Simon D. Bamforth

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.

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

BackgroundCongenital 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.ResultsWe 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.ConclusionsOur 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.

Transcriptional Coactivator Cited2 Induces Bmi1 and Mel18 and Controls Fibroblast Proliferation via Ink4a/ARF

ABSTRACT Cited2 (CBP/p300 interacting transactivator with ED-rich tail 2) is required for embryonic development, coactivation of transcription factor AP-2, and inhibition of hypoxia-inducible factor 1 transactivation. Cited2 is induced by multiple growth factors and cytokines and oncogenically transforms cells. Here, we show that the proliferation of Cited2 −/− mouse embryonic fibroblasts ceases prematurely. This is associated with a reduction in growth fraction, senescent cellular morphology, and increased expression of the cell proliferation inhibitors p16INK4a, p19ARF, and p15INK4b. Deletion of INK4a/ARF (encoding p16INK4a and p19ARF) completely rescued the defective proliferation of Cited2−/− fibroblasts. However, the deletion of INK4a/ARF did not rescue the embryonic malformations observed in Cited2 −/− mice, indicating that INK4a/ARF-independent pathways are likely to be involved here. We found that Cited2 −/− fibroblasts had reduced expression of the polycomb-group genes Bmi1 and Mel18, which function as INK4a/ARF and Hox repressors. Complementation with CITED2-expressing retrovirus enhanced proliferation, induced Bmi1/Mel18 expression, and decreased INK4a/ARF expression. Bmi1- and Mel18-expressing retroviruses enhanced the proliferation of Cited2 −/− fibroblasts, indicating that they function downstream of Cited2. Our results provide genetic evidence that Cited2 controls the expression of INK4a/ARF and fibroblast proliferation, at least in part via the polycomb-group genes Bmi1 and Mel18.

Rapid identification and 3D reconstruction of complex cardiac malformations in transgenic mouse embryos using fast gradient echo sequence magnetic resonance imaging.

Developmental malformations of the heart in mouse embryos are commonly studied by histological sectioning. This is slow, labour intensive, and results in the loss of three-dimensional (3D) information. Magnetic resonance studies of embryos typically use spin-echo sequences, using prolonged acquisition times (>36 h) or perfusion with contrast agents to enhance resolution and contrast. This is technically difficult, and requires significant amounts of operator time. We imaged paraformaldehyde fixed embryos using a fast spoiled 3D gradient echo sequence with T(1)-weighting, in unattended overnight runs of less than 9 h. In wild-type embryos, we visualised normal cardiac structures, including cardiac chambers, the ventricular septum, primary and secondary atrial septa, valves, superior and inferior vena cava, aorta, pulmonary artery, and ductus arteriosus. In embryos lacking Cited2 (a transcriptional co-activator required for normal heart development), we identified cardiac malformations including atrial and ventricular septal defects, cono-truncal defects, and aortic arch malformations. We generated 3D reconstructions of normal and mutant hearts using contour identification and surface rendering computer software. The malformations were confirmed by histological sectioning. Our data indicate that fast gradient echo sequence magnetic resonance imaging can be used to rapidly and accurately identify complex cardiovascular malformations in transgenic and mutant mouse embryos.

Normal and abnormal development of the intrapericardial arterial trunks in humans and mice

AIMS The definitive cardiac outflow channels have three components: the intrapericardial arterial trunks; the arterial roots with valves; and the ventricular outflow tracts (OFTs). We studied the normal and abnormal development of the most distal of these, the arterial trunks, comparing findings in mice and humans. METHODS AND RESULTS Using lineage tracing and three-dimensional visualization by episcopic reconstruction and scanning electron microscopy, we studied embryonic day 9.5-12.5 mouse hearts, clarifying the development of the OFTs distal to the primordia of the arterial valves. We characterize a transient aortopulmonary (AP) foramen, located between the leading edge of a protrusion from the dorsal wall of the aortic sac and the distal margins of the two outflow cushions. The foramen is closed by fusion of the protrusion, with its cap of neural crest cells (NCCs), with the NCC-filled cushions; the resulting structure then functioning transiently as an AP septum. Only subsequent to this closure is it possible to recognize, more proximally, the previously described AP septal complex. The adjacent walls of the intrapericardial trunks are derived from the protrusion and distal parts of the outflow cushions, whereas the lateral walls are formed from intrapericardial extensions of the pharyngeal mesenchyme derived from the second heart field. CONCLUSIONS We provide, for the first time, objective evidence of the mechanisms of closure of an AP foramen that exists distally between the lumens of the developing intrapericardial arterial trunks. Our findings provide insights into the formation of AP windows and the variants of common arterial trunk.

High-resolution imaging of normal anatomy, and neural and adrenal malformations in mouse embryos using magnetic resonance microscopy

An efficient investigation of the effects of genetic or environmental manipulation on mouse development relies on the rapid and accurate screening of a substantial number of embryos for congenital malformations. Here we demonstrate that it is possible to examine normal organ development and identify malformations in mouse embryos by magnetic resonance microscopy in a substantially shorter time than by conventional histology. We imaged embryos in overnight runs of under 9 h, with an operator time of less than 1 h. In normal embryos we visualized the brain, spinal cord, ganglia, eyes, inner ear, pituitary, thyroid, thymus, trachea, bronchi, lungs, heart, kidneys, gonads, adrenals, oesophagus, stomach, intestines, spleen, liver and pancreas. Examination of the brain in embryos lacking the transcriptional coactivator Cited2 showed cerebellar and midbrain roof agenesis, in addition to exencephaly. In these embryos we were also able to detect agenesis of the adrenal gland. We confirmed all malformations by histological sectioning. Thus magnetic resonance microscopy can be used to rapidly identify developmental and organ malformations in mutant mouse embryos generated by transgenic techniques, in high‐throughput mutagenesis screens, or in screens to identify teratogenic compounds and environmental factors contributing to developmental malformations.

TGFβ signaling and congenital heart disease: Insights from mouse studies

Transforming growth factor β (TGFβ) regulates one of the major signaling pathways that control tissue morphogenesis. In vitro experiments using heart explants indicated the importance of this signaling pathway for the generation of cushion mesenchymal cells, which ultimately contribute to the valves and septa of the mature heart. Recent advances in mouse genetics have enabled in vivo investigation into the roles of individual ligands, receptors, and coreceptors of this pathway, including investigation of the tissue specificity of these roles in heart development. This work has revealed that (1) cushion mesenchyme can form in the absence of TGFβ signaling, although mesenchymal cell numbers may be misregulated; (2) TGFβ signaling is essential for correct remodeling of the cushions, particularly those of the outflow tract; (3) TGFβ signaling also has a role in ensuring accurate remodeling of the pharyngeal arch arteries to form the mature aortic arch; and (4) mesenchymal cells derived from the epicardium require TGFβ signaling to promote their differentiation to vascular smooth muscle cells to support the coronary arteries. In addition, a mouse genetics approach has also been used to investigate the disease pathogenesis of Loeys-Dietz syndrome, a familial autosomal dominant human disorder characterized by a dilated aortic root, and associated with mutations in the two TGFβ signaling receptor genes, TGFBR1 and TGFBR2. Further important insights are likely as this exciting work progresses.

Clarification of the identity of the mammalian fifth pharyngeal arch artery

The remodeling of the pharyngeal arch arteries is a complex process that occurs across vertebrates, although the specific number of arteries varies across species, with six in fish, but only five in birds and mammals, although they are numbered one through four, and six. The existence of a fifth arch artery in mammals has been debated for more than a century. Although some have doubted, and continue to doubt, its existence, several cardiovascular malformations can be explained only on the basis of its presence. We have analyzed the developing pharyngeal arch arteries in mouse and human embryos, using high‐resolution episcopic microscopy. We have then created three‐dimensional models, allowing us to identify any structures that would satisfy the descriptions of fifth arch arteries. This detailed examination revealed collateral channels connecting the fourth and sixth pharyngeal arch arteries in approximately half of the mouse embryos examined. Such collateral channels were seen in only one human embryo of eight examined by high‐resolution episcopic microscopy, although we had previously identified such collateral channels using wax plate reconstruction. An extra vessel, occupying a discrete component of the pharyngeal mesenchyme, and therefore resembling a true fifth pharyngeal arch artery, was observed in one Carnegie Stage 14 human embryo. The pharyngeal mesenchyme in the human, therefore, can contain a fifth arch, with a contained artery, albeit transiently. Persistence of this structure, and the observed collateral channels, provides mechanisms to explain the congenital cardiovascular malformations described as persistent fifth aortic arch, and double‐barreled aorta. Clin. Anat. 2013.

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.

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 (Cited2Nkx) 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 Cited2Nkx 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 Cited2Nkx vs. controls, n= 11 vs. n= 7, respectively) concomitant with a 1.5-fold reduction in Vegfa expression (P< 0.02, Cited2Nkx 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.