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Sci Rep
2017 Feb 14;7:42506. doi: 10.1038/srep42506.
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Analysis of Craniocardiac Malformations in Xenopus using Optical Coherence Tomography.
Deniz E
,
Jonas S
,
Hooper M
,
N Griffin J
,
Choma MA
,
Khokha MK
.
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Birth defects affect 3% of children in the United States. Among the birth defects, congenital heart disease and craniofacial malformations are major causes of mortality and morbidity. Unfortunately, the genetic mechanisms underlying craniocardiac malformations remain largely uncharacterized. To address this, human genomic studies are identifying sequence variations in patients, resulting in numerous candidate genes. However, the molecular mechanisms of pathogenesis for most candidate genes are unknown. Therefore, there is a need for functional analyses in rapid and efficient animal models of human disease. Here, we coupled the frog Xenopus tropicalis with Optical Coherence Tomography (OCT) to create a fast and efficient system for testing craniocardiac candidate genes. OCT can image cross-sections of microscopic structures in vivo at resolutions approaching histology. Here, we identify optimal OCT imaging planes to visualize and quantitate Xenopus heart and facial structures establishing normative data. Next we evaluate known human congenital heart diseases: cardiomyopathy and heterotaxy. Finally, we examine craniofacial defects by a known human teratogen, cyclopamine. We recapitulate human phenotypes readily and quantify the functional and structural defects. Using this approach, we can quickly test human craniocardiac candidate genes for phenocopy as a critical first step towards understanding disease mechanisms of the candidate genes.
Figure 1. Imaging the Xenopus tropicalis heart with OCT.(a) Stage 46 tadpole embedded in 1% agarose and positioned so that the ventral side faces the OCT lens. Next, using a 5-axis stage, the embryo is oriented to the reference axis-mid eye plane. Finally, the heart is scanned from various angles to capture the three imaging planes. To capture the Ventral Three Chamber View (VTCV) and Outflow Tract View (OTV) we set the OCT imaging plane ± 0–15° to the reference axis and manually move the embryo posteriorly towards the tailon the y-axis until we capture the ventricle and the outflow tract. Similarly, to capture the Lateral View (LV) we adjusted the imaging plane ± 30–50° to the reference axis (b) Ventral Three Chamber View: Visualizes the single ventricle and both atria. Red dashed line marks the left atrium, right atrium, atrial septum, ventricle, atrioventricular valve, and trabecula in different stages of the cardiac cycle from systole to diastole (left to right). (c) Outflow Tract View: By moving the imaging plane anteriorly to the cranium, OCT captures the distaloutflow tract including the spiral valve and systemic arteries. (d) Lateral View: Rotating the OCT beam angle 30–50 °C reveals the proximaloutflow tract and the ventricle visualized during mid-systole. (e) Cardiac structures Resolved by OCT and Fate Mapping: Aortic arches; outflow tract, spiral valve and atrioventricular valve and ventricle; ventricle populated by primary and secondary heart field. (f) Xenopus tropicalis wildtype cardiac measurements by OCT and Histology. EDD: end diastolic diameter, ESD: end systolic diameter AV: atrioventricular, OFT: outflow tract, P: proximal, D: distal, StdD: standard deviation, SEM: Standard error of the mean, CoV: Coefficient of variation. Y-X Scale bar: 100 μm.
Figure 2. Quantitative assessment of the tadpole hearts using OCT with cardiomyopathy and heterotaxy.Cardiomyopathy model generated by myosin heavy chain 6 (MYH6) knockdown and heterotaxy model generated by dynein heavy chain 9 knockdown (DNAH9). Measurement of (a) EDD, (b) ESD (c) SF (d) AV valve excursion distance (e) OFT wall-to-diameter (f) OFT excursion distance. UIC: uninjected control, EDD: end diastolic diameter, ESD: end systolic diameter, OFT: outflow tract, SF: shortening fraction. (Mann-Whitney test; p < 0.05) (*p < 0.05/**p < 0.01/****p < 0.0001) (Bars represent mean with 95% confidence interval).
Figure 3. Imaging Xenopus craniofacial structures with OCT (a) We create a slit at the center of the clay with the Stage 46 tadpole positioned ventrally towards the OCT imaging plane (XZ-plane). Then we adjust the 5-axis stage to capture a zero- degree reference axis where a plane intersects the tadpole’s eyes symmetrically and the tip of the tail. This plane defines the 0° orthogonal plane. We then move this imaging plane in y, x and z axes to capture transverse, sagittal and coronal sections respectively. Imaging planes are adjusted on all three axes to capture distinct facial structures. (b) Top panel shows the schematic representation of the three neural crest streams: mandibular (green), hyoid (pink) and branchial (yellow) which form Meckel, ceratohyal and gill cartilages respectively. Each structure is highlighted under simple stereomicroscopy image (middle panel) and after alcian blue stain (bottom panel) (c) OCT images of craniofacial structures in all three planes: Transverse, Sagittal and Coronal. Meckel’s cartilage (green label) is best viewed in sagittal (yz-axis) and coronal (xy-axis) sections. The ceratohyalcartilage can be easily resolved by the coronal sections. The gill cartilages are more posterior compared to Meckel’s and ceratohyalcartilage, and the gill cartilages occupies most of the craniofacial space. Qualitative analyses can be made in all axes.
Figure 4. Qualitative neural crest defects in Xenopus with cyclopamine treatment.Stage 46 uninjected control tadpole compared to cyclopamine treated tadpoles and demonstrated the teratogenic effects. (a–c) Left column shows the control and the right column shows the cyclopamine treated tadpole. In cyclopamine treated tadpoles both jaws were short and thick. Meckel and ceratohyalcartilage were smaller and gill cartilages were nearly lost. (Scale Bar on x and y axis: 0–2 mm).
Figure 5. Quantitative neural crest defects in Xenopus with cyclopamine treatment.(a/a′–c/c′) Stage 46 control tadpole compared to cyclopamine (2.5 mg) treated tadpoles. The tadpole is positioned on the ventral side up, and the OCT imaging plane scrolled along the z-axis until the largest mid-lateral portion of the cartilages is visualized. We acquired a set of 3D data. On these coronal sections a line between two points traveling along the mid-corpus of the Meckel’s and ceratohyalcartilage is measured. Then the largest middle and lateral borders along the corpus of the most anteriorgill are marked, and the midline length is measured. Quantitative analysis demonstrated reduction in length and area in treated tadpoles on the coronal plane. L: left, (Mann-Whitney test; p < 0.05) (*p < 0.05/**p < 0.01/****p < 0.0001) (Bars represent mean with 95% confidence interval).
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