Fat cadherins have critical and conserved roles in coordinating tissue growth and tissue organization. How these cell-adhesion molecules coordinately control growth and patterning is still not well understood. To address this need, we have initiated comprehensive CRISPR-based mutagenesis of Drosophila ft in vivo, and used endogenous Ft tagged with GFP to identify co-immunoprecipitating proteins with mass spectrometry. Fine-scale mutagenesis of endogenous ft indicated Ft has critical roles in control of bilateral symmetry. Proteomic studies of endogenous complexes revealed nuclear proteins that interact with Ft, suggesting novel functions of Ft in transcriptional regulation. To investigate these roles of Ft we propose three specific Aims. 1. Delineate growth and PCP domains and define protein interactors in vivo. We will use in vivo gene editing approaches to define domains of Ft that regulate planar cell polarity (PCP) tissue organization and growth. Using endogenous Ft-GFP, we identified novel in vivo Ft binding partners. We will confirm these interactors and investigate their functional relevance. 2. Define the function of Ft in fluctuating asymmetry (FA). Deletion of a region of Fat that is highly conserved in Drosophila and humans results in viable flies that have lost fine control of bilateral symmetry. This implies Ft has a role in mechanisms underlying bilateral symmetry, and functions to sense or implement fine-scale organ checkpoints. We will test if alterations in known outputs of Ft (PCP, Hippo or mitochondrial function) are responsible for FA, and test if genes implicated in FA are affected by Ft. We will determine the tissue specificity of Ft in restricting FA, and investigate proteins that bind the D region for function in FA. 3. Define the function of Ft in the nucleus. We found the cytoplasmic domain of Ft can localize to the nucleus and interact with chromatin remodeling complexes. We will define NLS and NES sequences, and determine the consequences of deleting those sequences in the endogenous locus. We will determine if nuclear Ft specifically affects Hippo, PCP signaling or metabolism, and investigate Ft transcriptional regulation of target genes. RNAseq, ChIP-seq and in vivo reporter analyses will be used to determine the function of nuclear Ft. If successful, these studies will illuminate novel functions of Ft in control of bilateral symmetry and transcription, and provide mechanistic insight into regulation and integration of Ft?s diverse functions in vivo
Fat cadherins are large cell-cell adhesion molecules that control tissue growth and tissue organization in flies and man. Mutations in Fat cadherins in humans result in devastating diseases such as Van Maldergem syndrome, characterized by intellectual disabilty, and a range of developmental defects that include craniofacial defects, limb malformations and kidney defects. Mutations in Fat cadherins are also found in many cancers. We use Drosophila as a powerful genetic model to understand the molecular mechanisms by which Fat cadherins regulate development, and why mutations lead to human disease.
