Cell-cell communications in neural circuit assembly

项目摘要 神经生物学中的一个关键问题是单个神经元如何精确地与每个神经元连接。 另一个在开发过程中形成功能电路。了解的机制 哺乳动物大脑中的神经回路组装可以提供对 人类大脑紊乱在哺乳动物的大脑中，平均每个神经元 与成千上万的其他神经元连接。这些复杂电路的组装 在神经发育的许多步骤中依赖于细胞间的通讯。在 在此赠款的前两个周期，我们专注于开发MADM（马赛克 双标记分析）。带有两种不同颜色的MADM标签 共享共同谱系的单个神经元或神经元组。与此同时， MADM可以使标记有一种颜色的神经元纯合突变体， 目标基因和用第二颜色野生型标记的神经元作为内部对照。 MADM使研究人员能够检查哺乳动物神经细胞中的基因功能， 开发（以及其他过程）与单细胞分辨率，并使许多 新发现在这次更新中，我们将利用MADM和我们拥有的其他工具， 在以前的资助周期中开发，用于研究神经回路中的细胞间通信 组装在小鼠大脑中。具体来说，我们专注于两类蛋白质： 神经营养素受体和端神经素。 使用神经营养因子受体TrkC的MADM分析，我们先前已经表明 浦肯野细胞中TrkC的稀疏但非整体敲除减少了树突状细胞的生长， 和分支，表明竞争机制的树突形态发生。我们 将研究TrkC介导的竞争性树突的细胞机制 使用体内成像的形态发生，测试突触后活动是否需要 浦肯野细胞树突发育及其活性与TrkC的关系 信号我们还将探讨神经营养因子受体TrkB在神经元细胞中的作用。 形态发生从我们对果蝇嗅觉回路组装的研究中，我们发现 两种端神经素，它们是进化上保守的II型跨膜蛋白， 通过同性吸引来指导突触伴侣匹配。我们将测试 在小鼠脑中，端神经元蛋白还介导神经回路组装中的细胞-细胞相互作用 使用MADM分析、条件性敲除、病毒介导的 错误表达和体外测定。

PROJECT SUMMARY A key question in neurobiology is how individual neurons precisely connect with each other to form functional circuits during development. Understanding the mechanisms of neural circuit assembly in the mammalian brain may provide insights into the etiology of human brain disorders. In the mammalian brain, each neuron on average forms connection with thousands of other neurons. The assembly of these complex circuits depends on cell-cell communication during many steps of neural development. In the previous two cycles of this grant, we have focused on developing MADM (Mosaic Analysis with Double Markers) in mice. MADM labels with two distinct colors isolated individual neurons or groups of neurons that share a common lineage. At the same time, MADM can render neurons labeled with one of the colors homozygous mutant for a gene of interest and neurons labeled with the second color wild type as internal controls. MADM has allowed researchers to examine gene function in mammalian neural development (as well as other processes) with single-cell resolution, and enabled many new discoveries. In this renewal, we will utilize MADM and other tools we have developed in the previous grant cycles to study cell-cell communications in neural circuit assembly in the mouse brain. Specifically, we focus on two classes of proteins: neurotrophin receptors and teneurins. Using MADM analysis of the neurotrophin receptor TrkC, we have previously shown that sparse but not global knockout of TrkC in Purkinje cells reduces dendritic growth and branching, suggesting a competitive mechanism for dendrite morphogenesis. We will investigate the cellular mechanisms by which TrkC-mediated competitive dendrite morphogenesis using in vivo imaging, test whether postsynaptic activity required for Purkinje cell dendrite development, and the relationship between activity and TrkC signaling. We will also explore the function of neurotrophin receptor TrkB in neuronal morphogenesis. From our studies in Drosophila olfactory circuit assembly, we identified two teneurins, which are evolutionally conserved type II transmembrane proteins, that instruct synaptic partner matching via homophilic attraction. We will test whether teneurins also mediate cell-cell interaction in neural circuit assembly in the mouse brain using a combination of MADM analysis, conditional knockout, virus-mediated misexpresssion, and in vitro assays.