a, Haematoxylin and eosin stained sections of E12.5 control (left) and p5325,26,53,54/+ embryos (right). Examination confirmed neural tube closure defects (arrow). Original magnification, ×10. b, Close-up image of an ultraviolet-radiation-illuminated, ethidium bromide stained E15.5 p5325,26,53,54/+ embryo (right), highlighting the short lower jaw phenotype with protruding tongue (arrow) compared with control littermates (left). Seventy-four per cent of p5325,26,53,54/+ embryos (n = 27) exhibited a short lower jaw. Cleft lip is not shown. Original magnification, ×40. c, Top, Alizarin red (bone) and Alcian blue (cartilage) stained whole mount of an E15.0 p5325,26,53,54/+ embryo (right), showing reduced bone density in the cranium (c), nasal cavity (n), shorter ulna (u), humerus (h), mandible (m) and femur (f), as well as reduced bone formation in the ribs (R), where fewer vertebrae are undergoing ossification than in littermate controls (left). The number of vertebrae with bone formation was 19 in the controls (arrow; V19) and 18 in p5325,26,53,54/+ embryos (arrow; V18). The severity of the bone and cartilage defects was variable, with the most severe defects evident in embryos with exencephaly and severe craniofacial defects (n = 7). Bottom, Quantification of bone lengths shown as percentage of E14.5–15.0 littermate controls. The bone lengths of the mandible, humerus, ulna and femur were measured using the ruler function in Adobe Photoshop on images acquired at 6.3× magnification. Only litters with detectable bone formation in p5325,26,53,54/+ embryos were included in bone length analyses: Student’s t-test; **, P = 0.008 (mandible); **, P = 0.005 (humerus). d, Representative images of haematoxylin and eosin stained sagittal sections of E12.5 control hearts (left) and p5325,26,53,54/+ hearts (right), showing all three cardiac cell types in both genotypes. en, endocardium; ep, epicardium; myo, myocardium (arrows). Original magnification, ×200. e, A haematoxylin and eosin stained E12.5 p5325,26,53,54/+ heart exhibiting persistent truncus arteriosus (PTA) (33%, n = 6). The cardiac outflow tract in the control embryo (left) is septated into the aorta (Ao) and main pulmonary artery (MPA), whereas the cardiac outflow tract (truncus arteriosus (TA)) in the p5325,26,53,54/+ embryo (right) remains unseptated, resulting in PTA. Original magnification, ×100. f, Illustration of a control heart (left) and a p5325,26,53,54/+ embryo heart (right), highlighting double outlet right ventricle (DORV) and atrioventricular cushion defects. Both the aorta (Ao) and the MPA flow out of the right ventricle (RV), resulting in mixed oxygenated and deoxygenated blood in the systemic circulation when combined with concurrent ventricular septal defects (VSDs). The atrioventricular cushions remain bulbous and fail to elongate into mature valve leaflets. Red denotes oxygenated blood; blue denotes deoxygenated blood; and purple (pink) denotes mixed oxygenated and deoxygenated blood. mv, mitral valve; tv, tricuspid valve. Original magnification, ×100. g, Representative haematoxylin and eosin stained transverse section of thymus from a p5325,26,53,54/+ E15.5 embryo (right) reveals a smaller thymus than in littermate controls (left) (63% of controls; n = 4). Original magnification, ×200. h, Representative haematoxylin and eosin analysis of liver sections from E12.5 controls (left) and p5325,26,53,54/+ embryos (right), showing normal liver architecture in both genotypes (top). High magnification image (bottom) of the region of the liver that is outlined by the white box in the top panel shows the presence of nucleated erythrocytes (arrows), indicating proper haematopoiesis. Original magnification top, ×100; bottom, ×400. i, Top, Table summarizing the incidence (%) and sample size (n) of phenotypes assessed qualitatively in p5325,26,53,54/+ embryos. The occurrence of these phenotypes in CHARGE syndrome is also indicated (+, present; −, absent). Bottom, Table summarizing the phenotypes assessed quantitatively in p5325,26,53,54/+ embryos relative to controls, shown as the percentage average size of the control (%), with sample size (n) also indicated. The occurrence of these phenotypes in CHARGE syndrome is also shown (+, present). A detailed description of the bone and cartilage defects is provided in c.