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To regulate brain function, peripheral compounds must traverse the blood-brain barrier (BBB), an interface between the brain and the circulatory system. To determine whether specific transport mechanisms are relevant for sleep, we conducted a BBB-specific inducible RNAi knockdown (iKD) screen for genes affecting sleep in Drosophila . We observed reduced sleep with knockdown of solute carrier CG6126 , a carnitine transporter, as determined by isotope flux. Our findings suggest that CG6126 regulation of sleep is through the role of the carnitine shuttle in regulating fatty acid metabolism as lipid droplets accumulate in the brains of CG6126 BBB iKD flies. Knocking down mitochondrial carnitine transferases in non-BBB glial cells mimicked the reduced sleep of the CG6126 BBB iKD flies, while bypassing the necessity of carnitine transport with dietary medium-chain fatty acids or palmitoylcarnitine rescued sleep. We propose that carnitine transport via CG6126 promotes brain fatty acid metabolism necessary for maintaining sleep., Competing Interests: Competing interests statement:The authors declare no competing interest.

The target of rapamycin(TOR)gene is closely related to metabolism and cellular aging, but it is unclear whether the TOR pathways mediate endurance exercise against the accelerated aging of skeletal muscle induced by high salt intake. In this study, muscular TOR gene overexpression and RNAi were constructed by constructing MhcGAL4/TOR-overexpression and MhcGAL4/TORUAS-RNAi systems in Drosophila. The results showed that muscle TOR knockdown and endurance exercise significantly increased the climbing speed, climbing endurance, the expression of autophagy related gene 2(ATG2), silent information regulator 2(SIR2), and pparγ coactivator 1(PGC-1α) genes, and superoxide dismutases(SOD) activity, but it decreased the expression of the TOR gene and reactive oxygen species(ROS) level, and it protected the myofibrillar fibers and mitochondria of skeletal muscle in Drosophila on a high-salt diet. TOR overexpression yielded similar results to the high salt diet(HSD) alone, with the opposite effect of TOR knockout found in regard to endurance exercise and HSD-induced age-related skeletal muscle degradation. Therefore, the current findings confirm that the muscle TOR gene plays an important role in endurance exercise against HSD-induced age-related skeletal muscle degeneration, as it determines the activity of the mammalian target of rapamycin(MTOR)/SIR2/PGC-1α and MTOR/ATG2/PGC-1α pathways in skeletal muscle., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Metabolic and neurological disorders commonly display dysfunctional branched-chain amino acid (BCAA) metabolism, though it is poorly understood how this leads to neurological damage. We investigated this by generating Drosophila mutants lacking BCAA-catabolic activity, resulting in elevated BCAA levels and neurological dysfunction, mimicking disease-relevant symptoms. Our findings reveal a reduction in neuronal AMP-activated protein kinase (AMPK) activity, which disrupts autophagy in mutant brain tissues, linking BCAA imbalance to brain dysfunction. Mechanistically, we show that excess BCAA-induced mitochondrial reactive oxygen species (ROS) triggered the binding of protein phosphatase 2 A catalytic subunit (PP2Ac) to AMPK, suppressing AMPK activity. This initiated a dysregulated feedback loop of AMPK-mitochondrial interactions, exacerbating mitochondrial dysfunction and oxidative neuronal damage. Our study identifies BCAA imbalance as a critical driver of neuronal damage through AMPK suppression and autophagy dysfunction, offering insights into metabolic-neuronal interactions in neurological diseases and potential therapeutic targets for BCAA-related neurological conditions., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The movements that give rise to the body's structure are powered by cell shape changes and rearrangements that are coordinated at supracellular scales. How such cellular coordination arises and integrates different morphogenetic programs is unclear. Using quantitative imaging, we found a complex pattern of adherens junction (AJ) levels in the ectoderm prior to gastrulation onset in Drosophila. AJ intensity exhibited a double-sided gradient, with peaks at the dorsal midline and ventral neuroectoderm. We show that this dorsal-ventral AJ pattern is regulated by epidermal growth factor (EGF) signaling and that this signal is required for ectoderm cell movement during mesoderm invagination and axis extension. We identify AJ levels and junctional actomyosin as downstream effectors of EGFR signaling. Overall, our study demonstrates an EGF-patterned mechanical feedback mechanism that coordinates tissue folding and convergent extension to facilitate embryo-wide gastrulation movements., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Sevenless, the Drosophila homologue of ROS1 (University of Rochester Sarcoma) (herein, dROS1) is a receptor tyrosine kinase (RTK) essential for the differentiation of Drosophila R7 photoreceptor cells. Activation of dROS1 is mediated by binding to the extracellular region (ECR) of the GPCR (G protein coupled receptor) BOSS (Bride Of Sevenless) on adjacent cells. Activation of dROS1 by BOSS leads to subsequent downstream signaling pathways including SOS (Son of Sevenless). However, the physical basis for how dROS1 interacts with BOSS has long remained unknown. Here we provide a cryo-EM structure of dROS1's extracellular region, which mediates ligand binding. We show that the extracellular region of dROS1 adopts a folded-over conformation stabilized by an N-terminal domain comprised of two disulfide stapled helical hairpins. We further narrowed down the interacting binding epitopes on both dROS1 and BOSS using hydrogen-deuterium exchange mass spectrometry (HDX-MS). This includes beta-strands in dROS1's third Fibronectin type III (FNIII) domain and a C-terminal peptide in BOSS' ECR. Our mutagenesis studies, coupled with AlphaFold complex predictions, support a binding interaction mediated by a hydrophobic interaction and beta-strand augmentation between these regions. Our findings provide a fundamental understanding of the regulatory function of dROS1 and further provide mechanistic insight into the human ortholog and oncogene ROS1., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Toxic protein aggregates are associated with various neurodegenerative diseases, including Huntington's disease (HD). Since no current treatment delays the progression of HD, we develop a mechanistic approach to prevent mutant huntingtin (mHttex1) aggregation. Here, we engineer the ATP-independent cytosolic chaperone PEX19, which targets peroxisomal membrane proteins to peroxisomes, to remove mHttex1 aggregates. Using yeast toxicity-based screening with a random mutant library, we identify two yeast PEX19 variants and engineer equivalent mutations into human PEX19 (hsPEX19). These variants effectively delay mHttex1 aggregation in vitro and in cellular HD models. The mutated hydrophobic residue in the α4 helix of hsPEX19 variants binds to the N17 domain of mHttex1, thereby inhibiting the initial aggregation process. Overexpression of the hsPEX19-FV variant rescues HD-associated phenotypes in primary striatal neurons and in Drosophila. Overall, our data reveal that engineering ATP-independent membrane protein chaperones is a promising therapeutic approach for rational targeting of mHttex1 aggregation in HD., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Mitotic bookmarking has been posited as an important strategy for cells to faithfully propagate their fate memory through cell generations. However, the physiological significance and regulatory mechanisms of mitotic bookmarking in neural development remain unexplored. Here, we identified TATA-binding protein (TBP) as a crucial mitotic bookmarker for preserving the fate memory of Drosophila neural stem cells (NSCs). Phosphorylation by the super elongation complex (SEC) is important for TBP to retain as discrete foci at mitotic chromosomes of NSCs to effectively transmit their fate memory. TBP depletion leads to drastic NSC loss, whereas TBP overexpression enhances the ability of SEC to induce neural progenitor dedifferentiation and tumorigenesis. Importantly, TBP achieves its mitotic retention through recruiting the chromatin remodeler EP400, which in turn increases local chromatin accessibility via depositing H2A.Z. Thus, local chromatin remodeling ensures mitotic bookmarking, which may represent a general principle underlying the preservation of cell fate memory., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The elongation of tissues and organs is important for proper morphogenesis in animal development. In Drosophila ovaries, the elongation of egg chambers involves aligned Collagen IV fiber-like structures, a gradient of extracellular matrix stiffness and actin-based protrusion-driven collective cell migration, leading to the rotation of the egg chamber. Egg chamber elongation and rotation depend on the atypical cadherin Fat2. Fat2 contains in its intracellular region three WRC interacting receptor sequence (WIRS) motifs, which previously had been shown to bind to the WAVE regulatory complex (WRC), a conserved actin regulator. Here, we show that in fat2 mutant flies lacking the WIRS motifs, egg chambers fail to rotate and Collagen IV fiber-like structures are impaired, yet a gradient of extracellular matrix stiffness is established and egg chambers properly elongate. We conclude that the WIRS motifs are required for egg chamber rotation and that egg chamber rotation might be a prerequisite for proper formation of Collagen IV fiber-like structures. Egg chamber rotation, however, is dispensable for extracellular matrix stiffness gradient formation and for egg chamber elongation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

Noncoding satellite DNA repeats are abundant at the pericentromeric heterochromatin of eukaryotic chromosomes. During interphase, sequence-specific DNA-binding proteins cluster these repeats from multiple chromosomes into nuclear foci known as chromocenters. Despite the pivotal role of chromocenters in cellular processes like genome encapsulation and gene repression, the associated proteins remain incompletely characterized. Here, we use 2 satellite DNA-binding proteins, D1 and Prod, as baits to characterize the chromocenter-associated proteome in Drosophila embryos, ovaries, and testes through quantitative mass spectrometry. We identify D1- and Prod-associated proteins, including known heterochromatin proteins as well as proteins previously unlinked to satellite DNA or chromocenters, thereby laying the foundation for a comprehensive understanding of cellular functions enabled by satellite DNA repeats and their associated proteins. Interestingly, we find that multiple components of the transposon-silencing piRNA pathway are associated with D1 and Prod in embryos. Using genetics, transcriptomics, and small RNA profiling, we show that flies lacking D1 during embryogenesis exhibit transposon expression and gonadal atrophy as adults. We further demonstrate that this gonadal atrophy can be rescued by mutating the checkpoint kinase, Chk2, which mediates germ cell arrest in response to transposon mobilization. Thus, we reveal that a satellite DNA-binding protein functions during embryogenesis to silence transposons, in a manner that is heritable across later stages of development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Chavan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hox proteins, a sub-group of the homeodomain (HD) transcription factor family, provide positional information for axial patterning in development and evolution. Hox protein functional specificity is reached, at least in part, through interactions with Pbc (Extradenticle (Exd) in Drosophila) and Meis/Prep (Homothorax (Hth) in Drosophila) proteins. Most of our current knowledge of Hox protein specificity stems from the study of anterior and central Hox proteins, identifying the molecular and structural bases for Hox/Pbc/Meis-Prep cooperative action. Posterior Hox class proteins, Abdominal-B (Abd-B) in Drosophila and Hox9-13 in vertebrates, have been comparatively less studied. They strongly diverge from anterior and central class Hox proteins, with a low degree of HD sequence conservation and the absence of a core canonical Pbc interaction motif. Here we explore how Abd-B function interface with that of Exd/Hth using several developmental contexts, studying mutual expression control, functional dependency and intrinsic protein requirements. Results identify cross-regulatory interactions setting relative expression and activity levels required for proper development. They also reveal organ-specific requirement and a binary functional interplay with Exd and Hth, either antagonistic, as previously reported, or synergistic. This highlights context specific use of Exd/Hth, and a similar context specific use of Abd-B intrinsic protein requirements., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Curt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Myosin storage myopathy (MSM) is a rare skeletal muscle disorder caused by mutations in the slow muscle/β-cardiac myosin heavy chain (MHC) gene. MSM missense mutations frequently disrupt the tail's stabilizing heptad repeat motif. Disease hallmarks include subsarcolemmal hyaline-like β-MHC aggregates, muscle weakness, and, occasionally, cardiomyopathy. We generated transgenic, heterozygous Drosophila to examine the dominant physiological and structural effects of the L1793P, R1845W, and E1883K MHC MSM mutations on diverse muscles. The MHC variants reduced lifespan and flight and jump abilities. Moreover, confocal and electron microscopy revealed that they provoked indirect flight muscle breaks and myofibrillar disarray/degeneration with filamentous inclusions. Incorporation of GFP-myosin enabled in situ determination of thick filament lengths, which were significantly reduced in all mutants. Semiautomated heartbeat analysis uncovered aberrant cardiac function, which worsened with age. Thus, our fly models phenocopied traits observed among MSM patients. We additionally mapped the mutations onto a recently determined, 6 Å resolution, cryo-EM structure of the human cardiac thick filament. The R1845W mutation replaces a basic arginine with a polar-neutral, bulkier tryptophan, while E1883K reverses charge at critical filament loci. Both would be expected to disrupt the core and the outer shell of the backbone structure. Replacing L1793 with a proline, a potent breaker of α-helices, could disturb the coiled-coil of the myosin rod and alter the tail-tail interactome. Hence, all mutations likely destabilize and weaken the filament backbone. This may trigger disease in humans, while potentially analogous perturbations are likely to yield the observed thick filament and muscle disruption in our fly models., Competing Interests: Conflicts of interest: The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The Drosophila PAX6 homolog twin of eyeless (toy) sits at the pinnacle of the genetic pathway controlling eye development, the retinal determination network. Expression of toy in the embryo is first detectable at cellular blastoderm stage 5 in an anterior-dorsal band in the presumptive procephalic neuroectoderm, which gives rise to the primordia of the visual system and brain. Although several maternal and gap transcription factors that generate positional information in the embryo have been implicated in controlling toy, the regulation of toy expression in the early embryo is currently not well characterized. In this study, we adopt an integrated experimental approach utilizing bioinformatics, molecular genetic testing of putative enhancers in transgenic reporter gene assays and quantitative analysis of expression patterns in the early embryo, to identify 2 novel coacting enhancers at the toy gene. In addition, we apply mathematical modeling to dissect the regulatory landscape for toy. We demonstrate that relatively simple thermodynamic-based models, incorporating only 5 TF binding sites, can accurately predict gene expression from the 2 coacting enhancers and that the HUNCHBACK TF plays a critical regulatory role through a dual-modality function as an activator and repressor. Our analysis also reveals that the molecular architecture of the 2 enhancers is very different, indicating that the underlying regulatory logic they employ is distinct., Competing Interests: Conflicts of interest: The authors report no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Norepinephrine in vertebrates and its invertebrate analog, octopamine, regulate the activity of neural circuits. We find that, when hungry, Drosophila larvae switch activity in type II octopaminergic motor neurons (MNs) to high-frequency bursts, which coincide with locomotion-driving bursts in type I glutamatergic MNs that converge on the same muscles. Optical quantal analysis across hundreds of synapses simultaneously reveals that octopamine potentiates glutamate release by tonic type Ib MNs, but not phasic type Is MNs, and occurs via the G q -coupled octopamine receptor (OAMB). OAMB is more abundant in type Ib terminals and acts through diacylglycerol and its target Unc13A, a key component of the glutamate release machinery. Potentiation varies significantly-by up to 1,000%-across synapses of a single Ib axon, with synaptic Unc13A levels determining both release probability and potentiation. We propose that a dual molecular mechanism-an upstream neuromodulator receptor and a downstream transmitter release controller-fine-tunes catecholaminergic modulation so that strong tonic synapses exhibit large potentiation, while weaker tonic and all phasic synapses maintain consistency, yielding a sophisticated regulation of locomotor behavior., Competing Interests: Competing interests statement:The authors declare no competing interest.

During development, cells of the nervous system begin as unspecified precursors and proceed along one of two developmental paths to become either neurons or glia. Work in the fruit fly Drosophila melanogaster has established the role of the transcription factor Glial cells missing (Gcm) in directing neuronal precursor cells to assume a glial cell fate. Gcm acts on many target genes, one of which is reversed polarity (repo). repo encodes a homeodomain transcription factor and is necessary for the terminal differentiation of glial cells. Transient Gcm expression is followed by maintained expression of repo. Evidence supports autoregulation to be one of the mechanisms that maintains repo expression, as ectopic repo expression in embryos can activate repo-lacZ reporter constructs. In this paper we further explore the ability of repo to activate reporter constructs in transgenic embryos and in cultured S2 cells. We provide further evidence that Repo protein acts as a transcription factor on its own regulatory DNA sequence. We report that three canonical Repo binding sites (RBSs) are located within the upstream 4.3 kilobase repo cis-regulatory DNA (CRD). The upstream 2 kb within the repo CRD has remarkable repo-dependent gene expression activity, and mutagenesis of RBS1 in this 2 kb region results in a significant decrease in repo-induced reporter gene expression in both systems. Our results in cell culture experiments also show that RBS2 and/or RBS3 can affect repo-dependent gene expression in the context of the whole upstream repo CRD. Mutagenesis of both RBS2 and RBS3 in the repo CRD, leaving RBS1 intact, significantly reduces repo-induced reporter gene expression. These results suggest that all three canonical RBSs may be cooperatively involved in autoregulation of repo expression., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Cell-cell communication through direct contact, or juxtacrine signaling, is important in development, disease, and many areas of physiology. Synthetic forms of juxtacrine signaling can be precisely controlled and operate orthogonally to native processes, making them a powerful reductionist tool with which to address fundamental questions in cell-cell communication in vivo. Here, we investigate how cell-cell contact length and tissue growth dynamics affect juxtacrine signal responses through implementing a custom synthetic gene circuit in Drosophila wing imaginal discs alongside mathematical modeling to determine synthetic Notch (synNotch) activation patterns. We find that the area of contact between cells largely determines the extent of synNotch activation, leading to the prediction that the shape of the interface between signal-sending and signal-receiving cells will impact the magnitude of the synNotch response. Notably, synNotch outputs form a graded spatial profile that extends several cell diameters from the signal source, providing evidence that the response to juxtacrine signals can persist in cells as they proliferate away from source cells, or that cells remain able to communicate directly over several cell diameters. Our model suggests that the former mechanism may be sufficient, since it predicts graded outputs without diffusion or long-range cell-cell communication. Overall, we identify that cell-cell contact area together with output synthesis and decay rates likely govern the pattern of synNotch outputs in both space and time during tissue growth, insights that may have broader implications for juxtacrine signaling in general., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Collective cell migration is fundamental in development, wound healing, and metastasis. During Drosophila oogenesis, border cells (BCs) migrate collectively inside the egg chamber, controlled by the Ste20-like kinase Misshapen (Msn). Msn coordinates the restriction of protrusion formation and contractile forces within the cluster. Here, we demonstrate that Tao acts as an upstream activator of Msn in BCs. Depleting Tao significantly impedes BC migration, producing a phenotype similar to Msn loss of function. Furthermore, we show that the localization of Msn relies on its citron homology (CNH) domain, which interacts with the small GTPase Rap2l. Rap2l promotes the trafficking of Msn to the endolysosomal pathway. Depleting Rap2l elevates Msn levels by reducing its trafficking into late endosomes and increases overall contractility. These data suggest that Tao promotes Msn activation, while global Msn protein levels are controlled via Rap2l and the endolysosomal degradation pathway. Thus, two mechanisms ensure appropriate Msn levels and activation in BCs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The syncytial mammalian muscle fiber contains a heterogeneous population of (myo)nuclei. At the neuromuscular junction (NMJ), myonuclei have specialized positioning and gene expression. However, it remains unclear how myonuclei are recruited and what regulates myonuclear output at the NMJ. Here, we identify specific properties of myonuclei located near the Drosophila larval NMJ. These synaptic myonuclei have increased size in relation to their surrounding cytoplasmic domain (size scaling), increased DNA content (ploidy), and increased levels of transcription factor pMad, a readout for BMP signaling activity. Our genetic manipulations show that local BMP signaling affects muscle size, nuclear size, ploidy, and NMJ size and function. In support, RNA sequencing analysis reveals that pMad regulates genes involved in muscle growth, ploidy (i.e., E2f1), and neurotransmission. Our data suggest that muscle BMP signaling instructs synaptic myonuclear output that positively shapes the NMJ synapse. This study deepens our understanding of how myonuclear heterogeneity supports local signaling demands to fine tune cellular function and NMJ activity., (© 2024 von Saucken et al.)

Microtubule nucleation is important for microtubule organization in dendrites and for neuronal injury responses. The core nucleation protein, γTubulin (γTub), is localized to dendrite branch points in Drosophila sensory neurons by Wnt receptors and scaffolding proteins on endosomes. However, whether Wnt ligands are important is unknown. We found that Wnt secretion from epithelial cells was required for γTub localization to dendrite branch points. Using RNAi and mutant approaches, we demonstrated that Wnt4 and wntD both position γTub. Moreover, injury-induced increases in neuronal microtubule dynamics required Wnt secretion from epithelial cells. Overexpression of Wnts in epithelial cells increased microtubule dynamics to the same extent as axon injury indicating surrounding cells have an instructive role in neuronal nucleation. To determine how Wnt ligands concentrate microtubule nucleation at dendrite branch points, we tested whether endocytosis is restricted to specific regions of dendrites. Markers of clathrin-mediated endocytosis localized to puncta at branch points. Behavior of these puncta was sensitive to inhibition of endocytosis suggesting they represented endocytic sites. In addition to previously described colocalization of Wnt receptors and scaffolds with Rab5 endosomes, we identified a separate set of Wnt signaling puncta that colocalized with clathrin in dendrites. Moreover, γTub and Wnt scaffolding protein recruitment to branch points was reduced by clathrin RNAi, and injury-induced up-regulation of microtubule dynamics was sensitive to clathrin reduction. We propose that the localization of Wnt endocytic sites to dendrite branch points results in the local generation of microtubule nucleating endosomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Thyagarajan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Female reproduction comes at great expense to energy metabolism compensated by extensive organ adaptations including intestinal size. Upon mating, endocrine signals orchestrate a 30% net increase of absorptive epithelium. Mating increases production of the steroid hormone Ecdysone released by the Drosophila ovaries that stimulates intestinal stem cell (ISC) divisions. Here, we uncover the transcription factor crooked legs (crol) as an intraepithelial coordinator of Ecdysone-induced ISC mitosis. For the precise investigation of non-autonomous factors on ISC behaviour, we establish Rapport, a spatiotemporally-controlled dual expression and tracing system for the analysis of paracrine genetic manipulation while tracing ISC behaviour. Rapport tracing reveals that Ecdysone-induced Crol controls mitogenic Wnt/Wg-ligand expression from epithelial enterocytes activating ISC mitosis. Paracrine Wg stimulation is counterbalanced by Crol-repression of string/CDC25 and CyclinB autonomously in ISC. Rapport-based ISC tumours confirm paracrine stimulation through the Ecdysone-Crol-Wg axis on mitotic behaviour, whereas the autonomous anti-proliferative role of Crol in ISC is conserved in models of colorectal cancer. Finally, mathematical modelling corroborates increasing enterocyte numbers and Wnt/Wg-degradation to set a stable post-mating intestinal size. Together, our findings provide insights into the complex endocrine growth control mechanisms during mating-induced adaptations and might help untangling pleiotropic hormonal effects observed in gastrointestinal tumorigenesis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Developmental neuronal remodeling is extensive and mechanistically diverse across the nervous system. We sought to identify Drosophila pupal neurons that underwent mechanistically new types of neuronal remodeling and describe remodeling Beat-VaM and Beat-VaL neurons. We show that Beat-VaM neurons produce highly branched neurites in the CNS during larval stages that undergo extensive local pruning. Surprisingly, although the ecdysone receptor (EcR) is essential for pruning in all other cell types studied, Beat-VaM neurons remodel their branches extensively despite cell autonomous blockade EcR or caspase signaling. Proper execution of local remodeling in Beat-VaM neurons instead depends on extrinsic signaling from astrocytes converging with intrinsic and less dominant EcR-regulated mechanisms. In contrast, Beat-VaL neurons undergo steroid hormone-dependent, apoptotic cell death, which we show relies on the segment-specific expression of the Hox gene Abd-B. Our work provides new cell types in which to study neuronal remodeling, highlights an important role for astrocytes in activating local pruning in Drosophila independent of steroid signaling, and defines a Hox gene-mediated mechanism for segment-specific cell elimination., (© 2024 Lehmann et al.)

Preserving a large number of essential yet highly unstable ribosomal DNA (rDNA) repeats is critical for the germline to perpetuate the genome through generations. Spontaneous rDNA loss must be countered by rDNA copy number (CN) expansion. Germline rDNA CN expansion is best understood in Drosophila melanogaster, which relies on unequal sister chromatid exchange (USCE) initiated by DNA breaks at rDNA. The rDNA-specific retrotransposon R2 responsible for USCE-inducing DNA breaks is typically expressed only when rDNA CN is low to minimize the danger of DNA breaks; however, the underlying mechanism of R2 regulation remains unclear. Here we identify the insulin receptor (InR) as a major repressor of R2 expression, limiting unnecessary R2 activity. Through single-cell RNA sequencing, we find that male germline stem cells (GSCs), the major cell type that undergoes rDNA CN expansion, have reduced InR expression when rDNA CN is low. Reduced InR activity in turn leads to R2 expression and CN expansion. We further find that dietary manipulation alters R2 expression and rDNA CN expansion activity. This work reveals that the insulin pathway integrates rDNA CN surveying with environmental sensing, revealing a potential mechanism by which diet exerts heritable changes to genomic content., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Deciphering how genes interpret information from transcription factor (TF) concentrations within the cell nucleus remains a fundamental question in gene regulation. Recent advancements have revealed the heterogeneous distribution of TF molecules, posing challenges to precisely decoding concentration signals. Using high-resolution single-cell imaging of the fluorescently tagged TF Bicoid in living Drosophila embryos, we show that Bicoid accumulation in submicrometer clusters preserves the spatial information of the maternal Bicoid gradient. These clusters provide precise spatial cues through intensity, size, and frequency. We further discover that Bicoid target genes colocalize with these clusters in an enhancer-binding affinity-dependent manner. Our modeling suggests that clustering offers a faster sensing mechanism for global nuclear concentrations than freely diffusing TF molecules detected by simple enhancers.

Lysosomal pH dysregulation is a critical element of the pathophysiology of neurodegenerative diseases, cancers, and lysosomal storage disorders (LSDs). To study the role of lysosomes in pathophysiology, probes to analyze lysosomal size, positioning, and pH are indispensable tools. Here, we developed and characterized a ratiometric genetically encoded lysosomal pH probe, RpH-ILV, targeted to a subpopulation of lysosomal intraluminal vesicles. This subpopulation behaves similarly to the general population of LAMP1-positive vesicles in terms of pH response to pharmacological stresses. In addition, RpH-ILV, which is trafficked to the lysosome via a different cytosolic motif than our previous ratiometric sensor, RpH-LAMP1, is well tolerated by the model organism Drosophila melanogaster , exhibits minimal plasma membrane fluorescence, and reveals sensitivity to the lysosomal damaging agent LLOMe, adding a valuable tool to our repertoire of lysosomal pH sensors.

Sensory disabilities have been identified as significant risk factors for dementia but underlying molecular mechanisms are unknown. In different Drosophila models with loss of sensory input, we observe non-autonomous induction of the integrated stress response (ISR) deep in the brain, as indicated by eIF2α S50 phosphorylation-dependent elevated levels of the ISR effectors ATF4 and XRP1. Unlike during canonical ISR, however, the ATF4 and XRP1 transcription factors are enriched in cytosolic granules that are positive for RNA and the stress granule markers Caprin, FMR1, and p62, and are reversible upon restoration of vision for blind flies. Cytosolic restraint of the ATF4 and XRP1 transcription factors dampens expression of their downstream targets including genes of cell death pathways activated during chronic cellular stress and thus constitutes a chronic stress protective response (CSPR). Cytosolic granules containing both p62 and ATF4 are also evident in the thalamus and hippocampus of mouse models of congenital or degenerative blindness. These data indicate a conserved link between loss of sensory input and curbed stress responses critical for protein quality control in the brain., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Long noncoding RNAs known as roX (RNA on the X) are crucial for male development in Drosophila, as their loss leads to male lethality from the late larval stages. While roX RNAs are recognized for their role in sex-chromosome dosage compensation, ensuring balanced expression of X-linked genes in both sexes, their potential influence on autosomal gene regulation remains unexplored. Here, using an integrative multi-omics approach, we show that roX RNAs not only govern the X chromosome but also target genes on autosomes that lack male-specific lethal (MSL) complex occupancy, together with Polycomb repressive complexes (PRCs). We observed that roX RNAs colocalize with MSL proteins on the X chromosome and PRC components on autosomes. Intriguingly, loss of roX function reduces X-chromosomal H4K16ac levels and autosomal H3K27me3 levels. Correspondingly, X-linked genes display reduced expression, whereas many autosomal genes exhibit elevated expression upon roX loss. Our findings propose a dual role for roX RNAs: activators of X-linked genes and repressors of autosomal genes, achieved through interactions with MSL and PRC complexes, respectively. This study uncovers the unconventional epigenetic repressive function of roX RNAs with PRC interaction., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Propagation of intercellular calcium waves through tissues has been found to coordinate different multicellular responses. Nevertheless, our understanding of how calcium waves operate remains limited. In this study, we explore the real-time dynamics of intercellular calcium waves in Drosophila adipose tissues. We identify Adipokinetic Hormone (AKH), the fly functional homolog of glucagon, as the key factor driving Ca 2+ activities in adipose tissue. We find that AKH, which is released into the hemolymph from the AKH-producing neurosecretory cells, stimulates calcium waves in the larval fat by a previously unrecognized gap-junction-independent mechanism to promote lipolysis. In the adult fat body, however, gap-junction-dependent intercellular calcium waves are triggered by a presumably uniformly diffused AKH. Additionally, we discover that amino acids activate the AKH-producing neurosecretory cells, leading to increased intracellular Ca 2+ and AKH secretion. Altogether, we show that dietary amino acids regulate the AKH release from the AKH-producing neurosecretory cells in the brain, which subsequently stimulates gap-junction-independent intercellular calcium waves in adipose tissue, enhancing lipid metabolism., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

At presynaptic active zones (AZs), scaffold proteins are critical for coordinating synaptic vesicle release and forming essential nanoarchitectures. However, regulatory principles steering AZ scaffold assembly, function, and plasticity remain insufficiently understood. We here identify an additional Drosophila AZ protein, "Blobby", essential for proper AZ nano-organization. Blobby biochemically associates with the ELKS family AZ scaffold protein Bruchpilot (BRP) and integrates into newly forming AZs. Loss of Blobby results in fewer AZs forming, ectopic AZ scaffold protein accumulations ("blobs") and disrupts nanoscale architecture of the BRP-AZ scaffold. Functionally, blobby mutants show diminished evoked synaptic currents due to reduced synaptic vesicle release probability and fewer functional release sites. Blobby is also present in adult brain synapses, and post-developmental knockdown of Blobby in the mushroom body impairs olfactory aversive memory consolidation. Thus, our analysis identifies an additional layer of AZ regulation critical for developmental AZ assembly but also for AZ-mediated plasticity controlling behavior., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Zinc homeostasis contributes significantly to numerous physiological processes. Drosophila ZnT35C protein, a zinc transporter encoded by CG3994, is chiefly located on the cell membrane and facilitates the transport of zinc from the cytoplasm to the extracellular space to sustain zinc homeostasis within the organism. Previous studies about ZnT35C have involved diverse structures such as the Malpighian tubules, adult brain, and sensory nervous system. Nonetheless, the role of ZnT35C in Drosophila spermatogenesis remained unclear. In our study, we discovered that ZnT35C plays a pivotal role in Drosophila spermatogenesis. Its knockdown resulted in sperm loss and male infertility. When ZnT35C was knocked down in cyst cells, zinc was concentrated within cyst cells, inhibiting the proper development of germ cells and thereby causing the incapacity of flies to generate mature sperms. Zinc supplementation can effectively rescue this failure of spermatogenesis. Our research outcomes suggest that ZnT35C, through modulating the zinc environment within the testes, impacts the male fertility of Drosophila, occupying a crucial position in the spermatogenesis process., (© 2025 Wiley Periodicals LLC.)

Hox genes specify identities mainly in the anteroposterior axis in various animal tissues, some of them forming part of the internal organs and systems. The expression and activity of these genes have been analyzed mainly in Drosophila melanogaster, the fruit fly, and in mouse; in the former, the functional study of Hox genes has been detailed predominantly in epidermal structures, but their role in internal organs poses some challenges, particularly in pupae. One of these genes, Abdominal-B, dictates the development of many internal organs in the posterior abdomen of the fly, yet techniques for its analysis, like in vivo time-lapse, have long been impractical. The protocol outlined in this chapter presents a simple and effective method that not only addresses this issue but also opens up new approaches to previously inaccessible in vivo developmental questions, such as the spatial challenges of abdominal organ packing and the minimal requirements for the coordinated growth of organs., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The process of aging is a natural phenomenon characterized by gradual deterioration in biological functions and systemic homeostasis, which can be modulated by both genetic and environmental factors. Numerous investigations conducted on model organisms, including nematodes, flies, and mice, have elucidated several pivotal aging pathways, such as insulin signaling and AMPK signaling. However, it remains uncertain whether the regulation of the aging process is uniform or diverse across different tissues and whether manipulating the same aging factor can result in consistent outcomes in various tissues. In this study, we utilize the Drosophila organism to investigate tissue-specific proteome signatures during the aging process. Although distinct proteins undergo changes in aged tissues, certain common altered functional networks are constituently identified across different tissues, including the decline of the mitochondrial ribosomal network, autophagic network, and anti-ROS defense networks. Furthermore, downregulation of insulin receptor (InR) in the midguts, muscle, and central nervous system (CNS) of flies leads to a significant extension in fly lifespans. Notably, despite manipulating the same aging gene InR, diverse alterations in proteins are observed across different tissues. Importantly, knockdown of InR in the midguts leads to a distinct proteome compared with other tissues, resulting in enhanced actin nucleation and glutathione metabolism, while attenuating age-related elevation of serine proteases. Consequently, knockdown of InR results in rejuvenation of the integrity of the midgut barrier and augmentation of anti-ROS defense capabilities. Our findings suggest that the barrier function of the midgut plays a pivotal role in defending against aging, underscoring the paramount importance of maintaining optimal gut physiology to effectively delay the aging process. Moreover, when considering age-related changes across various tissues, it is more reasonable to identify functional networks rather than focusing solely on individual proteins., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Ex vivo calcium imaging in Drosophila opens an expansive amount of research avenues for the study of live signal propagation through complex tissue. Here, we describe how to isolate Drosophila organs of interest, like the developing wing imaginal disc and larval brain, culture them for extended periods, up to 10 h, and how to image the calcium dynamics occurring within them using genetically encoded biosensors like GCaMP. This protocol enables the study of complex calcium signaling dynamics, which is conserved throughout biology in such processes as cell differentiation and proliferation, immune reactions, wound healing, and cell-to-cell and organ-to-organ communication, among others. These methods also allow pharmacological compounds to be tested to observe effects on calcium dynamics with the applications of target identification and therapeutic development., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Homeobox genes (HOX), the master regulators, deploy a unique set of target genes to coordinate and orchestrate the spatiotemporal development of an organism. HOX encoded transcriptional factors regulate the expression of target genes by binding to the specific sequences on the genome. Chromatin Immunoprecipitation (ChIP) and Chromatin Immunoprecipitation with Sequencing (ChIP-Seq) are widely used to map and understand specific gene locus and global regulatory regions on the genome. ChIP is a powerful technique of cross-linking the proteins bound to the DNA, fragmenting DNA to the desired size, and pulling them down using specific antibodies to enrich and analyze the protein-bound DNA. Based on the mapping information, a differential ChIP can be performed to understand cis-regulatory modulations at a defined locus by two developmental stages. This chapter describes the differential ChIP used to identify new targets by comparing two different developmental stages simultaneously using Drosophila melanogaster., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The ability to add bioactivities, such as cell signaling or ligand recognition, to biomaterials has generated the potential to include multiple bioactivities into a single material. In some cases, it is desirable to localize these activities to different areas of the biomaterial, creating functional patterns. While photolithography and 3D printing have been effective techniques for patterning functions in many materials, patterning remains a challenge in materials composed of protein, in part due to how these materials are artificially assembled. Protein fibers are often produced from protein films that co-acervate at the air-water interface. This chapter describes methods to leverage this coacervation process to pattern materials, using the Drosophila melanogaster Hox protein Ultrabithorax (Ubx) as a model self-assembling protein. Through gene fusion, Ubx and a functional protein are produced as a single polypeptide, capable of both forming materials and performing the activity of interest. This functionality is retained in the final materials. In this chapter, we describe how to use multiple Ubx fusion proteins to not only imbue the final materials with multiple functions, but also to create macroscale patterns of the appended proteins in fibrous protein-based materials. These patterned materials include striped fibers, bifunctional-faced fibers, gradient fibers, and core-shell fibers., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3' untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3' UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The mechanism of volatile general anaesthetics has long been a mystery. Anaesthetics have no structural motifs in common, beyond lipid solubility, yet all exert a similar effect. The fact that the inert gas xenon is an anaesthetic suggests their common mechanism might relate to physical rather than chemical properties. Electron transfer through chiral proteins can induce spin polarization. Recent work suggests that anaesthetics dissipate spin polarization during electron transfer to oxygen, slowing respiration. Here we show that the volatile anaesthetics isoflurane and sevoflurane specifically disrupt complex I-linked respiration in the thoraces of Drosophila melanogaster, with less effect on maximal respiration. Suppression of complex I-linked respiration was greatest with isoflurane. Using high-resolution tissue fluorespirometry, we show that these anaesthetics simultaneously increase mitochondrial membrane potential, implying reversal of the ATP synthase. Inhibition of ATP synthase with oligomycin prevented respiration and increased membrane potential back to the maximal (LEAK state) potential. Magnesium-green fluorescence predicted a collapse in ATP availability following a single anaesthetic dose, consistent with ATP hydrolysis through reversal of the ATP synthase. Raised membrane potential corresponded to a rise in ROS flux, especially with isoflurane. Anaesthetic doses causing respiratory suppression were in the same range as those that induce anaesthesia, although we could not establish tissue concentrations. Our findings show that anaesthetics suppress complex I-linked respiration with concerted downstream effects. But we cannot explain why only mutations in complex I, and not elsewhere in the electron-transfer system, confer hypersensitivity to anaesthetics., Competing Interests: Declaration of competing interest The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Financial support was provided by the Bill and Melinda Gates Foundation and by the Biotechnology and Biological Sciences Research Council., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Many cellular functions are compartmentalized within the optimized environments of organelles. However, processing or storage of metabolites from the same pathway can occur in multiple organelles. Thus, spatially separated organelles need to cooperate functionally. Coordination between organelles in different specialized cells is also needed, with shared metabolites passed via circulation. Peroxisomes are membrane-bounded organelles responsible for cellular redox and lipid metabolism in eukaryotic cells. Peroxisomes coordinate with other organelles including mitochondria, endoplasmic reticulum, lysosomes, and lipid droplets. This functional coordination requires, or is at least enhanced by, direct contact between peroxisomes and other organelles. Peroxisome dysfunction in humans leads to multiorgan effects including neurological, metabolic, developmental, and age-related diseases. Thus, increased understanding of peroxisome coordination with other organelles, especially cells in various organs is essential. Drosophila melanogaster (fruit fly) has emerged recently as an effective animal model for understanding peroxisomes. Here we review current knowledge of pathways regulating coordination between peroxisomes with other organelles in flies, speculating about analogous roles for conserved Drosophila genes encoding proteins with known organelle coordinating roles in other species., Competing Interests: The authors declare there are no competing interests.

In the Drosophila testis, developing germ cells are encapsulated by somatic support cells throughout development. Soma-germline interactions are essential for successful spermiogenesis. However, it is still not fully understood what signaling events take place between the soma and the germline. In this study, we found that a Bone Morphogenetic Protein (BMP) ligand, Glass bottom boat (Gbb), secreted from somatic cyst cells (CCs), signals to differentiating germ cells to maintain proper spermiogenesis. Knockdown of Gbb in CCs or the type I BMP receptor Saxophone (Sax) in germ cells leads to a defect in sperm head bundling and decreased fertility. Our Transmission Electron Microscopy (TEM) analyses revealed that the mutant germ cells have aberrant morphology of mitochondria throughout the stages of spermiogenesis and exhibit a defect in nebenkern formation. Elongating spermatids show uncoupled nuclei and elongating mitochondrial derivatives, suggesting that improper mitochondrial development may cause sperm bundling defects. Taken together, we propose a new role of soma-derived BMP signaling, which is essential for spermiogenesis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Internal organ development requires cell internalization, which can occur individually or collectively. The best characterized mode of collective internalization is epithelial invagination. Alternate modes involving collective mesenchymal behaviours at the embryo surface have been documented, but their prevalence is unclear. The Drosophila embryo has been a major model for the study of epithelial invaginations. However, internalization of the Drosophila anterior midgut primordium is incompletely understood. Here, we report that an epithelial-mesenchymal transition (EMT) occurs across the internalizing primordium when it is still at the embryo surface. At the earliest internalization stage, the primordium displays less junctional DE-cadherin than surrounding tissues but still exhibits coordinated epithelial structure as it invaginates with the ventral furrow. This initial invagination is transient, and its loss correlates with the activation of an associated mitotic domain. Activation of a subsequent mitotic domain across the broader primordium results in cell divisions with mixed orientations that deposit some cells within the embryo. However, cell division is non-essential for primordium internalization. Post-mitotically, the surface primordium displays hallmarks of EMT: loss of adherens junctions, loss of epithelial cell polarity, and gain of cell protrusions. Primordium cells extend over each other as they internalize asynchronously as individuals or small groups, and the primordium becomes enclosed by the reorganizations of surrounding epithelial tissues. We propose that collective EMT at the embryo surface promotes anterior midgut internalization through both inwardly-directed divisions and movements of its cells, and that the latter process is facilitated by surrounding tissue remodeling., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Compelling evidence has strongly linked unregulated sugar levels to developing Alzheimer's disease, suggesting Alzheimer's to be 'diabetes of the brain or 'type 3 diabetes. Insulin resistance contributes to the pathogenesis of Alzheimer's disease due to uncontrolled and unchecked blood glucose, though the interrelatedness between Alzheimer's disease and type 2 diabetes is debatable. Here we describe the consequences of inducing type 3 diabetes by feeding Drosophila on a high sucrose diet, which effectively mimics the pathophysiology of diabetes. A high sucrose diet increases glycogen and lipid accumulation. Inducing type 3 diabetes worsened neurodegeneration and accelerated disease progression in Drosophila expressing the Alzheimer's Familial Arctic mutation. High sucrose milieu also negatively affected locomotor ability and reduced the lifespan in the Alzheimer's disease model of Drosophila. The results showed that creating diabetic conditions by using insulin receptor (InR) knockdown in the eyes of Drosophila led to a degenerative phenotype, indicating a genetic interaction between the insulin signaling pathway and Alzheimer's disease. The expression of PERK reflects disruption in the endoplasmic reticulum homeostasis due to amyloid-β (Aβ) under a high sucrose diet. These observations demonstrated an association between type 3 diabetes and Alzheimer's disease, and that a high sucrose environment has a degenerating effect on Alzheimer's disease condition., Competing Interests: Declaration of competing interest All the authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hematopoiesis is crucial for organismal health, and Drosophila serves as an effective genetic model due to conserved regulatory mechanisms with vertebrates. In larvae, hematopoiesis primarily occurs in the lymph gland, which contains distinct zones, including the cortical zone, intermediate zone, medullary zone, and posterior signaling center (PSC). Rab1 is vital for membrane trafficking and maintaining the localization of cell adhesion molecules, yet its role in hematopoietic homeostasis is not fully understood. This study investigates the effects of Rab1 dysfunction on β-integrin trafficking within circulating hemocytes and lymph gland cells. Rab1 impairment disrupts the endosomal trafficking of β-integrin, leading to its abnormal localization on cell membranes, which promotes lamellocyte differentiation and alters progenitor dynamics in circulating hemocytes and lymph glands, respectively. We also show that the mislocalization of β-integrin is dependent on the adhesion protein DE-cadherin. The reduction of β-integrin at cell boundaries in PSC cells leads to fewer PSC cells and lamellocyte differentiation. Furthermore, Rab1 regulates the trafficking of β-integrin via the Q-SNARE protein Syntaxin 17 (Syx17). Our findings indicate that Rab1 and Syx17 regulate distinct trafficking pathways for β-integrin in different hematopoietic compartments and maintain hematopoietic homeostasis of Drosophila., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Glucose is the main energy source of the brain, yet recent studies demonstrate that fatty acid oxidation (FAO) plays a relevant role in the pathogenesis of central nervous system disorders. We evaluated FAO in brain mitochondria under physiological conditions, in the aging brain, and after stroke. Using high-resolution respirometry we compared medium-chain (MC, octanoylcarnitine) and long-chain (LC, palmitoylcarnitine) acylcarnitines as substrates of β-oxidation in the brain. The protocols developed avoid FAO overestimation by malate-linked anaplerotic activity in brain mitochondria. The capacity of FA oxidative phosphorylation (F-OXPHOS) with palmitoylcarnitine was up to 4 times higher than respiration with octanoylcarnitine. The optimal concentration of palmitoylcarnitine was 10 μM which corresponds to the total concentration of LC acylcarnitines in the brain. Maximal respiration with octanoylcarnitine was reached at 20 μM, however, this concentration exceeds MC acylcarnitine concentrations in the brain 15 times. F-OXPHOS capacity was highest in mouse cerebellum, intermediate in cortex, prefrontal cortex, and hypothalamus, and hardly detectable in hippocampus. F-OXPHOS capacity was 2-fold lower and concentrations of LC acylcarnitines were 2-fold higher in brain of aged rats. A similar trend was observed in the rat model of endothelin-1-induced stroke, but reduction of OXPHOS capacity was not limited to FAO. In conclusion, although FAO is not a dominant pathway in brain bioenergetics, it deserves specific attention in studies of brain metabolism., Competing Interests: Declaration of competing interest Luiza Helena Daltro Cardoso and Cristiane Cecatto are employees and Erich Gnaiger is the founder and CEO of Oroboros Instruments, Innsbruck, Austria. The other authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Mitosis is largely controlled by the reversible phosphorylation of effector proteins. The addition or removal of phosphate groups alters the activities of these proteins, resulting in changes in chromosome structure, cytoskeletal dynamics, nuclear envelope integrity, and other transformations that must occur as a cell progresses through mitosis. Drosophila has been instrumental in the elucidation of the molecular mechanisms of mitosis, which are mostly conserved among animals. In this model system, sophisticated genetic tools can be used to study mitosis in different tissues during development in vivo. Drosophila cell culture affords complementary possibilities. In this chapter, we present a phosphoproteomic protocol using Drosophila cell culture to identify phosphorylation sites that depend on mitotic kinases and phosphatases. We also provide protocols to dissect the roles of the identified sites in the regulation of protein interactions and localization during mitosis, using Drosophila embryos. We emphasize the advantages of the selected methods compared to possible alternatives in Drosophila or in other systems., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cachexia and systemic organ wasting are metabolic syndrome often associated with cancer. However, the exact mechanism of cancer associated cachexia like syndrome still remain elusive. In this study, we utilized a scribble (scrib) knockdown induced hindgut tumor to investigate the role of JNK kinase in cachexia like syndrome. Scrib, a cell polarity regulator, also acts as a tumor suppressor gene. Its loss and mis-localization are reported in various type of malignant cancer-like breast, colon and prostate cancer. The scrib knockdown flies exhibited male lethality, reduced life span, systemic organ wasting and increased pJNK level in hindgut of female flies. Interestingly, knocking down of human JNK Kinase analogue, hep, in scrib knockdown background in hindgut leads to restoration of loss of scrib mediated lethality and systemic organ wasting. Our data showed that scrib loss in hindgut is capable of inducing cancer associated cachexia like syndrome. Here, we firstly report that blocking the JNK signaling pathway effectively rescued the cancer cachexia induced by scrib knockdown, along with its associated gut barrier disruption. These findings have significantly advanced our understanding of cancer cachexia and have potential implications for the development of therapeutic strategies. However, more research is needed to fully understand the complex mechanisms underlying this condition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gastrulation involves multiple, physically-coupled tissue rearrangements. During Drosophila gastrulation, posterior midgut (PMG) invagination promotes both germband extension and hindgut invagination, but whether the normal epithelial rearrangement of PMG invagination is required for morphogenesis of the connected tissues has been unclear. In steppke mutants, epithelial organization of the PMG primordium is strongly disrupted. Despite this disruption, germband extension and hindgut invagination are remarkably effective, and involve myosin network inductions known to promote their wild-type remodelling. Known tissue-autonomous signaling could explain the planar-polarized, junctional myosin networks of the germband, but pushing forces from PMG invagination have been implicated in inducing apical myosin networks of the hindgut primordium. To confirm that the wave of hindgut primordium myosin accumulations is due to mechanical effects, rather than diffusive signalling, we analyzed α-catenin RNAi embryos, in which all of the epithelial tissues initially form but then lose cell-cell adhesion, and observed strongly diminished hindgut primordium myosin accumulations. Thus, alternate mechanical changes in steppke mutants seem to circumvent the lack of normal PMG invagination to induce hindgut myosin networks and invagination. Overall, both germband extension and hindgut invagination are robust to experimental disruption of the PMG invagination, and, although the processes occur with some abnormalities in steppke mutants, there is remarkable redundancy in the multi-tissue system. Such redundancy could allow complex morphogenetic processes to change over evolutionary time., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Social isolation (SI) in humans can lead to various psychological and physical abnormalities. However, the molecular mechanisms and potential drug treatments for this illness are not well understood. Drosophila, a social organism, exhibits distinct behavioral defects under SI conditions, such as reduced sleep and loss of sugar intake preference. By examining the transcriptional profiles of SI flies, we discovered significant impacts on metabolic pathways. Notably, serotonin (5-HT) levels were reduced in the brains of SI flies. Treatment with 5-HT reversed the behavioral defects in SI flies. 5-HT is known to regulate mitochondrial synthesis in mouse brain, and we found it also enhances mitochondrial biogenesis in flies. Further investigation revealed that the 5-HT 7 receptor subtype was involved in SI behavior. To activate mitochondrial metabolism, we overexpressed phosphoglycerate kinase (Pgk), an enzyme in the glycolytic pathway, in neurons. This overexpression rescued the behavioral defects in SI flies. Additionally, terazosin, an alpha-1 adrenergic receptor antagonist known to activate Pgk, produced a similar rescue effect. Our study elucidates a key principle of SI-induced psychological damage and proposes a drug targeting strategy for future validation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Taking advantage of the good mechanical strength of expanded Drosophila brains and to tackle their relatively large size that can complicate imaging, we apply potassium (poly)acrylate-based hydrogels for expansion microscopy (ExM), resulting in a 40x plus increased resolution of transgenic fluorescent proteins preserved by glutaraldehyde fixation in the nervous system. Large-volume ExM is realized by using an axicon-based Bessel lightsheet microscope, featuring gentle multi-color fluorophore excitation and intrinsic optical sectioning capability, enabling visualization of Tm5a neurites and L3 lamina neurons with photoreceptors in the optic lobe. We also image nanometer-sized dopaminergic neurons across the same intact iteratively expanded Drosophila brain, enabling us to measure the 3D expansion ratio. Here we show that at a tile scanning speed of ~1 min/mm 3 with 10 12 pixels over 14 hours, we image the centimeter-sized fly brain at an effective resolution comparable to electron microscopy, allowing us to visualize mitochondria within presynaptic compartments and Bruchpilot (Brp) scaffold proteins distributed in the central complex, enabling robust analyses of neurobiological topics., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Mitochondrial biogenesis requires the expression of genes encoded by both the nuclear and mitochondrial genomes. However, aside from a handful transcription factors regulating specific subsets of mitochondrial genes, the overall architecture of the transcriptional control of mitochondrial biogenesis remains to be elucidated. The mechanisms coordinating these two genomes are largely unknown. We performed a targeted RNAi screen in developing eyes with reduced mitochondrial DNA content, anticipating a synergistic disruption of tissue development due to impaired mitochondrial biogenesis and mitochondrial DNA (mtDNA) deficiency. Among 638 transcription factors annotated in the Drosophila genome, 77 were identified as potential regulators of mitochondrial biogenesis. Utilizing published ChIP-seq data of positive hits, we constructed a regulatory network revealing the logic of the transcription regulation of mitochondrial biogenesis. Multiple transcription factors in core layers had extensive connections, collectively governing the expression of nearly all mitochondrial genes, whereas factors sitting on the top layer may respond to cellular cues to modulate mitochondrial biogenesis through the underlying network. CG1603, a core component of the network, was found to be indispensable for the expression of most nuclear mitochondrial genes, including those required for mtDNA maintenance and gene expression, thus coordinating nuclear genome and mtDNA activities in mitochondrial biogenesis. Additional genetic analyses validated YL-1, a transcription factor upstream of CG1603 in the network, as a regulator controlling CG1603 expression and mitochondrial biogenesis., Competing Interests: FZ, AL, AF, JH, ZW, SG, ZC, HX No competing interests declared

cGAS-like receptor (cGLR)-stimulator of interferon genes (STING) recently emerged as an important pathway controlling viral infections in invertebrates. However, its exact contribution at the organismal level remains uncharacterized. Here, we use STING::GFP knockin reporter Drosophila flies to document activation of the pathway in vivo. Four tissues strongly respond to injection of the cyclic dinucleotide 3'2'- cyclic guanosine monophosphate-adenosine monophosphate (cGAMP): the central nervous system, midgut, Malpighian tubules, and genital ducts. The pattern of STING::GFP induction in flies injected with 3'2'-cGAMP or infected by two viruses with different tropism suggests that the reporter is induced by a systemic signal produced in virus-infected cells. Accordingly, ectopic expression of cGLR2 in the fat body induces STING signaling in remote tissues and a cGLR1/2-dependent activity is transferred to females during mating. Furthermore, viral infection can alter sleep in a cGLR1/2- and STING-dependent manner. Altogether, our results reveal a contribution of cyclic dinucleotide signaling to a systemic host response to viral infection in Drosophila., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Piwi-interacting RNAs (piRNAs) are the main repressors of transposable elements (TEs) in animal germlines. In Drosophila, Piwi-piRNA complexes associate with nascent TE transcripts to drive heterochromatin formation and TE repression. However, previous studies have shown that Piwi also associates with large numbers of mRNAs, raising the question of how Piwi discriminates between mRNAs and TEs. To answer this question, we performed a comprehensive analysis of Piwi-associated RNAs, compositionally and functionally, to decipher the targeting rules of Piwi-piRNA complexes. While Piwi initially identifies its targets through the seed sequence, it requires pairing well beyond the seed, nearly a perfect match, to elicit a repressive response. In addition to the complementarity of piRNAs to their targets, their abundance must reach a certain threshold to be functional. Together, these findings explain large differences in the target repression of Piwi-associated RNAs and reveal how Piwi efficiently distinguishes TEs from mRNAs despite associating with both., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here, we present the culmination of the efforts of the modENCODE (model organism Encyclopedia of DNA Elements) and modERN (model organism Encyclopedia of Regulatory Networks) consortia to systematically assay TF binding events in vivo in two major model organisms, Drosophila melanogaster (fly) and Caenorhabditis elegans (worm). These data sets comprise 605 TFs identifying 3.6 M sites in the fly and 356 TFs identifying 0.9 M sites in the worm, and represent the majority of the regulatory space in each genome. We demonstrate that TFs associate with chromatin in clusters termed "metapeaks," that larger metapeaks have characteristics of high-occupancy target (HOT) regions, and that the importance of consensus sequence motifs bound by TFs depends on metapeak size and complexity. Combining ChIP-seq data with single-cell RNA-seq data in a machine-learning model identifies TFs with a prominent role in promoting target gene expression in specific cell types, even differentiating between parent-daughter cells during embryogenesis. These data are a rich resource for the community that should fuel and guide future investigations into TF function. To facilitate data accessibility and utility, all strains expressing green fluorescent protein (GFP)-tagged TFs are available at the stock centers for each organism. The chromatin immunoprecipitation sequencing data are available through the ENCODE Data Coordinating Center, GEO, and through a direct interface that provides rapid access to processed data sets and summary analyses, as well as widgets to probe the cell-type-specific TF-target relationships., (© 2024 Kudron et al.; Published by Cold Spring Harbor Laboratory Press.)