Quantifying three-dimensional morphology and RNA from individual embryos.
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.
Department of Cell Biology and Anatomy, Alberta Children's Hospital Research Institute, McCaig Institute for Bone and Joint Health, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.
Rebecca M
0000-0001-7319-0755
RM
Green
Department of Cell Biology and Anatomy, Alberta Children's Hospital Research Institute, McCaig Institute for Bone and Joint Health, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.
Courtney L
CL
Leach
Department of Cell Biology and Anatomy, Alberta Children's Hospital Research Institute, McCaig Institute for Bone and Joint Health, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.
Natasha
N
Hoehn
Zuckerberg San Francisco General Hospital, Orthopaedic Trauma Institute, University of California, San Francisco, San Francisco, CA.
Ralph S
RS
Marcucio
Department of Cell Biology and Anatomy, Alberta Children's Hospital Research Institute, McCaig Institute for Bone and Joint Health, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada.
Benedikt
B
Hallgrímsson
D000818:Animals; D004622:Embryo, Mammalian; D047108:Embryonic Development; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008722:Methods; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009024:Morphogenesis / Q000235:genetics / Q000502:physiology; D012313:RNA / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017421:Sequence Analysis; D055114:X-Ray Microtomography
D012313:RNA
PaxGene; RNA genotype-phenotype relationship; iodine; microCT