Phenotyping structural abnormalities in mouse embryos using high-resolution episcopic microscopy

The arrival of simple and reliable methods for 3D imaging of mouse embryos has opened the possibility of analysing normal and abnormal development in a far more systematic and comprehensive manner than has hitherto been possible. This will not only help to extend our understanding of normal tissue and organ development but, by applying the same approach to embryos from genetically modified mouse lines, such imaging studies could also transform our knowledge of gene function in embryogenesis and the aetiology of developmental disorders. The International Mouse Phenotyping Consortium is coordinating efforts to phenotype single gene knockouts covering the entire mouse genome, including characterising developmental defects for those knockout lines that prove to be embryonic lethal. Here, we present a pilot study of 34 such lines, utilising high-resolution episcopic microscopy (HREM) for comprehensive 2D and 3D imaging of homozygous null embryos and their wild-type littermates. We present a simple phenotyping protocol that has been developed to take advantage of the high-resolution images obtained by HREM and that can be used to score tissue and organ abnormalities in a reliable manner. Using this approach with embryos at embryonic day 14.5, we show the wide range of structural abnormalities that are likely to be detected in such studies and the variability in phenotypes between sibling homozygous null embryos.


Supplementary Fig. 2.
Virtual sagittal re-sections through HREM data of wild-type E14.5 mouse embryos. Selected important features are labelled. a. Resection through the embryo displayed in the inlay on the left. Note the section plane (p). Scalebar = 1 mm. Magnifications of the boxed areas are displayed as inlays on the right. Most cranial box corresponds to uppermost inlay. Note the internal carotid artery (ica), vertebral artery (va), spinal ganglion (g2) and spinal nerve (sn2) C2, diaphragm (dp), and ureter (ur) in the center of the pelvis of the kidney (ki). b. Anterior cerebral artery (aca) and pineal gland anlage (pg). Note the commissural fibers (cf), and adenohypohysis (ah). c. Nerve fibers innervating the vibrissae (vf) and cranial nerves IX, X, XI inside and below the jugular foramen. Note the ganglion of the vagus nerve (arrowhead), trigeminal ganglion (tg), and the eye muscles that surround the optic stalk (os). d. Urethra (ura), rectum (rec), and urinary bladder (ub). Note the distally enlarged mesonephric duct (md). e. Larynx (la). Note the laryngopharynx (lp) and the remnants of the notochord (nco) in the forming vertebral column. f. Lumbar part of diaphragm (pld). Note the openings for esophagus (e) and descending aorta (da). g. Eye (eb) and shoulder. Note the infraspinatus muscle below the spina scapulae (scs). h. Radius (ra) and ulna (ua), tibia (ti) and fibula (fi). Note the cubital joint and the knee joint.

Supplementary Fig. 3.
Virtual coronal re-sections through HREM data of wild-type E14.5 mouse embryos. Selected important features are labelled. a. Resection through the embryo displayed in the inlay on the left. Note the section plane (p). Scalebar = 1 mm. b. Magnification of the brain as boxed in a. c. Magnification of the region boxed in b. Note the fibers of the internal capsule (ic), choroid plexus (cp) of the 2 nd ventricle, and middle cerebral artery (mca). d. Magnification of the pelvis as boxed in a. Note the pancreas (pa), kidneys (ki), and right gonad (go). e. Magnification of the region boxed in d. Note the para-aortic bodies (pmg), ureter (ur), and abdominal aorta (aa). f. brain, palatine (pa), and eye (eb). Note the forming caudate nucleus (ca) and thalamus (th). g. cranial nerve 7 (VII). Inlay shows a close up of the nerve (arrowhead), where it leaves the rhombencephalon (rho). h. Cranial nerve 8 (VIII). Pars cochlearis fibers enter the modiolus of the cochlea (co).

Supplementary Fig. 4.
Flow diagram of phenotyping protocol. Analysis proceeds from initial examination of embryo external appearance from 3D modelling to systematic examination of the HREM section image stack (in an overall rostrocaudal sequence). Organs and tissues to be assessed are listed in Table1. After completion of the axial sequence, Virtual resection data corresponding to sagittal and coronal section planes are similarly reviewed, following the checklists outlined (Table 1).

Supplementary Table 1.
Abnormalities diagnosed in the 34 different mouse strains analysed. Note that, according to their developmental stage, all embryos feature palatine cleft. We have therefore only used this term for embryos of developmental stage 23 (Theiler) with palatine plates lateral to their tongues. In addition, many embryos had remnants of the interventricular foramen, an observation that can be considered normal for the examined stages and is not therefore listed in this Abnormal cerebral anterior artery 1 Anal atresia 1

Pds5b
Growth retardation of single organs 3