Identification of long non-coding RNAs in mammalian brain development

我们的第一个NSERC发现奖期间是特殊的，导致了一系列重要的论文，并为当前的应用奠定了基础。它支持我们与哺乳动物基因组功能注释（FANTOM 5）项目的合作，以创建定量标记转录起始位点的数据集，使我们能够在胚胎发生期间以24小时的间隔和出生后早期每72小时在小鼠中创建小脑转录组的时间序列。该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：长链非编码RNA（lncRNA）在帮助指导哺乳动物大脑发育中的作用。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作用的试验台。lncRNA在大脑发育中的功能作用是理解大脑发育遗传学的一个未被探索和令人兴奋的方向。 本提案的主题是三个目标和三套可交付成果。 目的1：我们的FANTOM 5（Zenbu）数据库的生物信息学分析，以确定lncRNA作为小脑发育的候选者。表达水平（每百万个转录本）、小脑特异性（与RIKEN数据集中的其他~400个细胞和组织相比，小脑中转录本的富集）以及从我们的小脑时程转录组数据集中出现的动态发育表达的过滤器将提供更小的lncRNA集来探索功能。我们希望产生一个目录的lncRNA是潜在的重要在小鼠小脑发育。 目的2：详细验证参与颗粒细胞发育的新型候选lncRNA。我们在这一目标中的重点是从目标1中获得最佳候选物，并将其提交给验证步骤，其中包括qRT-PCR以量化基因表达的变化，以及原位杂交以可视化整个胚胎/发育时间轴中表达的空间和时间参数。为了鉴定在颗粒细胞中表达的lncRNA，我们计划进行双荧光（原位杂交和免疫组织化学）。可交付的成果将是一个图谱，它将绘制出Aim 1中出现的“过滤”lncRNA的表达。 目标三：在目标1&2中鉴定的一小组候选lncRNA将使用功能获得和丧失实验来探索，以确定lncRNA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的分析中进行，使用颗粒细胞培养、小脑平板培养、子宫内敲除和小鼠敲除作为功能分析中的顺序步骤。颗粒细胞发育表型如细胞增殖、细胞特化、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.