Expression & function of micro RNAs in neural stem cells

描述（由申请人提供）：最近的研究已经鉴定出一小组微小RNA（miRNA），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。 miRNA 被认为是多个靶基因的转录后阻遏物，可以调节蛋白质表达和发育程序。我们的假设是神经发育的早期事件是由特定的 miRNA 调节的。因此，神经干细胞及其后代的不同命运的限制预计将受到可识别的 miRNA 组的调节。我们的初步结果表明，某些 miRNA 组在神经干细胞及其一些后代中短暂表达，但在胚状体或成体组织中未发现。我们提出两个具体目标：（1）识别在皮质分化过程中受到调节的 miRNA。我们将通过在定制设计的 miRNA 微阵列上测试不同年龄的 miRNA 来确定大鼠和小鼠大脑发育 (E10.5-E18.5) 期间 miRNA 的相对表达水平。然后，那些在发育过程中或在受限解剖位置短暂表达的细胞将通过激光捕获显微切割进一步绘制，以识别代表不同发育命运的离散细胞池。 (2)通过反义抑制或特定miRNA的过度表达来调节神经干细胞和放射状胶质细胞的分化。功能获得和丧失技术将用于操纵培养的神经干细胞中的 miRNA 行为。来自 E13.5 大鼠皮质的两种不同的永生化克隆，即神经源性 (L2.2) 或神经胶质源性 (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 miRNA 调节已知调节神经元和神经胶质发育的选定转录因子表达的能力。该项目代表了一种分析中枢神经系统发育过程中 miRNA 表达并探索 miRNA 表达调控如何影响神经元和神经胶质发育的新方法。

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.