Discovering miRNA-Mediated Mechanisms of Interneuron Development

项目摘要 抑制性中间神经元(IN)的发育需要成功完成一系列 工艺，包括长距离迁移、层压、分子规格、电路 整合，功能成熟。功能障碍与神经发育密切相关 和精神障碍，所以了解INS是如何实现其成熟的分布，数量， 而身份认同是理解疾病的关键。细胞内在基因表达程序，包括 对大量基因的精确调控，与环境线索相互作用以控制 发展。MicroRNAs(MiRNAs)是已知的基因表达的转录后调节因子 是智能网发展的多个方面所必需的，但通过哪些具体机制 MiRNAs做到这一点仍然未知，部分原因是特定于IN的miRNA活性模式 未知。我们通过进行ago交联和随后的免疫沉淀来弥合这一差距。 通过测序(AGO CLIPSEQ)全面描述发育中的IN靶组 时间点。然而，识别受单个miRNAs调控的发育途径 一直具有挑战性，因为靶组极其复杂-miRNAs同时调节 许多基因，并可以通过共同靶向关系相互补偿。我们建议 在基因水平上研究miRNA调控的另一种方法。穿过 对CLIPseq数据的生物信息学分析，我确定了miRNA热点，或共同靶向的基因 多个miRNAs，我们假设这是由 许多关键的发育基因。有了这些候选基因，我现在准备解开 MiRNA对特定基因的调节控制着IN发育的特定方面。我会用新的 基于CRISPR的方法在特定细胞类型中干扰候选基因的miRNA调节 方式和发育缺陷筛查，首先使用一组体外和体内读数 在迁徙、生存和亚型规范中。然后，我将评估总体上的扰动 使用靶向扰动序列的发育轨迹，它将转录组作为一种 单细胞成熟状态的复杂读数。最终，有了这条实验性的管道，我 将发现控制IN发育的基因和miRNA机制。

Project Summary Inhibitory interneuron (IN) development requires successful completion of a series of processes, which include long-range migration, lamination, molecular specification, circuit integration, and functional maturation. IN dysfunction is highly implicated in neurodevelopmental and psychiatric disorders, so understanding how INs achieve their mature distribution, number, and identity is critical to understanding disease. Cell-intrinsic gene expression programs, involving the precise regulation of huge sets of genes, interact with environmental cues to control development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression known to be necessary for multiple aspects of IN development, yet the specific mechanisms by which miRNAs do this remain unknown, in part because IN-specific patterns of miRNA activity were unknown. We bridged this gap by performing Ago cross-linking and immunoprecipitation followed by sequencing (Ago CLIPseq) to comprehensively profile the IN targetome at developmental timepoints. However, identifying developmental pathways regulated by individual miRNAs has been challenging because the targetome is extremely complex—miRNAs simultaneously regulate many genes and can compensate for each other through co-targeting relationships. We propose an alternative approach of investigating miRNA regulation on a gene-by-gene level. Through bioinformatic analysis of the CLIPseq data, I identified miRNA hotspots, or genes co-targeted by multiple miRNAs, which we hypothesize is a signature of strong miRNA regulation shared by many key developmental genes. With these candidate genes, I am now poised to untangle how miRNA regulation of specific genes controls particular aspects of IN development. I will use new CRISPR-based methods for disrupting miRNA regulation of candidate genes in a cell type-specific manner and screen for developmental defects, first using a set of in vitro and in vivo readouts for IN migration, survival, and subtype specification. Then, I will assess perturbations of overall developmental trajectory using targeted Perturb-seq, which harnesses the transcriptome as a complex readout of maturation state in single cells. Ultimately, with this experimental pipeline I will discover genes and miRNA mechanisms that control IN development.