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 3µm/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.

Keywords: Embryonic lethal screening; IMPC; MicroCT.

Fig. 1

Early postnatal to early post-implantation mouse samples imaged by iodine contrast microCT. (A–F) Surface rendering and (A′–F′) a sagittal cross-section of mouse postnatal at P3 to embryonic at E9.5 samples generated by iodine contrast 3D volumes acquired on microCT.

Fig. 2

High resolution 2D virtual sectioning of reconstructed E12.5 and E9.5 microCT datasets through different body planes and through the developing heart. Surface rendering and digital sections of the 3D volumes from sagittal, coronal, and transverse planes of both (A – D) E12.5 and (I – L) E9.5 embryos, as well as transverse sections through the heart at four levels from the anterior to the posterior on both (E – H) E12.5 and (M – P) E9.5 embryos. The high spatial resolution and clear contrast images make is possible to clearly define anatomical features for both E12.5 and E9.5 stages. V: ventricles of the brain. FB: forebrain. MB: midbrain. HB: hindbrain. IP: infundibulum of pituitary. RP: Rathke's pouch. PC: primordial cartilage of the vertebrae. L: liver. SC: spinal cord. DRG: dorsal root ganglion. RA: right atrium. LA: left atrium. RV: right ventricle. LV: left ventricle. TA: thoracic aorta. PT: pulmonary trunk. OFT: outflow tract. TM: trabeculated myocardium. ACV: anterior cardinal veins. S: interventricular septum. PC: pericardial cavity. IG: interventricular groove. HT: heart tube. OV: otic vesicle. BA: first branchial arches. NT: neural tube. DA: dorsal aorta. AT: common atrium. Arrowhead: trabeculae.

Fig. 3

MicroCT imaging of E10.5 to E8.5 mouse embryos within extra-embryonic structures. Iodine contrast E10.5 embryo within yolk sac imaged on microCT revealed (A) the remodeled vasculature on the yolk sac and (B) the orientation of the embryo within the yolk sac. E9.5 embryo imaged within decidua shows (C) the connection of vitelline vein/artery (pseudo-colored in blue/pink) and umbilical vein/artery (pseudo-colored in pink/blue) to yolk sac and placenta and (D) 2D virtual section of the E9.5 embryo within decidua reveals the diameter and length of remodeled umbilical vessels. (E) E8.5 embryo imaged within decidua shows the original orientation of the unturned embryo with the allantois extending from the tail toward the chorion. (F) 2D virtual section of an E8.5 embryo shows the sharp boundary of the somites (pseudo-colored in yellow) and how the allantois extends to the chorion (pseudo-colored in purple).

Fig. 4

Rad9a knockout mouse is embryonic lethal with severe developmental defects at E9.5. (A) Schematic diagram of the wild type and the knockout alleles of Rad9a. Red line under each allele indicates the amplicon region used for genotyping primer design. (B) PCR result of the iodine contrast sample to confirm genotypes. (C and D) The entire litter from a heterozygote × heterozygote Rad9a^{tm1.1(KOMP)Wtsi} cross were imaged within deciduum. (E and F) Embryos within yolk sacs and (G and H) by themselves along with the umbilical cord connecting to the placenta were digitally segmented out from the original data set. (E and F) Rad9a^{tm1.1(KOMP)Wtsi} null embryo is smaller in size with un-remodeled yolk sac vasculature compared to heterozygous littermate. (G and H) At E9.5, Rad9a null embryo did not turn, the anterior neural folds were still open, the ventral midgut region remained open, and the posterior part of the null embryo is severely dysmorphic. (I and J) The heart tube in Rad9a^{tm1.1(KOMP)Wtsi} remained linear without undergoing further looping process compared to the left/right atrium and ventricles formed in heterozygote.