Orb2 enables rare-codon-enriched mRNA expression during Drosophila neuron differentiation.

Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent mRNA stability in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for mRNA stability and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA stability and protein expression.

Senescent glia link mitochondrial dysfunction and lipid accumulation.

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species1,2. Acutely, senescent cells promote wound healing3,4 and prevent tumour formation5; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy6-11, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear12,13. Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence14,15, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

Centrosome-organized plasma membrane infoldings linked to growth of a cortical actin domain.

Regulated cell shape change requires the induction of cortical cytoskeletal domains. Often, local changes to plasma membrane (PM) topography are involved. Centrosomes organize cortical domains and can affect PM topography by locally pulling the PM inward. Are these centrosome effects coupled? At the syncytial Drosophila embryo cortex, centrosome-induced actin caps grow into dome-like compartments for mitoses. We found the nascent cap to be a collection of PM folds and tubules formed over the astral centrosomal MT array. The localized infoldings require centrosome and dynein activities, and myosin-based surface tension prevents them elsewhere. Centrosome-engaged PM infoldings become specifically enriched with an Arp2/3 induction pathway. Arp2/3 actin network growth between the infoldings counterbalances centrosomal pulling forces and disperses the folds for actin cap expansion. Abnormal domain topography with either centrosome or Arp2/3 disruption correlates with decreased exocytic vesicle association. Together, our data implicate centrosome-organized PM infoldings in coordinating Arp2/3 network growth and exocytosis for cortical domain assembly.

Descending networks transform command signals into population motor control.

To convert intentions into actions, movement instructions must pass from the brain to downstream motor circuits through descending neurons (DNs). These include small sets of command-like neurons that are sufficient to drive behaviours1-the circuit mechanisms for which remain unclear. Here we show that command-like DNs in Drosophila directly recruit networks of additional DNs to orchestrate behaviours that require the active control of numerous body parts. Specifically, we found that command-like DNs previously thought to drive behaviours alone2-4 in fact co-activate larger populations of DNs. Connectome analyses and experimental manipulations revealed that this functional recruitment can be explained by direct excitatory connections between command-like DNs and networks of interconnected DNs in the brain. Descending population recruitment is necessary for behavioural control: DNs with many downstream descending partners require network co-activation to drive complete behaviours and drive only simple stereotyped movements in their absence. These DN networks reside within behaviour-specific clusters that inhibit one another. These results support a mechanism for command-like descending control in which behaviours are generated through the recruitment of increasingly large DN networks that compose behaviours by combining multiple motor subroutines.

Drosophila immune cells transport oxygen through PPO2 protein phase transition.

Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells1,2. Here we describe that Drosophila crystal cells-myeloid-like immune cells called haemocytes-control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, we propose that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.

Connectomic reconstruction of a female Drosophila ventral nerve cord.

A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC)1, which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines2 and X-ray holographic nanotomography3. With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.

Synaptic architecture of leg and wing premotor control networks in Drosophila.

Animal movement is controlled by motor neurons (MNs), which project out of the central nervous system to activate muscles1. MN activity is coordinated by complex premotor networks that facilitate the contribution of individual muscles to many different behaviours2-6. Here we use connectomics7 to analyse the wiring logic of premotor circuits controlling the Drosophila leg and wing. We find that both premotor networks cluster into modules that link MNs innervating muscles with related functions. Within most leg motor modules, the synaptic weights of each premotor neuron are proportional to the size of their target MNs, establishing a circuit basis for hierarchical MN recruitment. By contrast, wing premotor networks lack proportional synaptic connectivity, which may enable more flexible recruitment of wing steering muscles. Through comparison of the architecture of distinct motor control systems within the same animal, we identify common principles of premotor network organization and specializations that reflect the unique biomechanical constraints and evolutionary origins of leg and wing motor control.

Mapping model units to visual neurons reveals population code for social behaviour.

The rich variety of behaviours observed in animals arises through the interplay between sensory processing and motor control. To understand these sensorimotor transformations, it is useful to build models that predict not only neural responses to sensory input1-5 but also how each neuron causally contributes to behaviour6,7. Here we demonstrate a novel modelling approach to identify a one-to-one mapping between internal units in a deep neural network and real neurons by predicting the behavioural changes that arise from systematic perturbations of more than a dozen neuronal cell types. A key ingredient that we introduce is 'knockout training', which involves perturbing the network during training to match the perturbations of the real neurons during behavioural experiments. We apply this approach to model the sensorimotor transformations of Drosophila melanogaster males during a complex, visually guided social behaviour8-11. The visual projection neurons at the interface between the optic lobe and central brain form a set of discrete channels12, and prior work indicates that each channel encodes a specific visual feature to drive a particular behaviour13,14. Our model reaches a different conclusion: combinations of visual projection neurons, including those involved in non-social behaviours, drive male interactions with the female, forming a rich population code for behaviour. Overall, our framework consolidates behavioural effects elicited from various neural perturbations into a single, unified model, providing a map from stimulus to neuronal cell type to behaviour, and enabling future incorporation of wiring diagrams of the brain15 into the model.

Rapid Preparation of Living Drosophila Pupal Macrophages for Ex Vivo Imaging.

The fruit fly Drosophila is a well-established invertebrate model that enables in vivo imaging of innate immune cell (e.g., macrophage) migration and signaling at high spatiotemporal resolution within the intact, living animal. While optimized methods already exist to enable flow cytometry-based macrophage isolation from Drosophila at various stages of development, there remains a need for more rapid and gentle methods to isolate living macrophages for downstream ex vivo applications. Here, we describe techniques for rapid and direct isolation of living macrophages from mature Drosophila pupae and their downstream ex vivo preparation for live imaging and immunostaining. This strategy enables straightforward access to physiologically relevant innate immune cells, both circulating and tissue-resident populations, for subsequent imaging of signal transduction.

Transient loss of Polycomb components induces an epigenetic cancer fate.

Although cancer initiation and progression are generally associated with the accumulation of somatic mutations1,2, substantial epigenomic alterations underlie many aspects of tumorigenesis and cancer susceptibility3-6, suggesting that genetic mechanisms might not be the only drivers of malignant transformation7. However, whether purely non-genetic mechanisms are sufficient to initiate tumorigenesis irrespective of mutations has been unknown. Here, we show that a transient perturbation of transcriptional silencing mediated by Polycomb group proteins is sufficient to induce an irreversible switch to a cancer cell fate in Drosophila. This is linked to the irreversible derepression of genes that can drive tumorigenesis, including members of the JAK-STAT signalling pathway and zfh1, the fly homologue of the ZEB1 oncogene, whose aberrant activation is required for Polycomb perturbation-induced tumorigenesis. These data show that a reversible depletion of Polycomb proteins can induce cancer in the absence of driver mutations, suggesting that tumours can emerge through epigenetic dysregulation leading to inheritance of altered cell fates.

Mechanical stress combines with planar polarised patterning during metaphase to orient embryonic epithelial cell divisions.

The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.

InR and Pi3K maintain intestinal homeostasis through STAT/EGFR and Notch signaling in enteroblasts.

To maintain the integrity of the adult gut, the proliferation and differentiation of stem cells must be strictly controlled. Several signaling pathways control the proliferation and differentiation of Drosophila intestinal epithelial cells. Although the modulatory effects of insulin pathway components on cell proliferation have been characterized, their specific role in which cell type and how these components interact with other regulatory signaling pathways remain largely unclear. In this study, we found that InR/Pi3K has major functions in enteroblasts (EBs) that were not previously described. The absence of InR/Pi3K in progenitors leads to a decrease in the number of EBs, while it has no significant effect on intestinal stem cells (ISCs). In addition, we found that InR/Pi3K regulates Notch activity in ISCs and EBs in an opposite way. This is also the reason for the decrease in EB. On the one hand, aberrantly low levels of Notch signaling in ISCs inhibit their proper differentiation into EBs; on the other hand, the higher Notch levels in EBs promote their excessive differentiation into enterocytes (ECs), leading to marked increases in abnormal ECs and decreased proliferation. Moreover, we found that Upd/JAK/STAT signaling acts as an effector or modifier of InR/Pi3K function in the midgut and cooperates with EGFR signaling to regulate cell proliferation. Altogether, our results demonstrate that InR and Pi3K are essential for coordinating stem cell differentiation and proliferation to maintain intestinal homeostasis.

Converting an allocentric goal into an egocentric steering signal.

Neuronal signals that are relevant for spatial navigation have been described in many species1-10. However, a circuit-level understanding of how such signals interact to guide navigational behaviour is lacking. Here we characterize a neuronal circuit in the Drosophila central complex that compares internally generated estimates of the heading and goal angles of the fly-both of which are encoded in world-centred (allocentric) coordinates-to generate a body-centred (egocentric) steering signal. Past work has suggested that the activity of EPG neurons represents the fly's moment-to-moment angular orientation, or heading angle, during navigation2,11. An animal's moment-to-moment heading angle, however, is not always aligned with its goal angle-that is, the allocentric direction in which it wishes to progress forward. We describe FC2 cells12, a second set of neurons in the Drosophila brain with activity that correlates with the fly's goal angle. Focal optogenetic activation of FC2 neurons induces flies to orient along experimenter-defined directions as they walk forward. EPG and FC2 neurons connect monosynaptically to a third neuronal class, PFL3 cells12,13. We found that individual PFL3 cells show conjunctive, spike-rate tuning to both the heading angle and the goal angle during goal-directed navigation. Informed by the anatomy and physiology of these three cell classes, we develop a model that explains how this circuit compares allocentric heading and goal angles to build an egocentric steering signal in the PFL3 output terminals. Quantitative analyses and optogenetic manipulations of PFL3 activity support the model. Finally, using a new navigational memory task, we show that flies expressing disruptors of synaptic transmission in subsets of PFL3 cells have a reduced ability to orient along arbitrary goal directions, with an effect size in quantitative accordance with the prediction of our model. The biological circuit described here reveals how two population-level allocentric signals are compared in the brain to produce an egocentric output signal that is appropriate for motor control.

Transforming a head direction signal into a goal-oriented steering command.

To navigate, we must continuously estimate the direction we are headed in, and we must correct deviations from our goal1. Direction estimation is accomplished by ring attractor networks in the head direction system2,3. However, we do not fully understand how the sense of direction is used to guide action. Drosophila connectome analyses4,5 reveal three cell populations (PFL3R, PFL3L and PFL2) that connect the head direction system to the locomotor system. Here we use imaging, electrophysiology and chemogenetic stimulation during navigation to show how these populations function. Each population receives a shifted copy of the head direction vector, such that their three reference frames are shifted approximately 120° relative to each other. Each cell type then compares its own head direction vector with a common goal vector; specifically, it evaluates the congruence of these vectors via a nonlinear transformation. The output of all three cell populations is then combined to generate locomotor commands. PFL3R cells are recruited when the fly is oriented to the left of its goal, and their activity drives rightward turning; the reverse is true for PFL3L. Meanwhile, PFL2 cells increase steering speed, and are recruited when the fly is oriented far from its goal. PFL2 cells adaptively increase the strength of steering as directional error increases, effectively managing the tradeoff between speed and accuracy. Together, our results show how a map of space in the brain can be combined with an internal goal to generate action commands, via a transformation from world-centric coordinates to body-centric coordinates.

Cell-type-directed design of synthetic enhancers.

Transcriptional enhancers act as docking stations for combinations of transcription factors and thereby regulate spatiotemporal activation of their target genes1. It has been a long-standing goal in the field to decode the regulatory logic of an enhancer and to understand the details of how spatiotemporal gene expression is encoded in an enhancer sequence. Here we show that deep learning models2-6, can be used to efficiently design synthetic, cell-type-specific enhancers, starting from random sequences, and that this optimization process allows detailed tracing of enhancer features at single-nucleotide resolution. We evaluate the function of fully synthetic enhancers to specifically target Kenyon cells or glial cells in the fruit fly brain using transgenic animals. We further exploit enhancer design to create 'dual-code' enhancers that target two cell types and minimal enhancers smaller than 50 base pairs that are fully functional. By examining the state space searches towards local optima, we characterize enhancer codes through the strength, combination and arrangement of transcription factor activator and transcription factor repressor motifs. Finally, we apply the same strategies to successfully design human enhancers, which adhere to enhancer rules similar to those of Drosophila enhancers. Enhancer design guided by deep learning leads to better understanding of how enhancers work and shows that their code can be exploited to manipulate cell states.

A novel antidiuretic hormone governs tumour-induced renal dysfunction.

Maintenance of renal function and fluid transport are essential for vertebrates and invertebrates to adapt to physiological and pathological challenges. Human patients with malignant tumours frequently develop detrimental renal dysfunction and oliguria, and previous studies suggest the involvement of chemotherapeutic toxicity and tumour-associated inflammation1,2. However, how tumours might directly modulate renal functions remains largely unclear. Here, using conserved tumour models in Drosophila melanogaster3, we characterized isoform F of ion transport peptide (ITPF) as a fly antidiuretic hormone that is secreted by a subset of yki3SA gut tumour cells, impairs renal function and causes severe abdomen bloating and fluid accumulation. Mechanistically, tumour-derived ITPF targets the G-protein-coupled receptor TkR99D in stellate cells of Malpighian tubules-an excretory organ that is equivalent to renal tubules4-to activate nitric oxide synthase-cGMP signalling and inhibit fluid excretion. We further uncovered antidiuretic functions of mammalian neurokinin 3 receptor (NK3R), the homologue of fly TkR99D, as pharmaceutical blockade of NK3R efficiently alleviates renal tubular dysfunction in mice bearing different malignant tumours. Together, our results demonstrate a novel antidiuretic pathway mediating tumour-renal crosstalk across species and offer therapeutic opportunities for the treatment of cancer-associated renal dysfunction.

Flexible circuit mechanisms for context-dependent song sequencing.

Sequenced behaviours, including locomotion, reaching and vocalization, are patterned differently in different contexts, enabling animals to adjust to their environments. How contextual information shapes neural activity to flexibly alter the patterning of actions is not fully understood. Previous work has indicated that this could be achieved via parallel motor circuits, with differing sensitivities to context1,2. Here we demonstrate that a single pathway operates in two regimes dependent on recent sensory history. We leverage the Drosophila song production system3 to investigate the role of several neuron types4-7 in song patterning near versus far from the female fly. Male flies sing 'simple' trains of only one mode far from the female fly but complex song sequences comprising alternations between modes when near her. We find that ventral nerve cord (VNC) circuits are shaped by mutual inhibition and rebound excitability8 between nodes driving the two song modes. Brief sensory input to a direct brain-to-VNC excitatory pathway drives simple song far from the female, whereas prolonged input enables complex song production via simultaneous recruitment of functional disinhibition of VNC circuitry. Thus, female proximity unlocks motor circuit dynamics in the correct context. We construct a compact circuit model to demonstrate that the identified mechanisms suffice to replicate natural song dynamics. These results highlight how canonical circuit motifs8,9 can be combined to enable circuit flexibility required for dynamic communication.

Dopaminergic systems create reward seeking despite adverse consequences.

Resource-seeking behaviours are ordinarily constrained by physiological needs and threats of danger, and the loss of these controls is associated with pathological reward seeking1. Although dysfunction of the dopaminergic valuation system of the brain is known to contribute towards unconstrained reward seeking2,3, the underlying reasons for this behaviour are unclear. Here we describe dopaminergic neural mechanisms that produce reward seeking despite adverse consequences in Drosophila melanogaster. Odours paired with optogenetic activation of a defined subset of reward-encoding dopaminergic neurons become cues that starved flies seek while neglecting food and enduring electric shock punishment. Unconstrained seeking of reward is not observed after learning with sugar or synthetic engagement of other dopaminergic neuron populations. Antagonism between reward-encoding and punishment-encoding dopaminergic neurons accounts for the perseverance of reward seeking despite punishment, whereas synthetic engagement of the reward-encoding dopaminergic neurons also impairs the ordinary need-dependent dopaminergic valuation of available food. Connectome analyses reveal that the population of reward-encoding dopaminergic neurons receives highly heterogeneous input, consistent with parallel representation of diverse rewards, and recordings demonstrate state-specific gating and satiety-related signals. We propose that a similar dopaminergic valuation system dysfunction is likely to contribute to maladaptive seeking of rewards by mammals.

A single photoreceptor splits perception and entrainment by cotransmission.

Vision enables both image-forming perception, driven by a contrast-based pathway, and unconscious non-image-forming circadian photoentrainment, driven by an irradiance-based pathway1,2. Although two distinct photoreceptor populations are specialized for each visual task3-6, image-forming photoreceptors can additionally contribute to photoentrainment of the circadian clock in different species7-15. However, it is unknown how the image-forming photoreceptor pathway can functionally implement the segregation of irradiance signals required for circadian photoentrainment from contrast signals required for image perception. Here we report that the Drosophila R8 photoreceptor separates image-forming and irradiance signals by co-transmitting two neurotransmitters, histamine and acetylcholine. This segregation is further established postsynaptically by histamine-receptor-expressing unicolumnar retinotopic neurons and acetylcholine-receptor-expressing multicolumnar integration neurons. The acetylcholine transmission from R8 photoreceptors is sustained by an autocrine negative feedback of the cotransmitted histamine during the light phase of light-dark cycles. At the behavioural level, elimination of histamine and acetylcholine transmission impairs R8-driven motion detection and circadian photoentrainment, respectively. Thus, a single type of photoreceptor can achieve the dichotomy of visual perception and circadian photoentrainment as early as the first visual synapses, revealing a simple yet robust mechanism to segregate and translate distinct sensory features into different animal behaviours.