Beyond Transcription - microRNA Regulation of Neuronal Development - ABSTRACT As the brain matures, each neuronal cell type must execute a specialized developmental program to build a cell with the appropriate features. These programs are composed of waves of gene expression that turn on and off with exquisite precision as the cell goes through sequential developmental stages. In fact, failure of these pro- grams to unfold correctly is linked to several neurodevelopmental disorders. Modern transcriptomics has allowed us to map developmental trajectories of different neuronal subtypes at increasingly high resolution. However, this information alone is not sufficient. In fact, layers of sophisticated post-transcriptional regulation of gene ex- pression by microRNAs (miRNA) help execute these incredibly complex developmental programs. MiRNAs act as post-transcriptional repressors, ensuring that their mRNA targets are not expressed at an inappropriate time or place. We and others demonstrated that removal of a single brain-enriched miRNA can have profound con- sequences for brain development, but because each miRNA represses hundreds of different targets, it is hard to pinpoint precise molecular mechanisms. In light of this information, it is necessary to radically change the way we study miRNAs. Here, we propose two novel strategies that do not focus on single miRNAs and are designed to immediately identify molecular mechanisms of post-transcriptional regulation of gene expression. The first approach is based on the fact that, at times, de-repression of a single miRNA-target interaction (MTI) can be sufficient to induce a phenotype. Hence, in Aim 1 we establish a pipeline to perform a large-scale test of all MTIs in excitatory principal neurons (PN), independently of which miRNA is binding to them, to identify which ones are critical for their development. To do so, we engineered tools to map and manipulate cell type-specific MTIs, and fluorescent reporters that function as fast readouts of the developmental stage of PNs. The second approach takes advantage of the fact that, often, multiple miRNAs converge on the same target to ensure tight regulation. If evolution imposed multiple layers of repression on the same target, then controlling its protein levels must be essential to maintain the proper developmental trajectory. Thus, in Aim 2 we establish a pipeline to identify the targets most heavily repressed by miRNAs and study the functional consequences of their complete de-repres- sion on the developmental trajectory of PNs. For both aims, we propose to investigate how de-repression of a single MTI or of a single miRNA target affects the structure, function, and connectivity of developing cortical PNs both in vitro and in vivo. With this proposal we expect to greatly expand our understanding of broad post-tran- scriptional mechanisms of PN development, both at single MTI resolution and at the level of single target re- pressed by many miRNAs. Such knowledge will be key not only for basic neurobiology, but also to identify how failure in miRNA repression could lead to neurodevelopmental disorders.
