Dynamic imaging of the growth plate cartilage reveals multiple contributors to skeletal morphogenesis

The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here, we perform a quantitative analysis of cell behaviors in the growth plate cartilage, the template for long bone (limb skeleton) formation, to gain insights into this process. The proliferative zone (PZ) is comprised of elliptical chondrocytes aligned perpendicular to the proximal-distal axis (PDA) of the tissue, suggesting some coordination between oriented cell behaviors and oriented tissue growth. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe PZ cell behaviors during cartilage elongation in 4D (xyz and t). These time-lapse movies reveal distributed growth in the PZ, resulting in cell trajectories with a spreading displacement mainly along the PDA. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular morphogenetic processes that contribute to the cell trajectories. Surprisingly, convergent extension (CE) and cellular column formation are not the major contributors of the stages examined; instead, the analyses reveal important roles of extracellular matrix (ECM) deposition and cell volume enlargement