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

项目总结 相邻细胞之间的信号和黏附相互作用在发育过程中发挥着重要作用 过程，帮助确定细胞的命运和身份，迁移路径，并最终确定细胞功能。 目前，表征细胞间接触的最好方法是通过超微结构研究。这些 提供丰富的信息，但技术复杂、执行起来单调乏味，并且需要广泛的分析 以确定感兴趣的特定交互。 我们正在努力开发一种细胞对细胞接触追踪的方法，该方法在光学上提供有用的信息 显微镜水平。利用人工合成的缺口受体和它们识别的配体，现在可以设计 并从基因上编码一种在体内运行的触敏检测系统。我们选择了 CD19/synNotch受体系统在CART治疗中用于检测细胞之间的接触。在我们的版本中， 显示在一个细胞表面的CD19配体的识别激活了相反细胞表面的synNotch受体 表面，诱导转录激活物的切割和释放，从而诱导一种 荧光标记蛋白。 我们正在努力完善正在开发的嗅觉系统中的细胞对细胞接触追踪方法。 斑马鱼。斑马鱼在这项研究中有许多优势。其中包括极好地访问所有 发展阶段，快速的生成时间，卓越的成像光学特性，以及容易 转基因。嗅觉系统有简单的、特征明确的电路和细胞特异性启动子 可用于推动特定类别的嗅觉感觉神经元的转基因表达。这个项目的目的是 是确定嗅觉轴突从其起源延伸到嗅觉上皮时接触的细胞 到达它在嗅球中的目标。这一信息对于理解这些感觉轴突是如何回家的至关重要。 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.