Gata4 regulates hedgehog signaling and Gata6 expression for outflow tract development

Dominant mutations of Gata4, an essential cardiogenic transcription factor (TF), were known to cause outflow tract (OFT) defects in both human and mouse, but the underlying molecular mechanism was not clear. In this study, Gata4 haploinsufficiency in mice was found to result in OFT defects including double outlet right ventricle (DORV) and ventricular septum defects (VSDs). Gata4 was shown to be required for Hedgehog (Hh)-receiving progenitors within the second heart field (SHF) for normal OFT alignment. Restored cell proliferation in the SHF by knocking-down Pten failed to rescue OFT defects, suggesting that additional cell events under Gata4 regulation is important. SHF Hh-receiving cells failed to migrate properly into the proximal OFT cushion, which is associated with abnormal EMT and cell proliferation in Gata4 haploinsufficiency. The genetic interaction of Hh signaling and Gata4 is further demonstrated to be important for OFT development. Gata4 and Smo double heterozygotes displayed more severe OFT abnormalities including persistent truncus arteriosus (PTA). Restoration of Hedgehog signaling renormalized SHF cell proliferation and migration, and rescued OFT defects in Gata4 haploinsufficiency. In addition, there was enhanced Gata6 expression in the SHF of the Gata4 heterozygotes. The Gata4-responsive repressive sites were identified within 1kbp upstream of the transcription start site of Gata6 by both ChIP-qPCR and luciferase reporter assay. These results suggested a SHF regulatory network comprising of Gata4, Gata6 and Hh-signaling for OFT development.


Introduction
Congenital Heart Defects (CHDs) occur in approximately 1% of live births [1] and are the most common serious birth defects in humans [2,3]. Approximately one third of the CHDs involve malformations of the outflow tract (OFT), which leads to significant morbidity and mortality of children and adults [4]. Multiple OFT abnormalities involve the defective relationship between the Aorta and Pulmonary Artery to the underlying left and right ventricles. For example, double-outlet right ventricle (DORV) is an anomaly in which the Aorta and Pulmonary Artery originate from the right ventricle [4]. A key characteristic of DORV that distinguishes it from other OFT defects is that the aorta and pulmonary trunk are well separated but are improperly aligned over the right ventricle. The molecular basis of OFT misalignment has remained unclear.
SHF-derived cells migrate into the developing poles of the heart tube, to direct the morphogenesis of the cardiac inflow and outflow. The anterior SHF (aSHF) is essential for OFT and great artery development [5][6][7][8][9]. The failure of the aSHF-derived myocardial and endocardial contributions to the arterial pole of the heart causes a shortened OFT and arterial pole misalignment, resulting in inappropriate connections of the great arteries to the ventricular mass [10][11][12]. Deletion of genes responsible for SHF morphogenesis, such as Isl1, Mef2c, and Jag-ged1, leads to abnormal OFT formation including DORV [5,6,[12][13][14][15][16][17][18][19]. These observations lay the groundwork for investigating the molecular pathways required for OFT development in SHF cardiac progenitor cells.
In this study, the mechanistic requirement for Gata4 in OFT development was investigated. Gata4-dependent pathways were revealed to be contributors to OFT development in Gata4 heterozygous mouse embryos.

Gata4 is required for OFT alignment
Gata4 is strongly expressed in the heart, pSHF and OFT at E9.5 [20,35,39]. There is a gap in expression between the OFT and the pSHF at embryonic day 9.5 ( Fig 1A, indicated by a "#"). IHC staining for Gata4 at later stages during OFT development showed strong Gata4 expression in the heart, the developing OFT and the pSHF, but only in a limited subset of aSHF cells at E10.5 (Fig 1B, indicated by a "#"). At E11.5, both the chamber myocardium and the developing OFT had strong Gata4 expression. However, Gata4 expression was absent from the cardiac neural crest (CNC)-derived distal OFT (Fig 1C, indicated by a "#").
Previous studies have shown that Hh signal-receiving progenitors localized in both the aSHF and the pSHF, and regulate the migration of SHF toward the OFT and inflow tract (IFT) to form the pulmonary artery and the atrial septum respectively [46][47][48]. We combined Gli1--CreER T2/+ with Gata4 fl/+ to create Gata4 haploinsufficiency in SHF Hh signal-receiving progenitors. CreER T2 was activated by TMX administration at E7.5 and E8.5 in Gli1-CreER T2/+ ; Gata4 fl/+ embryos. The reduced expression of Gata4 by the deletion of Gli1-CreER T2 recombination was confirmed by realtime-PCR using the SHF tissue of E9.5 embryos (Fig 1S-1G). With TMX administration at E7.5 and E8.5, 66.7% of Gli1-CreER T2/+ ; Gata4 fl/+ embryos displayed DORV, while the littermate control Gata4 fl/+ embryos displayed normal OFT alignment ( Fig 2K and 2K' vs. 2H, 2H', 8/12 vs. 0/15, P = 0.0002). Blue ink was injected in the pulmonary artery of the Gata4 fl/+ E14.5 embryos, resulting in the staining of the pulmonary artery and the right ventricle. However, the Gli1-CreER T2/+ ; Gata4 fl/+ embryos showed staining in not only the right ventricle and the pulmonary artery, but also the aortic artery, which confirms the phenotype of DORV in these embryos (Fig 2L and 2M). In addition, when the embryos were given TMX at E8.5 and E9.5, normal OFT alignment was observed in all Gli1--CreER T2/+ ; Gata4 fl/+ embryos (Table 1). To exclude the possibility that the phenotype might be due to the double heterozygosity for Gata4 and Gli1, the phenotype of the Gli1-CreER T2/+ ; Gata4 fl/+ embryos without TMX treatment was examined. There were no heart defects observed in the embryos at E14.5 ( Table 1). Considering that TMX activates expression 12 h after injection and that the action lasts for 36 hours [49,50], we concluded that Gata4 is required in the SHF Hedgehog (Hh) signal-receiving progenitors from E8 to E10.5 for proper OFT alignment.

Rescue of SHF proliferation by disruption of Pten does not rescue DORV in Gata4 mutant embryos
Our previous study demonstrated that Gata4 mutants disrupted cell cycle progression in the pSHF cardiac precursors resulting in atrial septal defects and genetically targeted downregulation of Pten rescued the proliferation defects in SHF of the Gata4 heterozygotes [35]. Would the defected cell cycle by Gata4 mutants lead to OFT alignment defects? In order to answer this question, the analysis was conducted to qualify if Pten downregulation (TMX at E7.5 and E8.5), could also rescue DORV in Hh-receiving cell-specific Gata4 heterozygotes. Decreased dosage of Pten caused DORV in only 1 of the 20 embryos, and none with ASD ( Fig 3G, 3H and 3I).   Gli1-CreER T2/+ ; Pten fl/+ ). Consistently, expression of the cell proliferation genes including Cdk2, Cdk4 and Ccnd2 was lower in the Gli1-CreER T2/+ ; Gata4 fl/+ embryos but was restored to normal levels with a Pten knockdown ( Fig 3W). This data suggested that correction of the SHF proliferation defects failed to rescue the OFT misalignment of the Gata4 mutant embryos, and thus different mechanisms were involved in the regulations of Gata4 for atrial septal and OFT.

Gata4 acts upstream of Hh signaling in OFT development
We have previously reported that Gata4 acts upstream of Hh-signaling for atrial septation [35]. We tested the hypothesis that Gata4 acts upstream of Hh-signaling for OFT development using a genetic epistasis study. The purpose was to understand if increased Hh-signaling via a constitutively activated Smo mutant, SmoM2 [35], induced by TMX administration at E7.5 and E8.5, could rescue the OFT misalignment in Gata4-heterozygotes. DORV was observed in 28.6% of littermate control Gli1-CreER T2/+ ; SmoM2 fl/+ embryos (2/7) (Fig 4G and 4G') and   Table 1). This results demonstrated rescue of DORV in Gata4-mutant embryos by constitutive Hh signaling.

Gata4 is required for the contribution of Hh-receiving cells to the OFT
Hh signaling has been reported to regulate the migration of SHF Hh-receiving cells toward the arterial pole of the heart [46]. We therefore hypothesized that Gata4 drives SHF Hh-receiving cells migration toward the developing OFT. This hypothesis was tested by using genetic inducible fate mapping (GIFM) [51]. The Hh-receiving lineage cells were marked by TMX administration at E7.5 and E8.5 (Gli1-CreER T2/+ ; R26R fl/+) and β-gal expression was evaluated at E11.5. We assessed if there was less migrating Hh-receiving SHF cells migrating through the distal OFT (dOFT) towards the proximal OFT (pOFT) in the Gata4 haploinsufficient embryos To examine if a Gata4 heterozygous influenced the SHF cell recruitment within the proximal OFT, we analyzed the fate map of SHF lineage cells in the OFT of the Gata4 heterozygotes. Defined by Mef2cAHF::Cre driven β-galactosidase-expressing cells, the total number of the SHF lineage cells within the proximal and distal half of the OFT were compared between the Mef2cAHF::Cre; R26R fl/+ ;Gata4 +/-; and the Mef2cAHF::Cre;R24R fl/+ embryos at E10.5. The number of SHF lineage cells populating the pOFT of the Mef2cAHF::Cre;Gata4 +/-; R26R fl/+ embryos was significantly less than those in the control Mef2cAHF::Cre; R26R fl/+ embryos ( Fig  5M vs. 5P); however, this decrement was not observed in the distal OFT (Fig 5N vs. 5Q). The distribution pattern of the SHF lineage was not different in the Mef2cAHF::Cre;Gata4 +/-; R26R fl/+ and the Mef2cAHF::Cre;R26R fl/+ embryos (Fig 5O vs. 5R). Fewer cells were observed to populate the developing dorsal mesocardium protrusion (DMP) in Mef2cAHF::Cre; Gata4 +/-; R26R fl/+ (red arrow, Fig 5O vs. 5R). This was consistent with the previous report that Gata4 is required in the SHF for the DMP [35]. These results demonstrated the requirement of Gata4 for the SHF lineage cells populating in the developing OFT.

Gata4 is involved in the endothelial-to-mesenchymal transformation (EMT) and mesenchymal cell proliferation for OFT cushion development
The role of Gata4 in the EMT for the endocardial cushion development has been well described previously [20,23,52]. Since less SHF lineage cells populating in the developing OFT myocardium were observed, we asked if this defect would affect the sequential events such as EMT, cell proliferation or cell survival in the developing OFT via a non-cell autonomous manner. Expression of mesenchymal marker N-cadherin was used to label the cushion cells undergoing EMT. LacZ staining was performed before the IHC staining, which indicated the active Cre recombination for specifically Gata4 knocking-down. The Hh-receiving cells were shown to populate at the conal OFT as early as E14.5. Significantly less N-cadherin staining of the conal OFT cushion cells in the Gli1-CreER T2/+ ; Gata4 fl/+ ; R26R fl/+ embryos versus the Gata4 fl/+ ; R26R fl/+ littermate control embryos at E10.5 (Fig 6A-6C vs. 6D-6E) was observed, suggesting that the EMT process of the OFT cushion was inhibited by the lower Gata4 expression in Hh-receiving cells. Cell proliferation was examined by BrdU incorporation at E11.5. Gli1-CreER T2/+ ; Gata4 fl/+ embryos demonstrated 17% fewer BrdU-positive SHF cells in the OFT conal cushion (Fig 6G vs. 6J and 6I; P = 0.0134), but not the OFT truncal cushion (Fig 6H vs. 6K and 6L; P = 0.1998), when compared to the littermate Gata4 fl/+ embryos at E11.5. Cell death were assessed by TUNEL staining and no differences in either the conal or truncal cushion between Gli1-CreER T2/+ ; Gata4 fl/+ and the Gata4 fl/+ embryos was observed (Fig 6M-6P). Together, these results demonstrated that Gata4 is required for normal cell EMT and proliferation in OFT conal cushion development, possibly through a non-cell autonomous manner.

Gata6 was overexpressed in the SHF of the Gata4 transgenic mouse embryos
Because Gata4 and Gata6 double mutant embryos display PTA [33], Gata6 expression in Gata4 mutants was examined. Gata6 was expressed in the heart, the OFT and strongly in the splanchnic mesoderm (Fig 7A, arrow), but not neural crest cell derivatives (Fig 7A, arrowhead) of the Gata4 fl/+ embryo at E9.5. In Gata4 knockdown embryos specifically in the Hh-receiving cells, the Gata6 expression domain was strongly enhanced in the OFT and the splanchnic mesoderm. Consistently, enhanced expression of Gata6 in the OFT and the SHF of the Gata4 fl/fl ; Gli1-CreER T2/+ was further confirmed by the real-time PCR at the mRNA level ( Fig  7B). The Gata6 expression in the SHF of Gata4 fl/fl ; Gli1-CreER T2/+ mouse embryo was increased by 1.7-fold comparing to the control Gata4 fl/+ embryos (P<0.05). Gata6 expression in the OFT of the Gata4 fl/fl ; Gli1-CreER T2/+ mouse embryo was increased by 3.4-fold comparing to the littermate control (P<0.01). These results suggested a negative association between the expression of Gata4 and Gata6 in the SHF and developing OFT.
We tested the possibility of Gata4 regulating the expression of Gata6 in the SHF as a repressor by ChIP-qPCR using the microdissected SHF from the E9.5 wildtype mouse embryos. The Gata6 loci was bioinformatically interrogated for potential Gata4-responsive elements using the overlap of evolutionary conservation and Gata4 occupancy in HL-1 cells or embryonic mouse hearts [53,54] (Fig 7C). Eight potential Gata4-binding regions for Gata6 were screened and our previously identified Gata4 responsive Gli1 loci [35] was used as the positive control (Gli1-ctrl). The results showed enrichments of the region Gli1-ctrl and the region Gata6-1b, but not the others (Fig 7D). This suggested that the region Gata6-1b, within 1 Kbp upstream of Gata6 start codon, was responsive to Gata4 binding. Luciferase reporter assay showed no changes of firefly luciferase activity under Gata6-1b expression in HEK293 cells (Fig 7E, Gata4 +G6-luc vs. Gata4+pGL3, P>0.05). However, three mutant constructs for Gata6-1b, each ablating one Gata4-binding site, significantly enhanced the luciferase activity (Fig 7E, P<0.001 for all three mutants versus the Gata4+pGL3). Together, these results place Gata4 upstream of Gata6 as a repressor in the SHF.

Discussion
The requirement of Gata4 for OFT development has been reported in mice and humans, and Gata4 mutations causing DORV have been reported in mice [19,20,33]. Here Gata4 is demonstrated required in the SHF Hh-receiving cells for OFT alignment. Our previous study has demonstrated that Gata4 is required for Hh signaling in the SHF for cell proliferation. However, the current study suggested that the cell proliferation defects in the SHF caused by Gata4 mutation may not be the only mechanism underlying the OFT misalignment. Another important contributing factor is the migration defect of the SHF cells, which were associated with disrupted Hh-signaling, as proved by the rescue of over-activating Hh-signaling. As subsequent events, Gata4 haploinsufficency in the Hh-receiving cells disrupted EMT process and cell proliferation in the conal OFT cushion, suggesting that both the cell-autonomous and non-cell autonomous effects of Gata4 drive OFT development. In addition, we demonstrated Gata4 as a repressor of Gata6 in the SHF by identifying Gata4-responsive binding sites in its promoter regions. This result provides a molecular explanation for the severity of OFT defects observed in Gata4 and Gata6 double mutant embryos. These data suggested that breaking down the threshold of GATA including Gata4 and Gata6, and Hh signaling tone might be associated with the severity of OFT defects.
The SHF was initially described as a progenitor field for the cardiac OFT and a rich literature has established the requirement of aSHF contributions for OFT development [5, 12, 35, [55][56][57][58][59][60][61][62][63][64][65]. More recently, the contribution of pSHF cardiac progenitors to the OFT and the future subpulmonary myocardium has been reported; however, the mechanistic requirement for this contribution is not well understood [46,[66][67][68]. The cell lineage in which Gata4 is required for OFT development has not been reported. Gata4 is expressed in both the aSHF and pSHF, although its expression is much stronger in the pSHF [35]. The decreased number of Mef2-C-AHF::Cre positive cells in the proximal OFT cushion of E10.5 Gata4 −/+ embryos demonstrated that Gata4 plays a role in adding the SHF progenitor cells to the developing OFT. Surprisingly, OFT defects were not observed in either aSHF-specific (Mef2c-AHF::Cre) or pSHF-specific (Osr1-CreER T ) Gata4 haploinsufficiency. Instead, it was found that the severity of the OFT defects and incidence rate in embryos with Gata4 haploinsufficiency in Hh-receiving cells were identical to those in Gata4 −/+ embryos. Because Hh-receiving cells are located throughout the SHF, these observations suggested that Gata4 is required in both pSHF and aSHF progenitor cells for OFT alignment.
Evidence was provided that Gata4 acts upstream of Hh-signaling in the SHF for OFT development. The Gata4 −/+ embryos have combined phenotypes of ASD and DORV [35]. This study, adding new knowledge to the previous [35], have disclosure that Gata4-Hh-signaling plays active, but different, roles at the venous and atrial pole of the developing heart. In the SHF, Gata4-Hh-signaling controls cell cycle progression and thereby the proliferation of the cardiac progenitors. At the venous pole, diminished Gata4-Hh signaling for cell cycle regulation is balanced by Pten through transcriptional inhibition of Cyclin D4 and Cdk4 [13,35], as DMP hypoplasia and SHF cell cycle defects are rescued by Pten knockdown [13,35]. At the atrial pole, the Pten knockdown was able to rescue the cell cycle defects in the SHF, but failed to rescue DORV or OA defects in Gata4 heterozygous mutants. This observation suggests that correction of SHF cell proliferation is not sufficient to support a normal OFT development in Gata4 mutants. In this study, increased apoptosis was not observed in the SHF of Gata4 heterozygote mutant embryos [35]. Indeed, cell migration under the regulation of Gata4-Hh signaling is important for OFT development. However, fate mapping of the SHF using either Mef2c-AHF::Cre or the Gli1-CreER T2 disclosed less SHF-derived cells in the distal OFT in Gata4 mutant embryos. Specifically, there was a decreased number of SHF Hh-receiving cells throughout the migration route from the SHF into the OFT, which traveled from the dorsal mesocardium and continued through the rostral splanchnic mesoderm, past the distal OFT and reached the proximal OFT. Hh-receiving progenitors have been found to migrate from the aSHF to populate the pulmonary trunk between E9.5 to E11.5 [46], suggesting that Hh-signaling is required for SHF cell migration. The observation that DORV in Gata4 mutant embryos can be rescued by constitutive Hh-signaling is correlated with restored cell migration and cell proliferation in the SHF. Therefore, this data suggested that the normal Gata4 regulation of both the proliferation and the migration of the SHF cardiac progenitors are required for OFT development.
During development, the ventricular outlets are aligned to the ventricles by the fusion of conal cushions with the interventricular septum [69], and improper lengthening of the conal cushion may cause rotation problems resulting in misalignment. We demonstrated that Gata4 Results are presented as mean ± SEM; n = 4; �� P < 0.05 vs. Gata6-ctrl, ��� P < 0.01, compared with Gata6-ctrl. (E) Gata4-stimulated firefly luciferase activity in wild-type Gata6 and Gata6 mutant fragments. Results are presented as mean ± SEM; n = 4; ��� P < 0.01, compared with Gata4+pGL3. https://doi.org/10.1371/journal.pgen.1007711.g007 Gata4 regulates Hh-signaling and Gata6 for outflow tract alignment plays a non-cell autonomous role in the EMT process to give rise to the conal cushions mesenchyme. It is still unknown what specific signals the SHF-derived OFT Gli1+ myocardial cells provide for EMT. Future studies should aim to identify the ligands secreted by Hh-receiving cells. Moreover, a smaller percentage of BrdU+ cells in the conal cushion of the OFT was found at E11.5 of the Gata4 fl/+ ; Gli1 Cre-ERT2/+ embryos, suggesting Gata4 plays a role in regulating the OFT cushion cell proliferation. Inactivating N-Cadherin in the SHF resulted in hypoplastic OFT and right ventricle, associated with decreased proliferation [70,71]. Thus, the lower number of proliferating cells might be associated with the lower expression of N-Cadherin. Overall, cellular, molecular and genetic evidence proved that Gata4-Hh signaling is required in OFT alignment via both the cell-autonomous and non-cell autonomous manners.
Although important Gata4 transcriptional targets in the heart have been identified [13,18,37], Gata4-dependent molecular pathways required for OFT development remain unknown. Gli1 was previously identified as a downstream target of Gata4 in the pSHF for atrial septation [35]. In the current study, it was further demonstrated that Gata4 controls Hh-signaling though Gli1 transcriptional regulation for cell migration and OFT alignment. In addition, Gata4 was demonstrated to be a transcriptional repressor of Gata6 in the SHF, and the Gata4-responsive sites in the Gata6 promoter region were identified. Previously, several downstream targets of Gata4 including the Mef2c and Gli1 have been recognized [13,15,16,18,36,37], while none of them respond to the inhibitory effects. Gata6 is the first identified repressing target of Gata4, providing direct evidence that Gata4, as a transcription factor, is not only an activator but also a repressor. Enhanced Gata6 expression in Gata4 mutants might illustrate a compensatory feedback loop, given that Gata6 and Gata4 are redundant for cardiac myocyte differentiation [72,73]. Gata4/Gata6 compound heterozygotes displayed persistent truncus arteriosus (PTA), a severe OFT defect caused by combined alignment and OFT septation defects [33]. This study shows that Gata4/Smo compound heterozygotes show a similar phenotype. Gata4 heterozygote alone does not display PTA, which might be due to the partial recovery of GATA function from enhanced Gata6 expression. Together with previous study [33], these data suggest a threshold of Gata4, Gata6, and Hh signaling and that is required for OFT development. This implies that GATA TFs may be essential for the quantitative regulation of Hh signaling, and diminished GATA function or reduced GATA and Hh signaling together may cause more severe OFT defects. Future studies will focus on the quantitative relationship between GATA tone and Hh signaling tone, as well as the Gata4 dependent gene regulatory network (GRN) [74] for OFT development.

Ethics statement
Mouse experiments were completed according to a protocol reviewed and approved by the Institutional Animal Care and Use Committee of Texas A&M University (#2015-0398), in compliance with the USA Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Tamoxifen administration and X-gal staining
TMX-induced activation of CreER T2 was accomplished by oral gavage with two doses of 75 mg/kg TMX at E7.5 and E8.5 [44,46]. X-gal staining of embryos was performed as described [46]. The total number of β-gal positive cells was obtained by counting those on each individual sections and adding up all through the SHF and the OFT.

BrdU incorporation and Immunohistochemistry Staining (IHC)
Standard procedures were used for histology and IHC. For BrdU incorporation, pregnant mice were given 100mg BrdU per kg bodyweight at 10mg/mL concentration solutions at E11.25 with two doses, 3 hours and 6 hours before sacrifice, respectively. The BrdU staining was performed using a BrdU In-Situ detection kit (EMD Millipore). For TUNNEL staining, an ApopTag plus peroxidase In-Situ apoptosis detection kit was used (EMD Millipore). IHC was performed using the following antibodies: anti-Gata4 (Abcam, #ab84593), anti-Gata6 (Abcam, #ab175349), and anti-N-cadherin (Abcam, #ab18203). After incubating with the first antibody, a VECTASTAIN ABC HRP Kit (LifeSpan BioSciences, Inc) was used for detecting the protein expression signal. For counting the ratio of proliferating cells, a total of 100 random cells within the SHF and the specific OFT regions per each section were counted using the Particle Analysis tool of ImageJ and the ratio of positively stained cells was recorded. For each sample, five equivalent serial sections were counted and the averages were taken for statistical analysis.

Micro-dissection of pSHF and RNA extraction
To obtain the pSHF splanchnic mesoderm for use in quantitative realtime-PCR, E9.5 embryos were dissected as described before [45,75]. The heart, aSHF, and pSHF were collected separately in RNA-later, and then stored at −20˚C until genotyping was completed.

Realtime-PCR
Total RNA was extracted from the PSHF regions of mouse embryos hearts using RNeasy Mini Kit (QIAGEN), according to the manufacturer's instructions. Two hundred ng of total RNA was reverse transcribed using a SuperScript TM III Reverse Transcriptase kit from Invitrogen. qPCR was performed using a POWER SYBER Green PCR mater mix from Applied Biosystems. Results were analyzed using the delta-delta Ct method with GAPDH as a normalization control [76].

Chromatin immunoprecipitation
Chromatin Immunoprecipitation was performed as described previously [35]. The ChIP assay was performed using a Gata4 antibody (Santa Cruz, #sc-1237 X). Genomic regions with potential Gata4-binding sites and negative control sites are listed in Table 2. Primers used for evaluating the enrichment of the Gata4 pull-down fragments via realtime-PCR are listed in Table 3.

Dual Luciferase Reporter Assay
Dual Luciferase Reporter Assay was performed as described previously [35,44,45]. Genomic regions with potential Gata4-binding sites tested are listed in Table 2. Primers used for sitespecific mutation and subclone are listed in Table 4. Supporting information