This allows the generation of high-definition time-lapse movies to explore the cell dynamics of embryonic mouse and chick limb mesenchyme as it undergoes cartilage formation. To visualize the dynamic cellular behaviors of undifferentiated limb mesenchyme as it undergoes chondrogenesis, we modified the micromass system to use lower density cultures to follow individual cells (see Experimental Procedures). Results Generation of a Live Imaging System to Visualize Dynamics of Limb Mesenchymal Cells Undergoing Chondrogenesis This has enabled the functional characterization of limb-skeletal progenitor cells and identified cellular events that are critically required for the formation of a cartilage template controlled by key molecular regulators found mutated in human chondrodysplasia syndromes. Thus, we set out to generate high-definition time-lapse movies to visualize embryonic mouse and chick limb mesenchyme as it undergoes cartilage formation and combined this dynamic imaging with loss-of-function approaches and functional assessment of unique cellular properties of labeled mesenchymal progenitor cells. Despite the potential of this system as a tool for understanding skeletal development, it has not been utilized to study the dynamic cellular events required for skeletal morphogenesis. Over a 3 day period, these cells form cartilage nodules that accurately recapitulate the formation and maturation of the cartilage anlagen during embryonic skeletal development in vivo.
In the micromass system, limb mesenchyme is dissociated into single cells and plated at high density. These findings shed light on the cellular basis for chondrodysplasia syndromes and formation of the vertebrate skeleton. Moreover, we visualized labeled progenitor cells from different regions of the limb bud and identified unique cellular properties that may direct their contribution toward specific skeletal elements such as the humerus or digits. In contrast, Bmp signaling regulates a cellular program we term “compaction” in which mesenchymal cells acquire a cohesive cell behavior required to delineate the boundaries and size of cartilage elements. We uncovered an unsuspected role for Sox9 in control of cell morphology, independent from its major downstream target ColIIa, critically required for the mesenchyme-to-chondrocyte transition. We generated an imaging system to dynamically visualize limb mesenchymal cells undergoing successive phases in cartilage formation and to delineate the cellular function of key regulators of chondrogenesis found mutated in chondrodysplasia syndromes. The cellular events underlying skeletal morphogenesis and the formation of cartilage templates are largely unknown.
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