Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage

Abstract

Cartilaginous structures are at the core of embryo growth and shaping before the bone forms. Here we report a novel principle of vertebrate cartilage growth that is based on introducing transversally-oriented clones into pre-existing cartilage. This mechanism of growth uncouples the lateral expansion of curved cartilaginous sheets from the control of cartilage thickness, a process which might be the evolutionary mechanism underlying adaptations of facial shape. In rod-shaped cartilage structures (Meckel, ribs and skeletal elements in developing limbs), the transverse integration of clonal columns determines the well-defined diameter and resulting rod-like morphology. We were able to alter cartilage shape by experimentally manipulating clonal geometries. Using in silico modeling, we discovered that anisotropic proliferation might explain cartilage bending and groove formation at the macro-scale.DOI: http://dx.doi.org/10.7554/eLife.25902.001

Phosphotungstic acid‐enhanced microCT: Optimized protocols for embryonic and early postnatal mice

“…Analysis of the early development of the chondrocranium and its association with the forming skull has been hampered by the difficulty in visualizing embryonic cartilage in 3D. The recent Kaucka and Tesařová protocols demonstrated that PTA staining could successfully be used to visualize and analyze the chondrocranium, both in its entirety and with specific focus on the nasal capsule, in embryonic mice . Our expansion of the Kaucka and Tesařová protocols allows the segmentation of bone from the same PTA‐enhanced microCT scan, allowing the creation of high‐resolution 3D models of the forming dermatocranium and chondrocranium (Figure ).…”

“…Furthermore, the majority of published protocols provide only minimal details, requiring extensive experimentation to reach an optimized protocol, particularly if using a different model organism, specimen age, or tissue type. One research group has published detailed PTA protocols for use in embryonic mice from E12.5 to 18.5 . The Kaucka and Tesařová protocols added several significant steps to Metscher's original PTA staining method: a slow graded ethanol and methanol dehydration series, different suggested staining times for several ages of embryonic mice, and a final graded methanol rehydration series.…”

“…The Kaucka and Tesařová protocols added several significant steps to Metscher's original PTA staining method: a slow graded ethanol and methanol dehydration series, different suggested staining times for several ages of embryonic mice, and a final graded methanol rehydration series. The slow dehydration and rehydration steps were added to help minimize soft tissue shrinkage, addressing many of the concerns with iodine staining. With these modifications, the Kaucka and Tesařová protocols provided excellent contrast enhancement, particularly for embryonic murine cranial soft tissues and cartilage .…”

“…The slow dehydration and rehydration steps were added to help minimize soft tissue shrinkage, addressing many of the concerns with iodine staining. With these modifications, the Kaucka and Tesařová protocols provided excellent contrast enhancement, particularly for embryonic murine cranial soft tissues and cartilage . However, these protocols vary between publications, making it difficult to elect an optimum method.…”

“…However, these protocols vary between publications, making it difficult to elect an optimum method. For example, E15.5 embryos were either stained for 6 days with 0.7% PTA solution or 3 weeks with 1.0% to 1.5% PTA solution . These differences may reflect differences in the specific research goals for the stained specimens.…”