Abstract
Dominant mutations of Gata4, an essential cardiogenic transcription factor (TF), cause outflow tract (OFT) defects in both human and mouse. We investigated the molecular mechanism underlying this requirement. Gata4 happloinsufficiency in mice caused OFT defects including double outlet right ventricle (DORV) and conal ventricular septum defects (VSDs). We found that Gata4 is required within Hedgehog (Hh)-receiving second heart field (SHF) progenitors for normal OFT alignment. Increased Pten-mediated cell-cycle transition, rescued atrial septal defects but not OFT defects in Gata4 heterozygotes. SHF Hh-receiving cells failed to migrate properly into the proximal OFT cushion in Gata4 heterozygote embryos. We find that Hh signaling and Gata4 genetically interact for OFT development. Gata4 and Smo double heterozygotes displayed more severe OFT abnormalities including persistent truncus arteriosus (PTA) whereas restoration of Hedgehog signaling rescued OFT defects in Gata4-mutant mice. In addition, enhanced expression of the Gata6 was observed in the SHF of the Gata4 heterozygotes. These results suggested a SHF regulatory network comprising of Gata4, Gata6 and Hh-signaling for OFT development. This study indicates that Gata4 potentiation of Hh signaling is a general feature of Gata4-mediated cardiac morphogenesis and provides a model for the molecular basis of CHD caused by dominant transcription factor mutations.
Author Summary Gata4 is an important protein that controls the development of the heart. Human who possess a single copy of Gata4 mutation display congenital heart defects (CHD), including the double outlet right ventricle (DORV). DORV is an alignment problem in which both the Aorta and Pulmonary Artery originate from the right ventricle, instead of originating from the left and the right ventricles, respectively. To study how Gata4 mutation causes DORV, we used a Gata4 mutant mouse model, which displays DORV. We showed that Gata4 is required in the cardiac precursor cells for the normal alignment of the great arteries. Although Gata4 mutation inhibits the rapid increase in number of the cardiac precursor cells, rescuing this defects does not recover the normal alignment of the great arteries. In addition, there is a movement problem of the cardiac precursor cells when migrating toward the great arteries during development. We further showed that a specific molecular signaling, Hh-signaling, is responsible to the Gata4 action in the cardiac precursor cells. Importantly, over-activating the Hh-signaling rescues the DORV in the Gata4 mutant embryos. This study provides an explanation for the ontogeny of CHD.
Introduction
Congenital Heart Defects (CHDs) CHDs occurr 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 relationship of 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 in DORV has remained unclear.
SHF-derived cells migrate into the developing poles of the heart tube, to effect morphogenesis of the cardia cinflow and outflow. The anterior SHF is essential for OFT and great artery development [5-9]. The failure of the anterior SHF-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 [10-12]. Deletion of genes responsible for SHF morphogenesis, such as Isl1, Mef2c, and Jagged1, leads to abnormal OFT formation including DORV [5, 6, 8, 12-19]. These observations lay the groundwork for investigating the molecular pathways required for OFT development in SHF cardiac progenitor cells.
Gata4, a member of the GATA family of zinc finger transcription factors, is an essential cardiogenic transcriptional regulator implicated in many aspects of cardiac development and function [20-34]. Human genetic studies have implicated haploinsufficiency of GATA4 in human CHDs, to date including atrial septal defects (ASD), ventral septal defects (VSD), and tetralogy of Fallot (TOF) [21, 35-39]. In mouse models, decreased expression of Gata4 results in the development of common atrioventricular canal (CAVC), DORV, and hypoplastic ventricular myocardium in a large proportion of mouse embryos [27, 40]. Multiple studies have demonstrated the molecular requirement of Gata4 in the endocardium for normal cardiac valve formation [24, 30, 41]. Furthermore, we previously demonstrated that Gata4 is required in the posterior SHF for atrial septation. Both Hedgehog (Hh) signaling and Pten-mediated cell-cycle progression were shown to be downstream of Gata4 in atrial septation [42]. However, the mechanistic requirement for Gata4 in OFT development is less clear. For example, from the multiple Gata4 transcriptional targets that have been identified in the context of heart development, including Nppa, α-MHC, α-CA, B-type natriuretic peptide (BNP), Ccnd2, and Cyclin D2, and Mef2c [20, 23, 24, 26, 43, 44], only Mef2c has a functional role in OFT development [12].
In this study, we investigated the mechanistic requirement for Gata4 in OFT development. We found that Gata4 heterozygosity in SHF hedgehog (Hh)-receiving cells recapitulates the OFT misalignment observed in Gata4 germline heterozygotes in mice. Gata4 heterozygous embryos had decreased numbers of SHF-derived cells populating the anterior SHF and the developing OFT at E10.5. By genetic inducible fate mapping (GIFM), Hh-receiving cells fail to migrate properly into the OFT of Gata4 mutant mice. We have previously reported that Gata4 acts upstream of Hh-signaling for atrial septation [42]. Here we observed more severe OFT defects observed in embryos with SHF-specific heterozygosity of Gata4 and Smo, the obligate Hh signaling receptor. Furthermore, rescue of Gata4-mediated OFT misalignment by constitutive activation of Hh-signaling indicated a consistent epistatic relationship between Gata4 and Hh signaling in OFT development. Furthermore, upregulation of Gata6 in the Gata4 mutant SHF may provide an explanation for the severity of OFT defects observed in Gata4 mutant embryos. Our study thereby revealed Gata4-dependent pathways contributing to OFT development in Gata4 heterozygous mouse embryos.
Results
GATA4 is required for OFT alignment
Gata4 is strongly expressed in the heart, pSHF and OFT at E9.5 [27, 42, 50]. 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 “↓”).
Gata4 was previously reported to be required for OFT alignment [27]. To study the role of Gata4 in OFT development, we re-examined Gata4 heterozygotes for OFT defects. As described previously [42], Gata4 heterozygotes were generated by crossing Gata4fl/+ with EllaCre, which drives Cre expression in the germline [51] to effect germline Gata4 deletion. The Gata4 germline deletion was ensured by genotyping using the embryo tail DNA. Whereas Gata4fl/+ (n = 13) and EllaCre/+ (n = 12) embryos demonstrated normal heart at E14.5 (Figs. 2A and A’, 2B and B’), 61.1% of Gata4+/-; EllaCre/+ embryos demonstrated VSD and DORV (Figs. 2C’, 11/18, P=0.0004). Consistent with our prior work, we observed primum ASDs with absence of the DMP in 8/18 Gata4+/-; EllaCre/+ embryos [42] (Figs. 2C).
To determine the lineage requirement for Gata4 in AV septation, we analyzed mouse embryos haploinsufficient for Gata4 in the myocardium, CNC, endocardium or SHF. We combined Tnt: Cre [52] with Gata4fl/+ to create Gata4 haploinsufficiency in the myocardium. Normal OFT alignment was observed in all TntCre/+; Gata4fl/+ (12/12) and littermate control Gata4fl/+ embryos (9/9) at E13.5 (P=1) (Figs. 2E and E’ vs. 2D and D’, P=1). We combined Wnt1: Cre [53, 54] with Gata4fl/+ create Gata4 haploinsufficiency in the CNC. Normal OFT alignment was observed in all Wnt1Cre/+; Gata4fl/+ mutant embryos (24/24) and littermate control Gata4fl/+ embryos (16/16) at E13.5 (Figs. 2F and F’ vs. 2D and D’, P=1). We combined Nfat1c: Cre [53, 54] with Gata4fl/+ create Gata4 haploinsufficiency in the endocardium. Normal OFT alignment was observed in nearly all Nfatc1Cre/+; Gata4fl/+ mutant embryos (14/15) and littermate control Gata4fl/+ embryos (10/10) at E13.5 (Figs. 2G and G’ vs. 2D and D’, P=1). These results demonstrated that Gata4 haploinsufficiency in the myocardium, CNC or endocardium supported normal OFT alignment.
Gata4 is required in the SHF Hedgehog (Hh) signal-receiving progenitors for OFT alignment
We hypothesized that Gata4 is required in the aSHF for OFT alignment in aSHF-specific Gata4 heterozygous mice. We tested this hypothesis by combining Mef2cAHF: Cre with Gata4fl/+. Surprisingly, OFT misalignment with DORV was only observed in 1 out of 22 embryos and none in the littermate controls (Fig. 2I and I’ vs. 2H and H’, P=1). We next tested if Gata4 is required in the pSHF for OFT alignment in in pSHF-specific Gata4 heterozygous mice by crossing Osr1 CreERT2/+ [46, 47] with Gata4fl/+. Similarly, neither Gata4fl/+; Osr1 CreERT2/+ embryos (0/5) nor littermate control Gata4fl/+ embryos (0/6) demonstrated OFT misalignments at E14.5 (Fig. 2J and J’ vs. 2H and H’, P=1). These results demonstrated that Gata4 haploinsufficiency in either aSHF or pSHF supported normal OFT alignment.
Previous studies have shown that SHF Hh signal-receiving progenitors localized in both the aSHF and the pSHF, and regulated the migration of SHF toward the OFT and inflow tract (IFT) to form the pulmonary artery and the atrial septum separately [45, 55, 56]. We combined Gli1Cre-ERT2 with Gata4fl/+ to create Gata4 haploinsufficiency in SHF Hh signal-receiving progenitors. CreERT2 was activated by tamoxifen (TM) administration at E7.5 and E8.5 in Gli1Cre-ERT2; Gata4fl/+ embryos. With TM administration at E7.5 and E8.5, 66.7% of Gli1Cre-ERT2; Gata4fl/+ embryos displayed DORV, while the littermate control Gata4fl/+ embryos displayed normal OFT alignment (Figure 2K and K’ vs. 2H, 2H’, 8/12 vs. 0/15, P=0.0002). We concluded that Gata4 is required in the SHF Hedgehog (Hh) signal-receiving progenitors for OFT alignment.
Gata6 was overexpressed in the SHF of the Gata4 heterozygotes
Gata4 and Gata6 double mutant embryos display PTA [40]. We examined Gata6 expression in Gata4 mutants. Gata6 was expressed in the heart, the OFT and strongly in the splanchnic mesoderm (Fig. 3A, arrow), but not neural crest cell derivatives (Fig. 3A, arrowhead) of the Gata4fl/+ embryo at E9.5. In Gata4 knockdown embryos specifically in the Hh-receiving cells, 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 Gata4fl/fl; Gli1Cre-ERT2/+ was further confirmed by the real-time PCR at the mRNA level (Fig. 3B). The Gata4 expression in the SHF of Gata4fl/fl; Gli1Cre-ERT2/+ mouse embryo was 2.7-fold that observed in control Gata4fl/+ embryos (P<0.05). Gata6 expression in the OFT of the Gata4fl/fl; Gli1Cre-ERT2/+ mouse embryo was 4.4-fold that of the littermate control (P<0.01). Our results suggested a negative association between the expression of Gata4 and Gata6 in the SHF and developing OFT.
Gata4 regulates cell proliferation in the OFT conal cushion
We wonder if Gata4 is required for proliferation during the OFT cushion development. Cell proliferation was examined by BrdU incorporation at E11.5. Gata4fl/+; Gli1Cre-ERT2/+ embryos demonstrated 17% fewer BrdU-positive SHF cells in the OFT conal cushion (Fig. 4C vs. 4A and 4E; P =0.0134), but not the OFT truncal cushion (Fig. 4D vs. 4B and 4F; P =0.1998), compared to the littermate Gata4fl/+embryos at E11.5. This result demonstrate that Gata4 is required for normal cell proliferation in OFT conal cushion development. We assessed cell death by TUNEL staining and observed no differences in either the conal or truncal cushion between Gata4fl/+; Gli1Cre-ERT2/+and the Gata4fl/+embryos (Fig. 4G - 4J). Together, these findings define a requirement for Gata4 in the proliferation but not in the survival of OFT conal cushion cells.
Rescue of SHF proliferation by disruption of Pten does not rescue DORV in Gata4 mutant embryos
Our previous study demonstrated that Gata4 regulates the cell cycle progression in posterior SHF cardiac precursors and that genetically targeted disruption of Pten rescued the proliferation defects in SHF of the Gata4 heterozygotes [57]. Hence, we examined whether proliferation rescue in SHF, by Pten downregulation (TMX at E7.5 and E8.5), could rescue DORV in Hh-receiving cell-specific Gata4 heterozygotes. We observed that decreased Pten dose caused only one DORV, but no ASD, in 20 embryos (Fig. 5A-C). Consistent with our previous report, although the ASD in Gli1Cre-ERT2/+;Gata4fl/+ embryos was rescued by Pten downregulation (Fig. 5C vs. 5B, 1/20 in Gli1Cre-ERT2/+;Gata4fl/+;Ptenfl/+ vs. 14/29 in Gli1Cre-ERT2/+;Gata4fl/+, P = 0.0013), Gli1Cre-ERT2/+;Gata4fl/+;Ptenfl/+ embryos still displayed DORV with an incidence rate unchanged from Gli1Cre-ERT2/+;Gata4fl/+ embryos (Fig. 5E vs. 5F, 12/29 vs. 6/20, Table 1, P = 0.5495). This data suggested to us that correction of the SHF proliferation defects was not able to rescue the OFT misalignment of the Gata4 mutant embryos.
Gata4 acts upstream of Hh signaling in OFT development
We have previously reported that Gata4 acts upstream of Hh-signaling for atrial septation [42]. The requirement of Gata4 in Hh-receiving cells for OFT alignment suggested that Gata4 and Hh signaling may interact genetically in the SHF for OFT development. We tested this hypothesis in the Gata4 and Smo compound heterozygotes (Gata4fl/+;Smofl/+;Gli1Cre-ERT2/+) versus littermate controls (Gata4fl/+; Gli1Cre-ERT2/+ or Smofl/+;Gli1Cre-ERT2/+). Consistent OFT defects were observed in compound Gata4; Smo embryos (Gata4fl/+;Smofl/+;Gli1Cre-ERT2/+) (5/9, Fig 6C - 6E) whereas no OFT defects were observed in Smofl/+;Gli1Cre-ERT2/+embryos (0/7, Fig 6B and B’; P= 0.0337). The total incidence of OFT defects occured in the Gata4fl/+;Smofl/+;Gli1Cre-ERT2/+ was not different than in the Gata4fl/+; Gli1Cre-ERT2/+ embryos (Fig 6C-E, 5/9 vs. 4/6, P= 0.7326). However, more severe range of OFT defects was observed in Gata4fl/+;Smofl/+;Gli1Cre-ERT2/+ embryos, including DORV (3 out of 5, Figs. 6C and C’), OA (1 out of 5, Figs. 6D and D’) and persistent truncus arteriosus (PTA) (1 out of 5, Figs. 6E and E’). PTA, caused by a combined defect of alignment and separation, was only observed in Gata4fl/+;Smofl/+;Gli1Cre-ERT2/+. This result suggest an interaction between Gata4 and Hh-signaling in OFT development.
We tested the hypothesis that Gata4 actis upstream of Hh-signaling for OFT development using a genetic epistasis study. We tested whether increased Hh-signaling via a constitutively activated Smo mutant, SmoM2 [58], could rescue the OFT misalignment in Gata4-heterozygotes. DORV was observed in 28.6% of littermate control Gli1Cre-ERT2/+;R26-SmoM2fl/+embryos (2/7) (Fig. 6G and G’) and 58.3% of littermate control Gli1Cre-ERT2/+;Gata4fl/+embryos at E14.5 (7/12) (Fig. 6H and H’). In contrast, none of Gata4fl/+;Gli1Cre-ERT2/+;R26-SmoM2fl/+ embryos showed DORV (Fig. 6I and I’), indicating significant rescue by R26-SmoM2fl/+, Gli1Cre-ERT2/+(Fig. 6I vs Fig. 6H, P = 0.0071, 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 [45]. We therefore hypothesized that Gata4 is required for the SHF Hh-receiving cells migration toward the developing OFT. We tested this hypothesis using genetic inducible fate mapping (GIFM) [59]. The Hh-receiving lineage cells were marked in R26Rfl/+;Gli1Cre-ERT2/+embryos by TM administration at E7.5 and E8.5 and β-gal expression was evaluated at E10.5 in Gata4 heterozygotes. 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. We have previously reported decreased number of Hh-receiving cells in the pSHF at E9.5 associated with developing defects of DMP in the Gata4fl/+;R26Rfl/+;Gli1Cre-ERT2/+embryos [57]. We observed that there were also significantly less Hh-receiving cells within the aSHF region (Fig. 7A vs. 7D and Fig. 7G, 334.0 ± 1.4 vs. 186.7 ± 4.9, P=0.009) of the Gata4fl/+;R26Rfl/+;Gli1Cre-ERT2/+embryos. The cells of Hh-receiving lineage were observed in the developing OFT at this stage. By counting the number of β-galactosidase-expressing cells in the proximal half (Fig. 7B vs. 7E and 7H, 49.7 ± 9.6 vs. 26.7 ± 6.7, P=0.097) and the distal half of the OFT (Fig.7C vs. 7F and 7I, 91.7 ± 9.2 vs. 57.0 ± 1.4, P=0.0362), we found that both of the regions of the Gata4 heterozygotes had less β-galactosidase-expressing cells than the littermate controls (Figs. 7E and 7F).
To examine if Gata4 haploinsufficency 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 expression: β-galactosidase-expressing cells, the total number of the SHF lineage cells within the proximal half and the distal half of the OFT were compared between the Mef2cAHF::Cre;Gata4fl/+; R24Rfl/+ and the Mef2cAHF::Cre;R24Rfl/+embryos at E10. The number of SHF lineage cells populating the proximal OFT of the Mef2cAHF::Cre;Gata4+/-; R24Rfl/+ embryos was significantly less than that those in control Mef2cAHF::Cre; R24Rfl/+ embryos (Fig. 7J vs. 7M); however, this decrement was not observed in the distal OFT (Fig. 7K vs. 7N). The distribution pattern of the SHF lineage was not different in the Mef2cAHF::Cre;Gata4+/-; R24Rfl/+ and the Mef2cAHF::Cre;R24Rfl/+embryos (Figs. 7L vs. 7O). AS a control, we observed fewer cells populating the developing dorsal mesocardium protrusion (DMP) in Mef2cAHF::Cre;Gata4+/-; R24Rfl/+(red arrow, Fig. 7L vs. 7O), consistent with our previous report that Gata4 is required in the SHF for the DMP [42]. These results demonstrated the requirement of Gata4 for the SHF lineage cells populating in the developing OFT.
Discussion
The requirement of Gata4 for OFT development has been reported in mice and human, and mouse Gata4 mutations cause DORV [22, 27, 40]. Here we demonstrate that Gata4 is required in the SHF Hh-receiving cells for OFT alignment in the SHF. 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 directly associate with the OFT misalignment; instead, the migration defects of the SHF cells is. And the migration defects were associated with disrupted Hh-signaling, because the OFT misalignment was rescued by over-activating of Hh-signaling. In addition, our data suggested 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 anterior SHF contributions for OFT development [5, 10-19, 60-63]. More recently, the contribution of posterior SHF cardiac progenitors to the OFT and the future subpulmonary myocardium has been reported, however, the mechanistic requirement for this contribution is not well understood [45, 64-66]. 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 than in the aSHF [57]. The decreased number of Mef2C-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. However, surprisingly, OFT defects were not observed in either aSHF-specific or pSHF-specific Gata4 happloinsufficiency. Instead, we found that OFT defects severity and incidence rate in embryos with Gata4 haploinsufficienc in Hh-receiving cells were identical to those in Gata4−/+ embryos. Because Hh-receiving cells are located throughout the SHF, these observations suggest Gata4 is required in both pSHF and aSHF progenitor cells for OFT alignment.
We provided evidence that Gata4 acts upstream of Hh-signaling in the SHF for OFT development. The Gata4−/+ embryos have combined phenotypes of ASD and DORV [57]. We previously reported the Gata4-Hh-signaling regulation in atrial septation and identified Gli1 as the direct target of GATA4 [42]. Here, our data of less percentile of BrdU+ cells in the conal cushion of the OFT at E11.5 of the Gata4fl/+; Gli1Cre-ERT2/+ embryos, suggesting a role of Gata4 in regulating the OFT cushion cell proliferation. In the posterior SHF, Gata4-Hh-signaling controls cell cycle progression and thereby the proliferation of the cardiac progenitors. Diminished Gata4-Hh signaling causes a failure of development of the DMP, the anlage of the atrial septum, resulting in ASDs [57]. The effect of this pathway on the cell cycle is balanced by Pten via transcriptional inhibition of Cyclin D4 and Cdk4 [20, 57], as DMP hypoplasia and SHF cell cycle defects are rescued by Pten knockdown [57]. In the current study, Pten knockdown was unable 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, and that Gata4 plays a distinct role in the anterior SHF.
Endodermal Hh signaling is required for the survival of the pharyngeal endoderm, which cell non-autonomously affects SHF survival and OFT lengthening [55]. In our study, increased apoptosis was not observed in the SHF of Gata4 heterozygote mutant embryos [57]. However, fate mapping of the SHF using either Mef2c::Cre or the Gli1Cre:ERT2 disclosed less SHF-derived cells in the distal OFT in Gata4 mutant embryos. Specifically, there was decreased number of SHF Hh-receiving cells throughout the migration route from the SHF into the OFT: from the dorsal mesocardium through the rostral splanchnic mesoderm, past the distal OFT to 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 [45], 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 implies correction of both the proliferation and the migration defects of the SHF cardiac progenitors, not proliferation defects only. Overall, here we provide cellular, molecular and genetic evidence that Gata4-Hh signaling hierarchy is required in OFT alignment, with specific regulation of both proliferation and migration of SHF progenitors.
Although important Gata4 transcriptional targets in the heart have been identified [20, 26, 44], Gata4-dependent molecular pathways required for OFT development have remained unknown. We previously identified Gli1 as a downstream target of Gata4 in the posterior SHF for atrial septation [42]. In the current study we further demonstrated that Gata4 regulated Hh-signaling via transcriptional regulation through Gli1 in the anterior SHF for cell migration and OFT alignment. In addition, we provide evidence that Gata6 expression is negatively regulated by Gata4 in the OFT. Enhanced Gata6 expression in Gata4 mutants might illustrate a compensatory feedback loop, given that Gata6 and Gata4 are redundant for cardiac myocyte differentiation [67, 68]. Gata4/Gata6 compound heterozygotes displayed persistnat truncus ateriosus (PTA), a severe OFT defect caused by combined alignment and OFT septation defects (40). Here we find that Gata4/Smo compound heterozygotes show a similar phenotype. Gata4 heterozygotes alone do not display PTA, which might be due to the partial recovery of GATA function from enhanced Gata6 expression. Together with previous study [40], these data suggest a threshold of Gata4, Gata6, and Hh signaling and that is required for OFT development. This suggests that GATA TFs may be essential for the quantitative regulation of Hh signaling, and that strongly diminished GATA function or diminished GATA and Hh signaling together may cause worse OFT defects through regulation of OFT Hh signaling. Future studies will focus on the quantitative relationship between GATA tone and Hh signaling tone and on the Gata4 dependent gene regulatory network (GRN) [69] for OFT development.
Materials and methods
Mouse lines
All mouse experiments were performed in a mixed B6/129/SvEv background. Gata4fl/+, Gli1CreERT2/+, Mef2cAHF::Cre, Tie2Cre/+, Smofl/+ mouse lines were kind gifts from Dr. Ivan Moskowitz lab (University of Chicago, Chicago). TnTCre/+ mouse line was from Dr. Yiping Chen lab (Tulane University, New Orleans). Nfat1cCre/+ mouse line was from Dr. Bin Zhou lab (Albert Einstein College of Medicine, Bronx, NY). The SmoM2fl/+, Osr1Cre-ERT2/+ and EIIacre/+mouse lines were purchased from the Jackson Laboratory. Mouse experiments were completed according to a protocol reviewed and approved by the Institutional Animal Care and Use Committee of the Texas A&M University and the University of North Dakota, in compliance with the USA Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Tamoxifen administration and X-gal staining
Tamoxifen (TM) -induced activation of CreERT2 was accomplished by oral gavage with two doses of 75 mg/kg TM at E7.5 and E8.5 [45, 46]. X-gal staining of embryos was performed as described [45].
BrdU incorporation and Immunohistochemistry Staining (IHC)
Standard procedures were used for histology and IHC. IHC was performed using the following antibodies: anti-Gata4 (Abcam), anti-Gata6 (Abcam). 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).
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 [47, 48]. 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™ 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 [49].
Acknowledgements
We would specifically like to acknowledge the support of Dr. Boon Chew for the study.