Introduction Cardiovascular malformations are a major cause of mortality and morbidity, but their genetic basis is poorly understood. The mouse, due to its anatomical and genetic similarities to humans, is an ideal model for understanding the genetic basis of congenital malformations. Both gene knockout and ethylnitrosourea mutagenesis are commonly used to create novel mouse models. Unlike genotype-driven knockout approaches, ethylnitrosourea mutagenesis makes no a priori assumptions about the nature of the genes. Ethylnitrosourea-induced random point mutations result in creating the alleles that are similar to the spectrum observed in human disease, and provide insight into the molecular mechanisms of protein function. Here we have combined ethylnitrosourea mutagenesis with magnetic resonance imaging (MRI) to screen for mouse mutations affecting cardiovascular development.

Methods Male mice (Balb/c or C57BL/6), injected with two doses of ethylnitrosourea (80 mg/kg of body weight), were crossed with C3H females. Male progeny were crossed with their daughters. Third-generation embryos were dissected at 15.5 dpc, fixed in paraformaldehyde and gadolinium-diethylene triamine pentaacetic acid, embedded in agarose and imaged using MRI at a resolution of 25.4 × 25.4 × 24.4 μm per voxel in high-throughput format. 3D MRI data were reconstructed into TIFF files and analysed with Amira 3.1.1 software. Mutations were mapped by genotyping single nucleotide polymorphisms that differentiate parental strains.

Results We have screened 71 mutated mouse lines and analysed over 3150 embryos. We have identified seven lines exhibiting abnormal heart development. The Vcc (Vacterl, caudal regression, Currarino syndrome) line shows vertebral, anorectal, cardiac, tracheoesophageal, renal and limb malformations. The Cdl mutation results in cardiac malformation, ductus venosus agenesis and lymphatic cystic hygroma. The George mutation resembles 22q11 deletion syndrome, with cardiac malformations, palatal cleft, absent thymus and cochlea. The Cat mutation results in common arterial trunk, septal defect and hypoplastic thymus. The Raa mutation results in right-sided aortic arches. The Iso1 and Iso2 mutations result in cardiac and/or pulmonary isomerism. Genetic and molecular analysis indicates that Vcc results from a mutation in Pcsk5, and provides novel insights for the role of proprotein convertases in embryonic signalling, patterning and heart development. Mutations in PCSK5 were identified in a proportion of patients with VACTERL/caudal regression syndromes (see Genes Dev 2008). The Cdl and George mutations will provide novel insights into cardiovascular, lymphatic and venous development, and will be described. Genetic analysis of the other mutations is continuing.

Implications Our results indicate that the combination of ethylnitrosourea mutagenesis and MRI allows for the identification of cardiovascular anomalies of the types observed in the human population. Genetic analyses of these mutations and the molecular mechanisms underlying the defects will let us understand the genetic bases of human cardiovascular developmental malformations.