Recent studies have identified a small set of micro RNAs (miRNAs) that are differentially expressed early during CNS development and in cultured stem cells but are lost with differentiation. MiRNAs are thought to act as post-transcriptional repressors of multiple target genes that can regulate protein expression and programs of development. Our hypothesis is that early events in neural development are regulated by specific miRNAs. Thus the restriction of neural stem cells and their progeny to different fates are predicted to be regulated by identifiable sets of miRNAs. Our preliminary results demonstrate that certain sets of miRNAs are expressed transiently in neural stem cells and some of their progeny but are not found in embryoid bodies or adult tissues. We propose two specific aims: (1) Identify miRNAs that are regulated during cortical differentiation. We will determine the relative expression levels of miRNAs during rat and mouse brain development (E10.5-E18.5) by testing miRNAs from different ages on custom-designed miRNA microarrays. Those that are expressed transiently during development or in restricted anatomic locations will then be mapped further by laser capture microdissection to identify discrete pools of cells representing different developmental fates. (2) Modulate differentiation of neural stem cells and radial glia by antisense inhibition or overexpression of specific miRNAs. Gain and loss of function techniques will be used to manipulate miRNA actions in cultured neural stem cells. Two different immortalized clones derived from E13.5 rat cortices that are neurogenic (L2.2) or gliogenic (L2.3) will be used to analyze patterns of neuronal and glial differentiation, respectively. Finally, we will analyze the ability of miRNAs to regulate expression of selected transcription factors that are known to regulate neuronal and glial development. This project represents a novel approach to analyze expression of miRNA during CNS development and to explore how regulation of miRNA expression affects neuronal and glial development. ? ?
