Detecting cell to cell contacts in zebrafish with a synthetic receptor methodology

项目摘要 邻近细胞之间的信号传导和粘附相互作用在发育中发挥着重要作用 过程，帮助确定细胞的命运和身份，迁移途径，并最终细胞功能。 目前，表征细胞与细胞接触的最佳方法是通过超微结构研究。这些 提供了丰富的信息，但技术复杂，执行繁琐，需要大量的分析 以识别感兴趣的特定交互。 我们正在努力开发一种细胞与细胞接触追踪的方法， 显微镜水平。使用合成的notch受体和它们识别的配体，现在可以设计 并对在体内操作的触敏检测系统进行遗传编码。我们选择了 CD 19/synNotch受体系统用于CART治疗以检测细胞之间的接触。在我们的版本中， 识别展示在一个细胞表面上的CD 19配体激活相对表面上的synNotch受体。 表面，诱导转录激活因子的切割和释放，所述转录激活因子诱导转录因子的表达。 荧光标记蛋白 我们正在努力完善一个细胞与细胞接触追踪方法，在发展中的嗅觉系统， 斑马鱼斑马鱼在这项研究中有许多优势。其中有很好的访问所有 开发阶段，快速生成时间，出色的成像光学特性， 转基因嗅觉系统具有简单、特征良好的电路和细胞特异性启动子 可用于驱动特定类别的嗅觉感觉神经元中的转基因表达。这个项目的目的 识别嗅觉感觉轴突从其嗅上皮起源延伸时接触的细胞 到嗅球中的目标这些信息对于理解这些感觉轴突如何回家至关重要 在灯泡内的特定目标区域。

PROJECT SUMMARY Signaling and adhesive interactions between neighboring cells play an important role in developmental processes, helping determine cell fates and identities, migration pathways, and ultimately cellular function. Currently the best method for characterizing cell to cell contacts is through ultrastructural studies. These provide a wealth of information, but are technically complex, tedious to perform, and require extensive analysis to identify specific interactions of interest. We are working to develop a method of cell to cell contact tracing that provides useful information at the optical microscopy level. Using synthetic notch receptors and the ligands they recognize, it is now possible to design and genetically encode a touch sensitive detection system that operates in vivo. We have chosen the CD19/synNotch receptor system utilized in CART therapy to detect contacts between cells. In our version, recognition of the the CD19 ligand displayed on one cell surface activates a synNotch receptor on an opposing surface, inducing the cleavage and release of a transcriptional activator that induces the expression of a fluorescent marker protein. We are working to perfect a cell to cell contact tracing methodology in the developing olfactory system of the zebrafish. Zebrafish have a number of advantages for this study. Among them are excellent access to all developmental stages, a rapid generation time, outstanding optical properties for imaging, and easy transgenesis. The olfactory system has simple, well characterized circuitry and cell-specific promoters are available to drive transgenic expression in specific classes of olfactory sensory neurons. The aim of this project is to identify the cells an olfactory sensory axon contacts as it extends from its origin in the olfactory epithelium to its target in the olfactory bulb. This information is crucial for understanding how these sensory axons home in on specific target regions within the bulb.