Identification of long non-coding RNAs in mammalian brain development

我们的第一个NSERC发现奖获得期是特殊的-导致了一系列重要的论文，并为当前的申请奠定了基础。它支持了我们与哺乳动物基因组功能注释(FANTOM5)项目的合作，创建了一个定量的、带标签的转录起始点的数据集，使我们能够在小鼠胚胎发育期间每隔24小时，在出生后早期每隔72小时创建小脑转录组的时间序列。这个数据集的详细使用将应用于基因调控中一个独特而令人兴奋的主题：长非编码RNA(LncRNAs)在帮助指导哺乳动物大脑发育方面的作用。小脑，凭借我们对其细胞类型和发育动力学的精湛知识，将成为探索这些分子在大脑发育中的作用的试验台。在脑发育的背景下，lncRNAs的功能作用在很大程度上是一个尚未被探索的和令人兴奋的方向，以了解大脑的发育遗传学。这项提案的主题是三个目标和一套可交付成果。目的1：对我们的FANTOM5(ZENBU)数据库进行生物信息学分析，以确定lncRNAs是小脑发育的候选基因。筛选表达水平(每百万条转录数)、小脑特异性(与RIKEN数据集中的其他400个细胞和组织相比，小脑中丰富的转录本)，以及从我们的小脑时间进程转录切割数据集中产生的动态发育表达，将提供一组更小的LncRNA来探索其功能。我们希望制作一份在小鼠小脑发育中潜在重要的lncRNA目录。目的2：详细验证参与颗粒细胞发育的新候选LncRNAs。我们在这个目标中的重点是从Aim 1中挑选出最好的候选基因，并将他们提交给验证步骤，其中包括量化基因表达变化的qRT-PCR和可视化整个胚胎/发育时间线上表达的空间和时间参数的原位杂交。为了确定在颗粒细胞中表达的lncRNAs，我们计划进行双荧光(原位杂交和免疫组织化学)。可交付成果将是一份图谱，将绘制出从目标1出现的“过滤”的lncRNAs的表达。目标3：将使用获得和丧失功能的实验来探索在目标1和2中确定的一小部分候选lncRNAs，以确定lncRNAs在颗粒细胞发育中的作用。这项工作将在分子、细胞和整个系统水平上进行分析，使用颗粒细胞培养、小脑片培养、宫内敲除和小鼠敲除作为功能分析中的连续步骤。颗粒细胞的发育表型，如细胞增殖、细胞规格、细胞迁移和细胞分化，将是确定lncRNA扰动是否影响发育的终点。

Our first NSERC Discovery award period was exceptional - leading to a series of important papers and laying the groundwork for the current application. It supported our collaboration with the Functional Annotation of the Mammalian Genome (FANTOM5) project to create a dataset of quantitative, tagged transcription start sites that has enabled us to create a time-series of cerebellar transcriptome in the mouse at 24 hour intervals during embryogenesis and every 72 hours during early postnatal life. The detailed use of this dataset will be applied to a unique and exciting topic in gene regulation: The role of long non-coding RNAs (lncRNAs) in helping to direct mammalian brain development. The cerebellum, with our exquisite knowledge of its cells types and developmental dynamics, will be the test bed to explore the role of these molecules in brain development. The functional role of lncRNAs in the context of brain development is a largely unexplored and exciting direction in understanding the developmental genetics of the brain. Three objectives and sets of deliverables are the subject of this proposal. Aim 1: Bioinformatic analysis of our FANTOM5 (Zenbu) database to identify lncRNAs as candidates for cerebellar development. The filters of expression level (transcripts per million), cerebellar specificity (enrichment of transcripts in the cerebellum compared to the other ~400 cells and tissues in the RIKEN dataset), and a dynamic developmental expression that emerges from our cerebellar time-course transcriptomic dataset will provide a much smaller set of lncRNAs to explore function. We look to produce a catalogue of lncRNAs that are potentially important in mouse cerebellar development. Aim 2: Detailed validation of novel candidate lncRNAs involved in granule cell development. Our focus in this aim is to take the top candidates from Aim 1 and submit them to validation steps that will include qRT-PCR to quantify changes in gene expression and in situ hybridization to visualize the spatial and temporal parameters of expression across the embryonic/developmental timeline. To identify lncRNAs that are expressed in granule cells we plan to do double-fluorescence (in situ hybridization and immunohistochemistry). The deliverable will be an atlas that will map out the expression of the "filtered" lncRNAs that emerge from Aim 1. Objective 3: A small set of candidate lncRNAs identified in Aims 1&2 will be explored using gain- and loss-of-function experiments to determine lncRNAs' role in granule cell development. This work will be done at the molecular, cellular and whole system levels of analysis using granule cell culture, cerebellar slab culture, in utero knockdown and mouse knock outs as sequential steps in the functional analysis. Granule cell developmental phenotypes such as cell proliferation, cell specification, cell migration, and cell differentiation will be the endpoints to determine if lncRNA perturbations impact development.