In vivo platforms for exploring co-evolution of ligands, receptors, and their targets - Abstract: Cell signaling regulates information transfer across tissues through graded ligands that interact with receptors. Across evolution, ligand and receptor proteins co-evolve, with changes in one protein being counterbalanced by changes in its partner, thus preserving successful interactions that promote development rather than pathology. Despite the importance of co-evolution in speciation and disease, our understanding of these reciprocal processes primarily relies on bioinformatics predictive tools and in vitro systems like yeast two-hybrid assays. Currently, there is no robust in vivo system, at moderate cost, to validate the co-evolution of multiple cell-signaling components. Given the differences in physiological conditions across tissues that impact protein interactions, we will develop in vivo genetic and molecular platforms for investigating co-evolving proteins. Research using Drosophila melanogaster has greatly contributed to both basic and medical research at a low cost. Building on our extensive experience with Drosophila species, we plan to generate two new in vivo systems to study the co- evolution of Gurken (GRK), a TGF-alpha ligand that activates the epidermal growth factor receptor (EGFR), endogenously. Drosophila oogenesis is a powerful model system for studying EGFR-guided axis formation and eggshell morphology, providing simple phenotypic readouts. Although the pathway and the underlying mechanism, a localized source of GRK, are conserved among Drosophila species, we have observed dramatic variations in the ability of GRK orthologs to signal interchangeably across species, ranging from no signal to a striking overactivation of EGFR signaling. The new in vivo platforms will allow us to thoroughly explore the effects of a histidine-rich domain loss in GRK on EGFR activation across different subgenera. Additionally, we will investigate the causes of EGFR overactivation when both GRK and EGFR are substituted with their homologs. S2 culture cells will be engineered with biosensors for EGFR signaling, providing a robust system to prioritize computationally predicted co-evolving sequences in GRK and EGFR for in vivo studies. Furthermore, we will examine the evolution of EGFR signaling targets, including the Tbx-20 homologs, Midline (MID) and H15, in relation to changes in signaling intensity and duration. Finally, we will study the evolution of cis-regulatory modules (CRMs) that control the expression of Mid/H15 tandem paralogs in oogenesis and other fly tissues. Our comprehensive and multidisciplinary approach-- combining new in vivo systems, computational, biophysical, analytical, and CRISPR/Cas9 genome engineering tools-- provides an exciting new path for identifying the co- evolving domains in ligands and receptors that control signaling levels. By investigating these domains in vivo, under natural physiological conditions, we will advance our understanding of cell signaling mechanisms, which could lead to new strategies for addressing tissue pathologies. The research field will benefit from new in vivo platforms to explore co-evolving processes during development and homeostasis. The EGFR signaling field will gain high-resolution analyses of co-evolving sequences that control signaling levels and duration in vivo.
