(a) H&E-stained sections of E12.5 control (left) and
p5325,26,53,54/+ embryos (right). Examination
confirmed neural tube closure defects (arrow). (b) Close-up image of
UV-illuminated, ethidium bromide-stained E15.5
p5325,26,53,54/+ embryo (right) to highlight short
lower jaw phenotype with protruding tongue (arrow) compared to control littermate
(left). 74% (n=27) of p5325,26,53,54/+ embryos exhibited
short lower jaw. Cleft lip not shown. (c) Top: Alizarin Red (bone) and
Alcian Blue (cartilage) whole-mount stained E15.0
p5325,26,53,54/+ embryo (right) showing reduced bone
density in the cranium (c) and nasal cavity (n); shorter ulna (u), humerus (h),
mandible (m), and femur (f); and reduced bone formation in the ribs (R), where fewer
vertebrae are undergoing ossification relative to control littermate (left). Number
of vertebrae with bone formation: 19 in control (arrow; V19) versus 18 in
p5325,26,53,54/+ embryo (arrow; V18). The severity
of bone and cartilage defects is variable, with the most severe defects evident in
embryos with exencephaly and severe craniofacial defects. n=7. Bottom:
Quantification of bone lengths shown as percent of E14.5-15.0 littermate controls.
Bone lengths of the mandible, humerus, ulna, and femur were measured using the ruler
function in Adobe Photoshop on images taken at 6.3×. 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 H&E-stained
sagittal sections of E12.5 control (left) and
p5325,26,53,54/+ hearts (right) showing all three
cardiac cell types in both genotypes. en: endocardium; ep: epicardium; myo:
myocardium (arrows). (e) H&E-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 or TA) in the
p5325,26,53,54/+ embryo (right) remains unseptated,
resulting in PTA. (f) Illustration of control heart (left) and
p5325,26,53,54/+ embryo heart (right), highlighting
DORV and atrioventricular cushion defects. Both the aorta (Ao) and main pulmonary
artery (MPA) flow out from the right ventricle (RV), resulting in mixed oxygenated
and deoxygenated blood in systemic circulation when combined with concurrent VSDs
(ventricular septal defects). The atrioventricular cushions remain bulbous and fail
to elongate into mature valve leaflets (mitral valve: mv; tricuspid valve: tv). Red:
oxygenated blood; Blue: deoxygenated blood; Purple/Pink: mixed
oxygenated/deoxygenated blood. (g) Representative H&E-stained
transverse section of thymus in p5325,26,53,54/+ E15.5
embryo (right) reveals smaller thymus compared to control littermate (left) (63% of
control; n=4). (h) Representative H&E analysis of liver sections
from E12.5 control (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 outlined by the white box in
the top panel shows the presence of nucleated erythrocytes (arrows), indicating
proper hematopoiesis. (i) Top: Table summarizing the incidence (%) and
sample size (n) for 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 phenotypes assessed quantitatively in
p5325,26,53,54/+ embryos relative to controls, shown
as the percent average size of controls (%), with sample size (n) also indicated.
The occurrence of these phenotypes in CHARGE syndrome is also shown (+ present).
Detailed description of bone and cartilage defects can be found in Extended-Data Fig. 4c.