Expression & function of micro RNAs in neural stem cells

描述(申请人提供)：最近的研究已经确定了一小部分微小RNA(MiRNAs)，它们在中枢神经系统发育早期和培养的干细胞中差异表达，但随着分化而丢失。MiRNAs被认为是多个靶基因的转录后抑制因子，可以调节蛋白质的表达和发育程序。我们的假设是，神经发育的早期事件由特定的miRNAs调控。因此，神经干细胞及其后代对不同命运的限制被预测为受可识别的miRNAs集调控。我们的初步结果表明，某些miRNAs在神经干细胞及其一些后代中瞬时表达，但在类胚体或成人组织中不存在。我们提出了两个具体的目标：(1)识别在皮质分化过程中受到调控的miRNAs。我们将通过在定制设计的miRNA微阵列上测试不同年龄的miRNAs来确定大鼠和小鼠大脑发育过程中miRNAs的相对表达水平(E10.5-E18.5)。那些在发育过程中或在有限的解剖位置瞬时表达的基因将通过激光捕获显微切割进一步定位，以确定代表不同发育命运的离散细胞池。(2)通过反义抑制或过表达特定的miRNAs来调节神经干细胞和放射状胶质细胞的分化。功能获得和功能丧失技术将被用于操纵培养的神经干细胞中的miRNA行为。来自E13.5大鼠皮质的两个不同的永生化克隆分别是神经源性(L2.2)或胶质源性(L2.3)，将分别用于分析神经元和神经胶质分化的模式。最后，我们将分析miRNAs调节选定转录因子表达的能力，这些转录因子已知调节神经元和神经胶质发育。该项目提供了一种新的方法来分析miRNA在中枢神经系统发育过程中的表达，并探索miRNA表达的调节如何影响神经元和神经胶质的发育。

DESCRIPTION (provided by applicant): 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.