It's about Time: Ossification Center Formation in C57BL/6 Mice from E12⁻E16

Kevin Flaherty, Joan T Richtsmeier, Kevin Flaherty, Joan T Richtsmeier

Abstract

The establishment of precise, high-resolution temporal sequences for morphogenetic events in laboratory mice remains a vexing issue in developmental biology. Mouse embryos collected at the same period of gestation, even those from the same litter, show wide variation in individual levels of progress along their developmental trajectory. Therefore, age at harvest does not provide sufficient information about developmental progress to serve as the basis for forming samples for the study of rapidly or near-simultaneously occurring events such as the sequence of ossification center formation. Here, we generate two measures of individual developmental progress (developmental age) for a large sample of mouse embryos using crown⁻rump lengths that measures size, and limbstaging ages produced by the embryonic Mouse Ontogenetic Staging System (eMOSS) that measure shape. Using these measures, we establish fine-grained sequences of ossification center appearance for mouse embryos. The two measures of developmental progress generate slightly different sequences of ossification center formation demonstrating that despite their tight correlation throughout the developmental period, size and shape are aspects of form that are at least partially dissociated in development.

Keywords: OSX; eMOSS; embryogenesis; heterochrony; morphometrics; mouse models; ossification center; staging system.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Crown-rump length measures the maximum total length of an embryo using a sagittal view.
Figure 2
Figure 2
Plot of Crown-rump length by age at harvest. Dots represent individual specimens and are colored according to age at harvest.
Figure 3
Figure 3
Plot of limbstaging age by age at harvest. Dots represent individual specimens and are colored according to age at harvest.
Figure 4
Figure 4
Deviation between age at harvest and limbstaging age for E12–E15. Each column represents a litter, with each data point representing an individual embryo. Colors represent age at harvest. Scores on the y-axis represent the difference between the expected age of an embryo based on age at harvest and limbstaging age for each individual.
Figure 5
Figure 5
Graph of crown–rump length and limbstaging age. Dots represent individual specimens and are colored according to age at harvest.
Figure 6
Figure 6
Appearance of frontal and parietal bones in Osx-GFP mice ordered by age at harvest, CRL, and limbstaging age. Each image shows a lateral view of the cranial vault of a left-facing Osx-GFP embryo, with Osx expression revealed by green fluorescent protein. The frontal and parietal bones and the eye are outlined in white to show the extent of Osx expression in each bone. The boxes outlining each specimen are colored according to age at harvest. Each age group is represented by a single litter. (A) specimens ordered according to age at harvest; (B) specimens ordered according to CRL; (C) specimens ordered according to limbstaging age.
Figure 7
Figure 7
Timeline of ossification center appearance marked by osteoblast differentiation (expression of Osx) generated using Osx-GFP mice ordered according to: (A) age at harvest; (B) limbstaging age; and (C) CRL.
Figure 8
Figure 8
Timeline of ossification center appearance marked by mineralization of osteoid (marked by calcein staining) generated using C57BL/6-calcein mice ordered according to: (A) age at harvest; (B) limbstaging age; and (C) CRL.

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