The Role of MicroRNAs in Vertebrate Development

描述（由申请人提供）：微小RNA（miRNA）是一个丰富的内源性~ 21 nt小RNA家族，其在转录后抑制基因表达。超过4%的人类基因编码miRNA，目前的估计表明，miRNA可以靶向人类基因组中超过30%的蛋白质编码基因。尽管它们被预测为基因表达的广泛调节因子，但它们在发育和疾病过程中的功能仍然知之甚少。该提案的重点是在细胞谱系特化和细胞信号传导过程中保守的microRNA（miR-430）的功能分析，其广泛的长期目标是了解miRNAs在发育和疾病过程中如何调节基因表达。在早期发育过程中发生两个主要事件：原始生殖细胞的特化和原肠胚形成期间胚层、内胚层、中胚层和外胚层的形成。我们将使用microRNA缺陷的斑马鱼胚胎来研究microRNA在发育过程中在这些过程中的作用。该提案的具体目的是：1）确定microRNA如何调节体细胞与生殖细胞中的基因表达，以及2）确定microRNA如何调节原肠胚形成期间的Nodal信号传导。目标#1中的实验将检验miRNA抑制体细胞中生殖系特异性基因表达的假设。这些实验还旨在鉴定这些体细胞靶标用于逃避生殖细胞中miRNA介导的阻遏的分子机制。因此，目标#1将极大地扩展我们对miRNA如何调节基因表达程序以建立主要细胞谱系的知识，并将为microRNA功能的分子机制提供基本见解。目标#2中的实验将检查特定Nodal组分的miRNA介导调节的作用，以在原肠胚形成期间赋予稳健性。这些实验将探索miRNAs如何调节Nodal信号通路中的不同组分，并将分析与Nodal信号通路中其他元件的遗传相互作用。TGF β信号传导和microRNA功能的缺失与发育缺陷和人类癌症有关。此外，miR-430是人类miR-17和miR-372的斑马鱼同源物，这两种miRNA促进肿瘤进展。因此，在脊椎动物模型系统中鉴定miR-430靶点并检查其功能将为理解其在人类中的作用和了解其功能障碍如何导致人类出生缺陷并导致疾病提供必要的背景。

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are an abundant family of endogenous ~21nt small RNAs that repress gene expression post-transcriptionally. More than 4% of the human genes encode for miRNAs and current estimates suggest that miRNAs could target more than 30% of the protein coding genes in the human genome. Despite their predicted roles as widespread regulators of gene expression, their functions during development and disease are still poorly understood. This proposal focuses on the functional analysis of a conserved microRNA (miR-430) during cell lineage specification and cell signaling, with the broad long-term objective of understanding how miRNAs regulate gene expression during development and disease. During early development two major events take place: the specification of the primordial germ cells and the formation of the germ layers during gastrulation, endoderm, mesoderm and ectoderm. We will use microRNA deficient zebrafish embryos to investigate the role of microRNAs in these processes during development. The specific Aims of this proposal are: 1) to determine how microRNAs regulate gene expression in somatic cells vs. germ cells, and 2) to determine how microRNAs regulate Nodal signaling during gastrulation. Experiments in Aim #1 will test the hypothesis that miRNAs dampen germ line specific gene expression in somatic cells. These experiments also aim to identify the molecular mechanisms used by these somatic targets to escape miRNA mediated repression in the germ cells. Thus, Aim #1 will greatly expand our knowledge of how miRNAs regulate gene expression programs to establish major cell lineages, and will provide fundamental insight into the molecular mechanism of microRNA function. Experiments in Aim #2 will examine the roles for miRNA-mediate regulation of specific Nodal components to confer robustness during gastrulation. These experiments will explore how miRNAs regulate different components in the Nodal signaling pathway, and will analyze the genetic interactions with other elements in the Nodal signaling pathway. Abnormalities in TGFb signaling and microRNA function have been implicated with developmental defects and human cancer. Furthermore, miR-430 is the zebrafish homolog of human miR-17 and miR-372, two miRNAs that promote tumor progression. Thus identifying the miR-430 targets and examining its functions in a vertebrate model system will provide the necessary context for understanding its roles in humans and learning how their dysfunction might cause human birth defects and contribute to disease.