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.