Histidine is selectively required for the growth of Myc-dependent dedifferentiation tumours in the Drosophila CNS.

Rewired metabolism of glutamine in cancer has been well documented, but less is known about other amino acids such as histidine. Here, we use Drosophila cancer models to show that decreasing the concentration of histidine in the diet strongly inhibits the growth of mutant clones induced by loss of Nerfin-1 or gain of Notch activity. In contrast, changes in dietary histidine have much less effect on the growth of wildtype neural stem cells and Prospero neural tumours. The reliance of tumours on dietary histidine and also on histidine decarboxylase (Hdc) depends upon their growth requirement for Myc. We demonstrate that Myc overexpression in nerfin-1 tumours is sufficient to switch their mode of growth from histidine/Hdc sensitive to resistant. This study suggests that perturbations in histidine metabolism selectively target neural tumours that grow via a dedifferentiation process involving large cell size increases driven by Myc.

The transcription factor Nerfin-1 prevents reversion of neurons into neural stem cells.

Cellular dedifferentiation is the regression of a cell from a specialized state to a more multipotent state and is implicated in cancer. However, the transcriptional network that prevents differentiated cells from reacquiring stem cell fate is so far unclear. Neuroblasts (NBs), the Drosophila neural stem cells, are a model for the regulation of stem cell self-renewal and differentiation. Here we show that the Drosophila zinc finger transcription factor Nervous fingers 1 (Nerfin-1) locks neurons into differentiation, preventing their reversion into NBs. Following Prospero-dependent neuronal specification in the ganglion mother cell (GMC), a Nerfin-1-specific transcriptional program maintains differentiation in the post-mitotic neurons. The loss of Nerfin-1 causes reversion to multipotency and results in tumors in several neural lineages. Both the onset and rate of neuronal dedifferentiation in nerfin-1 mutant lineages are dependent on Myc- and target of rapamycin (Tor)-mediated cellular growth. In addition, Nerfin-1 is required for NB differentiation at the end of neurogenesis. RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) analysis show that Nerfin-1 administers its function by repression of self-renewing-specific and activation of differentiation-specific genes. Our findings support the model of bidirectional interconvertibility between neural stem cells and their post-mitotic progeny and highlight the importance of the Nerfin-1-regulated transcriptional program in neuronal maintenance.

Starvation resistance is associated with developmentally specified changes in sleep, feeding and metabolic rate.

Food shortage represents a primary challenge to survival, and animals have adapted diverse developmental, physiological and behavioral strategies to survive when food becomes unavailable. Starvation resistance is strongly influenced by ecological and evolutionary history, yet the genetic basis for the evolution of starvation resistance remains poorly understood. The fruit fly Drosophila melanogaster provides a powerful model for leveraging experimental evolution to investigate traits associated with starvation resistance. While control populations only live a few days without food, selection for starvation resistance results in populations that can survive weeks. We have previously shown that selection for starvation resistance results in increased sleep and reduced feeding in adult flies. Here, we investigate the ontogeny of starvation resistance-associated behavioral and metabolic phenotypes in these experimentally selected flies. We found that selection for starvation resistance resulted in delayed development and a reduction in metabolic rate in larvae that persisted into adulthood, suggesting that these traits may allow for the accumulation of energy stores and an increase in body size within these selected populations. In addition, we found that larval sleep was largely unaffected by starvation selection and that feeding increased during the late larval stages, suggesting that experimental evolution for starvation resistance produces developmentally specified changes in behavioral regulation. Together, these findings reveal a critical role for development in the evolution of starvation resistance and indicate that selection can selectively influence behavior during defined developmental time points.

Feedback regulation of Drosophila BMP signaling by the novel extracellular protein larval translucida.

The cellular response to the Drosophila BMP 2/4-like ligand Decapentaplegic (DPP) serves as one of the best-studied models for understanding the long-range control of tissue growth and pattern formation during animal development. Nevertheless, fundamental questions remain unanswered regarding extracellular regulation of the ligand itself, as well as the nature of the downstream transcriptional response to BMP pathway activation. Here, we report the identification of larval translucida (ltl), a novel target of BMP activity in Drosophila. Both gain- and loss-of-function analyses implicate LTL, a leucine-rich repeat protein, in the regulation of wing growth and vein patterning. At the molecular level, we demonstrate that LTL is a secreted protein that antagonizes BMP-dependent MAD phosphorylation, indicating that it regulates DPP/BMP signaling at or above the level of ligand-receptor interactions. Furthermore, based on genetic interactions with the DPP-binding protein Crossveinless 2 and biochemical interactions with the glypican Dally-like, we propose that LTL acts in the extracellular space where it completes a novel auto-regulatory loop that modulates BMP activity.

Developmental emergence of sleep rhythms enables long-term memory in Drosophila.

In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day. The mechanism through which sleep rhythms emerge, and consequent advantage conferred to a juvenile animal, is unknown. In the second-instar Drosophila larvae (L2), like in human infants, sleep is not under circadian control. We identify the precise developmental time point when the clock begins to regulate sleep in Drosophila, leading to emergence of sleep rhythms in early third-instars (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousal-promoting Dh44 neurons, bringing arousal under clock control to drive emergence of circadian sleep. Last, we demonstrate that L3 but not L2 larvae exhibit long-term memory (LTM) of aversive cues and that this LTM depends upon deep sleep generated once sleep rhythms begin. We propose that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.

The chromatin remodeler ISWI acts during Drosophila development to regulate adult sleep.

Sleep disruptions are among the most commonly reported symptoms across neurodevelopmental disorders (NDDs), but mechanisms linking brain development to normal sleep are largely unknown. From a Drosophila screen of human NDD-associated risk genes, we identified the chromatin remodeler Imitation SWItch/SNF (ISWI) to be required for adult fly sleep. Loss of ISWI also results in disrupted circadian rhythms, memory, and social behavior, but ISWI acts in different cells and during distinct developmental times to affect each of these adult behaviors. Specifically, ISWI expression in type I neuroblasts is required for both adult sleep and formation of a learning-associated brain region. Expression in flies of the human ISWI homologs SMARCA1 and SMARCA5 differentially rescues adult phenotypes, while de novo SMARCA5 patient variants fail to rescue sleep. We propose that sleep deficits are a primary phenotype of early developmental origin in NDDs and point toward chromatin remodeling machinery as critical for sleep circuit formation.

The CHD8/CHD7/Kismet family links blood-brain barrier glia and serotonin to ASD-associated sleep defects.

Sleep disturbances in autism and neurodevelopmental disorders are common and adversely affect patient's quality of life, yet the underlying mechanisms are understudied. We found that individuals with mutations in CHD8, among the highest-confidence autism risk genes, or CHD7 suffer from disturbed sleep maintenance. These defects are recapitulated in Drosophila mutants affecting kismet, the sole CHD8/CHD7 ortholog. We show that Kismet is required in glia for early developmental and adult sleep architecture. This role localizes to subperineurial glia constituting the blood-brain barrier. We demonstrate that Kismet-related sleep disturbances are caused by high serotonin during development, paralleling a well-established but genetically unsolved autism endophenotype. Despite their developmental origin, Kismet's sleep architecture defects can be reversed in adulthood by a behavioral regime resembling human sleep restriction therapy. Our findings provide fundamental insights into glial regulation of sleep and propose a causal mechanistic link between the CHD8/CHD7/Kismet family, developmental hyperserotonemia, and autism-associated sleep disturbances.

Identification of a molecular basis for the juvenile sleep state.

Across species, sleep in young animals is critical for normal brain maturation. The molecular determinants of early life sleep remain unknown. Through an RNAi-based screen, we identified a gene, pdm3, required for sleep maturation in Drosophila. Pdm3, a transcription factor, coordinates an early developmental program that prepares the brain to later execute high levels of juvenile adult sleep. PDM3 controls the wiring of wake-promoting dopaminergic (DA) neurites to a sleep-promoting region, and loss of PDM3 prematurely increases DA inhibition of the sleep center, abolishing the juvenile sleep state. RNA-Seq/ChIP-Seq and a subsequent modifier screen reveal that pdm3 represses expression of the synaptogenesis gene Msp300 to establish the appropriate window for DA innervation. These studies define the molecular cues governing sleep behavioral and circuit development, and suggest sleep disorders may be of neurodevelopmental origin.

Quantitative imaging of sleep behavior in Caenorhabditis elegans and larval Drosophila melanogaster.

Sleep is nearly universal among animals, yet remains poorly understood. Recent work has leveraged simple model organisms, such as Caenorhabditis elegans and Drosophila melanogaster larvae, to investigate the genetic and neural bases of sleep. However, manual methods of recording sleep behavior in these systems are labor intensive and low in throughput. To address these limitations, we developed methods for quantitative imaging of individual animals cultivated in custom microfabricated multiwell substrates, and used them to elucidate molecular mechanisms underlying sleep. Here, we describe the steps necessary to design, produce, and image these plates, as well as analyze the resulting behavioral data. We also describe approaches for experimentally manipulating sleep. Following these procedures, after ~2 h of experimental preparation, we are able to simultaneously image 24 C. elegans from the second larval stage to adult stages or 20 Drosophila larvae during the second instar life stage at a spatial resolution of 10 or 27 µm, respectively. Although this system has been optimized to measure activity and quiescence in Caenorhabditis larvae and adults and in Drosophila larvae, it can also be used to assess other behaviors over short or long periods. Moreover, with minor modifications, it can be adapted for the behavioral monitoring of a wide range of small animals.

A sleep state in Drosophila larvae required for neural stem cell proliferation.

Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. Here we identify a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, we find that sleep loss in larvae impairs cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation.

Germ line specific expression of a protein phosphatase Y interacting protein (PPYR1) in Drosophila.

PPYR1, the product of the CG15031 gene, was identified as a protein phosphatase Y (PPY) interacting protein in Drosophila melanogaster using a yeast two-hybrid screen. PPYR1 displays a biphasic expression pattern: the maternal protein is abundant in the developing egg chambers and in the early embryos, while the zygotic protein appears later in development and is localized specifically in the testes of the males. The maternal and zygotic gene products differ from each other in their size having apparent molecular masses of 47 and 66 kDa, respectively. The maternal PPYR1 is localized in the cytoplasm of the follicular and nurse cells and is deposited as a ribonucleoprotein complex in the oocyte. In the early embryos, the PPYR1 is distributed evenly, and it gradually diminishes during embryonic development. Zygotic PPYR1 is expressed exclusively in the testes, predominantly in the cytoplasm of the spermatocytes. PPY is localized in the nuclei of the same cells. Our results suggest that PPYR1 has two distinct developmental isoforms: a maternal protein the expression of which is independent of PPY and a zygotic protein which is co-expressed with PPY.

Identification of germ plasm-enriched mRNAs in Drosophila melanogaster by the cDNA microarray technique.

The development of embryonic germ cells in Drosophila depends on the germ plasm, the most posterior part of the ooplasm. The germ plasm is devoted to the formation of future germ cells and is known to contain all the factors that are necessary to induce germ cell fate. Besides having a characteristic organelle and protein distribution, the germ plasm also contains a large number of localized RNA species that have been shown to play crucial roles in germ cell determination. To identify germ plasm-enriched, localized transcripts, we used a two-step method composed of cDNA microarray (containing 3200 annotated Drosophila cDNAs) and in situ RNA hybridization techniques. We compared germ plasm deficient, normal and ectopic germ plasm conditions in the cDNA microarray experiments. RNA species whose concentration increased when ectopic germ plasm was present and decreased when the germ plasm was missing were selected. These candidates were then subjected to a second screen which compared the distribution of the given RNA in wild type embryos and in eggs with ectopic germ plasm. Finally, 17 RNA species were found to be enriched in the germ plasm. Based on these data, we estimate that around 1% of the Drosophila genes encode for germ plasm-enriched, localized transcripts. We conclude that this combination of microarray and in situ hybridization techniques is a simple but powerful experimental design for the genome-wide identification of genes coding for germ plasm localized transcripts.

Viability, longevity, and egg production of Drosophila melanogaster are regulated by the miR-282 microRNA.

The first microRNAs were discovered some 20 years ago, but only a small fraction of the microRNA-encoding genes have been described in detail yet. Here we report the molecular analysis of a computationally predicted Drosophila melanogaster microRNA gene, mir-282. We show that the mir-282 gene is the source of a 4.9-kb-long primary transcript with a 5' cap and a 3'-poly(A) sequence and a mature microRNA of ∼25 bp. Our data strongly suggest the existence of an independent mir-282 gene conserved in holometabolic insects. We give evidence that the mir-282 locus encodes a functional transcript that influences viability, longevity, and egg production in Drosophila. We identify the nervous system-specific adenylate cyclase (rutabaga) as a target of miR-282 and assume that one of the main functions of mir-282 is the regulation of adenylate cyclase activity in the nervous system during metamorphosis.