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First published online 17 November 2004
doi: 10.1242/dev.01543

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ENU induced mutations causing congenital cardiovascular anomalies

¹ Laboratory of Developmental Biology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8019, USA
² Department of Pediatrics, National Naval Medical Center, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-5000, USA
³ Pediatric Cardiology, Children's National Medical Center, Washington, DC 20010, USA
⁴ Department of Anatomy and Cell Biology, Medical University of South Carolina, Charleston, SC 29425, USA
⁵ Pediatric Cardiology, Medical University of South Carolina, Charleston, SC 29425, USA
⁶ Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA
⁷ The Jackson Laboratory, Bar Harbor, ME 04609, USA

View larger version (131K): [in a new window]   Fig. 1. Mouse fetal ultrasound imaging. (A) In utero 2D-ultrasound image of a fetus, showing two white arrows used to measure the crown to rump length. (B) A fetus showing pericardial effusion (red arrow) and hydrops (yellow arrows). (C,D) Color flow Doppler analysis showed outflow regurgitation in an E18.5 fetus. Aliasing (see arrow) associated with the outflow (C) indicated increased velocity. Superimposed on the outflow is a regurgitant diastolic flow (D). (E) Spectral Doppler analysis revealed an abnormal regurgitant flow. (F) M-mode images from an E17.5 fetus, obtained from a short axis view (see diagram), show the position of the right (RV) and left (LV) ventricular walls and the interventricular septum (IVS) through multiple cardiac cycles. Wall thickness, and chamber volume in diastole and systole can be obtained by measuring the distances between numbered positions (red color dots versus corresponding position in M-mode image).

View larger version (140K): [in a new window]   Fig. 2. Histological sections showing morphology of the normal neonatal mouse heart. (A) Low magnification view of the anterior portion of the heart, showing the normal configuration of the interventricular septum (IVS) and ventricular walls. Note the relative thickness of these components. (B,C) The right ventricular (RV) subpulmonary outflow tract (PA) is shown in B, whereas C illustrates the left ventricular (LV) outflow tract (Ao). Note the fibrous continuity of the mitroaortic valves. (D-F) Configuration of structures in the venous pole of the heart. The sequence of sections shows normal connection of the left superior vena cava (LSVC) to the right atrium (RA) via the coronary sinus (CS). Also shown are the right superior vena cava (RSVC) connection to the right atrium (RA; D,E) and the atrial septal complex. Same magnification used in panels B-F. MV, mitral valve; TV, tricuspid valve; FO, foramen ovale; FS, foramen secundum.

View larger version (66K): [in a new window]   Fig. 3. Family 26 with persistent truncus arteriosus and craniofacial anomalies. (A) A pup that died at birth had a short snout, low set ears, rounded head, and short neck. (B) A normal neonatal C57BL6/J pup. Alcian blue staining (E,F) showed the abnormal pup (E) had a shortened premaxilla (PM), maxilla (M) and nasal bone (N), while its frontal bone (F) was expanded. The shape of the mandible (MN) was also altered. The heart exhibited persistent truncus arteriosus (PTA in D), when compared with normal septated outflows (C), consisting of an aortic (Ao) and pulmonary (P) trunk. Histological sections of the abnormal heart (G,H) revealed a single outflow positioned over the RV and VSD. This vessel gave rise to the ascending aorta (AAo) with its brachiocephalic artery (BCA), the right and left pulmonary arteries (RPA, LPA), and the coronary arteries (LCA, RCA). A VSD with inlet extension can be seen in (H). Asterisk in H denotes abnormal AV valve. Panels A and B, E and F, and G and H are shown at the same magnification.

View larger version (128K): [in a new window]   Fig. 4. Transposition of the great arteries and heart laterality defects in family 182. (A-D). A pup that died at birth showed thymic (T) hypoplasia (A) and an enlarged heart (A,B) with two thin outflows vessels positioned anterior-posterior (see arrowheads in C). For comparison, the chest cavity of a normal newborn mouse is shown in D. (E-J) Histological sections presented anterior to posterior (E-G) showed the aorta (Ao) positioned anteriorly (E), giving rise to the coronary arteries (F) and connecting to the RV (E). Lumen of the left coronary artery was enlarged (LCA). Panel G shows the pulmonary outflow (P) positioned posteriorly and a VSD. (H,I) Also observed is a primum ASD, single AV valve, canal type VSD (see canal in H), and bridging leaflet (arrow in H). Right atrial isomerism is indicated as the left-sided atrium (L-mRA) receives the left superior vena cava (LSVC) directly, while the right-sided atrium (R-mRA) receives the right superior vena cava (RSVC) in the normal fashion (I). (J) Ectopic pigment granules in the interventricular septum (arrows). (K-N) A fetal heart exhibited inverted ventricles together with right atrial isomerism. Apex of the heart is abnormally pointed to the right of the chest cavity (K,L). Arrow in K denotes the ductus arteriosus. The aortic (Ao) and pulmonary (PA) outflows are positioned anterior-posterior (L), with the anteriorly positioned aorta connected to the morphological right ventricle (mRV in M). A large coronary artery (CoA) drains into a sinusoidal fistula in the septum (see arrow in N). mLV, morphological left ventricle.

View larger version (100K): [in a new window]   Fig. 5. Family 53 with outflow and aortic arch anomalies. (A-E). Persistent truncus arteriosus and DORV. Neonatal pups showed PTA together with IAA (A) or DORV with a duplicated left carotid artery (double arrowheads in B). Histology (C) and 3D reconstruction using episcopic fluorescence image capture (D,E) of a heart similar to that in shown in A revealed a single outflow tract that gave rise to the aorta and the pulmonary arteries (pa) and coronary arteries (ca) (arrowheads in C; white arrows in E). A VSD connects the RV and LV. L, lung; T, thymus. (F-K). Abnormal pigmentation. A homozygous Sema3CL605P pup exhibited skin hypopigmentation (F,H,K), when compared with an age matched control pup (G,I,K). Shown is skin from head (F,G), trunk (H,I) and rump (J,K). Ectopic pigmentation is seen in the chest cavity (L-N), including the heart (arrow, L), lung (not shown), trachea (Tr, M) and great vessels (arrow in N). LC, left carotid artery.

View larger version (58K): [in a new window]   Fig. 6. Mutation in a highly conserved Sema3C immunoglobulin domain. Family 53 pups with PTA are homozygous for a T to C substitution (m/m sequencing trace files, right) in the highly conserved immunoglobulin (Ig) domain of Sema3C, which caused a leucine to proline substitution, and also resulted in the loss of a PstI restriction site and the gain of an AciI site. PCR amplification using primers spanning the Sema3CL605P mutation, followed by PstI digestion was used to genotype a litter of fetuses obtained from intercrossing two heterozygous Sema3CL605P mutants (see bottom gel). Three homozygous Sema3CL605Pmutants showed PTA, whereas one heterozygous fetus had DORV.

View larger version (102K): [in a new window]   Fig. 7. Conotruncal heart defects and coronary anomalies elicited by a novel connexin43 mutation. (A-G) Conotrucal bulge (black arrows in A) and hypoplastic thymus (T) are evident in this homozygous Cx43W45X pup. At the base of the outflows is a prominent peritruncal coronary vessel (white arrowhead in panels B,C), and coronary aneurysms are seen in the wall of the aortic and pulmonary outflows (white arrows in panels B,C). These can be seen in histological sections (D-G). Also note sinusoidal trabeculae at the base of the outflow (E-G). Arrowhead in D and arrows in E-G denote coronary aneurysms, and arrow in D and arrowheads in E-G denote abnormal coronary plexuses. (H-K). Histology shows a large subepicardial coronary vessel (arrowhead in D) and an abnormally thinned compact layer (arrow in H). In one heart, a coronary artery inserts into the aorta below the level of the valves (I), and enlarges to form a sinus (arrow in panel I). Also observed are a VSD (J) and thickened valves (arrowheads, K). (L) Protein structure of connexin43 is shown on the left, indicating four transmemebrane (TM1, 2, 3 and 4) domains, two extracellular loops (EC1 and 2) and an intracellular loop (ICL). The region spanning EC1 is highly conserved in vertebrates. Sequence trace files (right) revealed a G to A substitution that generated a STOP codon at amino acid 45, which normally encodes tryptophan (W).

View larger version (134K): [in a new window]   Fig. 8. Forelimb and heart defects in Family 166. (A-D) Forelimbs are abnormally flexed towards the chest, with clubbed front paws. Typically three digits are seen (B), but in some pups, there appears to be a fusion of the third and fourth digits (C,D). (F,G) Whole-mount view shows enlarged atria and an abnormal heart shape indicative of hypertrophy (F,G). An abnormal large peritruncal coronary vein is observed at the base of the outflows (white arrow, G). (E,H-M) Histology shows biventricular hypertrophy (H,J,L), and abnormal coronaries around the outflow vessels (E, white arrow in H). Also evident are muscular VSDs (see arrows in J-M), and disorganized myocardium (E,I,K,M), which frequently shows gaps between the myofiber bundles (see arrowhead in E). (N-P) The heart shown in (G) was processed for episcopic fluorescence image capture, and the image stacks obtained were re-sectioned at a different pitch to visualize the VSDs (see white arrows). Sections in E, H and I are from heterozygous, whereas the rest are from homozygous animals.

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