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Rab GTPases are molecular switches that regulate membrane trafficking in all cells. Neurons have particular demands on membrane trafficking and express numerous Rab GTPases of unknown function. Here, we report the generation and characterization of molecularly defined null mutants for all 26 rab genes in Drosophila . In flies, all rab genes are expressed in the nervous system where at least half exhibit particularly high levels compared to other tissues. Surprisingly, loss of any of these 13 nervous system-enriched Rabs yielded viable and fertile flies without obvious morphological defects. However, all 13 mutants differentially affected development when challenged with different temperatures, or neuronal function when challenged with continuous stimulation. We identified a synaptic maintenance defect following continuous stimulation for six mutants, including an autophagy-independent role of rab26. The complete mutant collection generated in this study provides a basis for further comprehensive studies of Rab GTPases during development and function in vivo.

Isolation profoundly influences social behavior in all animals. In humans, isolation has serious effects on health. Drosophila melanogaster is a powerful model to study small-scale, temporally-transient social behavior. However, longer-term analysis of large groups of flies is hampered by the lack of effective and reliable tools. We built a new imaging arena and improved the existing tracking algorithm to reliably follow a large number of flies simultaneously. Next, based on the automatic classification of touch and graph-based social network analysis, we designed an algorithm to quantify changes in the social network in response to prior social isolation. We observed that isolation significantly and swiftly enhanced individual and local social network parameters depicting near-neighbor relationships. We explored the genome-wide molecular correlates of these behavioral changes and found that whereas behavior changed throughout the six days of isolation, gene expression alterations occurred largely on day one. These changes occurred mostly in metabolic genes, and we verified the metabolic changes by showing an increase of lipid content in isolated flies. In summary, we describe a highly reliable tracking and analysis pipeline for large groups of flies that we use to unravel the behavioral, molecular and physiological impact of isolation on social network dynamics in Drosophila.

The role of the central neuropeptide pigment-dispersing factor (PDF) in circadian timekeeping in Drosophila is remarkably similar to that of vasoactive intestinal peptide (VIP) in mammals. Like VIP, PDF is expressed outside the circadian network by neurons innervating the gut, but the function and mode of action of this PDF have not been characterized. Here we investigate the visceral roles of PDF by adapting cellular and physiological methods to the study of visceral responses to PDF signaling in wild-type and mutant genetic backgrounds. We find that intestinal PDF acts at a distance on the renal system, where it regulates ureter contractions. We show that PdfR, PDF's established receptor, is expressed by the muscles of the excretory system, and present evidence that PdfR-induced cAMP increases underlie the myotropic effects of PDF. These findings extend the similarities between PDF and VIP beyond their shared central role as circadian regulators, and uncover an unexpected endocrine mode of myotropic action for an intestinal neuropeptide on the renal system.

Animal behavior is individually variable, and this variability is often consistent over time, a phenomenon called individuality or personality when multiple traits are involved. However, most studies test individuality in only one environment, even though behavior is known to be context-dependent. Analogous to the human ‘person-situation debate,’ we asked whether and to what extent behavioral individuality persists across changing environmental situations in Drosophila melanogaster . Using established and new behavioral assays, we examined three individual traits, namely exploration, attention, and anxiety, across varying environmental contexts, including temperature, visual cues, and arena shape, in both walking and flying flies. We found that individuality is strongly context-dependent, but even under substantial environmental changes, at least one behavioral trait retained individual-specific variation. Different environmental features did not affect individuality equally; instead, they formed a hierarchy in their influence on behavioral consistency. This hierarchy was supported by generalized linear modeling and hierarchical linear mixed-model analysis. Our results show that, as in humans, individuality in flies persists across different situations, although less strongly than across repeated tests in the same context. These findings establish Drosophila as a model for dissecting the developmental, neural, and genetic mechanisms underlying consistent individual differences in behavior across variable environments.

A catalogue of neuronal cell types has often been called a ‘parts list’ of the brain 1 , and regarded as a prerequisite for understanding brain function 2 , 3 . In the optic lobe of Drosophila , rules of connectivity between cell types have already proven to be essential for understanding fly vision 4 , 5 . Here we analyse the fly connectome to complete the list of cell types intrinsic to the optic lobe, as well as the rules governing their connectivity. Most new cell types contain 10 to 100 cells, and integrate information over medium distances in the visual field. Some existing type families (Tm, Li, and LPi) 6 – 10 at least double in number of types. A new serpentine medulla (Sm) interneuron family contains more types than any other. Three families of cross-neuropil types are revealed. The consistency of types is demonstrated by analysing the distances in high-dimensional feature space, and is further validated by algorithms that select small subsets of discriminative features. We use connectivity to hypothesize about the functional roles of cell types in motion, object and colour vision. Connectivity with ‘boundary types’ that straddle the optic lobe and central brain is also quantified. We showcase the advantages of connectomic cell typing: complete and unbiased sampling, a rich array of features based on connectivity and reduction of the connectome to a substantially simpler wiring diagram of cell types, with immediate relevance for brain function and development. An analysis of the Drosophila connectome yields all cell types intrinsic to the optic lobe, and their rules of connectivity.

Animal behavior is individually variable, and this variability is often consistent over time, a phenomenon called individuality or personality when multiple traits are involved. However, most studies test individuality in only one environment, even though behavior is known to be context-dependent. Analogous to the human ‘person-situation debate,’ we asked whether and to what extent behavioral individuality persists across changing environmental situations in Drosophila melanogaster . Using established and new behavioral assays, we examined three individual traits, namely exploration, attention, and anxiety, across varying environmental contexts, including temperature, visual cues, and arena shape, in both walking and flying flies. We found that individuality is strongly context-dependent, but even under substantial environmental changes, at least one behavioral trait retained individual-specific variation. Different environmental features did not affect individuality equally; instead, they formed a hierarchy in their influence on behavioral consistency. This hierarchy was supported by generalized linear modeling and hierarchical linear mixed-model analysis. Our results show that, as in humans, individuality in flies persists across different situations, although less strongly than across repeated tests in the same context. These findings establish Drosophila as a model for dissecting the developmental, neural, and genetic mechanisms underlying consistent individual differences in behavior across variable environments.

The role of the central neuropeptide pigment-dispersing factor (PDF) in circadian timekeeping in Drosophila is remarkably similar to that of vasoactive intestinal peptide (VIP) in mammals. Like VIP, PDF is expressed outside the circadian network by neurons innervating the gut, but the function and mode of action of this PDF have not been characterized. Here we investigate the visceral roles of PDF by adapting cellular and physiological methods to the study of visceral responses to PDF signaling in wild-type and mutant genetic backgrounds. We find that intestinal PDF acts at a distance on the renal system, where it regulates ureter contractions. We show that PdfR, PDF's established receptor, is expressed by the muscles of the excretory system, and present evidence that PdfR-induced cAMP increases underlie the myotropic effects of PDF. These findings extend the similarities between PDF and VIP beyond their shared central role as circadian regulators, and uncover an unexpected endocrine mode of myotropic action for an intestinal neuropeptide on the renal system.

Over the past decade, several studies have demonstrated that idiosyncratic animal behaviors remain stable over long time periods. The stability of individually variable behaviors over time is often referred to as an animal's individuality, or personality. However, most experimental studies have focused on individuality in a single, well-defined environmental context, whereas it is well-established from population studies that animal behavior is highly context-dependent. The 'person-situation debate' in humans and decades of observations of animal individuality under intrinsically variable natural conditions raise the question of whether and to what extent animal behavior remains stable across different situations, such as changing environmental contexts. For instance, one individual might be generally more visually guided than another, or rely only on one particular visual cue, or even on this very cue only in a specific environmental context. Here, we use a combination of both well-established and novel behavioral assays to demonstrate the relationship between individual behavior and variable environmental context under tightly controlled laboratory conditions in the model system Drosophila melanogaster. The stability of three individual traits (termed exploration, attention, and anxiety) was investigated under changing environmental contexts (temperature, visual cues, arena shape), in both walking and flying flies. We find that individuality is highly context-dependent, but even under the most extreme environmental alterations tested, stability of behavior always persisted in at least one of the traits. Furthermore, our quantification reveals a hierarchical order of environmental features influencing individuality. In summary, our work demonstrates that, similar to humans, fly individuality persists across different contexts, and individual differences shape behavior across variable environments, thereby making the underlying developmental and functional mechanisms amenable to genetic dissection.Competing Interest StatementThe authors have declared no competing interest.Footnotes* We revised the manuscript to update and improve the text and to mainly improve the statistical modelling to obtain the data presented in the manuscript. All data has now been reanalized using a generalized linear model (GLM),

Brain wiring is remarkably precise, yet most neurons readily form synapses with incorrect partners when given the opportunity. Dynamic axon-dendritic positioning can restrict synaptogenic encounters, but the spatiotemporal interaction kinetics and their regulation remain essentially unknown inside developing brains. Here we show that the kinetics of axonal filopodia restrict synapse formation and partner choice for neurons that are not otherwise prevented from making incorrect synapses. Using 4D imaging in developing Drosophila brains, we show that filopodial kinetics are regulated by autophagy, a prevalent degradation mechanism whose role in brain development remains poorly understood. With surprising specificity, autophagosomes form in synaptogenic filopodia, followed by filopodial collapse. Altered autophagic degradation of synaptic building material quantitatively regulates synapse formation as shown by computational modeling and genetic experiments. Increased filopodial stability enables incorrect synaptic partnerships. Hence, filopodial autophagy restricts inappropriate partner choice through a process of kinetic exclusion that critically contributes to wiring specificity. The molecular mechanisms that restrict synapse formation with incorrect partners remain unclear. Here, authors use 4D imaging in developing Drosophila brains to show that filopodial kinetics are regulated by autophagy and this restricts inappropriate partner choice through a process of kinetic exclusion

Most animal tissues and organ systems are comprised of highly ordered arrays of varying cell types. The development of external sensory organs requires complex cell-cell communication in order to give each cell a specific identity and to ensure a regular distributed pattern of the sensory bristles. This involves both long and short range signaling mediated by either diffusible or cell anchored factors. In a variety of processes the heterophilic Irre Cell Recognition Module, consisting of the Neph-like proteins: Roughest, Kin of irre and of the Nephrin-like proteins: Sticks and Stones, Hibris, plays key roles in the recognition events of different cell types throughout development. In the present study these proteins are apically expressed in the adhesive belt of epithelial cells participating in sense organ development in a partially exclusive and asymmetric manner. Using mutant analysis the GAL4/UAS system, RNAi and gain of function we found an involvement of all four Irre Cell Recognition Module-proteins in the development of a highly structured array of sensory organs in the wing disc. The proteins secure the regular spacing of sensory organs showing partial redundancy and may function in early lateral inhibition events as well as in cell sorting processes. Comparisons with other systems suggest that the Irre Cell Recognition module is a key organizer of highly repetitive structures.