Coordination and propagation of cell fate choice in neural circuit assembly

项目摘要 大量的神经元如何被指定为正确的细胞命运，并与适当的目标连接， 发育代表了发育神经科学的一个迷人领域。随机和 已经广泛地研究了实现神经元多样性的确定性细胞特化程序。来 在过去的几十年中，还鉴定了许多介导轴突导向的细胞表面分子， 连通性。然而，很少有人知道的协调之间的神经元规格和具体的， 连接模式，特别是当两个突触伴侣经历两种不同的细胞特化模式时 （随机与确定性）。果蝇的色觉回路是解决这个问题的一个有吸引力的模型 由于我们对其发展的深入了解，其精确的神经元连接，以及 用于细胞类型特异性操作的强大遗传工具。在苍蝇视网膜中，苍白（p）和黄色（y）亚型 彩色光感受器（R7和R8）是随机指定的，而它们在视叶中的突触伙伴 都是通过高度确定性的程序产生的。随机确定的p/y R7和R8如何找到 视叶中确定的目标这个决定是如何传播给他们的 在电路形成过程中的下游目标？什么分子引导这些事件？我们实验室以前的工作 Dpr 11和DIPg是免疫球蛋白超家族相互作用网络的成员 蛋白质，作为yR 7和其下游靶标之间突触连接的关键调节剂。我们 假设不同的细胞粘附分子对介导其他突触伴侣的匹配。通过 利用单细胞RNA测序技术、CRISPR基因编辑和复杂的遗传操作， 果蝇的色觉回路，我们的目标是确定直接突触伴侣的细胞粘附分子 匹配和分子逻辑之间的协调细胞类型规范和突触连接 在系统层面。我们将定义突触连接并产生细胞类型特异性转基因 试剂和相关细胞类型的更高深度的转录组（Aim 1）。我们将使用候选人方法 结合对分选的彩色光感受器的靶向神经元的转录组分析， 突触伴侣匹配所需的分子（Aim 2）。我们将比较给定的神经元是否使用 相同或不同的分子密码来匹配它的突触前和突触后伴侣。最后，我们将研究如何 突触伴侣的选择通过扰乱R7的细胞命运传播到更下游的神经元， R8（目标3）。该提案的成功完成将揭示新的分子机制， 突触配对和探索的基本原则，传播细胞命运的选择， 电路组装这里确定的原则将是有意义的，并适用于其他神经元回路 面临着类似的发展挑战，如啮齿动物的嗅觉系统和人类的色觉。

Project Summary How vast numbers of neurons are specified into correct cell fates and connected with proper targets during development represents a fascinating area of developmental neuroscience. Mechanisms of stochastic and deterministic cell specification programs to achieve neuronal diversity have been extensively studied. Over the last decades, a number of cell surface molecules have also been identified that mediate axon guidance and connectivity. However, little is known about the coordination between neuronal specification and specific connectivity patterns, especially when two synaptic partners undergo two different modes of cell specification (stochastic vs. deterministic). The Drosophila color vision circuit is an appealing model to address this question due to our deep knowledge of its development, its precise neuronal connectivity, and the availability of powerful genetic tools for cell-type specific manipulations. In the fly retina, pale (p) and yellow (y) subtypes of color photoreceptors (R7 and R8) are stochastically specified, whereas their synaptic partners in the optic lobe are produced through highly deterministic programs. How do stochastically determined p/y R7 and R8 find their targets that are deterministically specified in the optic lobes? How is this decision propagated to their downstream targets during circuit formation? What molecules direct these events? Previous work from our lab has identified Dpr11 and DIPg, which are members of an interacting network of immunoglobulin superfamily proteins, as critical regulators of the synaptic connection between yR7 and its downstream target. We hypothesize that different pairs of cell adhesion molecules mediate the matching of other synaptic partners. By using single-cell RNA sequencing technology, CRISPR gene editing, and sophisticated genetic manipulation in the Drosophila color vision circuit, we aim to identify cell adhesion molecules that direct synaptic partner matching and the molecular logic for coordinating between cell-type specification and the synaptic connectivity at the system level. We will define the synaptic connectivity as well as generate cell-type specific transgenic reagents and higher-depth transcriptomes of relevant cell types (Aim 1). We will use a candidate approach combined with transcriptome analysis of sorted targeted neurons of color photoreceptors to identify the molecules required for synaptic partner matching (Aim 2). We will compare whether a given neuron uses the same or different molecular codes for matching its pre- and post-synaptic partners. Finally, we will study how the synaptic partner choices propagate to neurons further downstream by perturbating the cell fates of R7 and R8 (Aim 3). Successful completion of this proposal will uncover novel molecular mechanisms regulating synaptic pairing and probe the fundamental principles underlying the propagation of cell fate choices during circuit assembly. The principles identified here will be significant and applicable to other neuronal circuits facing similar developmental challenges, such as the olfactory system in rodents and color vision in humans.