Abstract
One of the greatest enigmas of modern biology is how the geometry of muscular and skeletal structures are created and how their development is controlled during growth and regeneration. Scaling and shaping of vertebrate muscles and skeletal elements has always been enigmatic and required an advanced technical level in order to analyse the cell distribution in 3D. In this work, synchrotron X-ray computed microtomography (µCT) and chemical contrasting has been exploited for a quantitative analysis of the 3D-cell distribution in tissues of a developing salamander (Pleurodeles waltl) limb – a key model organism for vertebrate regeneration studies. We mapped the limb muscles, their size and shape as well as the number and density of cells within the extracellular matrix of the developing cartilage. By using tomographic approach, we explored the polarity of the cells in 3D, in relation to the structure of developing joints. We found that the polarity of chondrocytes correlates with the planes in joint surfaces and also changes along the length of the cartilaginous elements. Our approach generates data for the precise computer simulations of muscle-skeletal regeneration using cell dynamics models, which is necessary for the understanding how anisotropic growth results in the precise shapes of skeletal structures.
Highlights
One of the greatest enigmas of modern biology is how the geometry of muscular and skeletal structures are created and how their development is controlled during growth and regeneration
The conventional X-ray μCT was exploited for overview experiments and the 3D visualization of developing P. waltl limbs stained with phosphotungstic acid (PTA)
We have demonstrated a novel, technical approach allowing for the quantitative analysis of polarization and 3D cell distribution inside the whole developing muscle-cartilaginous units from a regenerative animal model
Summary
One of the greatest enigmas of modern biology is how the geometry of muscular and skeletal structures are created and how their development is controlled during growth and regeneration. Advanced light microscopy techniques can image thicker biological specimens at a high spatial resolution such as confocal microscopy, multiphoton microscopy, and optical coherence tomography[1]. Confocal microscopy is considered to be one of the most convenient techniques for imaging cells in 3D This method has been used for the study of cell columns in the articular cartilage of rats[2]. The auto-fluorescent properties of the cells can provide sufficient contrast to allow for the identification of the desired structures[1] They cannot be used for all types of tissues, which can limit the utilization of this technique. Imaging techniques based on the scattering of light are not suitable for imaging larger samples, e.g. the whole limbs of vertebrates. Despite the high spatial resolution for the whole body of an embryo, the method is destructive and highly demanding in terms of sample preparation
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