Bidirectional signaling by FGF proteins

About This Grant

SUMMARY Fibroblast growth factor proteins (FGFs) are ligands for FGF receptors (FGFRs), regulating cell adhesion, motility, proliferation, and survival. FGFs share a core homology domain of approximately 120 amino acids and have a globular β-trefoil structure, which enables them to interact with receptors on neighboring cells and mediate cell non-autonomous responses. Activation of FGFRs initiates the MAPK (mitogen-activated protein kinase) pathway, leading to the regulation of gene expression. The overarching goal of this proposal is to investigate a noncanonical activity of the Drosophila FGF Pyramus (Pyr), a Type I protein with an extracellular FGF-homologous portion, transmembrane domain (TMD), and an extended intracellular domain. Cleavage near the TMD releases the FGF domain extracellularly and a fragment of unknown function (PyrIntra) inside cells, suggesting that Pyr signals bidirectionally: forward through the extracellular domain and reverse through the intracellular domain. While bidirectional signaling is documented in other pathways like Notch/Delta and ephrin/Eph, it has not been documented for FGF/FGFRs. The early Drosophila embryo provides a simpler model to study FGF signaling, particularly in mesoderm cell migration during gastrulation and heart development. In these contexts, Pyr is expressed in the ectoderm and activates the FGFR Heartless (Htl) in the mesoderm, demonstrating a canonical response; however, its potential cell-autonomous, non-canonical function(s) in the ectoderm remain unclear. We hypothesize that Pyr supports forward signaling through the Htl FGFR in the mesoderm and reverse signaling through its intracellular domain in the ectoderm: functioning at the membrane to support cell polarity when intact, and entering the nucleus to support gene expression when cleaved. The proposed study will investigate bidirectional FGF signaling through three specific aims, using an innovative approach that combines genetics, live imaging, single-cell RNA sequencing (scRNA-seq), and affinity chromatography. Aim 1 will: (i) assess whether reverse signaling influences FGFR-dependent forward signaling dynamics in embryo assays, (ii) track Pyr cleavage using a novel in vivo reporter to monitor reverse signaling, and (iii) evaluate a potential FGFR-independent, cell-autonomous role for Pyr in supporting planar cell polarity. Aim 2 will follow up gene expression changes associated with pyr mutants identified by scRNA-seq, with particular focus on differences linked to the loss of the Pyr intracellular domain. Lastly, Aim 3 will focus on identifying Pyr-interacting proteins that mediate reverse signaling through affinity chromatography and mass spectrometry and characterize their mutant phenotypes. This work is significant as it is poised to redefine our understanding of FGF signaling across species by uncovering both conserved mechanisms and novel functions. More broadly, demonstrating dual roles for an FGF provides critical insights into a conserved signaling pathway that governs cardiac development and broader developmental programs, with implications for understanding congenital heart defects and tissue patterning in higher organisms.