A population level atlas of Mus musculus craniofacial skeleton and automated image-based shape analysis

Laboratory mice are staples for evo/devo and genetics studies. Inbred strains provide a uniform genetic background to manipulate and understand gene-environment interactions, while their crosses have been instrumental in studies of genetic architecture, integration and modularity, and mapping of complex biological traits. Recently, there have been multiple large-scale studies of laboratory mice to further our understanding of the developmental basis, evolution, and genetic control of shape variation in the craniofacial skeleton (i.e. skull and mandible). These experiments typically use micro-computed tomography (micro-CT) to capture the craniofacial phenotype in 3D and rely on manually annotated anatomical landmarks to conduct statistical shape analysis. Although the common choice for imaging modality and phenotyping provides the potential for collaborative research for even larger studies with more statistical power, the investigator (or lab-specific) nature of the data collection hampers these efforts. Investigators are rightly concerned that subtle differences in how anatomical landmarks were recorded will create systematic bias between studies that will eventually influence scientific findings. Even if researchers are willing to repeat landmark annotation on a combined dataset, different lab practices and software choices may create obstacles for standardization beyond the underlying imaging data. Here, we propose a freely available analysis system that could assist in the standardization of micro-CT studies in the mouse. Our proposal uses best practices developed in biomedical imaging and takes advantage of existing open-source software and imaging formats. Our first contribution is the creation of a synthetic template for the adult mouse craniofacial skeleton from 25 inbred strains and five F1 crosses that are widely used in biological research. The template contains a fully segmented cranium, left and right hemi-mandibles, endocranial space, and the first few cervical vertebrae. We have been using this template in our lab to segment and isolate cranial structures in an automated fashion from a mixed population of mice, including craniofacial mutants, aged 4-12.5 weeks. As a secondary contribution, we demonstrate an application of nearly automated shape analysis, using symmetric diffeomorphic image registration. This approach, which we call diGPA, closely approximates the popular generalized Procrustes analysis (GPA) but negates the collection of anatomical landmarks. We achieve our goals by using the open-source advanced normalization tools (ANT) image quantification library, as well as its associated R library (ANTsR) for statistical image analysis. Finally, we make a plea to investigators to commit to using open imaging standards and software in their labs to the extent possible to increase the potential for data exchange and improve the reproducibility of findings. Future work will incorporate more anatomical detail (such as individual cranial bones, turbinals, dentition, middle ear ossicles) and more diversity into the template.