Using developmental rate variation to establish a fine‐grained sequence of ossification center differentiation

Abstract

Mouse embryos from inbred strains exhibit a large range of phenotypic diversity, despite tight controls placed on genetic variation and environmental conditions. Individuals vary in terms of body size and the progress of developmental morphology, even within litters. This indicates that individual embryos develop at different rates. Staging atlases such as those developed by Kaufman and Theiler provide details of the morphological features and developmental processes evident on successive days of development, but do not offer a systematic method for determining if two embryos represent similar developmental stages within more fine‐grained time periods. To ensure that studies of developmental processes like tissue interactions and gene expression events that occur within restricted periods of a few hours are not confounded by inter‐individual variation in rates of development, precise temporal control is needed to sequentially order and contextualize data collected from individual embryos. We collected and compared temporal sequences of ossification center appearance and development using two measures of individual development: size, measured as crown‐rump length, and shape, using the eMOSS staging system (http://www.limbstaging.crg.es/) that generates estimates of individual ages based on the 2D outline of the hindlimb.Using crown‐rump length and the eMOSS system, we demonstrate significant developmental variation within litters, and significant disparity between an embryo's age based on the age at collection and age generated from eMOSS. Using eMOSS, we are able to elucidate the sequence of bone development in unprecedented detail. We recorded the appearance of ossification centers by scoring the presence of the critical osteoblast differentiation gene Osterix in OSX‐GFP mice sacrificed at 6 hour increments from E12.0 to E15.0 and scored the appearance of calcified bone by staining C57Bl/6 embryos between E14.0 and E16.0 with calcein. We improved the understanding of the temporal sequence and precise timing of these developmental events by generating an ossification center development sequence that details the appearance of bones according to the overall body size, and more importantly, the developmental hour at which they appeared.Support or Funding InformationThis work is supported in part by the NIH: Craniosynostosis Network P01HD078233 & R01DE018500, R01DE022988; NSF BCS‐1650824; and the Burroughs Wellcome Fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.