gli resulted in increased association of brg with mef2c site c, we chose this site to further investigate the dependence of brg association on endogenous hh signalling during p ec cell cardiomyogenesis. kaad-cyclopamine treatment of p ec cells resulted in a  ±   % and  ±   % decrease in gli expression on days and of p ec cell differentiation, respectively, without significantly affecting the day or brg transcript levels, compared to treatment with a vehicle control. similar cyclopamine-mediated reductions of gli expression have been seen before in p p19cl and mes cells. by day differentiating p ec cells treated with kaad-cyclopamine expressed significantly lower mef2c, gata-, and tbx transcript levels, compared to differentiating cells treated with the vehicle control. these lower levels of cardiac progenitor-enriched transcripts were expected as cyclopamine-treated p ec cells have repressed gata- expression and cyclopamine-treated p19cl ec cells show lower levels of gata- and nkx2- transcripts and we showed that a similar treatment in mes cells has a comparable effect. neither the level of contractile protein transcripts in the day differentiated cells nor the number of myhc+ve cardiomyocytes significantly differed between kaad-cyclopamine and vehicle-treated p ec cells. this is supported by previous reports, where differentiating p 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 brg to associate with mef2c site c, we performed an anti-brg chip in day differentiating p ec cells treated with vehicle or kaad-cyclopamine. the association of brg with mef2c site c in p ec cells was significantly reduced upon hh inhibition. importantly, kaad-cyclopamine treatment had no significant effect on brg1’s association with β-actin a known target of brg in mes cells. a gene desert locus was used as a negative control and showed no significant association with brg therefore, hh signalling is required, at least to some extent, for the efficient association of brg to the gli2-specific mef2c gene element c. discussion in this study we have shown that 1) gli enhances the expression of cardiac progenitor-enriched genes while blocking hh signalling through cyclopamine decreases the expression of cardiac progenitor-enriched genes; 2) gli immunoprecipitates with brg during mes cell cardiomyogenesis; and 3) brg is recruited to a gli2-specific mef2c gene element in a hh-dependent manner. thus, we propose a model, where hh/gli regulates the expression of mef2c via the recruitment of brg to the mef2c gene element upstream of the shf elements to ultimately regulate cardiomyogenesis in stem cells.fig. a model summarizing the role of the hh signalling pathway and its primary transducer, gli during mes cell cardiomyogenesis. a in this study, gli expression was observed to positively regulate cardiac progenitor-enriched genes in mes cells. the solid black arrows indicate hh/gli2’s proposed direct regulation. the hollow green arrow marks the transition that is enhanced by gli b gli2-regulated enrichment of cardiac progenitor transcripts may be explained in part by the ability of the transcriptional activator form of gli - mediated via a functional hh signalling pathway - to enrich brg association at gli2-specific mef2c site c. this enrichment site is proximal to known shf enhancer regions. other cofactors, including the remaining baf complex members and other unidentified shf-related transcription factors may support this gli2-mediated association. c when kaad-cyclopamine inhibits hh signalling, gli is likely degraded, which leads to a reduction of brg association on mef2c site c. although we have not observed the formation of gli2r in our system, it is possible that gli may be processed into gli2r when hh signalling is blocked. also, gli3r may contribute to the repression of mef2c expression. the downstream binding of either gli2r or gli3r to mef2c has yet to be assessed hh signalling is important for cardiac progenitor gene expression during mes cell differentiation using a stabilized gli mutant, we have shown that gli enhances early expression of cardiac progenitor transcripts such as mef2c, nkx2- and tbx during mes cell cardiomyogenesis. in agreement, when hh signalling in mes cells is blocked with cyclopamine, this leads to a great reduction in the expression of these genes. this supports and expands previous reports in p ec cells. the effect of cardiac progenitor gene upregulation by exogenous gli is probably not due to an increase in mesodermal transcript expression as gli overexpression does not have any significant effect on the levels of brachyury or mesp on days and respectively. notably, the small upregulation of mesp transcript levels on day in mes cells is minor in comparison with day values. this is in agreement with previous studies, which demonstrate that there is no significant hh-dependent effect on mesoderm transcripts in p ec cells. the enhanced expression of nkx2- in mes cells correlates with the hh-mediated expression of nkx2- in vivo and in p ec cells. the upregulation of tbx transcript levels in differentiating mes cells follows the same pattern of tbx upregulation seen in differentiating p cells. when hh signalling is inhibited in p ec or mes cells by kaad-cyclopamine, tbx expression is reduced, similarly to p ec cells overexpressing dominant-negative gli/enr. interestingly, tbx 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 tbx expression by the hh pathway is yet to be determined in vivo, based on results from previous reports and our observations, tbx expression is regulated by hh signalling in vitro, during p ec and mes cell differentiation. in contrast to mef2c, nkx2- and tbx upregulation in mes cells, gata- expression is not affected when gli is overexpressed. importantly, gata- transcript levels were downregulated in mes cells treated with cyclopamine, similarly to p ec cells. this suggests that while exogenous gli cannot enhance gata- expression in differentiating mes cells, repression of endogenous hh signalling is sufficient to reduce its expression. it is possible that gata- induction is more efficient with other gli family members, that gata- 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- expression might be independently regulated from mef2c, nkx2- and/or tbx expression. 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- and mef2c expression while gata- levels remain unaffected. in mes cells, overexpression of the cardiac progenitor gene regulator, yin yang enhances nkx2- and tbx expression with no apparent effect on gata- expression. therefore, our observation of mef2c, nkx2- and tbx upregulation in mes cells with no observable effect on day gata- 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- tbx mef2c and gata- in differentiating mes and p 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. moreover, simultaneous expression of gata- and tbx 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 gli overexpression or application of cyclopamine regulates nkx2- mef2c and tbx cardiac progenitor gene expression in mes cells, this does not alter the percentage of cardiomyocytes. one explanation might lie in gata- expression. namely, gata- appears to be a limiting factor, in a transcription factor cocktail, to induce myhc expression in mes cells. therefore, the lack of gata- enhancement by exogenous gli may offer an explanation for the lack of cardiomyocyte number increase in mes cultures. hh signalling gain-of-function experiments in p cells showed both an induction and enhancement of cardiomyogenesis. p 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 and thus may not have as many mesoderm cells that are susceptible to hh-mediated regulation. it is possible that gli overexpression affects the differentiation of other cell types as hh signalling regulates many developmental programs in vivo, resulting in a lower percentage of cardiomyocytes in mes 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 p ec cells. lastly, hh signalling may enhance mechanisms in the neighbouring lineages that in turn negatively regulate cardiac differentiation in mes cells. gli immunoprecipitates with brg during mes cardiomyogenesis and active hh signalling regulates brg association with mef2c gene to determine the mechanism of gli during in vitro cardiomyogenesis, we used both mes and p differentiating cells. the co-immunoprecipitation of gli and brg in differentiating mes cells is in agreement with previous observations of ha-tagged gli immunoprecipitation with brg in nih 3 t cells. to investigate the significance of this interaction, we tested the hypothesis that gli and brg co-regulate cardiomyogenesis in vitro by modulating the expression of gli target genes. in this light, we focused on mef2c gene, a direct target of gli during p ec cell cardiomyogenesis. increased mef2c transcript levels in mes support and extend these results. moreover, brg 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. the anti-brg chip analysis from day differentiating p ec control cells demonstrates that brg associates with mef2c sites c, d, and g-i, which gli has been shown to bind under similar conditions. these results support and extend a previous report by showing that brg 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. of these two sites, brg is more efficiently recruited to mef2c site c in p cells overexpressing gli moreover, when hh signalling in p cells is blocked, there is a reduction of brg association with this site. thus, our findings indicate that brg is recruited to at least one mef2c gene regulatory element in a hh- and gli2-dependent manner. our hypothesis that gli and brg may share common chromatin targets is also supported by the identification of additional putative gli- and brg1-target genes through an in silico analysis, where brg 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, gli and ptch, amongst the theoretical targets, validating this bioinformatic assay. a gene ontology analysis identified a broad spectrum of biological processes that are significantly enriched for these genes, such as regulation of gene expression, cell differentiation and system development. as gli and brg are ubiquitously expressed proteins, it is possible that they might co-regulate a variety of cellular and developmental programs. notably, we identified nervous system development, where gli and brg are known to interact, and heart development. foxh tbx notch and wnt5a are amongst the potential targets in the heart development category. foxh and tbx are expressed in the shf field, while notch and wnt signalling pathways are known to regulate heart development. thus, gli and brg may co-regulate cardiac genes that have yet to be identified. moreover, we predict that this co-regulation may be common to other developmental programs, which is supported by the identification of additional hh-dependent go categories, such as osteoblast differentiation, nervous system development and cell cycle regulation.table selected gene ontology biological processes significantly enriched among genes within 50 kb of a brg1- associating site and gli consensus binding motifa atf meis ncoa ncor smad2 actn ctnna ctnnd mapt, myo1e angpt fgf fgf pdgfa creb dhh, mef2d, ptch rara, smo bdnf, neurod nkx2- pax6 hoxa hoxa hoxd yy1 atoh otx otx gata2 foxh notch tbx ttn, wnt5a cdc cdk cdk e2f e2f2 bmp gli gli2 epha fat lama lamc tubb2b hdac mll myst smarca smarcd1 aa complete list can be found in additional file 5: table s1  taken together, our results indicate that gli and brg co-operate to regulate cardiomyogenesis in vitro. we propose a mechanism where hh signaling, through activated gli recruits brg along with other potential co-factors to the mef2c gene site c to promote mef2c expression. inhibition of hh signaling results in reduced brg association at mef2c site c and reduced mef2c expression. since brg association correlates with chromatin accessibility, we predict that gli might recruit brg to increase chromatin accessibility on mef2c and other potential yet unidentified target genes to regulate in vitro cardiomyogenesis. abbreviations chip, chromatin immunoprecipitation; dhh, desert hedgehog; dmso, dimethylsulphoxide; ec, embryonal carcinoma; egs, ethylene glycol bis; 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 additional files additional file 1: figure s mrna expression levels of the gli2s662a-flag transgene in mesc clones stably transfected with a gli expression plasmid or with the empty vector. total rna was extracted from cells at various time points during differentiation. expression values are relative to those at day with the clone “mesc#1” and are normalized to β-actin. for each clone, n =  additional file 2: figure s gli may regulate early neurogenesis during mes cell differentiation. total rna was isolated from differentiating mes and mes cultures on days indicated and analyzed using qpcr for the expression of pax and ascl expression levels were normalized to β-actin, calibrated to day mes culture expression levels, and presented as a percentage of the highest expression level recorded, per gene. error bars represent +/- sem; n =  one-tailed student’s t-tests were used for statistical analyses. grey lines represent paired t-tests; black lines represent unpaired t-tests; p < . additional file 3: figure s modulation of hh signalling does not affect cell proliferation or survival. day cells were treated with edu for 1 h prior to staining. at least cells per sample, across fields of view, were counted. error bars represent +/- sem. student t-tests were used for statistical analyses. n =  the yields of total rna extracted from mes and mes are given as a proxy for cell numbers in the cultures. for each time point, n is between and and error bars represent +/- sem. the proportion of cells with highly condensed, apoptotic nuclei after hoechst staining at day is given. at least cells per condition per replicate were counted. n =  yields of rna in mes cultures treated with methanol vehicle or kaad-cyclopamine. for each time point, n =  and error bars represent +/- sem. the proportion of cells with highly condensed, apoptotic nuclei is given for vehicle- and kaad-cyclopamine-treated mes cultures at day n =  and at least cells per replicate and condition were counted. additional file 4: figure s expression of brg in mes and p cells overexpressing gli qpcr analysis of brg mrna expression levels in mes and mes cells, or p and p cells. for expression levels were normalized to β-actin, calibrated to day mes or p culture expression levels, and presented as a percentage of the highest expression level recorded, per gene. two-tailed student’s t-tests were used for the mrna statistical analyses; n =  additional file 5: table s a complete list of gene ontology biological processes significantly enriched among genes within 50 kb of a brg1-associating site and gli consensus binding motif. 
in contrast, pharmacological blockade of the hh pathway in mes cells resulted in lower expression of these genes. we then determined that brg is recruited to a gli2-specific mef2c gene element in a hh signalling-dependent manner during cardiomyogenesis in p ec cells, a mes cell model. thus, we propose a mechanism where hh/gli regulates the expression of mef2c by recruiting brg to the mef2c gene, most probably via chromatin remodelling, to ultimately regulate in vitro cardiomyogenesis. to further assess the transcriptional role of hh signalling during cardiomyogenesis in stem cells, we studied the effects of overexpressing gli a primary transducer of the hh signalling pathway, in mes cells. mass spectrometric analysis identified the chromatin remodelling factor brg as a protein which co-immunoprecipitates with gli in differentiating mes cells. stable gli overexpression resulted in an enhancement of cardiac progenitor-enriched genes, mef2c, nkx2- and tbx during mes cell differentiation.
 the hedgehog signalling pathway regulates cardiomyogenesis in vivo and in differentiating p embryonal carcinoma cells, a mouse embryonic stem cell model. to further assess the transcriptional role of hh signalling during cardiomyogenesis in stem cells, we studied the effects of overexpressing gli a primary transducer of the hh signalling pathway, in mes cells.  stable gli overexpression resulted in an enhancement of cardiac progenitor-enriched genes, mef2c, nkx2- and tbx 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 brg as a protein which co-immunoprecipitates with gli in differentiating mes cells. we then determined that brg is recruited to a gli2-specific mef2c gene element in a hh signalling-dependent manner during cardiomyogenesis in p ec cells, a mes cell model.  thus, we propose a mechanism where hh/gli regulates the expression of mef2c by recruiting brg to the mef2c gene, most probably via chromatin remodelling, to ultimately regulate in vitro cardiomyogenesis. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cardiac developmentcardiomyogenesishedgehog signalling pathwaygli2brg1http://dx.doi.org/ /501100000024canadian institutes of health researchmop-53277mop-119458blais alexandre skerjanc ilona sylvia http://dx.doi.org/ /501100000121ontario council on graduate studies, council of ontario universitieshttp://dx.doi.org/ /501100000024canadian institutes of health researchhttp://dx.doi.org/ /501100000121ontario council on graduate studies, council of ontario universitieshttp://dx.doi.org/ /501100000024canadian institutes of health researchhttp://dx.doi.org/ /100004411heart and stroke foundation of canadaissue-copyright-statement© the author 2016 