EMOSS: A High Resolution Staging System for Mouse Embryos Aged E10–E15
Authors: Flaherty Kevin , Musy Marco , Sharpe James , Richtsmeier Joan 
Published: November 2023

Mice represent a powerful tool for research and experimentation within the embryological period. However, previous researchers have found considerable inter‐individual variability in the rate of embryonic development, even within inbred strains. This leads to discrepancies between chronological age, an external measure of the time elapsed since conception for a litter, and developmental age, a relative measure of the degree of progress achieved by each individual along its ontogenetic trajectory. While this discrepancy between chronological and developmental age presents a potential confounding factor for experimental research within the embryonic period, it also represents an opportunity to provide context to the results of such research if developmental age can be measured with sufficient resolution. We present eMOSS (embryonic Mouse Ontogenetic Staging System), a robust, high‐resolution method for estimating the developmental age of mouse embryos aged between embryonic day 9 and embryonic day 15 (E9–E15). eMOSS uses two‐dimensional hindlimb outlines to generate a point estimate of the developmental age of an embryo in hours, as well as age range and error estimates. The tool is freely and publicly available as a web applet at http://limbstaging.crg.es/. eMOSS presents three crucial advantages over previous staging systems: 1) it offers vastly increased temporal resolution, measuring developmental age in embryonic hours instead of days, 2) it is non‐destructive, requiring only an image of the hindlimb, and 3) it is a quantitative examination of a simple anatomical shape, which makes the system resistant to inter‐observer error and easy to implement. This system offers particular utility for research into gene and tissue interactions that occur during brief intervals during the embryonic period that can consequently be obscured or confounded by developmental age variability between individuals.We present data from an analysis using the eMOSS system with a large sample (n=289) of C57Bl/6J mouse embryos produced via timed matings at 24 hour intervals, and aged between E12–E15. Our results reveal the structure of developmental age variation within this period, with relatively high age variation among the younger mice in the sample (E12) and greatly reduced age variation in the older mice in the sample (E15). Despite the fact that crown‐rump length (a commonly used developmental age estimate) and developmental age are highly correlated for the whole sample (r2=0.87), crown‐rump length is a relatively poor predictor of developmental age within each age group. This relationship is due to increased variation in crown‐rump length during the later portion of the sample period coupled with a concurrent decrease in developmental age variation.Support or Funding InformationThis work is supported by Public Health Service Awards R01‐DE018500, ARRA 3R01DE018500‐S1, R01‐DE022988, P01HD078233; the National Science Foundation BCS‐0725227; and the Burroughs Wellcome Collaborative Research Travel Grant (BW Fund 2013).