The conserved transcription factor UNC-30/PITX1-3 coordinates synaptogenesis and cell identity in C. elegans motor neurons

项目摘要 这项提案的总体目标是确定发生在神经元和神经元中的分子机制。 使它们能够建立和维持功能性突触。为此，突触前神经元的能力， 神经递质的合成、包装和释放必须与突触后的能力相协调。 神经元呈现正确的神经递质受体。揭示了 协调这些过程可能会产生生物医学影响，因为建立和 功能性突触的维持与人类严重的神经变性疾病有关。解决 知识差距，C。线虫是研究这些机制的理想模型， 连接体、强大的遗传学和单细胞分辨率分析。利用这些工具， 保守的转录因子（TF）PITX 1 -30/PITX 1 -3已被证明控制神经元通讯 在神经索GABA能（GABA）运动神经元（MN）和体壁肌肉之间，通过直接激活 GABA生物合成基因的表达（例如，unc-47/VGAT，unc-25/GAD）。我们得到的数据显示 由GABA MNs产生的一种分泌型突触组织者unc-30或madd-4S的遗传缺失， GABA受体（GABAR）在突触后结构域（肌肉）上聚集的显著减少。而且我们 初步数据显示，缺乏unc-30基因活性的动物显示， GABA MNs。因此，我们假设，β-30控制着建立和维持， 功能性突触通过直接激活madd-4S和GABA生物合成基因。评价这一 假设，在目标1中，我将使用madd-4S特异性报告基因来确定是否madd-30直接调节 madd-4S表达。我还将进行跨物种救援实验以确定 在调节madd-4S表达中的作用在整个胚胎发育中是保守的。在目标2中，我将利用 生长素诱导的降解决定子系统在整个发育的特定阶段选择性地消耗β-30， 确定是否需要EEG-30来维持突触的功能。此外，我们的初步 数据表明GABA-30还需要防止GABA MN中采用替代的神经元身份， 提示双重作用：madd-4S和GABA生物合成基因的激活剂，以及替代性 身份基因在目标3中，我将检验这样一个假设，即不同的调节因素决定了β-30的双重作用。 作用这一建议是重要的，因为人类PITX和GABA生物合成基因的突变与人类的基因突变有关。 神经退行性疾病此外，它还解决了神经元发育领域的一个长期存在的问题 和疾病：神经元如何建立和维持功能突触。这项工作在技术上是创新的， 它使用强大的遗传方法和实施新的方法来研究TF在体内的功能。这 项目将提供一个极好的培训机会，以支持我成为一个富有成效的， 独立研究员。

PROJECT SUMMARY The overall goal of this proposal is to determine the molecular mechanisms that take place in neurons and enable them to establish and maintain functional synapses. For this, the ability of presynaptic neurons to synthesize, package, and release a neurotransmitter must be coordinated with the ability of postsynaptic neurons to present the correct neurotransmitter receptors. Revealing the molecular mechanism that coordinates these processes could have biomedical implications since defects in the establishment and maintenance of functional synapses are linked to severe neurodegenerative disorders in humans. To address this knowledge gap, C. elegans represents an ideal model to study those mechanisms due to its known connectome, powerful genetics, and single-cell resolution analysis. Leveraging these tools, the evolutionarily conserved transcription factor (TF) UNC-30/PITX1-3 has been shown to control neuronal communication between nerve cord GABAergic (GABA) motor neurons (MNs) and body-wall muscle by directly activating the expression of GABA biosynthesis genes (e.g., unc-47/VGAT, unc-25/GAD). We obtained data that shows genetic loss of either unc-30 or madd-4S, a secreted synaptic organizer produced by GABA MNs, results in a dramatic reduction of GABA-Receptor (GABAR) clustering on the postsynaptic domain (muscle). Moreover, our preliminary data shows that animals lacking unc-30 gene activity show reduction of madd-4S expression in GABA MNs. Therefore, we hypothesize that UNC-30 controls both the establishment and maintenance of functional synapses by directly activating madd-4S and GABA biosynthesis genes. To evaluate this hypothesis, in Aim 1 I will use madd-4S specific reporters to determine whether UNC-30 directly regulates madd-4S expression. I will also conduct cross-species rescue experiments to determine whether UNC-30’s role in regulating madd-4S expression is conserved across phylogeny. In Aim 2 I will take advantage of the auxin-inducible degron system to selectively deplete UNC-30 at a particular stage throughout development and determine whether UNC-30 is required to maintain the functionality of synapses. Moreover, our preliminary data suggest UNC-30 is also required to prevent the adoption of alternative neuronal identities in GABA MNs, suggesting a dual role: activator of madd-4S and GABA biosynthesis genes, and repressor of alternative identity genes. In Aim 3, I will examine the hypothesis that distinct regulatory factors dictate UNC-30’s dual role. This proposal is significant because human mutations in PITX and GABA biosynthesis genes are linked to neurodegenerative disorders. Also, it addresses a long-standing question in the fields of neuron development and disease: how do neurons establish and maintain functional synapses. This work is technically innovative in its use of powerful genetic approaches and implementing novel methods to study TF function in vivo. This project will provide an excellent training opportunity to support my long-term goal of becoming a productive, independent researcher.