Reference genes to study the sex-biased expression of genes regulating Drosophila metabolism.

Sex is an important variable in biology. Notable differences have been observed between male and female Drosophila in regulation of metabolism, in response to nutritional challenges, and in phenotypes relevant for obesity and metabolic disorders. The differences between males and females can be expected to result from differences in gene expression. We observed that expression levels of reference genes commonly used for normalization of qRT-PCR results such as GAPDH, ß-actin, and 18SrRNA, show prominent sexual dimorphism. Since this will impact relative expression and conclusions related to that, we performed a systematic analysis of candidate reference genes with the objective of identifying reference genes with stable expression in male and female Drosophila. These reference genes (LamCa, ßTub60D and ßTub97EF) were then used to assess sex-specific differences in expression of metabolism associated genes. Additionally, we evaluated the utility of these reference genes following a nutritional challenge and showed that LamCa and ßtub97EF are stably expressed between sexes and under different nutritional conditions and are thus suitable as reference genes. Our results highlight the importance of evaluating the stability of reference genes when sex-specific differences in gene expression are studied, and identify structural genes as a category worth exploring as reference genes in other species. Finally, we also uncovered hitherto unknown sexually dimorphic expression of a number of metabolism-associated genes, information of interest to others working in the field of metabolic disorders.

Ectopic expression in commonly used transgenic Drosophila GAL4 driver lines.

Transgenic tools such as the GAL4/UAS system in Drosophila have been used extensively to induce spatiotemporally controlled changes in gene expression and tissue-specific expression of a range of transgenes. We previously discovered unexpected expression of the commonly used dilp2-GAL4 line in tracheal tissue which significantly impacted growth phenotypes. We realized that few GAL4 lines have been thoroughly characterized, particularly when considering transient activity that may have significant impact on phenotypic readouts. Here, we characterized a further subset of 12 reportedly tissue-specific GAL4 lines commonly used in genetic studies of development, growth, endocrine regulation, and metabolism. Ten out of 12 GAL4 lines exhibited ectopic activity in other larval tissues, with seven being active in the larval trachea. Since this ectopic activity may result in phenotypes that do not depend on the manipulation in the intended target tissue, it is recommended to carefully analyze the outcome while taking this aspect into consideration.

Genetic mechanism regulating diversity in the placement of eyes on the head of animals.

Despite the conservation of genetic machinery involved in eye development, there is a strong diversity in the placement of eyes on the head of animals. Morphogen gradients of signaling molecules are vital to patterning cues. During Drosophila eye development, Wingless (Wg), a ligand of Wnt/Wg signaling, is expressed anterolaterally to form a morphogen gradient to determine the eye- versus head-specific cell fate. The underlying mechanisms that regulate this process are yet to be fully understood. We characterized defective proventriculus (dve) (Drosophila ortholog of human SATB1), a K50 homeodomain transcription factor, as a dorsal eye gene, which regulates Wg signaling to determine eye versus head fate. Across Drosophila species, Dve is expressed in the dorsal head vertex region where it regulates wg transcription. Second, Dve suppresses eye fate by down-regulating retinal determination genes. Third, the dve-expressing dorsal head vertex region is important for Wg-mediated inhibition of retinal cell fate, as eliminating the Dve-expressing cells or preventing Wg transport from these dve-expressing cells leads to a dramatic expansion of the eye field. Together, these findings suggest that Dve regulates Wg expression in the dorsal head vertex, which is critical for determining eye versus head fate. Gain-of-function of SATB1 exhibits an eye fate suppression phenotype similar to Dve. Our data demonstrate a conserved role for Dve/SATB1 in the positioning of eyes on the head and the interocular distance by regulating Wg. This study provides evidence that dysregulation of the Wg morphogen gradient results in developmental defects such as hypertelorism in humans where disproportionate interocular distance and facial anomalies are reported.

Formation of recurring transient Ca2+-based intercellular communities during Drosophila hematopoiesis.

Tissue development occurs through a complex interplay between many individual cells. Yet, the fundamental question of how collective tissue behavior emerges from heterogeneous and noisy information processing and transfer at the single-cell level remains unknown. Here, we reveal that tissue scale signaling regulation can arise from local gap-junction mediated cell-cell signaling through the spatiotemporal establishment of an intermediate-scale of transient multicellular communication communities over the course of tissue development. We demonstrated this intermediate scale of emergent signaling using Ca2+ signaling in the intact, ex vivo cultured, live developing Drosophila hematopoietic organ, the lymph gland. Recurrent activation of these transient signaling communities defined self-organized signaling "hotspots" that gradually formed over the course of larva development. These hotspots receive and transmit information to facilitate repetitive interactions with nonhotspot neighbors. Overall, this work bridges the scales between single-cell and emergent group behavior providing key mechanistic insight into how cells establish tissue-scale communication networks.

Duox activation in Drosophila Malpighian tubules stimulates intestinal epithelial renewal through a countercurrent flow.

The gut must perform a dual role of protecting the host against toxins and pathogens while harboring mutualistic microbiota. Previous studies suggested that the NADPH oxidase Duox contributes to intestinal homeostasis in Drosophila by producing reactive oxygen species (ROS) in the gut that stimulate epithelial renewal. We find instead that the ROS generated by Duox in the Malpighian tubules leads to the production of Upd3, which enters the gut and stimulates stem cell proliferation. We describe in Drosophila the existence of a countercurrent flow system, which pushes tubule-derived Upd3 to the anterior part of the gut and stimulates epithelial renewal at a distance. Thus, our paper clarifies the role of Duox in gut homeostasis and describes the existence of retrograde fluid flow in the gut, collectively revealing a fascinating example of inter-organ communication.

Somatotopic organization among parallel sensory pathways that promote a grooming sequence in Drosophila.

Mechanosensory neurons located across the body surface respond to tactile stimuli and elicit diverse behavioral responses, from relatively simple stimulus location-aimed movements to complex movement sequences. How mechanosensory neurons and their postsynaptic circuits influence such diverse behaviors remains unclear. We previously discovered that Drosophila perform a body location-prioritized grooming sequence when mechanosensory neurons at different locations on the head and body are simultaneously stimulated by dust (Hampel et al., 2017; Seeds et al., 2014). Here, we identify nearly all mechanosensory neurons on the Drosophila head that individually elicit aimed grooming of specific head locations, while collectively eliciting a whole head grooming sequence. Different tracing methods were used to reconstruct the projections of these neurons from different locations on the head to their distinct arborizations in the brain. This provides the first synaptic resolution somatotopic map of a head, and defines the parallel-projecting mechanosensory pathways that elicit head grooming.

Cdk8/CDK19 promotes mitochondrial fission through Drp1 phosphorylation and can phenotypically suppress pink1 deficiency in Drosophila.

Cdk8 in Drosophila is the orthologue of vertebrate CDK8 and CDK19. These proteins have been shown to modulate transcriptional control by RNA polymerase II. We found that neuronal loss of Cdk8 severely reduces fly lifespan and causes bang sensitivity. Remarkably, these defects can be rescued by expression of human CDK19, found in the cytoplasm of neurons, suggesting a non-nuclear function of CDK19/Cdk8. Here we show that Cdk8 plays a critical role in the cytoplasm, with its loss causing elongated mitochondria in both muscles and neurons. We find that endogenous GFP-tagged Cdk8 can be found in both the cytoplasm and nucleus. We show that Cdk8 promotes the phosphorylation of Drp1 at S616, a protein required for mitochondrial fission. Interestingly, Pink1, a mitochondrial kinase implicated in Parkinson's disease, also phosphorylates Drp1 at the same residue. Indeed, overexpression of Cdk8 significantly suppresses the phenotypes observed in flies with low levels of Pink1, including elevated levels of ROS, mitochondrial dysmorphology, and behavioral defects. In summary, we propose that Pink1 and Cdk8 perform similar functions to promote Drp1-mediated fission.

Kalium channelrhodopsins effectively inhibit neurons.

The analysis of neural circuits has been revolutionized by optogenetic methods. Light-gated chloride-conducting anion channelrhodopsins (ACRs)-recently emerged as powerful neuron inhibitors. For cells or sub-neuronal compartments with high intracellular chloride concentrations, however, a chloride conductance can have instead an activating effect. The recently discovered light-gated, potassium-conducting, kalium channelrhodopsins (KCRs) might serve as an alternative in these situations, with potentially broad application. As yet, KCRs have not been shown to confer potent inhibitory effects in small genetically tractable animals. Here, we evaluated the utility of KCRs to suppress behavior and inhibit neural activity in Drosophila, Caenorhabditis elegans, and zebrafish. In direct comparisons with ACR1, a KCR1 variant with enhanced plasma-membrane trafficking displayed comparable potency, but with improved properties that include reduced toxicity and superior efficacy in putative high-chloride cells. This comparative analysis of behavioral inhibition between chloride- and potassium-selective silencing tools establishes KCRs as next-generation optogenetic inhibitors for in vivo circuit analysis in behaving animals.

Experience-dependent glial pruning of synaptic glomeruli during the critical period.

Critical periods are temporally-restricted, early-life windows when sensory experience remodels synaptic connectivity to optimize environmental input. In the Drosophila juvenile brain, critical period experience drives synapse elimination, which is transiently reversible. Within olfactory sensory neuron (OSN) classes synapsing onto single projection neurons extending to brain learning/memory centers, we find glia mediate experience-dependent pruning of OSN synaptic glomeruli downstream of critical period odorant exposure. We find glial projections infiltrate brain neuropil in response to critical period experience, and use Draper (MEGF10) engulfment receptors to prune synaptic glomeruli. Downstream, we find antagonistic Basket (JNK) and Puckered (DUSP) signaling is required for the experience-dependent translocation of activated Basket into glial nuclei. Dependent on this signaling, we find critical period experience drives expression of the F-actin linking signaling scaffold Cheerio (FLNA), which is absolutely essential for the synaptic glomeruli pruning. We find Cheerio mediates experience-dependent regulation of the glial F-actin cytoskeleton for critical period remodeling. These results define a sequential pathway for experience-dependent brain synaptic glomeruli pruning in a strictly-defined critical period; input experience drives neuropil infiltration of glial projections, Draper/MEGF10 receptors activate a Basket/JNK signaling cascade for transcriptional activation, and Cheerio/FLNA induction regulates the glial actin cytoskeleton to mediate targeted synapse phagocytosis.

Recognition of nonself is necessary to activate Drosophila's immune response against an insect parasite.

BACKGROUND: Innate immune responses can be activated by pathogen-associated molecular patterns (PAMPs), danger signals released by damaged tissues, or the absence of self-molecules that inhibit immunity. As PAMPs are typically conserved across broad groups of pathogens but absent from the host, it is unclear whether they allow hosts to recognize parasites that are phylogenetically similar to themselves, such as parasitoid wasps infecting insects. RESULTS: Parasitoids must penetrate the cuticle of Drosophila larvae to inject their eggs. In line with previous results, we found that the danger signal of wounding triggers the differentiation of specialized immune cells called lamellocytes. However, using oil droplets to mimic infection by a parasitoid wasp egg, we found that this does not activate the melanization response. This aspect of the immune response also requires exposure to parasite molecules. The unidentified factor enhances the transcriptional response in hemocytes and induces a specific response in the fat body. CONCLUSIONS: We conclude that a combination of danger signals and the recognition of nonself molecules is required to activate Drosophila's immune response against parasitic insects.

Drosophila Contributions towards Understanding Neurofibromatosis 1.

Neurofibromatosis 1 (NF1) is a multisymptomatic disorder with highly variable presentations, which include short stature, susceptibility to formation of the characteristic benign tumors known as neurofibromas, intense freckling and skin discoloration, and cognitive deficits, which characterize most children with the condition. Attention deficits and Autism Spectrum manifestations augment the compromised learning presented by most patients, leading to behavioral problems and school failure, while fragmented sleep contributes to chronic fatigue and poor quality of life. Neurofibromin (Nf1) is present ubiquitously during human development and postnatally in most neuronal, oligodendrocyte, and Schwann cells. Evidence largely from animal models including Drosophila suggests that the symptomatic variability may reflect distinct cell-type-specific functions of the protein, which emerge upon its loss, or mutations affecting the different functional domains of the protein. This review summarizes the contributions of Drosophila in modeling multiple NF1 manifestations, addressing hypotheses regarding the cell-type-specific functions of the protein and exploring the molecular pathways affected upon loss of the highly conserved fly homolog dNf1. Collectively, work in this model not only has efficiently and expediently modelled multiple aspects of the condition and increased understanding of its behavioral manifestations, but also has led to pharmaceutical strategies towards their amelioration.

Dynamic encoding of temperature in the central circadian circuit coordinates physiological activities.

The circadian clock regulates animal physiological activities. How temperature reorganizes circadian-dependent physiological activities remains elusive. Here, using in-vivo two-photon imaging with the temperature control device, we investigated the response of the Drosophila central circadian circuit to temperature variation and identified that DN1as serves as the most sensitive temperature-sensing neurons. The circadian clock gate DN1a's diurnal temperature response. Trans-synaptic tracing, connectome analysis, and functional imaging data reveal that DN1as bidirectionally targets two circadian neuronal subsets: activity-related E cells and sleep-promoting DN3s. Specifically, behavioral data demonstrate that the DN1a-E cell circuit modulates the evening locomotion peak in response to cold temperature, while the DN1a-DN3 circuit controls the warm temperature-induced nocturnal sleep reduction. Our findings systematically and comprehensively illustrate how the central circadian circuit dynamically integrates temperature and light signals to effectively coordinate wakefulness and sleep at different times of the day, shedding light on the conserved neural mechanisms underlying temperature-regulated circadian physiology in animals.

Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region.

New comparative genomic evidence supporting the proteomic diversification role of A-to-I RNA editing in insects.

Adenosine-to-inosine (A-to-I) RNA editing, resembling A-to-G mutation, confers adaptiveness by increasing proteomic diversity in a temporal-spatial manner. This evolutionary theory named "proteomic diversifying hypothesis" has only partially been tested in very few organisms like Drosophila melanogaster, mainly by observing the positive selection on nonsynonymous editing events. To find additional genome-wide evidences supporting this interesting assumption, we retrieved the genomes of four Drosophila species and collected 20 deep-sequenced transcriptomes of different developmental stages and neuron populations of D. melanogaster. We systematically profiled the RNA editomes in these samples and performed meticulous comparative genomic analyses. Further evidences were found to support the diversifying hypothesis. (1) None of the nonsynonymous editing sites in D. melanogaster had ancestral G-alleles, while the silent editing sites had an unignorable fraction of ancestral G-alleles; (2) Only very few nonsynonymous editing sites in D. melanogaster had corresponding G-alleles derived in the genomes of sibling species, and the fraction of such situation was significantly lower than that of silent editing sites; (3) The few nonsynonymous editing with corresponding G-alleles had significantly more variable editing levels (across samples) than other nonsynonymous editing sites in D. melanogaster. The proteomic diversifying nature of RNA editing in Drosophila excludes the restorative role which favors an ancestral G-allele. The few fixed G-alleles in sibling species might facilitate the adaptation to particular environment and the corresponding nonsynonymous editing in D. melanogaster would introduce stronger advantage of flexible proteomic diversification. With multi-Omics data, our study consolidates the nature of evolutionary significance of A-to-I RNA editing sites in model insects.

Interaction with B-type lamin reveals the function of Drosophila Keap1 xenobiotic response factor in nuclear architecture.

BACKGROUND: The Keap1-Nrf2 pathway serves as a central regulator that mediates transcriptional responses to xenobiotic and oxidative stimuli. Recent studies have shown that Keap1 and Nrf2 can regulate transcripts beyond antioxidant and detoxifying genes, yet the underlying mechanisms remain unclear. Our research has uncovered that Drosophila Keap1 (dKeap1) and Nrf2 (CncC) proteins can control high-order chromatin structure, including heterochromatin. METHODS AND RESULTS: In this study, we identified the molecular interaction between dKeap1 and lamin Dm0, the Drosophila B-type lamin responsible for the architecture of nuclear lamina and chromatin. Ectopic expression of dKeap1 led to an ectopic localization of lamin to the intra-nuclear area, corelated with the spreading of the heterochromatin marker H3K9me2 into euchromatin regions. Additionally, mis-regulated dKeap1 disrupted the morphology of the nuclear lamina. Knocking down of dKeap1 partially rescued the lethality induced by lamin overexpression, suggesting their genetic interaction during development. CONCLUSIONS: The discovered dKeap1-lamin interaction suggests a novel role for the Keap1 oxidative/xenobiotic response factor in regulating chromatin architecture.

Identification of secretory autophagy as a mechanism modulating activity-induced synaptic remodeling.

The ability of neurons to rapidly remodel their synaptic structure and strength in response to neuronal activity is highly conserved across species and crucial for complex brain functions. However, mechanisms required to elicit and coordinate the acute, activity-dependent structural changes across synapses are not well understood, as neurodevelopment and structural plasticity are tightly linked. Here, using an RNAi screen in Drosophila against genes affecting nervous system functions in humans, we uncouple cellular processes important for synaptic plasticity and synapse development. We find mutations associated with neurodegenerative and mental health disorders are 2-times more likely to affect activity-induced synaptic remodeling than synapse development. We report that while both synapse development and activity-induced synaptic remodeling at the fly NMJ require macroautophagy (hereafter referred to as autophagy), bifurcation in the autophagy pathway differentially impacts development and synaptic plasticity. We demonstrate that neuronal activity enhances autophagy activation but diminishes degradative autophagy, thereby driving the pathway towards autophagy-based secretion. Presynaptic knockdown of Snap29, Sec22, or Rab8, proteins implicated in the secretory autophagy pathway, is sufficient to abolish activity-induced synaptic remodeling. This study uncovers secretory autophagy as a transsynaptic signaling mechanism modulating synaptic plasticity.

Fitness surfaces and local thermal adaptation in Drosophila along a latitudinal gradient.

Local adaptation is commonly cited to explain species distribution, but how fitness varies along continuous geographical gradients is not well understood. Here, we combine thermal biology and life-history theory to demonstrate that Drosophila populations along a 2500 km latitudinal cline are adapted to local conditions. We measured how heat tolerance and viability rate across eight populations varied with temperature in the laboratory and then simulated their expected cumulative Darwinian fitness employing high-resolution temperature data from their eight collection sites. Simulations indicate a trade-off between annual survival and cumulative viability, as both mortality and the recruitment of new flies are predicted to increase in warmer regions. Importantly, populations are locally adapted and exhibit the optimal combination of both traits to maximize fitness where they live. In conclusion, our method is able to reconstruct fitness surfaces employing empirical life-history estimates and reconstructs peaks representing locally adapted populations, allowing us to study geographic adaptation in silico.

A small-molecule approach to restore female sterility phenotype targeted by a homing suppression gene drive in the fruit pest Drosophila suzukii.

CRISPR-based gene drives offer promising prospects for controlling disease-transmitting vectors and agricultural pests. A significant challenge for successful suppression-type drive is the rapid evolution of resistance alleles. One approach to mitigate the development of resistance involves targeting functionally constrained regions using multiple gRNAs. In this study, we constructed a 3-gRNA homing gene drive system targeting the recessive female fertility gene Tyrosine decarboxylase 2 (Tdc2) in Drosophila suzukii, a notorious fruit pest. Our investigation revealed only a low level of homing in the germline, but feeding octopamine restored the egg-laying defects in Tdc2 mutant females, allowing easier line maintenance than for other suppression drive targets. We tested the effectiveness of a similar system in Drosophila melanogaster and constructed additional split drive systems by introducing promoter-Cas9 transgenes to improve homing efficiency. Our findings show that genetic polymorphisms in wild populations may limit the spread of gene drive alleles, and the position effect profoundly influences Cas9 activity. Furthermore, this study highlights the potential of conditionally rescuing the female infertility caused by the gene drive, offering a valuable tool for the industrial-scale production of gene drive transgenic insects.

p24 family Tango(1) at the endoplasmic reticulum exit site to organize cargo exit.

The p24 family of proteins have been regarded as cargo receptors for endoplasmic reticulum (ER) to Golgi transport; however, their precise functions have yet to be revealed. In this issue, Pastor-Pareja and colleagues (https://doi.org/10.1083/jcb.202309045) show that the interaction of these proteins with Tango1 is critical for their localization at the ER exit site (ERES) and efficient transport of secretory proteins in Drosophila.

Wolbachia infection negatively impacts Drosophila simulans heat tolerance in a strain- and trait-specific manner.

The susceptibility of insects to rising temperatures has largely been measured by their ability to survive thermal extremes. However, the capacity for maternally inherited endosymbionts to influence insect heat tolerance has been overlooked. Further, while some studies have addressed the impact of heat on traits like fertility, which can decline at temperatures below lethal thermal limits, none have considered the impact of endosymbionts. Here, we assess the impact of three Wolbachia strains (wRi, wAu and wNo) on the survival and fertility of Drosophila simulans exposed to heat stress during development or as adults. The effect of Wolbachia infection on heat tolerance was generally small and trait/strain specific. Only the wNo infection significantly reduced the survival of adult males after a heat shock. When exposed to fluctuating heat stress during development, the wRi and wAu strains reduced egg-to-adult survival but only the wNo infection reduced male fertility. Wolbachia densities of all three strains decreased under developmental heat stress, but reductions occurred at temperatures above those that reduced host fertility. These findings emphasize the necessity to account for endosymbionts and their effect on both survival and fertility when investigating insect responses to heat stress.