Regulation of Synapse Morphogenesis in Drosophila

 描述(申请人提供)：化学突触的形成和成熟对神经系统的所有主要功能都是必不可少的。突触发生是一个由高度保守的信号通路和细胞内效应蛋白调控的复杂的协调形态发生过程。在过去的十年中，越来越多的证据表明，这一过程受到从RNA转运到局部翻译的各种转录后机制的调控，这些机制提供了神经元和神经回路复杂结构所需的空间敏锐度。在可能影响关键神经元mRNAs翻译和稳定性的序列特异性调控因子中，microRNAs(MiRs)最近已成为一个丰富的潜在来源。这种大而多样的非编码RNA在神经系统中大量表达，数百个MIR定位于不同的脑区、特定的神经元亚群和突触间隔。尽管在检测转录组中miRs及其靶点所需的测序和信息学技术方面取得了令人印象深刻的进展，但对突触发生所需miR功能的全面分析在很大程度上仅限于内源性细胞-细胞相互作用和发育信号丢失的体外系统。为了应对这一挑战，我们花了第一个周期的资金用于在一个模型系统中开发用于条件操作和检测miR功能的遗传工具，该模型系统已被证明是兴奋性谷氨酸能突触传递的一个强大模型：果蝇神经肌肉接头(NMJ)。我们使用我们的工具箱剖析了一种新的跨突触机制，通过该机制，miR-8在发育过程中调节突触前和突触后脑室的协调形态发生，此外，我们还通过实验证明了这些工具对各种实验问题的有效性。然后，我们创建了一组改进的试剂来分析141个miR的突触调节功能，并完成了一次筛查，揭示了NMJ形成的不同方面所需的数十个保守的miR。这一开创性的工作使我们处于一个独特的位置，可以绘制出该系统中突触形成和成熟的每个阶段的miR调控的时空图景。我们已经确定了两种特殊的miR，它们在突触的相反侧发挥强大的相互调节作用：肌肉需要miR-34来控制突触前突触的增加和间距，而神经元需要miR-137来限制突触前臂的整体生长。MiR-34和miR-137都显示出与神经和精神疾病有关的基因的显著保守性，证实了它们在系统发育中的功能相关，并表明我们模型中的更深层次分析可以用来询问下游机制是否也是保守的。与定义作用于运动神经元和肌肉以影响NMJ发育的miRs的实验平行，我们将对表型和miR靶基因网络进行多水平的分析，以了解保守的miRs控制突触发生的机制的细胞和分子逻辑。

 DESCRIPTION (provided by applicant): The formation and maturation of chemical synapses is essential to all major functions of the nervous system. Synaptogenesis represents a complex process of coordinated morphogenesis governed by highly conserved signaling pathways and intracellular effector proteins. Over the past decade, accumulated evidence revealed that this process is under regulatory control of various post-transcriptional mechanisms, from RNA trafficking to local translation, that offer the spatial acuity needed for the complex architectureof neurons and neural circuits. Amongst the sequence-specific regulators that may shape the translation and stability of key neuronal mRNAs, microRNAs (miRs) have recently emerged as a rich potential source. This large and diverse class of non-coding RNA is expressed abundantly in the nervous system, with hundreds of miRs localized to different brain regions, specific neuronal subpopulations and synaptic compartments. Despite impressive advances in the sequencing and informatics technologies required to detect miRs and their targets within the transcriptome, a comprehensive analysis of miR functions required for synaptogenesis was largely limited to in vitro systems where the endogenous cell-cell interaction and developmental signals are lost. In order to address this challenge, we spent the first cycle of funding on this grant developing genetic tools for conditional manipulation and detection of miR function in a model system that has proven to be a powerful model of excitatory glutamatergic synaptic transmission: the Drosophila neuromuscular junction (NMJ). We used our toolkit to dissect a novel trans-synaptic mechanism by which miR-8 regulates coordinated morphogenesis of pre- and post-synaptic compartments during development, in addition to experiments to prove the efficacy of these tools for a variety of experimental questions. We then created a collection of improved reagents to analyze synapse- regulatory function for 141 miRs, and completed a screen revealing that there are dozens of well-conserved miRs required for distinct aspects of NMJ formation. This pioneering effort has put us in a unique position to map out the spatio-temporal landscape of miR regulation for each of the stages of synapse formation and maturation in this system. We have identified two particular miRs that exert potent and reciprocal regulatory effects on opposite sides of the synapse: miR-34 is required in muscle to control addition and spacing of presynaptic boutons, whereas, miR-137 is required in neurons to restrict the overall growth of presynaptic arbors. Both miR-34 and miR-137 show striking conservation to genes implicated in neurological and psychiatric disease, confirming that they are functionally relevant across phylogeny, and suggesting that deeper analysis in our model can be used to ask if the downstream mechanisms are also conserved. In parallel with experiments to define the miRs that act in motor neurons and muscles to shape NMJ development, we will pursue a multi-level analysis of phenotypes and miR target gene networks in order to understand the cellular and molecular logic of the mechanisms by which conserved miRs control synaptogenesis.