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空间和时间控制产生的。这种成熟过程产生具有不同细胞形态和基因表达谱的结构域。遗传学研究提供的证据表明，适当的结构域结构对生长板功能至关重要，并且这种结构容易受到损伤或疾病的损害。软骨细胞成熟中的一个重要事件是圆形前体细胞重组为盘状增殖软骨细胞，所述盘状增殖软骨细胞通过克隆扩增形成柱状。以前的研究支持一个模型，其中柱形成增殖软骨细胞发生通过连续的平面分裂和细胞嵌入的方式类似于收敛扩展。一个重要的警告是，这些研究以及所提出的生长板中细胞行为的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，并限制了对遗传数据的机械解释。该建议利用了最近在实验室开发的体外活体成像系统来阐明在增殖软骨细胞中调节柱形成的机制。具体而言，目的是为了测试的假设，即柱形成发生在一个收敛的扩展过程中，是由子细胞的界面处的相互作用驱动的细胞嵌入。将使用活体成像以及遗传和化学-遗传方法来检验这一假设，以检测子细胞中板状足的存在，以确定肌动蛋白动力学和整合素功能是否是柱形成所需的，并确定子细胞的过早分离是否会阻止Piga突变增殖性软骨细胞中的细胞重排。这些研究的结果将为组织工程软骨结构的产生提供重要的新信息，并将建立这种创新的方法作为一个重要的新工具，研究生长控制的机制。

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.