Molecular Regulators of Synaptic Specificity

大脑中的特定回路决定了我们如何感知和对环境做出反应。这些高度连接的网络在发育过程中出现，神经元将投射延伸到指定的会议地点，确定合作伙伴，并开始突触发生。虽然最初的连接可能会在以后被修改，但连接的总体模式是可以预测的，因此表明伴侣的选择可以由基因组编码。因此，通过识别连接模式改变的突变，遗传分析可以有效地用于寻找突触特异性基因。为了识别新的、保守的目标选择蛋白，我将筛选破坏线虫独特行为的突变，这些突变依赖于线虫中神经元特有的突触。这项工作主要集中在PVD感觉神经元及其突触靶点Pvc和Ava。刺激PVD激活其主要伙伴聚氯乙烯，并触发向前运动。然而，如果PVC连接被移除，则AVA被激活，从而导致反向移动。因此，选择性地中断pvd→pvc或pvd→AVA连接的突变体可以从容易区分的行为(例如，正向和反向运动)中被识别出来。由于其短的生命周期和强大的遗传工具，线虫特别适用于无偏见的基因筛选。在目标1中，我将使用光遗传学策略在正向遗传EMS突变屏幕上激活PVD，该屏幕使用高通量视频记录系统(WormLab)来识别具有选择性运动改变的突变。具有这些特定运动表型的行为突变将用GGRAP(跨突触伙伴的GFP重组)标记进行筛选，以确认PVD→Pvc或Pvd→AVA突触在突触发生过程中被破坏。分子克隆方法将用于鉴定受影响的突触特异性基因。目的2采用一种独立的方法，该方法源于PVD中的突触特异性基因应该由细胞自主转录因子(TF)调控。我的策略利用了之前从RNA-Seq分析中获得的35个富含PVD的TF的列表。我将在GRAP标记实验中使用RNAi和可用的遗传突变体来测试这些TF中的每一个在PVD→PVC或PVD→AVA突触发生中的潜在作用。这种转铁蛋白筛查的优点是同时失调多个靶基因，以获得强大的突触特异性表型。我将使用PVD特异性RNA-Seq来识别突触特异性TF的靶点，然后使用行为分析和GRAP标记分别测试它们在PVD突触特异性中的作用。总之，线虫的这些方法有望揭示突触特异性的关键决定因素，这些决定因素可以测试更复杂的神经系统中的保守角色，以及与突触发生改变相关的神经疾病的联系，如自闭症谱系障碍(ASD)。

Specific circuits in the brain determine how we sense and respond to our environment. These highly connected networks emerge during development as neurons extend projections to defined meeting sites, identify partners, and begin synaptogenesis. Although initial connections may be modified later, the overall pattern of connectivity is predictable thus suggesting that partner selection can be encoded by the genome. It follows that genetic analysis can be powerfully employed to find synaptic specificity genes by identifying mutants with altered patterns of connectivity. With the goal of identifying novel, conserved target selection proteins, I will screen for mutations that disrupt distinctive behaviors that depend on neuron-specific synapses in C. elegans. This work focuses on the PVD sensory neuron and its synaptic targets, PVC and AVA. PVD stimulation activates PVC, its dominant partner, and triggers forward movement. If the PVC connection is removed, however, AVA is activated instead, resulting in reverse locomotion. Thus, mutants that selectively disrupt either PVD→PVC or PVD→AVA connections can be identified from readily distinguished behaviors (e.g., forward vs reverse movement). With its short life cycle and powerful genetic tools, C. elegans is especially useful for unbiased genetic screens. In Aim 1 I will use an optogenetic strategy to activate PVD in a forward genetic EMS mutagenesis screen that uses a high-throughput video recording system (WormLab) to identify mutants with selectively altered locomotion. Behavioral mutants with these specific locomotory phenotypes will be screened with GRASP (GFP Reconstitution Across Synaptic Partners) markers to confirm that either PVD→PVC or PVD→AVA synapses are disrupted during synaptogenesis. Molecular cloning methods will be used to identify the affected synaptic specificity genes. Aim 2 adopts an independent approach that stems from the expectation that synaptic specificity genes in PVD should be regulated by cell autonomous transcription factors (TFs). My strategy exploits a list of 35 PVD-enriched TFs previously derived from RNA-Seq profiling. I will use RNAi and available genetic mutants in the GRASP marker assay to test each of these TFs for potential roles in either PVD→PVC or PVD→AVA synaptogenesis. This TF screen has the advantage of dysregulating multiple target genes simultaneously for a robust synaptic specificity phenotype. I will use PVD-specific RNA-Seq to identify the targets of the synapse-specific TFs and then test them individually for roles in PVD synaptic specificity using the behavioral assay and GRASP markers. Together, these approaches in C. elegans are expected to reveal key determinants of synaptic specificity that can be tested for conserved roles in more complex nervous systems and for links to neurological disorders associated with altered synaptogenesis such as Autism Spectrum Disorder (ASD).