Three-dimensional microCT imaging of mouse development from early post-implantation to early postnatal stages

Abstract

In this work, we report the use of iodine-contrast microCT to perform high-throughput 3D morphological analysis of mouse embryos and neonates between embryonic day 8.5 to postnatal day 3, with high spatial resolution up to 3 μm/voxel. We show that mouse embryos at early stages can be imaged either within extra embryonic tissues such as the yolk sac or the decidua without physically disturbing the embryos. This method enables a full, undisturbed analysis of embryo turning, allantois development, vitelline vessels remodeling, yolk sac and early placenta development, which provides increased insights into early embryonic lethality in mutant lines. Moreover, these methods are inexpensive, simple to learn and do not require substantial processing time, making them ideal for high throughput analysis of mouse mutants with embryonic and early postnatal lethality.

Loss of Apela Peptide in Mice Causes Low Penetrance Embryonic Lethality and Defects in Early Mesodermal Derivatives

“…We also performed phenotypic analysis with three-dimensional micro computed tomography (3D microCT) of KO NEO-IN embryos at E8.25 and E9.5 (Hsu et al, 2016). Digital sections confirmed the heart looping defect at E9.5 (Figure 4C).…”

“…Iodine contrast was achieved by immersion in 0.1N (v/v) iodine solution (Sigma) O/N at room temperature. Images were captured with a SKYSCAN 1272 microCT scanner (Bruker) and digitally segmented on CTAn (Hsu et al, 2016). Decidua were isolated from Apela KO NEO-IN intercrosses (for mutant analysis) or Apela KO NEO-IN males crossed to CD-1 females (for controls).…”

Phosphotungstic acid‐enhanced microCT: Optimized protocols for embryonic and early postnatal mice

“…Both iodine and PMA have been reported to inadequately stain the brain and associated structures within the endocranial cavity . Additionally, iodine protocols have reported shrinkage that results in spacing and separation between the skull and brain . PTA's ability to successfully stain the brain and permit specific layer identifications was recognized early by Metscher and has further proven to provide superior contrast enhancement and definition of brain regions in a bumblebee, which far exceeded that provided by iodine …”

“…These clear boundaries permit the segmentation, quantification, and analysis of both the brain and the ventricular system. In contrast to the iodine protocol used by Hsu et al, our protocol produces specimens in which the brain remains closely approximated with the skull, suggesting a reduced degree of tissue shrinkage and/or lack of deformation of the brain.…”

“…Balint et al reported generalized soft tissue shrinkage with morphology altering deformation in iodine‐stained porcine ligaments, occurring to a greater extent relative to ligaments stained with either PTA or PMA . Even more recent protocols that involve a tissue‐stabilization step prior to iodine staining still note observable tissue shrinkage, which may affect morphological analyses …”