Epiblastic Cited2 deficiency results in cardiac phenotypic heterogeneity and provides a mechanism for haploinsufficiency

Abstract Aims Deletion of the transcription factor Cited2 causes penetrant and phenotypically heterogenous cardiovascular and laterality defects and adrenal agenesis. Heterozygous human CITED2 mutation is associated with congenital heart disease, suggesting haploinsufficiency. Cited2 functions partly via a Nodal→Pitx2c pathway controlling left–right patterning. In this present study we investigated the primary site of Cited2 function and mechanisms of haploinsufficiency. Methods and results A Cited2 conditional allele enabled its deletion in particular cell lineages in mouse development. A lacZ reporter cassette allowed indication of deletion. Congenic Cited2 heterozygous mice were used to investigate haploinsufficiency. Embryos were examined by magnetic resonance imaging, by sectioning and by quantitative real-time polymerase chain reaction (qRT-PCR). Epiblast-specific deletion of Cited2 using Sox2Cre recapitulated penetrant and phenotypically heterogenous cardiovascular and laterality defects. Neural crest-specific deletion using Wnt1Cre affected cranial ganglia but not cardiac development. Mesodermal deletion with Mesp1Cre resulted in low penetrance of septal defect. Mesodermal deletion with T-Cre resulted in adrenal agenesis, but infrequent cardiac septal and laterality defects. β-Galatactosidase staining and qRT-PCR demonstrated the efficiency and location of Cited2 deletion. Murine Cited2 heterozygosity is itself associated with cardiac malformation, with three of 45 embryos showing ventricular septal defect. Cited2 gene expression in E13.5 hearts was reduced 2.13-fold in Cited2+/− compared with wild-type (P = 2.62 × 10−6). The Cited2 target gene Pitx2c was reduced 1.5-fold in Cited2+/− (P = 0.038) hearts compared with wild-type, and reduced 4.9-fold in Cited2−/− hearts (P = 0.00031). Pitx2c levels were reduced two-fold (P = 0.009) in Cited2+/− embryos, in comparison with wild-type. Cited2 and Pitx2c expression were strongly correlated in wild-type and Cited2+/− hearts (Pearson rank correlation = 0.68, P = 0.0009). Cited2 expression was reduced 7474-fold in Sox2Cre deleted hearts compared with controls (P = 0.00017) and Pitx2c was reduced 3.1-fold (P = 0.013). Deletion of Cited2 with Mesp1Cre resulted in a 130-fold reduction in cardiac Cited2 expression compared with control (P = 0.0002), but Pitx2c expression was not affected. Conclusion These results indicate that phenotypically heterogenous and penetrant cardiac malformations in Cited2 deficiency arise from a primary requirement in epiblast derivatives for left–right patterning, with a secondary cell-autonomous role in the mesoderm. Cardiac malformation associated with Cited2 haploinsufficiency may occur by reducing expression of key Cited2 targets such as Pitx2c.


Introduction
The transcription factor CITED2 binds the histone acetyltransferase CREBBP/EP300 with high affinity, 1 acting as a co-activator for transcription factors, such as TFAP2, LHX2, PPARA, and SMAD2/3. [2][3][4][5] It also acts as a repressor of hypoxia-activated transcription. 1 Genetic evidence indicates that Cited2 is essential for cardiac, adrenal, neural, and placental development, and also for embryonic leftright patterning. [6][7][8] The cardiac malformations in mice lacking Cited2 are highly penetrant and phenotypically heterogeneic, and include atrial, atrioventricular, and ventricular septal defects (ASD, AVSD, VSD), outflow tract defects [double outlet right ventricle (DORV), common arterial trunk (CAT), tetralogy of Fallot (TOF), transposition of great arteries (TGA)], and interrupted and right-sided aortic arch. 6,9 Mutation in Cited2 also results in adrenal agenesis, fusion of cranial ganglia, abnormal cardiac neural crest migration, and exencephaly. 6 In addition, Cited2 2/2 mice show left-right patterning defects characterized by right atrial and pulmonary isomerism, and abnormal ventricular topology at a lower penetrance. 7,8 Cardiovascular left-right patterning is created, in part, by the left-determining Nodal!Pitx2c pathway (reviewed by Hamada et al. 10 and Ramsdell 11 ). Genetic evidence shows that Cited2 is necessary for expression of Pitx2c, Nodal, and Lefty2 in the left-lateral plate mesoderm and of Lefty1 in the prospective floor plate, and is present at the promoter of the endogenous Pitx2c gene during development. 7,8 Thus, Cited2 is necessary for Nodal-activated gene transcription. 7 The mechanism for the cardiovascular phenotypic heterogeneity and the partial penetrance of left-right patterning defect, which is observed even on a congenic Cited2-deficient background, 7 is not clear. Importantly, loss-of-function mutations in CITED2 have been found in patients with phenotypically variable congenital heart disease. 12 These mutations are heterozygous, suggesting haploinsufficiency, but the mechanism for this is unclear.
Cited2 is expressed before gastrulation in the visceral endoderm. 13 Following gastrulation it is present in the newly forming mesoderm, developing blood islands, cardiac crescent, presomitic and splanchnic mesoderm, cranial neuroectoderm, and migrating neural crest before becoming ubiquitously expressed by embryonic day (E)8.5. 13 In the heart, it is highly expressed in the AV endocardial cushions. 8 One possible mechanism for cardiac phenotypic heterogeneity in Cited2 deficiency is that not only is it required for the earlier process of left-right patterning but also independently for the later processes of atrioventricular septation, outflow tract, and aortic arch development. 8 Abnormal patterning of aortic arches and CAT observed in Cited2 deficiency could, for instance, arise from defective neural crest development. 6 An alternative possibility is that an abnormality in the earlier developmental process of left-right patterning may secondarily affect the subsequent processes of atrioventricular septal, outflow tract, or aortic arch development. 7 These different mechanisms were investigated with a conditional knockout approach. We also investigated the mechanism of haploinsufficiency using quantitative analysis of Cited2 and its target gene Pitx2c. We show that the phenotypic heterogeneity in Cited2 deficiency is because of a primary requirement in epiblast derivatives with a secondary cell-autonomous role in the mesoderm. Also, Cited2 haploinsufficiency may cause cardiac malformation by reducing expression of key Cited2 targets such as Pitx2c.

Molecular biology
Standard molecular biology procedures were used for plasmid constructions. 14

Generation of Cited2 flox/flox mice
The targeting vector strategy and generation of correctly targeted embryonic stem cells with a conditional Cited2 allele (Cited2 flox or Cited2 f ) is shown in Supplementary material online, Figure S1. Methods are also provided in detail in the Supplementary data. The allele was designed such that, after successful Cre recombination, a lacZ expression cassette came under the control of the endogenous Cited2 promoter. This allowed indication of efficient recombination by b-galactosidase staining.

Analysis of embryos
Magnetic resonance imaging (MRI) was performed on a horizontal 9.4 T/21 cm VNMRS Direct Drive TM MR system (Varian Inc., Palo Alto, CA, USA) essentially as described previously 20 on embryos at E15.5. Whole mount in situ immunostaining was performed on E9.5 embryos using anti-neurofilament antibody (clone 2H3, Developmental Studies Hybridoma Bank, University of Iowa, IA, USA). 21 X-Gal and nuclear fast red (Sigma, St. Louis, MO, USA) staining were performed according to standard procedures. 14,16

Quantitative real time-polymerase chain reaction
Embryo hearts (E13.5) were dissected out initially in DEPC treated phosphate-buffered saline (PBS), and then finely dissected in RNAlater (Ambion, Austin, TX, USA) to remove mediastinal tissue. Embryos at E8.5 were dissected free from the yolk sac in cold DEPC-PBS and flash-frozen in liquid nitrogen. RNA was isolated using a Nucleospin RNA purification kit (Clontech, Palo Alto, CA, USA), and an Archive cDNA kit (Ambion) used to synthesize complementary DNA. Quantitative RT-PCR (qRT-PCR) reactions were carried out using a Bio-Rad I-Cycler (Bio-Rad, CA, USA) and preoptimized TaqManw primer-probe sets from Applied Biosytems (Mus musculus assays Mm00516121_m1 (for NM_010828 Cited2), Cited2 deficiency and cardiac phenotypic heterogeneity Mm00440826_m1 (for NM_001042502 Pitx2c), and eukaryotic 18S rRNA). Expression levels were normalized to 18S rRNA using the R 0 method of analysis. 22,23 Multiplex reactions were performed with 18S rRNA and the TaqMan primer-probe set for the gene of interest. Data were normalized to the level obtained for 18S RNA in each case. All reactions were performed in triplicates and, as a quality control, only samples for which the threshold cycle (Ct) replicate values were within 1 Ct value of each other were used for subsequent analysis. 24 Mean values for each reaction triplicate were analysed.

Statistical analyses
We used the chi-squared test for deviation from expected Mendelian ratios (http://www.kursus.kvl.dk/shares/vetgen/_Popgen/genetik/ applets/ki.htm), and calculated probability of a type I error using the CHIDIST function in Microsoft Excel (Redmond, WA, USA). For analysis of the qRT-PCR data, we used a two-tailed, two-sample heteroscedastic t-test assuming unequal variance.

Deletion of Cited2 in the epiblast
To determine the role of Cited2 in the epiblast it was deleted using Sox2Cre which induces recombination in all epiblast cells by E6.5. 15 . Genotyping of mice at weaning showed a complete absence of Cited2 2/flox ;Sox2Cre embryos, but genotyping at E15.5 showed that these embryos were present in the expected Mendelian ratio (Supplementary material online, Table S1). This indicated that Cited2 2/flox ;Sox2Cre embryos die either in late gestation or perinatally. Two of 10 Cited2 2/flox ;Sox2Cre embryos at E15.5 had visible exencephaly (not shown). Analysis of Cited2 2/flox ;Sox2Cre embryos at E15.5 using MRI showed that they frequently had a classical cardiac laterality defect, i.e. right-atrial isomerism, associated with rightpulmonary isomerism (six of 10 embryos) ( Figure 1; see also see Supplementary material online, Table S2), indicating a defect in embryonic left-right patterning. VSDs were present in all embryos (see Supplementary material online, Table S2). The embryos invariably had adrenal agenesis (see Supplementary material online, Figure S2). No defects were seen in the 11 littermate control Cited2 þ/flox ;Sox2Cre or in eight Cited2 2/flox embryos.

Deletion of Cited2 in the neural crest
To determine the requirement for Cited2 in the neural crest it was deleted using Wnt1Cre, which drives expression of Cre-recombinase in all neural crest-derived tissues, including the cardiac neural crest, from E9. 16,25,26 There was a significant reduction in the numbers of Cited2 2/flox ;Wnt1Cre mice in comparison with Cited2 þ/flox ;Wnt1Cre mice at weaning [21 of 61 vs. 40 of 61, respectively (x 2 ¼ 5.9; P , 0.015). There was no observed reduction when genotyping was performed between E9.5 and E18.5. Examination of embryos at E15.5-E18.5 (n ¼ 16) using MRI revealed none with heart or great vessel defects, but one embryo had exencephaly ( Figure 2B). Exencephaly was not observed in Cited2 þ/flox ;Wnt1Cre embryos (n ¼ 16). Examination of cranial ganglia by anti-neurofilament staining in Cited2 2/ flox ;Wnt1Cre embryos showed that 11 of 14 sets had fusions of ganglia IX and X ( Figure 2E and F) and hypomorphic ganglia V and VII/VIII. In contrast, two of 28 sets of ganglia from Cited2 þ/flox ;Wnt1Cre embryos had fusions of ganglia IX and X (x 2 ¼ 22.28; P , 0.001). In both Cited2 2/flox ;Wnt1Cre and in Cited2 þ/flox ;Wnt1Cre embryos, we observed extensive lacZ staining in the cranial mesenchyme, and in the first and second pharyngeal arches ( Figure 2G and H ). Staining was also observed in the cardiac neural crest (the third and fourth/sixth pharyngeal arches). The staining pattern corresponded to that described with Wnt1Cre-driven recombination in R26R mice that express lacZ conditional to Cre-mediated recombination. 16

Deletion of Cited2 in the mesoderm
The role of Cited2 in the cranial, cardiac, and extraembryonic mesoderm was examined by deletion using crosses with Mesp1Cre mice, which deletes in these tissues by E7.5. 17,27 As this Cre allele is a 'knockin' and disrupts the endogenous gene, we analysed genotypic ratios of offspring born by crossing these lines with Cited2 þ/2 mice to determine if there was a genetic interaction. The number of Cited2 þ/2 ; Mesp1Cre þ/KI mice at weaning was similar to Cited2 þ/þ ; Mesp1Cre þ/KI mice indicating no significant genetic interaction (Supplementary material online, Table S3). Isolated septal defects (but no outflow or aortic arch defects) were seen in three of 18 Cited2 2/flox ;Mesp1Cre embryos and no defects were seen in the seven Cited2 þ/flox ;Mesp1Cre or nine Cited2 2/flox littermate control embryos studied (Supplementary material online, Figure S3). The deletion of Cited2 using a TCre driver was then investigated. This would be expected to delete extensively in the lateralplate, intermediate, and paraxial mesoderm and the hindgut endoderm, with earliest deletion being detected by E7.5 18 Table S4). Deletion of exon 2 by Cre-recombinase brings the lacZ cassette under control of the endogenous Cited2 promoter, so analysis of lacZ expression allowed the confirmation of Cited2 flox recombination in the embryonic lineages where both the Cre driver and Cited2 are expressed. In the heart, we observed staining resembling the Sox2Cre pattern using Mesp1Cre, consistent with effective recombination in the cardiogenic mesoderm. 17,27 Reduced lacZ staining was observed in the outer curvature of the left ventricle suggesting that Cited2 is not highly expressed here (Figure 4). With TCre we observed complete recombination in the common atrial chamber, but partial recombination in the atrioventricular canal, primitive ventricle, bulbus cordis, and outflow tract. The lateral plate mesoderm is thought to play a key role in establishing left-right patterning (reviewed in Hamada et al. 10 ). We therefore examined the expression of lacZ in structures derived from the lateral plate mesoderm: these include the mesenchyme of the body wall (somatopleure) and of the viscera (splanchnopleure). 28 In Cited2 þ/flox ;Sox2Cre embryos, strong lacZ expression was observed in the splanchnopleure, and weaker expression in the somatopleure. With TCre and Mesp1Cre, lacZ expression was detected chiefly in the posterior splanchnopleure surrounding the midgut. No lacZ staining was detected in the somatopleure. We also examined lacZ expression at earlier developmental stages (Supplementary material online, Figure S4). At earlier stages, in Cited2 þ/flox ;Sox2Cre embryos at E7.5, lacZ expression was present in all epiblast-derived tissues, including the extraembryonic mesoderm. In Cited2 þ/ flox ;TCre embryos, we observed lacZ staining in the extraembryonic mesoderm, amnion, and blood islands, and also to some extent in the nascent embryonic mesoderm. This became more extensive in the primitive streak and migrating embryonic mesoderm at slightly later stages. Examination of head-fold stage Cited2 þ/flox ;Mesp1Cre embryos showed extensive recombination in the cardiac crescent (Supplementary material online, Figure S4).

Quantitative real-time polymerase chain reaction
To verify efficient deletion of Cited2 in the Sox2Cre and Mesp1Cre conditional knockouts at the mRNA level, and to determine the effect on the Cited2 target gene Pitx2c, qRT-PCR was performed. Cited2 expression was reduced 7474-fold (P ¼ 0.00017) in Cited2 2/flox ;Sox2Cre hearts compared with littermate controls ( Figure 5A). There was also a 3.1-fold reduction in Pitx2c expression (P ¼ 0.013; Figure 5B). Cited2 expression was reduced 130-fold in Cited2 2/flox ;Mesp1Cre hearts compared with littermate controls ( Figure 5C). However, no reduction in Pitx2c expression was seen in these hearts ( Figure 5D).

Cited2 haploinsufficiency in the mouse
The mutations found in human congenital heart disease patients 12 suggested that Cited2 haploinsufficiency may occur. We tested for this in the mouse by examining the genotypic ratios of Cited2 þ/2 intercrosses where we obtained 500 wild-type and 782 Cited2 þ/2 pups (expected 855). The 8.5% reduction in Cited2 þ/2 pups was statistically significant (x 2 ¼ 18.5; P , 0.0001). A similar reduction was not observed however when the genotypic ratios of E13.5 or E15.5 embryos were examined (Supplementary material online, Table S5).
To determine a mechanism for reduced survival, we examined Cited2 þ/2 embryos at E15.5 by MRI ( Figure 6). This was followed by histological sectioning of embryos that had either oedema or a cardiac abnormality visible on MRI. We found that three of 45 Cited2 þ/2 embryos had a clearly identifiable VSD, and one presented with DORV with hypoplastic pulmonary trunk ( Figure 6). These defects were not seen in the wild-type littermate controls. Thus, heterozygosity for a Cited2 null allele is associated, at low penetrance, with cardiac malformation in the mouse, implying haploinsufficiency.

Quantitative real-time-polymerase chain reaction analysis of Pitx2c and Cited2 expression
To determine if haploinsufficiency in Cited2-deficient hearts is linked to abnormality in Pitx2c expression, we examined gene expression using qRT-PCR. Embryos and hearts on a C57BL/6J background, generated by backcrossing for 14 generations, were used to minimize the effects of genetic background. Hearts at E13.5 were used as this is a key timepoint in cardiac septation, and embryos at E8.5 were selected as Pitx2c is expressed asymmetrically in the left lateral plate mesoderm at this stage. In E13.5 hearts, Cited2 was reduced 2.13-fold in Cited2 þ/2 compared with wild-type (P ¼ 2.62 Â 10 26 ) ( Figure 7A). We found that the Cited2 mRNA in Cited2 2/2 hearts was either undetectable or, if detectable, had values ranging between 1.45 Â 10 212 and 3.47 Â 10 211 relative to 18S (Figure 7C), likely to represent the limits of the assay. The Cited2 target gene Pitx2c was reduced 1.5-fold in Cited2 þ/2 (P ¼ 0.038) hearts and 4.7-fold (P ¼ 0.00031) in Cited2 2/2 hearts compared with wild-type controls ( Figure 7B and D). Cited2 and Pitx2c mRNA were strongly correlated in wild-type and Cited2 þ/2 hearts (Pearson rank correlation ¼ 0.68, P ¼  Cited2 deficiency and cardiac phenotypic heterogeneity 0.0009; Figure 7F). In the E8.5 embryos, we found a two-fold reduction in Pitx2c levels in Cited2 þ/2 compared with wildtype (P ¼ 0.009; Figure 7E).

Discussion
In these experiments, we used a conditional knockout approach to understand how Cited2 deficiency results in cardiac phenotypic heterogeneity. To define the role of Cited2 in the epiblast, we deleted it with Sox2Cre. 15 This resulted in 100% penetrance of cardiovascular malformations and adrenal agenesis. The most common cardiac malformation was a VSD, which was present in all embryos, and was associated with DORV in some embryos. In addition we observed left-right patterning, atrial septal, outflow tract, and aortic arch defects. These observations are consistent with, and fully recapitulate, those previously reported with the global deletion of Cited2. [6][7][8][9]29 They indicate that Cited2 is essential in the epiblast or its derivatives for normal embryonic left-right patterning, and for cardiac and adrenal development. The complete penetrance of the defects, and the widespread expression of lacZ in embryos containing a Cited2 flox allele and the Cre transgene also indicate that efficient recombination occurs in response to Cre expression. This was further validated by the large reduction seen in Cited2 mRNA levels in the hearts of Cited2 2/flox ;Sox2Cre embryos. We observed reduced lacZ staining in the outer curvature of the left ventricle following conditional deletion of Cited2 with Sox2Cre (as well as with Mesp1Cre and TCre). This is consistent with the published data, 8 and indicates that Cited2 is expressed at a lower level in this region of the heart. To define the role of Cited2 in the neural crest, we deleted it with Wnt1Cre. 16,30 This resulted in cranial ganglia fusions and exencephaly, but did not affect cardiac septal, outflow tract, aortic arch or adrenal development, or left-right patterning. These results indicate that although Cited2 does have a cell autonomous role in the neural crest, as evidenced by fusion of the cranial ganglia, it is not required here for outflow tract septation or aortic arch patterning.
To determine the requirement of Cited2 in the cranial, cardiac, and extraembryonic mesoderm it was deleted with Mesp1Cre, which results in extensive recombination in these tissues by E7.5. 17,27 Subsequently, by E9.5, recombination is observed extensively in myocardium and endocardium including the mesenchyme of the cono-truncal and atrioventricular cushions, in the epicardium, and in the pharyngeal arch mesoderm and vascular endothelium. 17,27,31 Deletion of Cited2 using Mesp1Cre resulted in low penetrance of septal defect, and no outflow tract, aortic arch, or laterality defects. These results indicate that Cited2 function in the mesoderm does not explain the phenotypically heterogeneic cardiac malformations observed in the epiblast-specific deletion. Efficient recombination at the Cited2 locus in response to Cre expression as demonstrated by lacZ staining and qRT-PCR indicated that lack of a phenotypically heterogeneic phenotype is unlikely to be owing to inefficient recombination.
As Cited2 is expressed in the lateral-plate mesoderm, 8 and as its deficiency also leads to loss of Nodal-activated transcripts such as a Nodal, Lefty2, and Pitx2c in the left-lateral plate mesoderm, 7,8 we investigated its role here by deletion using TCre. 18 This resulted in infrequent septal and laterality defect. Deletion of Cited2 induced by TCre was predominantly in the posterior rather than the anterior extent of the lateral plate mesoderm, making this one possible mechanism for the infrequency of left-right patterning defect observed with a TCre-induced deletion. The lacZ staining pattern seen in Cited2 þ/flox ;TCre embryos was comparable with that seen with TCre Â R26R lacZ embryos. 18 Moreover, in the heart the staining pattern was highly reminiscent of that observed for Isl1 32 suggesting that TCre may also drive recombination in second heart field-derived regions of the heart. These results indicate that the penetrant and phenotypically heterogeneic cardiac malformations in Cited2 deficiency arise from a primary requirement in epiblast derivatives for left-right Figure 6 Oedema and cardiac malformations in Cited2 þ/2 embryos. Magnetic resonance imaging of wild-type and littermate Cited2 þ/2 embryos at E15.5. Sagittal sections of wild-type (A) and Cited2 þ/2 (B) embryos. Severe oedema (asterisks) is seen in the Cited2 þ/2 embryo. (C ) Transverse section of wild-type and Cited2 þ/2 embryos. The Cited2 þ/2 embryo has a ventricular septal defect (VSD) (D). (E, F) Three-dimensional reconstruction of the heart in the wild-type and Cited2 þ/2 embryos shown in the previous panel. The aorta (Ao) and pulmonary artery (PA) arise from the left ventricle and right ventricle (RV), respectively, in the wild-type embryo, but in the Cited2 þ/2 embryo they arise from the RV creating a double-outlet right ventricle (DORV). VSD is indicated. Scale bars, 500 mm. Axes are as shown in Figure 1. patterning with a secondary cell-autonomous role in the mesoderm for septation. A limitation of our studies, however, is that the role of Cited2 in the endoderm-which has a major role in cardiac induction 33 -could not be established. We are currently limited here by the lack of a suitable Cre driver that expresses in the endoderm at the appropriate time-point.
Our data also show that Cited2 haploinsufficiency occurs in the mouse and is associated with cardiac malformation. Although 8.5% of Cited2 heterozygous pups are lost by weaning, consistent with the numbers of cardiac malformations observed, no deviation from expected numbers was seen during development. The mechanistic link between cardiac malformations and Cited2 haploinsufficiency is unclear, and to address this, we quantitatively examined the expression of Cited2 and its target gene Pitx2c in embryonic hearts and E8.5 embryos. Using qRT-PCR we showed that Cited2 expression is significantly reduced in Cited2 heterozygous embryonic hearts. We also showed that this was accompanied by a significant reduction in Pitx2c expression, and that the two were strongly correlated. We also observed a significant reduction in Pitx2c levels in E8.5 Cited2 þ/2 embryos. As the dose of Pitx2c is crucial for normal left-right patterning and cardiac development, 34,35 we suggest that Pitx2c deficiency observed in Cited2 heterozygous hearts and embryos could explain, at least in part, the observed cardiac malformations.
In conclusion, these results indicate that phenotypically heterogeneic and penetrant cardiac malformations in Cited2 deficiency arise from a primary requirement in epiblast derivatives for left-right patterning with a secondary cell-autonomous role in the mesoderm for septation. Cardiac malformation associated with Cited2 haploinsufficiency may occur by reducing the expression of key Cited2 targets, such as Pitx2c from E8.5. Importantly, cardiovascular phenotypic heterogeneity is observed, for instance, with mutation in NKX2-5, 36-38 CFC1 39,40 or ZIC3. [41][42][43] Consistent with our proposed model, these genes, like Cited2, are also known to control or modulate left-right patterning pathways. 42,[44][45][46][47] Our results support the emerging idea 7,11 that genes controlling early left-right patterning are candidates for diverse forms of human congenital heart disease, even in the absence of a classical laterality defect such as isomerism. Figure 7 Cited2 and Pitx2c mRNA levels in E13.5 embryo hearts and E8.5 embryos. Sample mean and SEM are indicated. (A) A 2.1-fold reduction in Cited2 mRNA levels was seen in Cited2 þ/2 hearts compared with wild-type. (B) Pitx2c levels were reduced 1.5-fold in Cited2 þ/2 hearts compared with wild-type. (C) A 92 000-fold reduction in Cited2 mRNA is seen in the Cited2 2/2 hearts compared with wild-type. (D) Pitx2c mRNA levels were reduced 4.7-fold in the Cited2 2/2 hearts compared with wild-type. (E) Pitx2c levels in wild-type and Cited2 þ/2 E8.5 embryos. A two-fold reduction in Pitx2c mRNA is seen in the Cited2 þ/2 embryos. (F) Pearson correlation between Cited2 and Pitx2c E13.5 heart mRNA levels with line-of-best-fit and 95% confidence intervals (dotted lines) shown. Filled circles represent data from Cited2 þ/2 hearts and diamonds those from wild-type. Cited2 and Pitx2c mRNA levels were strongly correlated in wild-type and Cited2 þ/2 hearts.

Supplementary material
Supplementary material is available at Cardiovascular Research online.