Regulation of protein dynamics in Drosophila embryos' ectoderm germ layer - PROJECT SUMMARY/ ABSTRACT Gene expression is a complex process involving transcription, translation, and the turnover of mRNAs and proteins. Proper spatiotemporal regulation of gene expression and protein abundance plays a crucial role in guiding the differentiation of the fertilized egg into a complex animal. Understanding the regulation of protein dynamics can provide valuable insights into the differentiation of germ layers (endoderm, mesoderm, ectoderm), as well as the development of specialized cells and tissues. However, quantifying lineage-specific protein dynamics in developing embryos is challenging due to technical limitations in single-cell proteomics and the inherent heterogeneity of embryonic tissues. As a result, researchers have often used mRNA levels as a proxy for protein expression, but this approach has significant limitations due to the poor correlation between mRNA and protein levels. This discrepancy highlights the importance of posttranscriptional mechanisms, such as protein degradation and translation efficiencies, in determining cell fate. To address these issues and enhance our understanding of embryogenesis, it is crucial to measure proteome dynamics during cell differentiation directly. These measurements will provide a more comprehensive and accurate view of the intricate processes at play during embryonic development. This study proposes using genetically modified morphogen-flattened Drosophila embryos expressing four morphogens uniformly. These embryos contain a uniform cell type at gastrulation, different for each morphogen-flattened embryo. The focus will be on quantifying protein dynamics and posttranscriptional regulation within the ectoderm germ layer. My approach involves deep multiplexed proteomics and single-cell RNAseq to analyze proteome differentiation and stochastic cell fate diversification (Aim 1 & 2). Additionally, by achieving the desired outcomes of measuring posttranscriptional regulation (translation efficiency and protein turnover) in Aim 3, the proposed mathematical model will provide valuable insights into mRNA-protein dynamics during cell differentiation in developing embryos, which will address some of the discrepancies between the two. In conclusion, this proposal aims to decipher the mechanisms of embryonic cell-type differentiation by using morphogen-flattened embryos and employing an integrative analysis of genomics and proteomics measurements. This comprehensive approach will yield valuable insights into the intricate interplay of transcriptional and posttranscriptional regulation during development, enhancing our understanding of the fundamental principles in embryonic development and informing strategies for manipulating cell fate in regenerative medicine and tissue engineering.
