Identification of long non-coding RNAs in mammalian brain development

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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.