letter nature genetics • volume 29 • december 2001 469 Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator Simon D. Bamforth 1 *, José Bragança 1 *, Jyrki J. Eloranta 2 , Jennifer N. Murdoch 3 , Fatima I.R. Marques 1 , Kamil R. Kranc 1 , Hend Farza 1 , Deborah J. Henderson 3 , Helen C. Hurst 2 & Shoumo Bhattacharya 1 *These authors contributed equally to this work. 1 Department of Cardiovascular Medicine, University of Oxford, Wellcome Trust Center for Human Genetics, Roosevelt Drive, Oxford, OX3 7BN UK. 2 ICRF Molecular Oncology Unit, Hammersmith Hospital, London, UK. 3 Neural Development Unit, Institute of Child Health, London, UK. Correspondence should be addressed to S.B. (e-mail: sbhattac@well.ox.ac.uk). The protein EP300 and its paralog CREBBP (CREB-binding pro- tein) are ubiquitously expressed transcriptional co-activators and histone acetyl transferases 1 . The gene EP300 is essential for normal cardiac and neural development, whereas CREBBP is essential for neurulation, hematopoietic differentiation, angio- genesis and skeletal and cardiac development 2–5 . Mutations in CREBBP cause Rubinstein-Taybi syndrome, which is character- ized by mental retardation, skeletal abnormalities and congeni- tal cardiac defects 6,7 . The CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) binds EP300 and CREBBP with high affinity 8 and regulates gene transcription 8–10 . Here we show that Cited2 -/- embryos die with cardiac malformations, adrenal agenesis, abnormal cranial ganglia and exencephaly. The car- diac defects include atrial and ventricular septal defects, over- riding aorta, double-outlet right ventricle, persistent truncus arteriosus and right-sided aortic arches. We find increased apoptosis in the midbrain region and a marked reduction in ErbB3-expressing neural crest cells in mid-embryogenesis. We show that CITED2 interacts with and co-activates all isoforms of transcription factor AP-2 (TFAP2). Transactivation by TFAP2 iso- forms is defective in Cited2 -/- embryonic fibroblasts and is res- cued by ectopically expressed CITED2. As certain Tfap2 isoforms are essential in neural crest, neural tube and cardiac development 11–13 , we propose that abnormal embryogenesis in mice lacking Cited2 results, at least in part, from its role as a Tfap2 co-activator. We created a null allele of Cited2 by replacing it with a PGK- neo r cassette (Fig. 1); we found no Cited2 -/ - mice in over 350 weaned offspring of Cited2 +/ - intercrosses. This indicated either embry- onic or perinatal lethality. We therefore genotyped embryos from Cited2 +/– intercrosses and found that the ratio of live Cited2 +/+ to Cited2 -/ - embryos at or after 17.5 days post coitum (dpc) was 29:12. Upon genotyping all dead embryos after 10.5 dpc, we found a marked excess of Cited2 -/ - embryos (20 Cited2 -/ - versus 1 Cited2 +/+ ), confirming embryonic lethality. The presence of live Cited2 -/ - embryos at 18.5 dpc, however, suggests that some embryos may also die perinatally. Fifty-seven percent (31/54) of Cited2 -/ - embryos (Fig. 1c ) examined at or after 10.5 dpc had exencephaly or anterior open neural tube defects. Exencephaly usually extended from the fore- brain–midbrain boundary, sparing the face, to the midbrain–hindbrain boundary. As anterior neural tube defects are frequently associated with abnormal apoptosis in the devel- oping brain 14 , we examined embryos at 9.5 dpc by whole-mount Published online: 5 November 2001, DOI: 10.1038/ng768 Fig. 1 Exencephaly and midbrain apoptosis in Cited2 -/- embryos. Scale bars, 1 mm. a, Structure of the wildtype murine Cited2 locus, targeting vector and targeted allele. The targeting vector was constructed to replace all Cited2 exons with a neomycin-resistance gene (neo r ; N in fi gure) cassette. Locations of external probes used to confi rm correct targeting are shown. E, EcoRI; B, Bgl II; TK, thymi- dine kinase. b, Top, Southern blot of Bgl II-digested embryonic genomic DNA hybridized with probe 2. Middle, northern blot of total RNA isolated from E12.5 embryos of defined genotypes and hybridized with a CITED2 cDNA probe. Bot- tom, 28S and 18S RNA species. c , Cited2 +/+ and Cited2 -/- embryos at 17.5 dpc. The Cited2 -/- embryo has exencephaly (arrows). d, Whole-mount TUNEL staining of littermate embryos at 9.5 dpc. Arrows indicate increased staining in the midbrain region of the Cited2 -/- embryos. a b c d © 2001 Nature Publishing Group http://genetics.nature.com © 2001 Nature Publishing Group http://genetics.nature.com