Neurotransmitter classification from electron microscopy images at synaptic sites in Drosophila melanogaster.

High-resolution electron microscopy of nervous systems has enabled the reconstruction of synaptic connectomes. However, we do not know the synaptic sign for each connection (i.e., whether a connection is excitatory or inhibitory), which is implied by the released transmitter. We demonstrate that artificial neural networks can predict transmitter types for presynapses from electron micrographs: a network trained to predict six transmitters (acetylcholine, glutamate, GABA, serotonin, dopamine, octopamine) achieves an accuracy of 87% for individual synapses, 94% for neurons, and 91% for known cell types across a D. melanogaster whole brain. We visualize the ultrastructural features used for prediction, discovering subtle but significant differences between transmitter phenotypes. We also analyze transmitter distributions across the brain and find that neurons that develop together largely express only one fast-acting transmitter (acetylcholine, glutamate, or GABA). We hope that our publicly available predictions act as an accelerant for neuroscientific hypothesis generation for the fly.

Mitochondrial ROS promotes susceptibility to infection via gasdermin D-mediated necroptosis.

Although mutations in mitochondrial-associated genes are linked to inflammation and susceptibility to infection, their mechanistic contributions to immune outcomes remain ill-defined. We discovered that the disease-associated gain-of-function allele Lrrk2G2019S (leucine-rich repeat kinase 2) perturbs mitochondrial homeostasis and reprograms cell death pathways in macrophages. When the inflammasome is activated in Lrrk2G2019S macrophages, elevated mitochondrial ROS (mtROS) directs association of the pore-forming protein gasdermin D (GSDMD) to mitochondrial membranes. Mitochondrial GSDMD pore formation then releases mtROS, promoting a switch to RIPK1/RIPK3/MLKL-dependent necroptosis. Consistent with enhanced necroptosis, infection of Lrrk2G2019S mice with Mycobacterium tuberculosis elicits hyperinflammation and severe immunopathology. Our findings suggest a pivotal role for GSDMD as an executer of multiple cell death pathways and demonstrate that mitochondrial dysfunction can direct immune outcomes via cell death modality switching. This work provides insights into how LRRK2 mutations manifest or exacerbate human diseases and identifies GSDMD-dependent necroptosis as a potential target to limit Lrrk2G2019S-mediated immunopathology.

Amyloid-like Assembly Activates a Phosphatase in the Developing Drosophila Embryo.

Prion-like proteins can assume distinct conformational and physical states in the same cell. Sequence analysis suggests that prion-like proteins are prevalent in various species; however, it remains unclear what functional space they occupy in multicellular organisms. Here, we report the identification of a prion-like protein, Herzog (CG5830), through a multimodal screen in Drosophila melanogaster. Herzog functions as a membrane-associated phosphatase and controls embryonic patterning, likely being involved in TGF-ß/BMP and FGF/EGF signaling pathways. Remarkably, monomeric Herzog is enzymatically inactive and becomes active upon amyloid-like assembly. The prion-like domain of Herzog is necessary for both its assembly and membrane targeting. Removal of the prion-like domain impairs activity, while restoring assembly on the membrane using a heterologous prion-like domain and membrane-targeting motif can restore phosphatase activity. This study provides an example of a prion-like domain that allows an enzyme to gain essential functionality via amyloid-like assembly to control animal development.

A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster.

Drosophila melanogaster has a rich repertoire of innate and learned behaviors. Its 100,000-neuron brain is a large but tractable target for comprehensive neural circuit mapping. Only electron microscopy (EM) enables complete, unbiased mapping of synaptic connectivity; however, the fly brain is too large for conventional EM. We developed a custom high-throughput EM platform and imaged the entire brain of an adult female fly at synaptic resolution. To validate the dataset, we traced brain-spanning circuitry involving the mushroom body (MB), which has been extensively studied for its role in learning. All inputs to Kenyon cells (KCs), the intrinsic neurons of the MB, were mapped, revealing a previously unknown cell type, postsynaptic partners of KC dendrites, and unexpected clustering of olfactory projection neurons. These reconstructions show that this freely available EM volume supports mapping of brain-spanning circuits, which will significantly accelerate Drosophila neuroscience. VIDEO ABSTRACT.

LlamaTags: A Versatile Tool to Image Transcription Factor Dynamics in Live Embryos.

Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. Here, we pioneer a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, we discovered stochastic bursts in the concentration of transcription factors that are correlated with bursts in transcription. We further used LlamaTags to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; we speculate that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos.

Dendritic Integration of Sensory Evidence in Perceptual Decision-Making.

Perceptual decisions require the accumulation of sensory information to a response criterion. Most accounts of how the brain performs this process of temporal integration have focused on evolving patterns of spiking activity. We report that subthreshold changes in membrane voltage can represent accumulating evidence before a choice. αß core Kenyon cells (αßc KCs) in the mushroom bodies of fruit flies integrate odor-evoked synaptic inputs to action potential threshold at timescales matching the speed of olfactory discrimination. The forkhead box P transcription factor (FoxP) sets neuronal integration and behavioral decision times by controlling the abundance of the voltage-gated potassium channel Shal (KV4) in αßc KC dendrites. αßc KCs thus tailor, through a particular constellation of biophysical properties, the generic process of synaptic integration to the demands of sequential sampling.

Representations of Novelty and Familiarity in a Mushroom Body Compartment.

Animals exhibit a behavioral response to novel sensory stimuli about which they have no prior knowledge. We have examined the neural and behavioral correlates of novelty and familiarity in the olfactory system of Drosophila. Novel odors elicit strong activity in output neurons (MBONs) of the α'3 compartment of the mushroom body that is rapidly suppressed upon repeated exposure to the same odor. This transition in neural activity upon familiarization requires odor-evoked activity in the dopaminergic neuron innervating this compartment. Moreover, exposure of a fly to novel odors evokes an alerting response that can also be elicited by optogenetic activation of α'3 MBONs. Silencing these MBONs eliminates the alerting behavior. These data suggest that the α'3 compartment plays a causal role in the behavioral response to novel and familiar stimuli as a consequence of dopamine-mediated plasticity at the Kenyon cell-MBONα'3 synapse.

Chromatin Architecture Emerges during Zygotic Genome Activation Independent of Transcription.

Chromatin architecture is fundamental in regulating gene expression. To investigate when spatial genome organization is first established during development, we examined chromatin conformation during Drosophila embryogenesis and observed the emergence of chromatin architecture within a tight time window that coincides with the onset of transcription activation in the zygote. Prior to zygotic genome activation, the genome is mostly unstructured. Early expressed genes serve as nucleation sites for topologically associating domain (TAD) boundaries. Activation of gene expression coincides with the establishment of TADs throughout the genome and co-localization of housekeeping gene clusters, which remain stable in subsequent stages of development. However, the appearance of TAD boundaries is independent of transcription and requires the transcription factor Zelda for locus-specific TAD boundary insulation. These results offer insight into when spatial organization of the genome emerges and identify a key factor that helps trigger this architecture.

Golgi-Resident Gαo Promotes Protrusive Membrane Dynamics.

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.

Drosophila melanogaster Set8 and L(3)mbt function in gene expression independently of histone H4 lysine 20 methylation.

Monomethylation of lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of Set8 null , H4 K20R/A , and l(3)mbt mutant Drosophila melanogaster We found that the gene expression profiles of H4 K20A and H4 K20R larvae are markedly different than Set8 null larvae despite similar reductions in H4K20me1. Set8 null mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo, but these phenotypes are not recapitulated by mutation of H4 K20 , indicating that the developmental defects of Set8 null animals are largely due to H4K20me1-independent effects on gene expression. Furthermore, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in Set8 null and H4 K20 mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin and therefore that H4K20me1 does not play a large role in gene expression.

Beyond histones: the elusive substrates of chromatin regulators.

Gene transcription is intimately linked to chromatin state and histone modifications. However, the enzymes mediating these post-translational modifications have many additional, nonhistone substrates, making it difficult to ascribe the most relevant modification. In this issue of Genes & Development, Crain and colleagues (doi:10.1101/gad.351698.124) have combined a powerful histone replacement system with mutational analysis of a chromatin regulator and a chromatin reader in Drosophila melanogaster Importantly, they discovered that genes controlled by the histone 4 lysine 20 (H4K20) methyltransferase Set8 and the protein recognizing H4K20 monomethylation, L(3)mbt, differ substantially from those affected by mutation of H4K20 itself. This demonstrates that H4K20 is not the key substrate for Set8 but that methylation of other, unidentified proteins mediates its effects on transcription.

Tolerance thresholds underlie responses to DNA damage during germline development.

Selfish DNA modules like transposable elements (TEs) are particularly active in the germline, the lineage that passes genetic information across generations. New TE insertions can disrupt genes and impair the functionality and viability of germ cells. However, we found that in P-M hybrid dysgenesis in Drosophila, a sterility syndrome triggered by the P-element DNA transposon, germ cells harbor unexpectedly few new TE insertions despite accumulating DNA double-strand breaks (DSBs) and inducing cell cycle arrest. Using an engineered CRISPR-Cas9 system, we show that generating DSBs at silenced P-elements or other noncoding sequences is sufficient to induce germ cell loss independently of gene disruption. Indeed, we demonstrate that both developing and adult mitotic germ cells are sensitive to DSBs in a dosage-dependent manner. Following the mitotic-to-meiotic transition, however, germ cells become more tolerant to DSBs, completing oogenesis regardless of the accumulated genome damage. Our findings establish DNA damage tolerance thresholds as crucial safeguards of genome integrity during germline development.

Long ant life span is maintained by a unique heat shock factor.

Eusocial insect reproductive females show strikingly longer life spans than nonreproductive female workers despite high genetic similarity. In the ant Harpegnathos saltator (Hsal), workers can transition to reproductive "gamergates," acquiring a fivefold prolonged life span by mechanisms that are poorly understood. We found that gamergates have elevated expression of heat shock response (HSR) genes in the absence of heat stress and enhanced survival with heat stress. This HSR gene elevation is driven in part by gamergate-specific up-regulation of the gene encoding a truncated form of a heat shock factor most similar to mammalian HSF2 (hsalHSF2). In workers, hsalHSF2 was bound to DNA only upon heat stress. In gamergates, hsalHSF2 bound to DNA even in the absence of heat stress and was localized to gamergate-biased HSR genes. Expression of hsalHSF2 in Drosophila melanogaster led to enhanced heat shock survival and extended life span in the absence of heat stress. Molecular characterization illuminated multiple parallels between long-lived flies and gamergates, underscoring the centrality of hsalHSF2 to extended ant life span. Hence, ant caste-specific heat stress resilience and extended longevity can be transferred to flies via hsalHSF2. These findings reinforce the critical role of proteostasis in health and aging and reveal novel mechanisms underlying facultative life span extension in ants.

Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex.

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals.

piRNA-mediated gene regulation and adaptation to sex-specific transposon expression in D. melanogaster male germline.

Small noncoding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of the piRNA pathway in transposon silencing and therefore genome defense. However, the ability of the piRNA program to respond to different transposon landscapes and the role of piRNAs in regulating host gene expression remain poorly understood. Here, we comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, we identified new piRNA clusters and established complex satellites as dual-strand piRNA clusters. While sharing most piRNA clusters, the two sexes employ them differentially to combat the sex-specific transposon landscape. We found two piRNA clusters that produce piRNAs antisense to four host genes in testis, including CG12717/pirate, a SUMO protease gene. piRNAs encoded on the Y chromosome silence pirate, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pirate is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-coding gene.

Twist-dependent ratchet functioning downstream from Dorsal revealed using a light-inducible degron.

Graded transcription factors are pivotal regulators of embryonic patterning, but whether their role changes over time is unclear. A light-regulated protein degradation system was used to assay temporal dependence of the transcription factor Dorsal in dorsal-ventral axis patterning of Drosophila embryos. Surprisingly, the high-threshold target gene snail only requires Dorsal input early but not late when Dorsal levels peak. Instead, late snail expression can be supported by action of the Twist transcription factor, specifically, through one enhancer, sna.distal This study demonstrates that continuous input is not required for some Dorsal targets and downstream responses, such as twist, function as molecular ratchets.

A phylogenetic profiling approach identifies novel ciliogenesis genes in Drosophila and C. elegans.

Cilia are cellular projections that perform sensory and motile functions in eukaryotic cells. A defining feature of cilia is that they are evolutionarily ancient, yet not universally conserved. In this study, we have used the resulting presence and absence pattern in the genomes of diverse eukaryotes to identify a set of 386 human genes associated with cilium assembly or motility. Comprehensive tissue-specific RNAi in Drosophila and mutant analysis in C. elegans revealed signature ciliary defects for 70-80% of novel genes, a percentage similar to that for known genes within the cluster. Further characterization identified different phenotypic classes, including a set of genes related to the cartwheel component Bld10/CEP135 and two highly conserved regulators of cilium biogenesis. We propose this dataset defines the core set of genes required for cilium assembly and motility across eukaryotes and presents a valuable resource for future studies of cilium biology and associated disorders.

The CD36 scavenger receptor Bez regulates lipid redistribution from fat body to ovaries in Drosophila.

Lipid distribution in an organism is mediated by the interplay between lipoprotein particles, lipoprotein receptors and class B scavenger receptors of the CD36 family. CD36 is a multifunctional protein mediating lipid uptake, mobilization and signaling at the plasma membrane and inside of the cell. The CD36 protein family has 14 members in Drosophila melanogaster, which allows for the differentiated analysis of their functions. Here, we unravel a role for the so far uncharacterized scavenger receptor Bez in lipid export from Drosophila adipocytes. Bez shares the lipid binding residue with CD36 and is expressed at the plasma membrane of the embryonic, larval and adult fat body. Bez loss of function lowers the organismal availability of storage lipids and blocks the maturation of egg chambers in ovaries. We demonstrate that Bez interacts with the APOB homolog Lipophorin at the plasma membrane of adipocytes and trace the Bez-dependent transfer of an alkyne-labeled fatty acid from adipocytes to Lipophorin. Our study demonstrates how lipids are distributed by scavenger receptor-lipoprotein interplay and contribute to the metabolic control of development.

Epidermal tyrosine catabolism is crucial for metabolic homeostasis and survival against high-protein diets in Drosophila.

The insect epidermis forms the exoskeleton and determines the body size of an organism. How the epidermis acts as a metabolic regulator to adapt to changes in dietary protein availability remains elusive. Here, we show that the Drosophila epidermis regulates tyrosine (Tyr) catabolism in response to dietary protein levels, thereby promoting metabolic homeostasis. The gene expression profile of the Drosophila larval body wall reveals that enzymes involved in the Tyr degradation pathway, including 4-hydroxyphenylpyruvate dioxygenase (Hpd), are upregulated by increased protein intake. Hpd is specifically expressed in the epidermis and is dynamically regulated by the internal Tyr levels. Whereas basal Hpd expression is maintained by insulin/IGF-1 signalling, Hpd induction on high-protein diet requires activation of the AMP-activated protein kinase (AMPK)-forkhead box O subfamily (FoxO) axis. Impairment of the FoxO-mediated Hpd induction in the epidermis leads to aberrant increases in internal Tyr and its metabolites, disrupting larval development on high-protein diets. Taken together, our findings uncover a crucial role of the epidermis as a metabolic regulator in coping with an unfavourable dietary environment.

The foraging Gene and Its Behavioral Effects: Pleiotropy and Plasticity.

The Drosophila melanogaster foraging (for) gene is a well-established example of a gene with major effects on behavior and natural variation. This gene is best known for underlying the behavioral strategies of rover and sitter foraging larvae, having been mapped and named for this phenotype. Nevertheless, in the last three decades an extensive array of studies describing for's role as a modifier of behavior in a wide range of phenotypes, in both Drosophila and other organisms, has emerged. Furthermore, recent work reveals new insights into the genetic and molecular underpinnings of how for affects these phenotypes. In this article, we discuss the history of the for gene and its role in natural variation in behavior, plasticity, and behavioral pleiotropy, with special attention to recent findings on the molecular structure and transcriptional regulation of this gene.