A cell-autonomous role of Cited2 in controlling myocardial and coronary vascular development.

AIMS: Myocardial development is dependent on concomitant growth of cardiomyocytes and a supporting vascular network. The coupling of myocardial and coronary vascular development is partly mediated by vascular endothelial growth factor (VEGFA) signalling and additional unknown mechanisms. We examined the cardiomyocyte specific role of the transcriptional co-activator Cited2 on myocardial microstructure and vessel growth, in relation to Vegfa expression. METHODS AND RESULTS: A cardiomyocyte-specific knockout of mouse Cited2 (Cited2(Nkx)) was analysed using magnetic resonance imaging and histology. Ventricular septal defects and significant compact layer thinning (P < 0.02 at right ventricular apex, P < 0.009 at the left ventricular apex in Cited2(Nkx) vs. controls, n = 11 vs. n = 7, respectively) were found. This was associated with a significant decrease in the number of capillaries to larger vessels (ratio 1.56 ± 0.56 vs. 3.25 ± 1.63, P = 2.7 × 10(-6) Cited2(Nkx) vs. controls, n = 11 vs. n = 7, respectively) concomitant with a 1.5-fold reduction in Vegfa expression (P < 0.02, Cited2(Nkx) vs. controls, n = 12 vs. n = 12, respectively). CITED2 was subsequently found at the Vegfa promoter in mouse embryonic hearts using chromatin immunoprecipitation, and moreover found to stimulate human VEGFA promoter activity in cooperation with TFAP2 transcription factors in transient transfection assays. There was no change in the myocardial expression of the left-right patterning gene Pitx2c, a previously known target of CITED2. CONCLUSIONS: This study delineates a novel cell-autonomous role of Cited2 in regulating VEGFA transcription and the development of myocardium and coronary vasculature in the mouse. We suggest that coupling of myocardial and coronary growth in the developing heart may occur in part through a Cited2→Vegfa pathway.

Cited2 controls left-right patterning and heart development through a Nodal-Pitx2c pathway.

Malformations of the septum, outflow tract and aortic arch are the most common congenital cardiovascular defects and occur in mice lacking Cited2, a transcriptional coactivator of TFAP2. Here we show that Cited2(-/-) mice also develop laterality defects, including right isomerism, abnormal cardiac looping and hyposplenia, which are suppressed on a mixed genetic background. Cited2(-/-) mice lack expression of the Nodal target genes Pitx2c, Nodal and Ebaf in the left lateral plate mesoderm, where they are required for establishing laterality and cardiovascular development. CITED2 and TFAP2 were detected at the Pitx2c promoter in embryonic hearts, and they activate Pitx2c transcription in transient transfection assays. We propose that an abnormal Nodal-Pitx2c pathway represents a unifying mechanism for the cardiovascular malformations observed in Cited2(-/-) mice, and that such malformations may be the sole manifestation of a laterality defect.

A novel role for transcription factor Lmo4 in thymus development through genetic interaction with Cited2.

Deletion of the transcriptional modulator Cited2 in the mouse results in embryonic lethality, cardiovascular malformations, adrenal agenesis, cranial ganglia fusion, exencephaly, and left-right patterning defects, all seen with a varying degree of penetrance. The phenotypic heterogeneity, observed on different genetic backgrounds, indicates the existence of both genetic and environmental modifiers. Mice lacking the LIM domain-containing protein Lmo4 share specific phenotypes with Cited2 null embryos, such as embryonic lethality, cranial ganglia fusion, and exencephaly. These shared phenotypes suggested that Lmo4 may be a potential genetic modifier of the Cited2 phenotype. Examination of Lmo4-deficient embryos revealed partially penetrant cardiovascular malformations and hypoplastic thymus. Examination of Lmo4;Cited2 compound mutants indicated that there is a genetic interaction between Cited2 and Lmo4 in control of thymus development. Our data suggest that this may occur, in part, through control of expression of a common target gene, Tbx1, which is necessary for normal thymus development.

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

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. beta-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 x 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.

Identification of cardiac malformations in mice lacking Ptdsr using a novel high-throughput magnetic resonance imaging technique.

BACKGROUND: Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo. RESULTS: We developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging (MRI). Using this approach we identified cardiac malformations in phosphatidylserine receptor (Ptdsr) deficient embryos. These included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. These results indicate that Ptdsr plays a key role in cardiac development. CONCLUSIONS: Our novel multi-embryo MRI technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. The technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases.