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 整合和功能成熟。IN功能障碍与神经发育密切相关。 和精神疾病，因此了解IN如何实现其成熟的分布，数量， 身份对于理解疾病至关重要。细胞内在基因表达程序，包括 大量基因的精确调控，与环境因素相互作用， 发展microRNA（miRNAs）是已知的基因表达的转录后调节因子， 对于IN开发的多个方面都是必要的，但具体的机制， miRNAs做到这一点仍然是未知的，部分原因是由于IN特异性的miRNAs活性模式是 未知我们通过进行Ago交联和免疫沉淀， 通过测序（Ago CLIPseq）来全面地描绘发育时的IN靶组， 时间点。然而，识别由单个miRNA调控的发育途径， 因为靶组非常复杂，miRNA同时调节 许多基因，并可以通过共同靶向关系相互补偿。我们提出 一种在基因水平上研究miRNA调控的替代方法。通过 通过对CLIPseq数据的生物信息学分析，我确定了miRNA热点，或者说是共同靶向的基因。 多个miRNA，我们假设这是一个强大的miRNA调控共享的签名， 许多关键的发育基因。有了这些候选基因，我现在准备解开 特定基因的miRNA调控控制IN发展的特定方面。我会用新的 基于CRISPR的方法用于破坏细胞类型特异性表达中候选基因的miRNA调控 方法和筛选发育缺陷，首先使用一组体外和体内读数， IN迁移、存活和亚型规范。然后，我将评估整体的扰动， 发展轨迹使用靶向Perturb-seq，它利用转录组作为一个 单个细胞成熟状态的复杂读出。最终，有了这个实验性的管道， 将发现控制IN发展的基因和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.