The Role of MicroRNAs in Vertebrate Development

描述(申请人提供)：microRNAs(MiRNAs)是一个丰富的内源性~21nt小RNA家族，在转录后抑制基因表达。超过4%的人类基因编码miRNAs，目前的估计表明miRNAs可以靶向人类基因组中超过30%的蛋白质编码基因。尽管它们被预测为广泛的基因表达调节因子，但它们在发育和疾病过程中的功能仍然知之甚少。本提案侧重于对一个保守的microRNA(miR-430)在细胞谱系指定和细胞信号传递过程中的功能分析，以了解miRNAs如何在发育和疾病过程中调节基因表达。在早期发育过程中，发生了两个主要事件：原始生殖细胞的具体化和原肠胚、内胚层、中胚层和外胚层胚层的形成。我们将使用microRNA缺失的斑马鱼胚胎来研究microRNAs在这些发育过程中的作用。这一建议的具体目的是：1)确定microRNAs如何调控体细胞和生殖细胞中的基因表达，以及2)确定microRNAs如何调控原肠发育过程中的Nodal信号。Aim#1中的实验将验证miRNAs抑制体细胞中生殖系特异性基因表达的假设。这些实验还旨在确定这些体细胞靶点用来逃避miRNA介导的生殖细胞抑制的分子机制。因此，目标1将极大地扩展我们对miRNAs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的见解。目标2中的实验将检验miRNA对特定节部成分的调节在原肠形成过程中赋予稳健性的作用。这些实验将探索miRNAs如何调节Nodal信号通路中的不同成分，并将分析Nodal信号通路中与其他元件的遗传相互作用。TGFb信号和microRNA功能的异常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，这两个miRNA促进肿瘤进展。因此，识别miR-430靶点并在脊椎动物模型系统中检测其功能将为理解其在人类中的作用以及了解其功能障碍如何导致人类出生缺陷和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.