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Deficiency of the transcription factor Cited2 in mice results in cardiac malformation, adrenal agenesis, neural tube, placental defects and partially penetrant cardiopulmonary laterality defects resulting from an abnormal Nodal->Pitx2c pathway. Here we show that a maternal high-fat diet more than doubles the penetrance of laterality defects and, surprisingly, induces palatal clefting in Cited2-deficient embryos. Both maternal diet and Cited2 deletion reduce embryo weight and kidney and thymus volume. Expression profiling identified 40 embryonic transcripts including Pitx2 that were significantly affected by embryonic genotype-maternal diet interaction. We show that a high-fat diet reduces Pitx2c levels >2-fold in Cited2-deficient embryos. Taken together, these results define a novel interaction between maternal high-fat diet and embryonic Cited2 deficiency that affects Pitx2c expression and results in abnormal laterality. They suggest that appropriate modifications of maternal diet may prevent such defects in humans.

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., ((c) 2010 Wiley-Liss, Inc.)

Microscopic MRI (microMRI) is an emerging technique for high-throughput phenotyping of transgenic mouse embryos, and is capable of visualising abnormalities in cardiac development. To identify cardiac defects in embryos, we have optimised embryo preparation and MR acquisition parameters to maximise image quality and assess the phenotypic changes in chromodomain helicase DNA-binding protein 7 (Chd7) transgenic mice. microMRI methods rely on tissue penetration with a gadolinium chelate contrast agent to reduce tissue T(1), thus improving signal-to-noise ratio (SNR) in rapid gradient echo sequences. We investigated 15.5 days post coitum (dpc) wild-type CD-1 embryos fixed in gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) solutions for either 3 days (2 and 4 mM) or 2 weeks (2, 4, 8 and 16 mM). To assess penetration of the contrast agent into heart tissue and enable image contrast simulations, T(1) and T(*) (2) were measured in heart and background agarose. Compared to 3-day, 2-week fixation showed reduced mean T(1) in the heart at both 2 and 4 mM concentrations (p < 0.0001), resulting in calculated signal gains of 23% (2 mM) and 29% (4 mM). Using T(1) and T(*) (2) values from 2-week concentrations, computer simulation of heart and background signal, and ex vivo 3D gradient echo imaging, we demonstrated that 2-week fixed embryos in 8 mM Gd-DTPA in combination with optimised parameters (TE/TR/alpha/number of averages: 9 ms/20 ms/60 degrees /7) produced the largest SNR in the heart (23.2 +/- 1.0) and heart chamber contrast-to-noise ratio (CNR) (27.1 +/- 1.6). These optimised parameters were then applied to an MRI screen of embryos heterozygous for the gene Chd7, implicated in coloboma of the eye, heart defects, atresia of the choanae, retardation of growth, genital/urinary abnormalities, ear abnormalities and deafness (CHARGE) syndrome (a condition partly characterised by cardiovascular birth defects in humans). A ventricular septal defect was readily identified in the screen, consistent with the human phenotype., ((c) 2009 John Wiley & Sons, Ltd.)

Rapid and precise phenotyping analysis of large numbers of wild-type and mutant mouse embryos is essential for characterizing the genetic and epigenetic factors regulating embryogenesis. We present a novel methodology that permits precise high-throughput screening of the phenotype of embryos with both targeted and randomly generated mutations. To demonstrate the potential of this methodology we show embryo phenotyping results produced in a large-scale ENU-mutagenesis study. In essence this represents an analysis pipeline, which starts with simultaneous micro-magentic resonance imaging (microMRI) screening (voxel size: 25.4 x 25.4 x 24.4 microm) of 32 embryos in one run. Embryos with an indistinct phenotype are then cut into parts and suspect organs and structures are analysed with HREM (high-resolution episcopic microscopy). HREM is an imaging technique that employs 'positive' eosin staining and episcopic imaging for generating three-dimensional (3D) high-resolution (voxel size: 1.07 x 1.07 x 2 microm) digital data of near histological contrast and quality. The results show that our method guarantees the rapid availability of comprehensive phenotype information for high numbers of embryos in, if necessary, histological quality and detail. The combination of high-throughput microMRI with HREM provides an alternative screening pipeline with advantages over existing 3D phenotype screening methods as well as traditional histology. Thus, the microMRI-HREM phenotype analysis pipeline recommends itself as a routine tool for analysing the phenotype of transgenic and mutant embryos.

Developmental malformations are a major cause of childhood mortality and are typically characterized by lesions that allow survival of the embryo through gestation. The genetics of developmental malformations are powerfully studied by using high-throughput, phenotype-driven screens (e.g., following zebrafish or mouse mutagenesis) or by genotype-driven studies using transgenic or knockout mice. With regard to either approach, the mouse is anatomically and phylogenetically closer to humans than any other genetically tractable model organism. This is particularly important in the cardiovascular and respiratory systems, which have unique mammalian features. The identification of murine models of developmental malformations is, however, hindered by the opacity of the late gestational mouse embryo. In this review, we describe recent advances in magnetic resonance imaging that make it possible to rapidly identify malformations in the developing mouse embryo with high efficiency., ((c) 2004 Wiley-Liss, Inc.)

E type cyclins (E1 and E2) are believed to drive cell entry into the S phase. It is widely assumed that the two E type cyclins are critically required for proliferation of all cell types. Here, we demonstrate that E type cyclins are largely dispensable for mouse development. However, endoreplication of trophoblast giant cells and megakaryocytes is severely impaired in the absence of cyclin E. Cyclin E-deficient cells proliferate actively under conditions of continuous cell cycling but are unable to reenter the cell cycle from the quiescent G(0) state. Molecular analyses revealed that cells lacking cyclin E fail to normally incorporate MCM proteins into DNA replication origins during G(0)-->S progression. We also found that cyclin E-deficient cells are relatively resistant to oncogenic transformation. These findings define a molecular function for E type cyclins in cell cycle reentry and reveal a differential requirement for cyclin E in normal versus oncogenic proliferation.

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.

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.