Rapid and precise phenotyping analysis of large numbers of wild-type and mutant mouse embryos is essential for characterizing the genetic and epigenetic factors regulating embryogenesis. We present a novel methodology that permits precise high-throughput screening of the phenotype of embryos with both targeted and randomly generated mutations. To demonstrate the potential of this methodology we show embryo phenotyping results produced in a large-scale ENU-mutagenesis study. In essence this represents an analysis pipeline, which starts with simultaneous micro-magentic resonance imaging ( µ MRI) screening (voxel size: 25.4 × 25.4 × 24.4 µ m) of 32 embryos in one run. Embryos with an indistinct phenotype are then cut into parts and suspect organs and structures are analysed with HREM (high-resolution episcopic microscopy). HREM is an imaging technique that employs 'positive' eosin staining and episcopic imaging for generating three-dimensional (3D) high-resolution (voxel size: 1.07 × 1.07 × 2 µ m) digital data of near histological contrast and quality. The results show that our method guarantees the rapid availability of comprehensive phenotype information for high numbers of embryos in, if necessary, histological quality and detail. The combination of high-throughput µ MRI with HREM provides an alternative screening pipeline with advantages over existing 3D phenotype screening methods as well as traditional histology. Thus, the µ MRI-HREM phenotype analysis pipeline recommends itself as a routine tool for analysing the phenotype of transgenic and mutant embryos.
SummaryThe great arteries of embryos are small channels of a complex three-dimensional arrangement. Measurements of their diameters, as required for understanding cardiovascular morphogenesis and the genesis of malformations, cannot be performed in two-dimensional histological sections. We present and evaluate a quick and simple method for performing highly significant and objective measurements of the diameters of blood vessels in vertebrate embryos and used this method for providing statistics of the diameter of the semi-lunar valves and the lumina of the great arteries of early chick and mouse foetus. We employed the high-resolution episcopic microscopy technique for generating volume data and three-dimensional computer models of the arterial trees of 30 chick embryos (Hamburger Hamilton stage 34), 30 mouse embryos of the OF1 strain harvested on 14.5 dpc, 30 embryos of the OF1 strain harvested on 15.5 dpc and 28 mouse embryos of the PARKES strain harvested on 14.5 dpc. The three-dimensional models (voxel size 2 μm × 2 μm × 2 μm and 3 μm × 3 μm × 3 μm) were used for defining virtual resection planes perpendicular to the longitudinal axis of the blood vessels at comparable positions. In these planes, we measured the lumen areas and the lumen perimeters. We also calculated the lumen diameter and the true lumen area from the perimeter and present statistical analysis. Finally, we evaluate and discuss the reliability and reproducibility of our method and present all measurements in a form that minimizes the influence of specimen size variation, specimen processing and data generation methods.
Knowledge about the variable course of the perforating arteries near the body of the femur is essential during surgical procedures (e.g., percutaneous cerclage wiring, plate osteosynthesis, Ilizarov technique). Our aims were to determine the number of perforating arteries, and to identify safe zones along the body of the femur within which perforating arteries are unlikely to pass toward the back of the thigh. The number of perforating arteries was determined in both legs of 100 formalin-fixed anatomic specimens of both sexes. The level of passage of perforating arteries near the body of the femur was measured in reference to a line from the anterior superior iliac spine to the medial femoral condyle. In each leg, two to seven perforating arteries were present. In 64% of legs, at least one artery divided into two to four branches before entering the back of the thigh. Thus, the total number of branches passing near the body of the femur varied between two to nine. Perforating arteries passed to the back of the thigh at every level between 14.0 and 36.5 cm from the anterior superior iliac spine (16-39% of the leg length). Within this distance, no safe zones along the body of the femur could be identified. The present study shows the high variability regarding number and course of the perforating arteries. Surgeons can be faced with an artery at every level on the posteromedial aspect of the body of the femur between 14.0 and 36.5 cm distally to the anterior superior iliac spine. Clin. Anat. 33:507-515, 2020.
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