Quantifying muscle architecture in embryos using diceCT and algorithmic fascicle tracking

Advances in soft-tissue imaging and muscle reconstruction tools have greatly expanded our capacity to extract myological properties relating to function. Recently, the development of semi-autonomous fascicle tracking algorithms has permitted in situ measurements of fiber lengths and orientation. While these tools have been applied to postnatal, predominantly adult vertebrate specimens, their efficacy has not been demonstrated on embryonic specimens, which possess smaller and less developed muscle tissues. If fascicle tracking algorithms could be extended successfully to embryonic specimens, then life history changes to muscle action and function could be recorded in situ and in high fidelity from the onset of muscle contraction. In this study, we present a successful implementation of a fascicle tracking tool on jaw adductor and depressor muscles in a domestic chick embryo (Gallus gallus domesticus). Comparisons of algorithmic and manual fascicle reconstructions show visual and quantitative validation of the protocol. Compared with results from adult chickens, jaw muscles in embryos were not as uniformly oriented, and the muscles that close the jaw had relatively small physiological cross-sectional areas. This result implies that the growth trajectory is influenced by feeding requirements, such as bite force. We also report an artifact with the fascicle tracking method, where fascicle lengths appear shorter in smaller, thinner muscles relative to measurements based on manual segmentation of the image data. Nevertheless, fascicle orientations are congruent with those extracted from manual segmentation, even for the smallest muscles. Taken together, we demonstrate that an existing tool for semi-automated fascicle tracking is extensible to embryonic specimens. As such, the approach presented here paves a new path for investigating form-function relationships and the effect of muscle action on other tissues, such as bone, from the earliest stages of muscle contractions.