Beyond Transcription - microRNA Regulation of Neuronal Development

摘要 随着大脑的成熟，每种神经细胞类型都必须执行专门的发育程序来构建细胞 具有适当的特征。这些程序由一波又一波的基因表达组成，这些基因表达的开关 在细胞经历连续的发育阶段时具有精致的精确度。事实上，这些支持的失败- 克数的正确展开与几种神经发育障碍有关。现代转录组学已经允许 美国将以越来越高的分辨率绘制不同神经元亚型的发育轨迹。然而， 仅有这些信息是不够的。事实上，一层层复杂的转录后调控基因前- 通过microRNAs表达(MiRNA)帮助执行这些令人难以置信的复杂的发育程序。MiRNAs行为 作为转录后抑制物，确保它们的mRNA靶标不会在不适当的时间表达 或地点。我们和其他人证明，移除单个大脑丰富的miRNA可以产生深远的影响。 用于大脑发育的序列，但因为每个miRNA抑制数百个不同的靶点，所以很难 以确定精确的分子机制。鉴于这些信息，有必要从根本上改变这种方式 我们研究miRNA。在这里，我们提出了两种新的策略，它们不专注于单个miRNAs，并且是设计的 以立即确定转录后基因表达调控的分子机制。第一 该方法基于这样一个事实，即有时单个miRNA-靶相互作用(MTI)的抑制可以 足以产生表型的。因此，在目标1中，我们建立了一条管道来执行所有MTI的大规模测试 在兴奋性主神经元(PN)中，独立于哪些miRNA与它们结合，以识别哪些miRNA 对他们的发展至关重要。为此，我们设计了一些工具来绘制和操作特定细胞类型的MTI， 以及荧光记者，其功能是快速读出PNS的发展阶段。第二种方法 利用多个miRNAs往往会聚在同一个目标上以确保严格监管的事实。 如果进化对同一目标施加了多层抑制，那么控制其蛋白质水平必须是 对保持适当的发展轨迹至关重要。因此，在目标2中，我们建立了一个管道来识别 被miRNA抑制最严重的靶点，并研究其完全去表达的功能后果- Sion谈PNS的发展轨迹。为了这两个目标，我们建议调查如何解除对 单个MTI或单个miRNA靶点影响发育中的皮质PNS的结构、功能和连接性 在体外和体内都有。有了这一建议，我们预计将极大地扩大我们对广泛的后TRAN- 在单一MTI分辨率和单靶Re水平上PN发展的脚本机制 受到许多miRNA的压制。这些知识不仅是基础神经生物学的关键，也是确定 MiRNA抑制失败可能导致神经发育障碍。

ABSTRACT As the brain matures, each neuronal cell type must execute a specialized developmental program to build a cell with the appropriate features. These programs are composed of waves of gene expression that turn on and off with exquisite precision as the cell goes through sequential developmental stages. In fact, failure of these pro- grams to unfold correctly is linked to several neurodevelopmental disorders. Modern transcriptomics has allowed us to map developmental trajectories of different neuronal subtypes at increasingly high resolution. However, this information alone is not sufficient. In fact, layers of sophisticated post-transcriptional regulation of gene ex- pression by microRNAs (miRNA) help execute these incredibly complex developmental programs. MiRNAs act as post-transcriptional repressors, ensuring that their mRNA targets are not expressed at an inappropriate time or place. We and others demonstrated that removal of a single brain-enriched miRNA can have profound con- sequences for brain development, but because each miRNA represses hundreds of different targets, it is hard to pinpoint precise molecular mechanisms. In light of this information, it is necessary to radically change the way we study miRNAs. Here, we propose two novel strategies that do not focus on single miRNAs and are designed to immediately identify molecular mechanisms of post-transcriptional regulation of gene expression. The first approach is based on the fact that, at times, de-repression of a single miRNA-target interaction (MTI) can be sufficient to induce a phenotype. Hence, in Aim 1 we establish a pipeline to perform a large-scale test of all MTIs in excitatory principal neurons (PN), independently of which miRNA is binding to them, to identify which ones are critical for their development. To do so, we engineered tools to map and manipulate cell type-specific MTIs, and fluorescent reporters that function as fast readouts of the developmental stage of PNs. The second approach takes advantage of the fact that, often, multiple miRNAs converge on the same target to ensure tight regulation. If evolution imposed multiple layers of repression on the same target, then controlling its protein levels must be essential to maintain the proper developmental trajectory. Thus, in Aim 2 we establish a pipeline to identify the targets most heavily repressed by miRNAs and study the functional consequences of their complete de-repres- sion on the developmental trajectory of PNs. For both aims, we propose to investigate how de-repression of a single MTI or of a single miRNA target affects the structure, function, and connectivity of developing cortical PNs both in vitro and in vivo. With this proposal we expect to greatly expand our understanding of broad post-tran- scriptional mechanisms of PN development, both at single MTI resolution and at the level of single target re- pressed by many miRNAs. Such knowledge will be key not only for basic neurobiology, but also to identify how failure in miRNA repression could lead to neurodevelopmental disorders.