Project Summary Birth defects are the leading cause of infant death in the United States (Pediatrics 2012, 129:338-48. PMID: 22291121). Major human birth defects originate during early embryogenesis. Zebrafish is an important vertebrate model system for understanding early embryogenesis. Genome analysis shows that 71% of human genes have at least one ortholog in zebrafish and 82% of the known genes responsible for human disease are present in zebrafish (Nature 2013, 496: 498-503. PMID: 23594743). Research on zebrafish embryos could shed invaluable light on human early embryogenesis, thus leading to better understanding of human birth defects. There is a rich literature on transcriptome-wide changes that accompany zebrafish early embryogenesis. However, transcriptome-level information is limited because zygotic transcription is silent before the mid-blastula transition (MBT), because post- transcriptional regulation modulates gene expression, and because protein post- translational modifications influence protein function. We hypothesize that high time- and spatial-resolution studies of the early-stage zebrafish proteome will provide new insights into early embryogenesis. In this proposal, we will develop new techniques to improve the scale and sensitivity of bottom-up and top-down proteomics, and those technologies will enable us to discover the proteome dynamics in wild-type zebrafish early-stage embryos across twelve developmental stages with single-cell resolution. Results from this proposal are extremely important. First, the top-down proteomics technique should revolutionize the current workflow. The highly sensitive proteomics technique will be an invaluable tool for analysis of mass-limited complex proteome samples. Second, the proteome dynamics database will certainly provide new insights into important events during early embryogenesis, e.g., MBT and early cellular differentiation. Third, the proteome dynamics database will provide the zebrafish community with a list of important gene targets for further gene mutation studies for understanding how gene mutations lead to birth defects.