Live Growth Plate Cartilage Imaging to Study Proliferative Column Formation

长骨和软骨内颅底的生长是由生长板软骨中软骨细胞成熟的时空控制而产生的。这一成熟过程产生了具有不同细胞形态和基因表达谱的结构域。遗传学研究提供证据表明，适当的结构域结构对生长板的功能至关重要，而这种结构域结构容易受到损伤或疾病的损害。软骨细胞成熟的一个重要过程是圆形前体细胞重组为盘状增殖软骨细胞，通过克隆扩增形成柱状软骨细胞。先前的研究支持一种模型，其中增殖软骨细胞通过顺序平面分裂和细胞嵌入以类似于收敛延伸的方式形成柱。一个重要的警告是，这些研究，以及在生长板中提出的细胞行为模型，是基于对固定组织切片的静态图像的分析。动态信息的缺乏降低了模型的可信度，并限制了遗传数据的机械解释。本建议利用最近在实验室开发的体外实时成像系统来阐明调节增殖软骨细胞柱形成的机制。具体来说，目的是为了验证这样一个假设，即在子细胞界面上的相互作用驱动的收敛扩展过程中，细胞插入会形成柱。这一假设将通过实时成像以及遗传学和化学遗传学方法进行验证，以检测子细胞中板足的存在，确定肌动蛋白动力学和整合素功能是否需要柱的形成，并确定子细胞的过早分离是否会阻止Piga突变型增殖软骨细胞中的细胞重排。这些研究结果将为组织工程生成软骨结构提供重要的新信息，并将使这种创新方法成为研究生长控制机制的重要新工具。

The growth of long bones and the endochondral cranial base is generated by spatial and temporal control of chondrocyte maturation in the growth plate cartilage. This maturation process gives rise to domains with distinct cell morphologies and gene expression profiles. Genetic studies provide evidence that proper domain architecture is crucial to growth plate function, and this architecture is susceptible to damage via injury or disease. One important event in chondrocyte maturation is the reorganization of round precursor cells into discoid proliferative chondrocytes that form columns via clonal expansion. Previous studies support a model in which column formation by proliferative chondrocytes occurs through sequential planar division and cell intercalation in a manner similar to convergent extension. One important caveat is that these studies, and the proposed models of cell behavior in the growth plate, are based on the analysis of static images from sections of fixed tissue. The absence of dynamic information reduces confidence in the models and places limits on the mechanistic interpretation of genetic data. This proposal utilizes an in vitro live imaging system recently developed in the laboratory to elucidate the mechanisms that regulate column formation in proliferative chondrocytes. Specifically, the aims are designed to test the hypothesis that column formation occurs by cell intercalation in a convergent extension process that is driven by interactions at the interface of daughter cells. This hypothesis will be tested using live imaging, together with genetic and chemical-genetic methods, to detect the presence of lamellopodia in daughter cells, to determine if actin dynamics and integrin function are required for column formation, and to determine if premature separation of daughter cells prevents cell rearrangement in Piga mutant proliferative chondrocytes. Results of these studies will provide important new information for the generation of cartilage architecture by tissue engineering and will establish this innovative method as a crucial new tool for study of the mechanisms of growth control.