microMRI-HREM pipeline for high-throughput, high-resolution phenotyping of murine embryos

Abstract

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 (microMRI) screening (voxel size: 25.4 x 25.4 x 24.4 microm) 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 x 1.07 x 2 microm) 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 microMRI with HREM provides an alternative screening pipeline with advantages over existing 3D phenotype screening methods as well as traditional histology. Thus, the microMRI-HREM phenotype analysis pipeline recommends itself as a routine tool for analysing the phenotype of transgenic and mutant embryos.

Fig. 1

Virtual section planes through an HREM data volume. Note the uniformity of tissue staining in all parts of the embryo. Scale bar, 500 µm.

Fig. 2

Flow chart of the phenotyping pipeline.

Fig. 3

Examples of abnormal morphology detected by high-throughput µMRI and HREM of 15.5-dpc mouse embryos. (a–c) µMRI data showing a palatine cleft (Pc in a), nuchal oedema (arrows in b), and a ventricular septal defect (asterisk in c). (d–f) HREM data showing defects not detected by µMRI: large hemiazygos (Hv) and azygos (Av) veins (d), persistent vitelline vein segments proximal and distal to the liver (arrowheads) and an umbilical vein (Uv) running in the body wall (e), and a hypoplastic left lobe (lThy) of the thyroid gland (f). B, brain; Sc, spinal cord; Li, liver; C, heart; G, gut; Aa, aortic arch; Db, ductus arteriosus; Llv, lumen of left ventricle; Lrv, lumen of right ventricle; V, vertebra; A, aorta; K, kidney; L, lung; U, future bladder; D, diaphragm; P, pancreas; E, oesophagus. Scale bars, 500 µm.

Fig. 4

Synergy of µMRI and HREM in high-throughput phenotyping of 15.5-dpc mouse mutants. (a–d) µMRI data (a,b) suggest a right retro-oesophageal subclavian artery – HREM data (c,d) confirm it due to higher spatial resolution (inlay, arrowhead). (e–h) µMRI data (e,f) suggest thinning of the right ventricle myocardium – HREM data (g,h) confirm it (inlay). (i–l) µMRI data show lumbar oedema (arrow in i) but no obvious cardiac defects (j) – HREM data (k,l) reveal a ventricular septal defect. Sc, spinal cord; Aa, aortic arch; E, oesophagus; T, trachea; Vs, ventricular septum; lv, left ventricle; rv, right ventricle. Scale bars, 500 µm.