Phenotyping cardiac and structural birth defects in fetal and newborn mice

Abstract

Mouse models are invaluable for investigating the developmental etiology and molecular pathogenesis of structural birth defects. While this has been deployed for studying a wide spectrum of birth defects, mice are particularly valuable for modeling congenital heart disease, given they have the same four-chamber cardiac anatomy as in humans. We have developed the use of noninvasive fetal ultrasound together with micro-computed tomography (micro-CT) imaging for high throughput phenotyping of mice for congenital heart defects (CHD) and other developmental anomalies. We showed the efficacy of fetal ultrasound and micro-CT imaging for diagnosis of a wide spectrum of CHD. With fetal ultrasound, longitudinal scans can be conducted to track the developmental profile of disease pathogenesis, providing both structural information with two-dimensional (2D) imaging, as well as functional data from hemodynamic assessments with color flow and spectral Doppler imaging. In contrast, with micro-CT imaging, virtual necropsies can be conducted rapidly postmortem for diagnosis of not only CHD, but also other structural birth defects. To validate the CHD diagnosis, we further showed the use of a novel histological technique with episcopic confocal microscopy to obtain rapid 3D reconstructions for accurate diagnosis of even the most complex anatomical defect. The latter histological imaging technique when combined with the use of ultrasound and micro-CT imaging provides a powerful combination of imaging modalities that will be invaluable in meeting the accelerating demands for high throughput mouse phenotyping of genetically engineered mice in the coming age of functional genomics. Birth Defects Research 109:778-790, 2017. © 2017 Wiley Periodicals, Inc.

Keywords: congenital heart disease; fetal ultrasound imaging; forward genetic screen; micro-CT; mouse model; structural birth defects.

Figure 1. Ultrasound diagnosis of a variety of congenital heart defects

(A–F). Hypoplastic right heart syndrome. Acuson Doppler imaging detected an E17.5 fetus with increased velocity in the inflow tract (A, asterisks). Vevo2100 revealed a hypoplastic right ventricle (B and C) with hypoplastic pulmonary artery (PA). Necropsy (D) confirmed hypoplastic PA, with ECM imaging (E,F) showing a hypoplastic right ventricle, hypoplastic tricuspid valve (TV) compared with the mitral valve (MV; arrowheads in E), and hypoplastic PA (arrowhead in F). (G–M). Hypoplastic left heart syndrome. Vevo2100 imaging of E16.5 fetus in sagittal view (G) showed reversed aortic blood flow during diastole (H) and confirmed by spectral Doppler (I). Short-axis view (J) showed hypoplastic left ventricle. Necropsy of stillborn pup (K) revealed hypoplastic aorta (Ao) and small LV. ECM histology (L,M) revealed small mitral valve orifice, small LV, and hypoplastic aorta, confirming HLHS. DA, ductus artery; DAo, descending aorta (N–R). Persistent truncus arteriosus. Acuson spectral Doppler showed an E17.5 fetus with outflow tract (OFT) regurgitation (arrowhead in N; OFT shown by asterisks; forward flow). Vevo2100 revealed a single OFT overriding the ventricular septum with a ventricular septal defect (VSD; arrowhead in O). This was associated with OFT regurgitation, indicated by red forward and blue reverse blood flow (O and O′), further confirmed with necropsy (P) and ECM analysis (Q,R). Also observed is ventricular noncompaction (Q,R) and muscular VSDs (arrowheads in R). (S–Y). Double outlet right ventricle with hypoplastic pulmonary artery and atrioventricular septal defect. Acuson imaging detected an E16.5 fetus with outflow tract regurgitation (OFT R in S). Vevo2100 imaging (T,T′) revealed a RV–LV shunt, indicating a ventricular septal defect (VSD; arrowheads in T). Vevo2100 imaging in the transverse view (U,U′) showed inflow tract forward (U, arrow) and regurgitant (U′, arrow) flows, whereas the sagittal view (V) showed the aorta (Ao) mostly connects to the RV with an adjacent very hypoplastic pulmonary artery (arrowhead, PA) and a VSD. Necropsy (W) and ECM imaging (X,Y) confirmed a hypoplastic PA (V,Y) with large VSD (Y, arrowhead). A common atrioventricular valve (CV in X) was observed in the transverse view. Scale bar, 1 mm. A, anterior; Cd, caudal; Cr, cranial; L, left; P, posterior; R, right; CT, common trunk, CVF, common atrioventricular valve forward flow; CVR, common atrioventricular valve reverse flow. From Liu et al., 2014.

Figure 2. Diagnosis of a variety of noncardiac defects

(A–C), Ultrasound diagnosis of heterotaxy. Vevo2100 imaging showed heterotaxy with heart apex on left (A, arrowhead) and stomach to right (S in B), which was confirmed by necropsy (C). Panel (D) showed a fetus with body wall closure defect with an omphalocele (arrowhead). Panel (E) shows ultrasound detection of fetus with polydactyly (asterisks). In panel (G), ultrasound imaging showed a fetus with microcephaly with no lower jaw (arrowhead0 and beak-like face as compared to normal fetus (F). Fetal ultrasound imaging also can detect hydrops and exencephaly with brain tissue protruding from the cranium (H). Scale bar, 1 mm. A, anterior; L, left; P, posterior, R, right. Cd, caudal; Cr, cranial. S, the stomach.

Figure 3. Micro-CT detection of outflow tract defects

Micro-CT in the coronal viewing plane revealed outflow tract mal-alignment defects, including double outlet right ventricle (DORV; F), transposition of the great arteries (TGA; G), and persistent truncus arteriosus (PTA; H). Necropsy and EFIC images of each heart is shown in (A–D) and (E–L). Scale bars=1 mm (A–H), 0.5 mm (I–L). Ao indicates aorta; LA, left atrium, LV, left ventricle; PA, pulmonary artery; RA, right atrium, and RV, right ventricle. Reprinted from Kim et al. Microcomputed tomography provides high accuracy congenital heart disease diagnosis in neonatal and fetal mice. Circulation: Cardiovascular Imaging. 2013;6:551–559.

Figure 4. Micro-CT detection of visceral organ situs defects

Necropsy (A–C) and corresponding micro-CT images (D–I) of newborn mice with normal placement of visceral organs (A, D, and G), situs inversus totalis with complete mirror reversal of organ situs (B, E, and H), and heterotaxy with left-right randomized organ situs (C, F, and I). The mutant pup exhibiting situs inversus possesses a right-sided heart (dextrocardia), left-sided inferior vena cava, inversion of lung and liver lobes, and a right-sided stomach (A, D, and G), whereas the mutant pup exhibiting heterotaxy showed dextrocardia, duplicated inferior vena cavae, left-pulmonary isomerism, normal liver lobation, and a left-sided stomach (C, F, and I). Arrows denote the direction to which the heart apex is pointing; asterisks indicate mirrored organ positioning. Scale bars=2.5 mm. Stm = stomach. Reprinted from Kim et al. Microcomputed tomography provides high accuracy congenital heart disease diagnosis in neonatal and fetal mice. Circulation: Cardiovascular Imaging. 2013;6:551–559.

Figure 5. Micro-CT detection of other structural birth defects

Diaphragmatic herniation can be detected as illustrated by necropsy (A) and micro-CT (B) in the coronal viewing plane. Note the location of the diaphragm. A liver lobe and a portion of the small intestine are visible above the diaphragm and displace lung lobes. Right pulmonary isomerism, characterized by a mirror duplication of each lobe of the right lung (asterisks) is shown by necropsy (C) and micro-CT (D). Likewise, left pulmonary isomerism is illustrated by necropsy (E) and micro-CT (F) and is shown by the presence of a large, duplicated left lobe (asterisks). Hydronephrosis, as indicated by distended renal pelvis (asterisks), affects the right kidney and can be seen by necropsy (G, H), histology via H&E staining (I), and micro-CT (J). Note that the ureter (#) continuous to the renal pelvis is also distended (I, J). Cystic kidneys can also be detected, as illustrated by necropsy (K, L), histology (M, N), and micro-CT (O). Arrows highlight notably large cysts throughout both kidneys. Scale Bars = 1mm (A–H, J–L, O); 500μm (I, M, N). Diaph. = diaphragm; Ht = heart; L = lung lobe; LvL = liver lobe; Panc. = pancreas; Sm. Int. = small intestine; Stm = stomach.

Figure 6. Episcopic confocal microscopy set up for serial 2D and 3D histopathology

The episcopic confocal microscopy technique utilizes a Leica SM2500 heavy-duty sliding microtome and Lecia True Confocal Scanner (TCS) Large Scale Imaging (LSI) microscopy system equipped with 488, 532, 635 nm 10 mW lasers. This system is mounted on a TMC Active Vibration Isolation Table (pneumatic).

Figure 7. Diagnosis of structural birth defects by episcopic confocal microscopy

(A,B) ECM imaging of a wildtpe (A) vs. mutant (B) newborn mouse revealed abnormal cloacal septation defect in the mutant leading to abnormal communication between the intestine and bladder. From Cui et al., 2014. (C–F) ECM imaging revealed brain abnormalities in mutant (D,F,H) vs. control (C,E,G) newborn mice. Shown are the findings of severe alobar holoprosencephaly (D), cerebelleral dysplasia (F), and hippocampal abnormities (H).