Beyond Transcription - microRNA Regulation of Neuronal Development

摘要 随着大脑的成熟，每种神经元细胞类型都必须执行专门的发育程序来构建细胞 具有适当的功能。这些程序由基因表达的波组成， 随着细胞经历连续的发育阶段，事实上，这些亲... 克正确展开与几种神经发育障碍有关。现代转录组学使得 我们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，在这方面， 仅有这些信息是不够的。事实上，基因表达的复杂转录后调控层， microRNA（miRNA）的表达有助于执行这些令人难以置信的复杂发育程序。miRNAs作为 作为转录后抑制因子，确保其mRNA靶不在不适当的时间表达 或地点我们和其他人证明，去除单个脑富集的miRNA可以产生深刻的影响， 研究人员已经发现了大脑发育的基因序列，但由于每种miRNA都抑制数百种不同的靶点， 精确的分子机制。根据这些信息，有必要从根本上改变 我们研究miRNAs。在这里，我们提出了两个新的策略，不集中在单一的miRNA， 以立即确定基因表达的转录后调节的分子机制。第一 这种方法是基于这样的事实，即有时，单个miRNA-靶标相互作用（MTI）的去抑制可以是 足以诱导表型。因此，在目标1中，我们建立了一个管道来执行所有MTIs的大规模测试 在兴奋性主神经元（PN）中，独立于哪种miRNA与它们结合， 对他们的发展至关重要。为了做到这一点，我们设计了一些工具来绘制和操纵细胞类型特异性的MTI， 以及作为PN发育阶段的快速读出器的荧光报告物。第二种方法 它利用了这样一个事实，即多个miRNA通常会聚集在同一个靶标上，以确保严格的调控。 如果进化对同一个目标施加了多层抑制，那么控制其蛋白质水平就必须 保持正确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， 靶向最严重抑制的miRNA，并研究其完全去表达的功能后果， 锡永了PNs的发展轨迹。对于这两个目标，我们建议调查如何去压抑的一个 单个MTI或单个miRNA靶点影响发育中皮质PN的结构、功能和连接性 无论是在体外还是在体内。有了这个建议，我们希望大大扩大我们的理解广泛的后trans-transmitted- PN发展的脚本机制，无论是在单一的MTI分辨率和在单一目标的水平， 被许多miRNAs压制。这些知识不仅是基础神经生物学的关键， miRNA抑制的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.