Background Given the need for descriptive and increasingly mechanistic morphological analyses, contrast‐enhanced microcomputed tomography (microCT) represents perhaps the best method for visualizing 3D biological soft tissues in situ. Although staining protocols using phosphotungstic acid (PTA) have been published with beautiful visualizations of soft tissue structures, these protocols are often aimed at highly specific research questions and are applicable to a limited set of model organisms, specimen ages, or tissue types. We provide detailed protocols for micro‐level visualization of soft tissue structures in mice at several embryonic and early postnatal ages using PTA‐enhanced microCT. Results Our protocols produce microCT scans that enable visualization and quantitative analyses of whole organisms, individual tissues, and organ systems while preserving 3D morphology and relationships with surrounding structures, with minimal soft tissue shrinkage. Of particular note, both internal and external features of the murine heart, lungs, and liver, as well as embryonic cartilage, are captured at high resolution. Conclusion These protocols have broad applicability to mouse models for a variety of diseases and conditions. Minor experimentation in the staining duration can expand this protocol to additional age groups, permitting ontogenetic studies of internal organs and soft tissue structures within their 3D in situ position.

The cranial endo- and dermal skeletons, which comprise the vertebrate skull, evolved independently over 470 million years ago and form separately during embryogenesis. In mammals, much of the cartilaginous chondrocranium is transient, undergoing endochondral ossification or disappearing, so its role in skull morphogenesis is not well studied and it remains an enigmatic structure. We provide complete three-dimensional (3D) reconstructions of the laboratory mouse chondrocranium from embryonic day 13.5 through 17.5 using a novel methodology of uncertainty-guided segmentation of phosphotungstic enhanced 3D microcomputed tomography images with sparse annotation. We evaluate the embryonic mouse chondrocranium and dermatocranium in 3D and delineate the effects of a Fgfr2 variant on embryonic chondrocranial cartilages and on their association with forming dermal bones using the Fgfr2cC342Y/+ Crouzon syndrome mouse. We show that the dermatocranium develops outside of and in shapes that conform to the chondrocranium. Results reveal direct effects of the Fgfr2 variant on embryonic cartilage, on chondrocranium morphology, and on the association between chondrocranium and dermatocranium development. Histologically we observe a trend of relatively more chondrocytes, larger chondrocytes, and/or more matrix in the Fgfr2cC342Y/+ embryos at all timepoints before the chondrocranium begins to disintegrate at E16.5. The chondrocrania and forming dermatocrania of Fgfr2cC342Y/+ embryos are relatively large, but a contrasting trend begins at E16.5 and continues into early postnatal (P0 and P2) timepoints, with the skulls of older Fgfr2cC342Y/+ mice reduced in most dimensions compared to Fgfr2c+/+ littermates. Our findings have implications for the study and treatment of human craniofacial disease, for understanding the impact of chondrocranial morphology on skull growth, and potentially on the evolution of skull morphology.

Crouzon syndrome is an autosomal dominant condition characterized by craniofacial anomalies in the absence of major hand and foot abnormalities. Although premature closure of the coronal suture is the focus of most studies of Crouzon syndrome, cartilage elements, particularly within the braincase floor have also been described as anomalous. The Fgfr2cC342Y/+ Crouzon syndrome mouse model carries a cysteine to tyrosine substitution at amino acid position 342 (Cys342Tyr; C342Y) in Fgfr2 equivalent to one of the FGFR2 mutations commonly associated with Crouzon syndrome. This mutation results in constitutive activation of the receptor and is associated with up‐regulation of osteogenic differentiation. Skeletogenesis of the skull and facial bones which are affected in these mice is preceded by development of the chondrocranium, a cartilaginous skull whose elements either disappear, remain as cartilage, or ossify endochondrally. Consequently, normal growth and development of the skull relies on appropriate chondrocyte activity including controlled proliferation. We analyzed proliferative activity of chondrocytes of the braincase floor and vomeronasal region of E14.5 and E15.5 Fgfr2cC342Y/+ mice and unaffected littermates to determine the effects of the mutation on the proliferation of chondrocytes. Relative to chondrocytes of unaffected littermates, chondrocytes of Fgfr2cC342Y/+ mice are delayed in proliferative potential as there is less proliferation in mutant chondrocytes of the braincase floor (p≤0.001) and para‐septal cartilages (p≤0.001) at E14.5 and more proliferation at E15.5 (para‐septal cartilage p≤0.001). There are no statistically significant differences in proliferative potential within the nasal septum at either age investigated. Disruption to the tightly regulated developmental processes in the chondrocranium can have significant and lasting effects on craniofacial development. Thus, expanding this investigation with additional developmental ages will help ascertain how the function of this FGFR mutation in embryonic cartilage contributes to the abnormal craniofacial form associated with Crouzon Syndrome.

Acquiring three‐dimensional imaging of animal specimens utilized in genetic, developmental, and evolutionary studies has typically been based on microCT (bone and mineralized tissues) and magnetic resonance microscopy (soft and un‐mineralized tissues). However, recent advances in microCT imaging have allowed for 1–5 μm resolution for small biological specimens, while typical microMRI imaging can generally only produce data with a resolution of approximately 30–40 μm. In order to achieve high‐resolution 3D visualization of soft tissues, the use of various contrast agents have been proposed, including iodine, osmium, and phosphotungstic acid (PTA). We investigated protocols for using PTA to stain soft tissues of the head in embryonic and early postnatal mice to enhance visualization of soft tissues by microCT imaging. Protocols for mice between embryonic day 13.5 and postnatal day 7 have been developed that allow for the visualization, segmentation, quantification, and analysis of soft tissue structures. Stained specimens were mounted in a 50:50 mix of polyester and paraffin waxes within a small tube and scanned on the GE v|tome|x L300 scanner in the Center for Quantitative Imaging at Pennsylvania State University with voxel dimensions between 1 and 10 μm, depending on specimen size. Multiple soft tissue structures, including the eye, inner ear, cartilage, muscle, glandular tissue, and brain, are easily differentiated in the resultant scans. Images of PTA‐stained specimens can be superimposed with microCT images of the same specimen before PTA staining to enable the study of relationships between hard and soft tissues in the same individual. This approach to both 2D and 3D visualization of soft tissues in murine models has immediate implications for understanding developmental processes and integration between hard and soft tissues of the cranium.Support or Funding InformationNational Science Foundation Grant BCS‐1731909, and NIDCR R01DE027677 and R01 DE022988This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differences in material characteristics of cartilage and bone are primarily known through investigation of postnatal specimens but the mechanical properties of these tissue during embryogenesis are less well studied. Importantly, embryonic cartilage and bone have not been tested extensively in aqueous environments, limiting the validity of previous investigations. The purpose of this project is to characterize the stiffness and strength of embryonic cranial bone and cartilage using atomic force microscopy (AFM). We test the hypothesis that embryonic cartilage is stronger than embryonic bone against the null hypothesis that embryonic tissue response will be comparable to adult tissue in which bone is the stronger of the two tissues. In vivo conditions are simulated by immersing tissues in room‐temperature phosphate‐buffered saline before and during testing. Data are acquired using calibrated AFM probes which yield quantitative and qualitative data. Force‐distance curves include data on topography, adhesion, and DMTModulus, the latter of which can be transformed into Young’s Modulus through models which approximate the shape of the AFM probe, including the Sneddon model. Calcified tissue intended for testing is verified on the micro scale using AFM topography and on the macro scale using calcein dye. Each 2 square micron topographical image is accompanied by 16,384 force versus distance curves which yield a value for modulus of elasticity. Data show DMTModulus values ranging from approximately 200 kPa to 7.7 MPa for embryonic cartilage and 120 kPa to 3.0 MPa for embryonic bone providing preliminary data indicating that cartilage varies more than bone and may be significantly stronger than bone in mouse embryos.