Gut tumors in flies alter the taste valence of an anti-tumorigenic bitter compound.

The sense of taste is essential for survival, as it allows animals to distinguish between foods that are nutritious from those that are toxic. However, innate responses to different tastants can be modulated or even reversed under pathological conditions. Here, we examined whether and how the internal status of an animal impacts taste valence by using Drosophila models of hyperproliferation in the gut. In all three models where we expressed proliferation-inducing transgenes in intestinal stem cells (ISCs), hyperproliferation of ISCs caused a tumor-like phenotype in the gut. While tumor-bearing flies had no deficiency in overall food intake, strikingly, they exhibited an increased gustatory preference for aristolochic acid (ARI), which is a bitter and normally aversive plant-derived chemical. ARI had anti-tumor effects in all three of our gut hyperproliferation models. For other aversive chemicals we tested that are bitter but do not have anti-tumor effects, gut tumors did not affect avoidance behaviors. We demonstrated that bitter-sensing gustatory receptor neurons (GRNs) in tumor-bearing flies respond normally to ARI. Therefore, the internal pathology of gut hyperproliferation affects neural circuits that determine taste valence postsynaptic to GRNs rather than altering taste identity by GRNs. Overall, our data suggest that increased consumption of ARI may represent an attempt at self-medication. Finally, although ARI's potential use as a chemotherapeutic agent is limited by its known toxicity in the liver and kidney, our findings suggest that tumor-bearing flies might be a useful animal model to screen for novel anti-tumor drugs.

Mechanistic characterization of a Drosophila model of paraneoplastic nephrotic syndrome.

Paraneoplastic syndromes occur in cancer patients and originate from dysfunction of organs at a distance from the tumor or its metastasis. A wide range of organs can be affected in paraneoplastic syndromes; however, the pathological mechanisms by which tumors influence host organs are poorly understood. Recent studies in the fly uncovered that tumor secreted factors target host organs, leading to pathological effects. In this study, using a Drosophila gut tumor model, we characterize a mechanism of tumor-induced kidney dysfunction. Specifically, we find that Pvf1, a PDGF/VEGF signaling ligand, secreted by gut tumors activates the PvR/JNK/Jra signaling pathway in the principal cells of the kidney, leading to mis-expression of renal genes and paraneoplastic renal syndrome-like phenotypes. Our study describes an important mechanism by which gut tumors perturb the function of the kidney, which might be of clinical relevance for the treatment of paraneoplastic syndromes.

FIBCD1 is an endocytic GAG receptor associated with a novel neurodevelopmental disorder.

Whole-exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group-binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate-4S (CS-4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss-of-function by disrupting FIBCD1-CS-4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor-related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal-dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity.

FlyPhoneDB: an integrated web-based resource for cell-cell communication prediction in Drosophila.

Multicellular organisms rely on cell-cell communication to exchange information necessary for developmental processes and metabolic homeostasis. Cell-cell communication pathways can be inferred from transcriptomic datasets based on ligand-receptor expression. Recently, data generated from single-cell RNA sequencing have enabled ligand-receptor interaction predictions at an unprecedented resolution. While computational methods are available to infer cell-cell communication in vertebrates such a tool does not yet exist for Drosophila. Here, we generated a high-confidence list of ligand-receptor pairs for the major fly signaling pathways and developed FlyPhoneDB, a quantification algorithm that calculates interaction scores to predict ligand-receptor interactions between cells. At the FlyPhoneDB user interface, results are presented in a variety of tabular and graphical formats to facilitate biological interpretation. To illustrate that FlyPhoneDB can effectively identify active ligands and receptors to uncover cell-cell communication events, we applied FlyPhoneDB to Drosophila single-cell RNA sequencing data sets from adult midgut, abdomen, and blood, and demonstrate that FlyPhoneDB can readily identify previously characterized cell-cell communication pathways. Altogether, FlyPhoneDB is an easy-to-use framework that can be used to predict cell-cell communication between cell types from single-cell RNA sequencing data in Drosophila.

Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies.

Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (<15aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.

Defining cell types and lineage in the Drosophila midgut using single cell transcriptomics.

The Drosophila midgut has emerged in recent years as a model system to study stem cell renewal and differentiation and tissue homeostasis. Histological, genetic and gene expression studies have provided a wealth of information on gut cell types, regionalization, genes and pathways involved in cell proliferation and differentiation, stem cell renewal, and responses to changes in environmental factors such as the microbiota and nutrients. Here, we review the contribution of single cell transcriptomic methods to our understanding of gut cell type diversity, lineage and behavior.

Deterministic splicing of Dscam2 is regulated by Muscleblind.

Alternative splicing increases the proteome diversity crucial for establishing the complex circuitry between trillions of neurons. To provide individual cells with different repertoires of protein isoforms, however, this process must be regulated. Previously, we found that the mutually exclusive alternative splicing of Drosophila Dscam2 produces two isoforms (A and B) with unique binding properties. This splicing event is cell type specific, and the transmembrane proteins that it generates are crucial for the development of axons, dendrites, and synapses. Here, we show that Muscleblind (Mbl) controls Dscam2 alternative splicing. Mbl represses isoform A and promotes the selection of isoform B. Mbl mutants exhibit phenotypes also observed in flies engineered to express a single Dscam2 isoform. Consistent with this, mbl expression is cell type specific and correlates with the splicing of isoform B. Our study demonstrates how the regulated expression of a splicing factor is sufficient to provide neurons with unique protein isoforms crucial for development.

Regulated Alternative Splicing of Drosophila Dscam2 Is Necessary for Attaining the Appropriate Number of Photoreceptor Synapses.

How the brain makes trillions of synaptic connections using a genome of only 20,000 genes is a major question in modern neuroscience. Alternative splicing is one mechanism that can increase the number of proteins produced by each gene, but its role in regulating synapse formation is poorly understood. In Drosophila, photoreceptors form a synapse with multiple postsynaptic elements including lamina neurons L1 and L2. L1 and L2 express distinct isoforms of the homophilic repulsive protein Dscam2, and since these isoforms cannot bind to each other, cell-specific expression has been proposed to be necessary for preventing repulsive interactions that could disrupt the synapse. Here, we show that the number of synapses are reduced in flies that express only one isoform, and L1 and L2 dendritic morphology is perturbed. We propose that these defects result from inappropriate interactions between L1 and L2 dendrites. We conclude that regulated Dscam2 alternative splicing is necessary for the proper assembly of photoreceptor synapses.

Neuronal cell-type-specific alternative splicing: A mechanism for specifying connections in the brain?

Alternative splicing (AS) allows a single gene to generate multiple protein isoforms. It has been hypothesized that AS plays a role in brain wiring by increasing the number of cell recognition molecules necessary for forming connections between neurons. Many studies have characterized isoform expression patterns of various genes in the brain, but very few have addressed whether specific isoforms play a functional role in neuronal wiring. In our recent work, we reported the cell-type-specific AS of the cell recognition molecule Dscam2. Exclusive expression of Dscam2 isoforms allows tightly associated neurons to signal repulsion selectively within the same cell-types, without interfering with one another. We show that preventing cell-specific isoform expression in 2 closely associated neurons disrupts their axon terminal morphology. We propose that the requirement for isoform specificity extends to synapses and discuss experiments that can test this directly. Factors that regulate Dscam2 cell-type-specific AS likely regulate the splicing of many genes involved in neurodevelopment. These regulators of alternative splicing may act broadly to control many genes involved in the development of specific neuron types. Identifying these factors is a key step in understanding how AS contributes to the brain connectome.

Cell-specific alternative splicing of Drosophila Dscam2 is crucial for proper neuronal wiring.

How a finite number of genes specify a seemingly infinite number of neuronal connections is a central question in neurobiology. Alternative splicing has been proposed to increase proteome diversity in the brain. Here we show that cell-specific alternative splicing of a cell-surface protein is crucial for neuronal wiring. Down syndrome cell adhesion molecule 2 (Dscam2) is a conserved homophilic binding protein that can induce repulsion between opposing neurons. In the fly visual system, L1 and L2 neurons both require Dscam2 repulsion, but paradoxically, they also physically contact each other. We found that the cell-specific expression of two biochemically distinct alternative isoforms of Dscam2 prevents these cells from repelling each other. Phenotypes were observed in the axon terminals of L1 and L2 when they expressed the incorrect isoform, demonstrating a requirement for distinct isoforms. We conclude that cell-specific alternative splicing is a mechanism for achieving proper connectivity between neurons.