Quantifying three-dimensional morphology and RNA from individual embryos

Abstract

Quantitative analysis of morphogenesis aids our understanding of developmental processes by providing a method to link changes in shape with cellular and molecular processes. Over the last decade, many methods have been developed for 3D imaging of embryos using microCT scanning to quantify the shape of embryos during development. These methods generally involve a powerful, cross-linking fixative such as paraformaldehyde to limit shrinkage during the CT scan. However, the extended time frames that these embryos are incubated in such fixatives prevent use of the tissues for molecular analysis after microCT scanning. This is a significant problem because it limits the ability to correlate variation in molecular data with morphology at the level of individual embryos. Here we outline a novel method that allows RNA, DNA, or protein isolation following CT scan while also allowing imaging of different tissue layers within the developing embryo. We show shape differences early in craniofacial development (E11.5) between common mouse genetic backgrounds, and demonstrate that we are able to generate RNA from these embryos after CT scanning that is suitable for downstream real time PCR (RT-PCR) and RNAseq analyses. Developmental Dynamics 246:431-436, 2017. © 2017 Wiley Periodicals, Inc.

Keywords: PaxGene; RNA genotype-phenotype relationship; iodine; microCT.

Figure 1

Different fixation methods effect size. A) Canonical Variates Analysis of the two mouse strains by two fixation methods. The first Canonical Variate separates group by fixation and the second by strain. B–C) Heat maps showing the changes across CV1 (B) and CV2 (C). Changes are the difference between the −5 end of the CV and the +5 end of the CV. Note that there are large negative changes in the top of the head in CV1 as CV value moves toward the PFA fixation. D) Plot of tail somite number versus centroid size.

Figure 2

Shape differences between groups following size regression. A) Principal Components Analysis showing the overall variation of all data. B) Canonical Variates Analysis with grouping by the two mouse strains and the two fixation methods. The first Canonical Variate separates group by strain. C–D) Displacement maps showing the difference at each landmark position between CV1 −5 (blue) and CV +5 (Red). Dorsal (D) is up, Ventral (V) is down. C) Frontal view (L -left, R - right) and D) lateral view (A - anterior, P - posterior). E–F) Color map of the same changes.

Figure 3

Analysis of RNA following CT scanning. A) Graph of the amplification plots for each Gapdh, Camkmt and Six2. Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye. Negative control wells show no amplification. B) Relative levels of Camkmt and Six2 across the groups. C) RNA integrity analysis from the BioAnalyzer, showing the clear 18S, 26S bands as well as a lower marker.

Figure 4

Embryo images. A-C) Segment images taken at various angles, D) Projection view of embryo.