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The Hedgehog (Hh) pathway plays diverse roles in cellular processes by activating the transcription factor Cubitus interruptus (Ci). Abnormal regulation of this pathway has been linked to various human diseases. While previous studies have focused on how Ci is regulated in the cytoplasm, the control of nuclear Ci remains poorly understood. In this study, we have found that the transcriptional cofactor Taiman (Tai) functions as an inhibitor of the Hh pathway. Tai interferes with the response of Hh signal, rather than Hh secretion. Our epistatic analyses reveal that Tai works in parallel with Ci to reduce its activity, thereby counteracting organ overgrowth and the activation of target genes caused by Ci overexpression. Specifically, Tai interacts with Ci to decrease its binding to target gene promoters. The Hh signal weakens the interaction between Ci and Tai, releasing the inhibition on Ci. Importantly, this regulatory mechanism is conserved from Drosophila to mammalian cells. Moreover, NCOA1-3 are the mammalian ortholog of Drosophila protein Tai, but only NCOA2 plays a similar role in inhibiting the Hh pathway. These findings reveal an additional way to modulate the transcriptional activity of nuclear Ci., Competing Interests: Competing interests statement:The authors declare no competing interest.

Early embryos undergo profound changes in their genomic architecture to establish the totipotent state, enabling pioneer factors to access chromatin and drive zygotic genome activation (ZGA). However, the mechanisms by which the totipotent state is established and properly interpreted by pioneer factors to allow orderly ZGA remain unknown. Here, we identify the H3.3-specific chaperone HIRA as a factor involving establishing totipotent-state chromatin in Drosophila early embryos. Through cophase separation with HIRA, the pioneer factor GAGA factor (GAF) efficiently binds to H3.3-marked nucleosomes to activate major-wave zygotic genes. Importantly, dPCIF1, a chromatin-associated protein, antagonized the GAF-HIRA interaction by competitively binding to HIRA, thereby restricting GAF on earlier chromatin and avoiding premature ZGA. Hence, the coordinated action of HIRA and dPCIF1 ensures sequential ZGA from the minor to major wave in early embryos. This study provides insights into understanding how a totipotent state is established and properly controlled during ZGA., Competing Interests: Competing interests statement:The authors declare no competing interest.

The chemoproteomics technique, activity-based protein profiling (ABPP), has proven to be an invaluable tool in assigning functions to enzymes. The serine hydrolase (SH) enzyme superfamily, in particular, has served as an excellent example in displaying the versatility of various ABPP platforms and has resulted in a comprehensive cataloging of the biochemical activities associated within this superfamily. Besides SHs, in mammals, several other enzyme classes have been thoroughly investigated using ABPP platforms. However, the utility of ABPP platforms in fly models remains underexplored. Realizing this knowledge gap, leveraging complementary ABPP platforms, we reported the full array of SH activities during various developmental stages and adult tissues in the fruit fly ( Drosophila melanogaster ). Following up on this study, using ABPP, we mapped SH activities in adult fruit flies in an infection model and found that a gut-resident lipase CG17192 showed increased activity during infection. To assign a biological function to this uncharacterized lipase, we performed an untargeted lipidomics analysis and found that phosphatidylinositols were significantly elevated when CG17192 was depleted in the adult fruit fly gut. Next, we overexpressed this lipase in insect cells, and using biochemical assays, we show that CG17192 is a secreted enzyme that has phospholipase C (PLC) type activity, with phosphatidylinositol being a preferred substrate. Finally, we show during infection that heightened CG17192 regulates phosphatidylinositol levels and, by doing so, likely modulates signaling pathways in the adult fruit fly gut that might be involved in the resolution of this pathophysiological condition.

Alzheimer's disease is a complex neurodegenerative condition characterized by the accumulation of amyloid beta plaques, leading to memory loss, cognitive decline, and impaired autonomous behavior. Despite extensive research, an effective treatment remains elusive. The buildup of amyloid beta plaques (Aβ42) in the brain causes oxidative stress and disrupts normal molecular signaling, adversely affecting neuron function. Previous research has identified factors that can either exacerbate or mitigate neurodegenerative diseases. Our study aimed to uncover new factors involved in the pathogenesis of Alzheimer's disease. Using Drosophila as a model organism, we employed the Gal4/UAS system to express human Aβ42 in the flies' retinal neurons which led to neurodegenerative changes in their compound eyes. To identify genetic modifiers, we conducted a screen by co-expressing microRNAs and found that miR-282 acts as a suppressor. Overexpressing miR-282 in the GMR > Aβ42 background reduced Aβ42-induced neurodegeneration. Further analysis using prediction tools and RNA interference experiments identified three potential downstream targets of miR-282: calpain-B, knot, and scabrous. Downregulating these genes via RNA interference in the GMR > Aβ42 background mitigated neurodegeneration. Our research highlights miR-282 as a novel molecule that may influence the progression of Alzheimer's disease, offering potential avenues for future therapeutic or diagnostic developments., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Centromeres depend on chromatin containing the conserved histone H3 variant CENP-A for function and inheritance, while the role of centromeric DNA repeats remains unclear. Retroelements are prevalent at centromeres across taxa and represent a potential mechanism for promoting transcription to aid in CENP-A incorporation or for generating RNA transcripts to maintain centromere integrity., Results: In this study, we probe into the transcription and RNA localization of the centromere-enriched retroelement G2/Jockey-3 (hereafter referred to as Jockey-3) in Drosophila melanogaster, currently the only in vivo model with assembled centromeres. We find that Jockey-3 is a major component of the centromeric transcriptome and produces RNAs that localize to centromeres in metaphase. Leveraging the polymorphism of Jockey-3 and a de novo centromere system, we show that these RNAs remain associated with their cognate DNA sequences in cis, suggesting they are unlikely to perform a sequence-specific function at all centromeres. We show that Jockey-3 transcription is positively correlated with the presence of CENP-A and that recent Jockey-3 transposition events have occurred preferentially at CENP-A-containing chromatin., Conclusions: We propose that Jockey-3 preferentially inserts at the centromere to ensure its own selfish propagation, while contributing to transcription across these regions. Given the conservation of retroelements as centromere components through evolution, our findings may offer a basis for understanding similar associations in other species., Competing Interests: Declarations Ethics approval and consent to participate Ethics approval is not applicable for this study. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Insecticide resistance (IR) poses a significant global challenge to public health and welfare. Here, we develop a locally-acting unitary self-eliminating allelic-drive system, inserted into the Drosophila melanogaster yellow (y) locus. The drive cassette encodes both Cas9 and a single gRNA to bias inheritance of the favored wild-type (1014 L) allele over the IR (1014 F) variant of the voltage-gated sodium ion channel (vgsc) target locus. When enduring a fitness cost, this transiently-acting drive can increase the frequency of the wild-type allele to 100%, depending on its seeding ratio, before being eliminated from the population. However, in a fitness-neutral "hover" mode, the drive maintains a constant frequency in the population, completely converting IR alleles to wild-type, even at low initial seeding ratios., Competing Interests: Competing interests E.B. has equity interest in Synbal, Inc. and Agragene, Inc., companies that may potentially benefit from the research results. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. The other authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Due to their position on glomerular capillaries, podocytes are continuously counteracting biomechanical filtration forces. Most therapeutic interventions known to generally slow or prevent the progression of chronic kidney disease appear to lower these biomechanical forces on podocytes, highlighting the critical need to better understand podocyte mechano-signalling pathways. Here we investigated whether the mechanotransducer Piezo is involved in a mechanosensation pathway in Drosophila nephrocytes, the podocyte homologue in the fly. Loss of function analysis in Piezo depleted nephrocytes reveal a severe morphological and functional phenotype. Further, pharmacological activation of endogenous Piezo with Yoda1 causes a significant increase of intracellular Ca ++ upon exposure to a mechanical stimulus in nephrocytes, as well as filtration disturbances. Elevated Piezo expression levels also result in a severe nephrocyte phenotype. Interestingly, expression of Piezo which lacks mechanosensitive channel activity, does not result in a severe nephrocyte phenotype, suggesting the observed changes in Piezo wildtype overexpressing cells are caused by the mechanosensitive channel activity. Moreover, blocking Piezo activity using the tarantula toxin GsMTx4 reverses the phenotypes observed in nephrocytes overexpressing Piezo. Taken together, here we provide evidence that Piezo activity levels need to be tightly regulated to maintain normal pericardial nephrocyte morphology and function., Competing Interests: Declarations Competing interests The authors declare no conflicts of interest., (© 2024. The Author(s).)

G protein alpha q subunit (Gαq) can binds to the G protein-coupled receptor (GPCR) for signaling and is closely related to lipid metabolism. Endurance exercise is an effective means of combating acquired obesity and its complications, but the mechanisms by which endurance exercise modulates hereditary obesity and its complications are unknown. In this study, we achieved knockdown of Gαq in drosophila adipose tissue and skeletal muscle by constructing the Gαq-UAS-RNAi/Ppl-Gal4 and Gαq-UAS-RNAi/Mef2-GAl4 systems. Drosophila were subjected a three-week endurance exercise intervention, and changes in relevant indicators were detected and observed by RT-PCR, ELISA, oil red staining, immunofluorescence staining, and transmission electron microscopy. The results showed that knockdown of Gαq in both adipose tissue and skeletal muscle induced a significant increase in triglycerides accompanied by a decrease in rapid climbing ability, a decrease in Superoxide Dismutase (SOD) activity level, and a decrease in Mitochondrial respiratory chain complexI (MRCC I) content in Drosophila whole body and skeletal muscle, and down-regulated the expression of the G protein alpha q subunit (Gαq), the skeletal muscle myosin heavy chain expression gene (Mhc), mitochondrial biogenesis gene Spargal(the PGC-1alpha homologue in Drosophila). Endurance exercise significantly improved the triglyceride levels in the whole body and skeletal muscle of drosophila with Gαq knockdown in adipose tissue and skeletal muscle, as well as their ability to climb, increased SOD activity level and MRCCI content level, and up-regulated the expression of Gαq, Mhc, and Spargal(Srl). Thus, the present findings suggest that genetic defects in the Gαq gene in adipose and skeletal muscle tissues induce hereditary obesity and skeletal muscle dysfunction, and that endurance exercise attenuates this hereditary obesity and concomitant skeletal muscle dysfunction in drosophila by improving skeletal muscle fiber contractile proteins, mitochondrial function and function, and antioxidant capacity via mediating the Gαq/Mhc, Gαq/Srl/MRCC-I, and Gαq/SOD pathways., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Protein-rich diets shorten lifespan but increase fecundity in many organisms. Animals actively adjust their feeding behavior to meet their nutritional requirements. However, the neural mechanisms underlying the dynamic regulation of protein consumption remain unclear. Here we find that both sexes of fruit flies exhibit a preference for protein food before mating to prepare for reproduction. Mated female flies display an increased appetite for yeast to benefit their offspring, albeit at the cost of stress resistance and lifespan. In contrast, males show a momentarily reduced yeast appetite after mating likely to restore their fitness. This mating state-dependent switch between sexes is mediated by a sexually dimorphic neural circuit labeled with leucokinin in the anterior brain. Furthermore, intermittent yeast consumption benefits both the lifespan and fecundity of males, while maximizing female fecundity without compromising lifespan., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Ligands of the TGF-β/BMP superfamily are crucially involved in the regulation of growth, patterning and organogenesis and can act as long-range morphogens. Essential for understanding TGF-β/BMP signaling dynamics and regulation are tools that allow monitoring and manipulating pathway components at physiological expression levels and endogenous spatiotemporal patterns. We used genome engineering to generate a comprehensive library of endogenously epitope- or fluorescent-tagged versions of receptors, co-receptors, transcription factors and key feedback regulators of the Drosophila BMP and Activin signaling pathways. We demonstrate that the generated alleles are biologically active and can be used for assessing tissue and subcellular distribution of the corresponding proteins. Furthermore, we show that the genomic platforms can be used for in locus structure-function and cis-regulatory analyses. Finally, we present a complementary set of protein binder-based tools, which allow visualization as well as manipulation of the stability and subcellular localization of epitope-tagged proteins, providing new tools for the analysis of BMP signaling and beyond., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Drosophila female germline stem cells (GSCs) complete asymmetric mitosis in the presence of an intact, but permeable, nuclear envelope and nuclear lamina (NL). This asymmetric division requires a modified centrosome cycle, wherein mitotic centrosomes with mature pericentriolar material (PCM) embed in the NL and interphase centrosomes with reduced PCM leave the NL. This centrosome cycle requires Emerin, an NL protein required for GSC survival and germ cell differentiation. In emerin mutants, interphase GSC centrosomes retain excess PCM, remain embedded in the NL and nucleate microtubule asters at positions of NL distortion. Here, we investigate the contributions of abnormal interphase centrosomes to GSC loss. Remarkably, reducing interphase PCM in emerin mutants rescues GSC survival and partially restores germ cell differentiation. Direct tests of the effects of abnormal centrosomes were achieved by expression of constitutively active Polo kinase to drive enlargement of interphase centrosomes in wild-type GSCs. Notably, these conditions failed to alter NL structure or decrease GSC survival. However, coupling enlarged interphase centrosomes with nuclear distortion promoted GSC loss. These studies establish that Emerin maintains centrosome structure to preserve stem cell survival., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Throughout metazoa, germ cells assemble RNA-protein organelles (germ granules). In Drosophila ovaries, perinuclear nuage forms in the nurse cells, while compositionally similar polar granules form in the oocyte. A similar system appears to exist in the distantly related (∼350 million years) wasp Nasonia, with some surprising divergences. Nuage is similarly formed in Nasonia, except that anterior nurse cells accumulate significantly more nuage, in association with high levels of DNA double-strand breaks, suggesting that increased transposon activity anteriorly is silenced by high nuage levels. In the oocyte, the germ plasm forms a single granule that is 40 times larger than a homologous Drosophila polar granule. While conserved germ granule proteins are recruited to the oosome, they show unusual localization: Tudor protein forms a shell encapsulating the embryonic oosome, while small Oskar/Vasa/Aubergine granules coalesce interiorly. Wasp Vasa itself is unusual since it has an alternative splice form that includes a previously unreported nucleoporin-like phenylalanine-glycine repeat domain. Our work is consistent with the high degree of evolutionary plasticity of membraneless organelles, and describes a new experimental model and resources for studying biomolecular condensates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Ventral furrow (VF) formation in Drosophila melanogaster is an important model of epithelial folding. Previous models of VF formation require cell volume conservation to convert apically localized constriction forces into lateral cell elongation and tissue folding. Here, we have investigated embryonic morphogenesis in anillin knockdown (scra RNAi) embryos, where basal cell membranes fail to form and therefore cells can lose cytoplasmic volume through their basal side. Surprisingly, the mesoderm elongation and subsequent folding that comprise VF formation occurred essentially normally. We hypothesized that the effects of viscous shear may be sufficient to drive membrane elongation, providing effective volume conservation, and thus driving tissue folding. Since this hypothesis may not be possible to test experimentally, we turned to a computational approach. To test whether viscous shear is a dominant force for morphogenesis in vivo, we developed a 3D computational model incorporating both accurate cell and tissue geometry, and experimentally measured material parameters. Results from this model demonstrate that viscous shear generates sufficient force to drive cell elongation and tissue folding in vivo., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Functional divergence of transcription factors (TFs) has driven cellular and organismal complexity throughout evolution, but its mechanistic drivers remain poorly understood. Here we test for new mechanisms using CORONA (CNA) and PHABULOSA (PHB), two functionally diverged paralogs in the CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) family of TFs. We show that virtually all genes bound by PHB ( ~ 99%) are also bound by CNA, ruling out occupation of distinct sets of genes as a mechanism of functional divergence. Further, genes bound and regulated by both paralogs are almost always regulated in the same direction, ruling out opposite regulation of shared targets as a mechanistic driver. Functional divergence of CNA and PHB instead results from differential usage of shared binding sites, with hundreds of uniquely regulated genes emerging from a commonly bound genetic network. Regulation of a given gene by CNA or PHB is thus a function of whether a bound site is considered 'responsive' versus 'non-responsive' by each paralog. Discrimination between responsive and non-responsive sites is controlled, at least in part, by their lipid binding START domain. This suggests a model in which HD-ZIPIII TFs use information integrated by their START domain to generate paralog-specific transcriptional outcomes from a shared network architecture. Taken together, our study identifies a mechanism of HD-ZIPIII TF paralog divergence and proposes the ubiquitously distributed START evolutionary module as a driver of functional divergence., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The expression of a few key genes determines the body plan of the fruit fly. We show that the spatial expression patterns for several of these genes scale precisely with embryo size. Discrete positional markers such as the peaks in striped patterns or the boundaries of expression domains have positions along the embryo's major axis proportional to embryo length, accurate to within 1%. Further, the information (in bits) that graded patterns of expression provide about a cell's position can be decomposed into information about fractional or scaled position and information about absolute position or embryo length; all information available is about scaled position, with [Formula: see text]2% error. These findings imply that the underlying genetic network's behavior exhibits scale invariance in a more precise mathematical sense. We argue that models that can explain this scale invariance also have a "zero mode" in the dynamics of gene expression, and this connects to observations on the spatial correlation of fluctuations in expression levels., Competing Interests: Competing interests statement:The authors declare no competing interest.

Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA-modifying enzyme that methylates wobble uridines in a subset of tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Through in vivo studies in Drosophila , we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory. ALKBH8 null animals lack wobble uridine methylation and exhibit reduced protein synthesis in the nervous system, including a specific decrease in selenoprotein levels. Either loss of ALKBH8 or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth in ALKBH8 null animals, confirming oxidative stress as the underlying cause of dysregulation. ALKBH8 null animals also exhibit associative memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA wobble uridine modification in redox homeostasis in the developing nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability., Competing Interests: Competing interests statement:The authors declare no competing interest.

Alzheimer's disease (AD), caused by amyloid beta (Aβ) plaques and Tau tangles, is a neurodegenerative disease characterized by progressive memory impairment and cognitive dysfunction. High-fat diet (HFD), which induces type 2 diabetes, exacerbates Aβ plaque deposition in the brain. To investigate the function of HFD in Tau-mediated AD, we fed an HFD to the Drosophila Tau model and found that HFD aggravates Tau-induced neurological phenotypes. Since microRNAs (miRNAs) are biomarkers for diabetes and AD, we evaluated the expression levels of common miRNAs of HFD and AD in HFD-fed Tau model fly brains. Among the common miRNAs, the expression levels of Let-7 and miR-34 were increased. We found that the inhibition of these miRNAs alleviates Tau-mediated AD phenotypes. Our research provides valuable insights into how HFD accelerates tau toxicity. Additionally, our work highlights the therapeutic potential of targeting Let-7 and miR-34 to develop innovative treatment approaches for AD., Competing Interests: Declaration of competing interest The authors have no financial interests in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The essential Drosophila RNA-binding protein Brain Tumor (Brat) represses specific genes to control embryogenesis and differentiation of stem cells. In the brain, Brat functions as a tumor suppressor that diminishes neural stem cell proliferation while promoting differentiation. Though important Brat-regulated target mRNAs have been identified in these contexts, the full impact of Brat on gene expression remains to be discovered. Here, we identify the network of Brat-regulated mRNAs by performing RNA sequencing (RNA-seq) following depletion of Brat from cultured cells. We identify 158 mRNAs, with high confidence, that are repressed by Brat. De novo motif analysis identified a functionally enriched RNA motif in the 3' untranslated regions (UTRs) of Brat-repressed mRNAs that matches the biochemically defined Brat binding site. Integrative data analysis revealed a high-confidence list of Brat-repressed and Brat-bound mRNAs containing 3'UTR Brat binding motifs. Our RNA-seq and reporter assays show that multiple 3'UTR motifs promote the strength of Brat repression, whereas motifs in the 5'UTR are not functional. Strikingly, we find that Brat regulates expression of glycolytic enzymes and the vacuolar ATPase complex, providing new insight into its role as a tumor suppressor and the coordination of metabolism and intracellular pH., (Published by Oxford University Press on behalf of Nucleic Acids Research 2024.)

Neurexin, a molecule associated with autism spectrum disorders, is thought to function mainly in neurons. Recently, it was reported that Neurexin is also present in muscle, but the role of Neurexin in muscle is still poorly understood. Here, we demonstrate that the overexpression of Neurexin in muscles effectively restored the locomotor function of Drosophila neurexin mutants, while rescuing effects are observed within the nervous. Notably, the defects in muscle structure and function caused by Neurexin deficiency were similar to those caused by mutations in dystroglycan, a gene associated with progressive muscular dystrophy. The absence of Neurexin leads to muscle attachment defects, emphasizing the essential role of Neurexin in muscle integrity. Furthermore, Neurexin deficiency reduces Dystroglycan glycosylation on the cell surface, which is crucial for maintaining proper muscle structure and function. Finally, Neurexin guides Dystroglycan to the glycosyltransferase complex through interactions with Rotated Abdomen, a homolog of mammalian POMT1. Our findings reveal that Neurexin mediates muscle development and function through Dystroglycan glycosylation, suggesting a potential association between autism spectrum disorders and muscular dystrophy., (© 2024. The Author(s).)

Rb/E2f and DREAM complexes play vital roles in regulating cell cycle progression. To date, how they coordinate their functions to regulate cell cycle-dependent gene expression is not clear. Here, we identified a long noncoding RNA (lncRNA), which we named DREAMer , that bridges the interaction between E2f1 and the dREAM complex to regulate endoreplication specifically in Drosophila salivary gland. We show that E2f1 directly stimulates DREAMer expression, whereas DREAMer mediates the repression of e2f1 transcription by modulating the recruitment of the dREAM complex to the e2f1 promoter via a direct interaction with the dREAM component E2f2. The depletion of DREAMer impairs dREAM binding, leading to derepression of e2f1 transcription, which ultimately increases E2f1 activity and promotes the endoreplication. Furthermore, the transcriptomic analysis revealed profound changes in cell cycle-related gene expression in DREAMer KO salivary glands. Together, our findings reveal an lncRNA-mediated link between the dREAM complex and E2f1, which regulates endoreplication during development.

Defects at the neuromuscular junction (NMJ) are among the earliest hallmarks of amyotrophic lateral sclerosis (ALS). According to the "dying-back" hypothesis, NMJ disruption not only precedes but also triggers the subsequent degeneration of motoneurons in both sporadic (sALS) and familial (fALS) ALS. Using human induced pluripotent stem cells (iPSCs), we show that the RNA-binding protein HuD (ELAVL4) contributes to NMJ defects and apoptosis in FUS-ALS. HuD overexpression mimics the severe FUS P525L mutation, while its knockdown rescues the FUS P525L phenotypes. In Drosophila, neuronal overexpression of the HuD ortholog, elav, induces motor dysfunction, and its knockdown improves motor function in a FUS-ALS model. Finally, we report increased HuD levels upon oxidative stress in human motoneurons and in sALS patients with an oxidative stress signature. Based on these findings, we propose that HuD plays a role downstream of FUS mutations in fALS and in sALS related to oxidative stress., (© 2024. The Author(s).)

Notch signalling activity regulates hematopoiesis in Drosophila and vertebrates alike. Parasitoid wasp infestation of Drosophila larvae, however, requires a timely downregulation of Notch activity to allow the formation of encapsulation-active blood cells. Here, we show that the Drosophila CSL transcription factor Suppressor of Hairless [Su(H)] is phosphorylated at Serine 269 in response to parasitoid wasp infestation. As this phosphorylation interferes with the DNA binding of Su(H), it reversibly precludes its activity. Accordingly, phospho-deficient Su(H) S269A mutants are immune-compromised. A screen for kinases involved in Su(H) phosphorylation identified Pkc53E, required for normal hematopoiesis as well as for parasitoid immune response. Genetic and molecular interactions support the specificity of the Su(H)-Pkc53E relationship. Moreover, phorbol ester treatment inhibits Su(H) activity in vivo and in human cell culture. We conclude that Pkc53E targets Su(H) during parasitic wasp infestation, thereby remodelling the blood cell population required for wasp egg encapsulation., Competing Interests: SD, LF, JF, BG, MZ, HM, IP, AP, AN No competing interests declared, (© 2023, Deichsel et al.)

Ubiquitin controls many cellular processes via its posttranslational conjugation onto substrates. Its use is highly variable due to its ability to form poly-ubiquitin chains with various topologies. Among them, linear chains have emerged as important regulators of immune responses and protein degradation. Previous studies in Drosophila melanogaster found that expression of linear poly-ubiquitin that cannot be dismantled into single moieties leads to their ubiquitination and degradation or, alternatively, to their conjugation onto proteins. However, it remains largely unknown which proteins are sensitive to linear poly-ubiquitin. To address this question, here we expanded the toolkit to modulate linear chains and conducted ultra-deep coverage proteomics from flies that express noncleavable, linear chains comprising 2, 4, or 6 moieties. We found that these chains regulate shared and distinct cellular processes in Drosophila by impacting hundreds of proteins, such as the circadian factor Cryptochrome. Our results provide key insight into the proteome subsets and cellular pathways that are influenced by linear poly-ubiquitin chains with distinct lengths and suggest that the ubiquitin system is exceedingly pliable., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The human dual specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) is implicated in the pathology of Down syndrome, microcephaly, and cancer; however the exact mechanism through which it functions is unknown. Here, we have studied the role of the Drosophila ortholog of DYRK1A, Minibrain (Mnb), in brain development and organ growth. The neuroblasts (neural stem cells) that eventually give rise to differentiated neurons in the adult brain are formed from a specialized tissue in the larval optic lobe called the neuroepithelium, in a tightly regulated process. Molecular marker analysis of mnb mutants revealed alterations in the neuroepithelium and neuroblast regions of developing larval brains. Using affinity purification-mass spectrometry (AP-MS), we identified the novel Mnb binding partners Ral interacting protein (Rlip) and RALBP1 associated Eps domain containing (Reps). Rlip and Reps physically and genetically interact with Mnb, and the three proteins may form a ternary complex. Mnb phosphorylates Reps, and human DYRK1A binds to the Reps orthologs REPS1 and REPS2. Mnb also promotes re-localization of Rlip from the nucleus to the cytoplasm in cultured cells. Furthermore, Mnb engages the small GTPase Ras-like protein A (Rala) to regulate brain and wing development. This work uncovers a previously unrecognized role of Mnb in the neuroepithelium and defines the functions of the Mnb/Reps/Rlip/Rala signaling network in organ growth and neurodevelopment., 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 biological rhythms generated by the endogenous circadian clocks across the tree of life regulate numerous behavioral, metabolic, and physiological processes. Although evidence from various studies in Drosophila melanogaster indicates the importance of the core circadian clock genes in the intricate interplay between the circadian clock and metabolism, little is known about the contribution of the circadian photoreceptor/s in this process. The deep brain circadian photoreceptor CRYPTOCHROME (CRY) is essential for resetting the clock in response to light and is also highly expressed in metabolically active tissues in Drosophila. In this study, we sought to explore the possible roles played by CRY in triglyceride (TG) metabolism. We observed that the cry mutant (cry01) flies exhibited increased starvation resistance and TG levels under both 12-hour (h) light:12-h dark cycle (LD) and under constant light compared with the control w1118 flies. We also observed that cry01 flies had significantly increased food intake, glycogen concentrations, and lifespan under LD. In addition, cryptochrome seemed to affect TG levels in adult flies in response to calorie-restricted and high-fat diets. These results suggest a role for the circadian photoreceptor CRY in TG metabolism in Drosophila., 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.)

Ecdysteroids are major hormones in insects and control molting, growth, reproduction, physiology, and behavior. The biosynthesis of ecdysteroids such as 20-hydroxyecdysone (20E) from dietary sterols is well characterized, but ecdysteroid catabolism is poorly understood. Ecdysteroid kinases (EcKs) mediate the reversible phosphorylation of ecdysteroids, which has been implicated in ecdysteroid recycling during embryogenesis and reproduction in various insects. However, to date, only 2 EcK-encoding genes have been identified, in the silkworm Bombyx mori and the mosquito Anopheles gambiae. Previously, we identified 2 ecdysteroid kinase-like (EcKL) genes-Wallflower (Wall) and Pinkman (pkm)-in the model fruit fly Drosophila melanogaster that are orthologs of the ecdysteroid 22-kinase gene BmEc22K. Here, using gene knockdown, knockout, and misexpression, we explore Wall and pkm's possible functions and genetically test the hypothesis that they encode EcKs. Wall and pkm null mutants are viable and fertile, suggesting that they are not essential for development or reproduction, whereas phenotypes arising from RNAi and somatic CRISPR appear to derive from off-target effects or other artifacts. However, misexpression of Wall results in dramatic phenotypes, including developmental arrest, and defects in trachea, cuticle, and pigmentation. Wall misexpression fails to phenocopy irreversible ecdysteroid catabolism through misexpression of Cyp18a1, suggesting that Wall does not directly inactivate 20E. Additionally, Wall misexpression phenotypes are not attenuated in Cyp18a1 mutants, strongly suggesting that Wall is not an ecdysteroid 26-kinase. We hypothesize that the substrate of Wall in this misexpression experiment and possibly generally is an unknown, atypical ecdysteroid that plays essential roles in Drosophila development, and may highlight aspects of insect endocrinology that are as-yet uncharacterized. We also provide preliminary evidence that CG5644 encodes an ecdysteroid 22-kinase conserved across Diptera., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Drosophila Pins (and its mammalian homologue LGN) play a crucial role in the process of asymmetric cell division (ACD). Extensive research has established that Pins/LGN functions as a conformational switch primarily through intramolecular interactions involving the N-terminal TPR repeats and the C-terminal GoLoco (GL) motifs. The GL motifs served as binding sites for the α subunit of the trimeric G protein (Gα), which facilitates the release of the autoinhibited conformation of Pins/LGN. While LGN has been observed to specifically bind to Gα i ·GDP, Pins has been found to associate with both Drosophila Gα i ( d Gα i ) and Gα o ( d Gα o ) isoforms. Moreover, d Gα o was reported to be able to bind Pins in both the GDP- and GTP-bound forms. However, the precise mechanism underlying the influence of d Gα o on the conformational states of Pins remains unclear, despite extensive investigations into the Gα i ·GDP-mediated regulatory conformational changes in LGN/Pins. In this study, we conducted a comprehensive characterization of the interactions between Pins-GL motifs and d Gα o in both GDP- and GTP-loaded forms. Our findings reveal that Pins-GL specifically binds to GDP-loaded d Gα o . Through biochemical characterization, we determined that the intramolecular interactions of Pins primarily involve the entire TPR domain and the GL23 motifs. Additionally, we observed that Pins can simultaneously bind three molecules of d Gα o ·GDP, leading to a partial opening of the autoinhibited conformation. Furthermore, our study presents evidence contrasting with previous observations indicating the absence of binding between d Gα i and Pins-GLs, thus implying the pivotal role of d Gα o as the principal participant in the ACD pathway associated with Pins.

All organisms sense and respond to pathogenic challenge. Tissue-specific responses are required to combat pathogens infecting distinct cell types. Cyclic dinucleotides (CDNs) are produced endogenously downstream of pathogen recognition or by pathogens themselves which bind to STING to activate NF-kB-dependent antimicrobial gene expression programs. It remains unknown whether there are distinct immune responses to CDNs in Drosophila tissues. Here, we investigated tissue specific CDN-STING responses and uncovered differences in gene-induction patterns across tissues that play important roles in viral infections. Using tissue-and cell-specific genetic studies we found that dSTING in the fat body controls CDN-induced expression of dSTING-regulated gene 1 (Srg1) but not dSTING-regulated gene 2 (Srg2) or 3 (Srg3). In contrast, the gastrointestinal tract largely controls expression of Srg2 and Srg3. We found that Srg3 is antiviral against the natural fly pathogen Drosophila C virus and the human arthropod-borne Rift Valley Fever virus (RVFV), but not other arthropod-borne viruses including Sindbis virus and dengue virus. Furthermore, we found that Srg3 has an important role in controlling RVFV infection of the ovary which has important implications in understanding vertical transmission of viruses and RVFV in mosquitoes. Overall, our study underscores the importance of tissue-specific responses in antiviral immunity and highlights the complex tissue regulation of the CDN-STING pathway., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Segrist 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.)

Complex behaviors arise from neural circuits that assemble from diverse cell types. Sleep is a conserved behavior essential for survival, yet little is known about how the nervous system generates neuron types of a sleep-wake circuit. Here, we focus on the specification of Drosophila 23E10-labeled dorsal fan-shaped body (dFB) long-field tangential input neurons that project to the dorsal layers of the fan-shaped body neuropil in the central complex. We use lineage analysis and genetic birth dating to identify two bilateral type II neural stem cells (NSCs) that generate 23E10 dFB neurons. We show that adult 23E10 dFB neurons express ecdysone-induced protein 93 (E93) and that loss of ecdysone signaling or E93 in type II NSCs results in their misspecification. Finally, we show that E93 knockdown in type II NSCs impairs adult sleep behavior. Our results provide insight into how extrinsic hormonal signaling acts on NSCs to generate the neuronal diversity required for adult sleep behavior. These findings suggest that some adult sleep disorders might derive from defects in stem cell-specific temporal neurodevelopmental programs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Drosophila nephrocytes are specialised cells that share critical functional, morphological, and molecular features with mammalian podocytes. Accordingly, nephrocytes represent a preferred invertebrate model for human glomerular disease. Here, we established a method for cell-specific isolation of the two types of Drosophila nephrocytes, garland and pericardial cells, from animals of different developmental stages and ages. Mass spectrometry-based proteomics and RNA-Seq-based transcriptomics were applied to characterise the proteome and transcriptome of the respective cells in an integrated and complementary manner. We observed characteristic changes in the proteome and transcriptome due to cellular ageing. Furthermore, functional enrichment analyses suggested that larval and adult nephrocytes, as well as garland and pericardial nephrocytes, fulfil distinct physiological functions. In addition, the pericardial nephrocytes were characterised by transcriptomic and proteomic profiles suggesting an atypical energy metabolism with very low oxidative phosphorylation rates. Moreover, the nephrocytes displayed typical signatures of extensive immune signalling and showed an active antimicrobial response to an infection. Factor-specific comparisons identified novel candidate proteins either expressed and secreted by the nephrocytes or sequestered by them. The data generated in this study represent a valuable basis for a more specific application of the Drosophila model in analysing renal cell function in health and disease., (© 2024. The Author(s).)

Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain tumor found in adult humans with a poor prognosis and average survival of 14-15 months. In order to have a comprehensive understanding of proteome and identify novel therapeutic targets, this study focused mainly on the differentially abundant proteins (DAPs) of Ras V12 -induced GBM. Ras V12 is a constitutively active Ras mutant form essential for tumor progression by continuously activating signaling pathways leading to uncontrolled tumor growth. This study used a transgenic Drosophila model with Ras V12 overexpression using the repo-GAL4 driver line, specifically in glial cells, to study GBM. The high-resolution mass spectrometry (HRMS)-based proteomic analysis of the GBM larval central nervous system identified three novel DAPs specific to mitochondria. These DAPs, probable maleylacetoacetate isomerase 2 (Q9VHD2), bifunctional methylene tetrahydrofolate dehydrogenase (Q04448), and glutamine synthetase1 (P20477), identified through HRMS were further validated by qRT-PCR. The protein-protein interaction analysis revealed interactions between Ras V12 and DAPs, with functional links to mitochondrial dynamics regulators such as Drp1, Marf, Parkin, and HtrA2. Notably, altered expressions of Q9VHD2, P20477, and Q04448 were observed during GBM progression, which offers new insights into the involvement of mitochondrial dynamic regulators in Ras V12 -induced GBM pathophysiology.

Chemical detection is vital for animal survival, aiding in avoiding toxins and selecting nutritious foods. While Drosophila larvae exhibit appetitive feeding behavior toward ribose, an important sugar for RNA, nucleotide, and nucleoside synthesis, how adult Drosophila perceives ribose remains unclear. Through behavioral and electrophysiological investigations, we unexpectedly discovered that adult flies actively avoid ribose. Our external electrophysiological analysis revealed that ribose is detected through bitter-sensing gustatory receptor neurons in S-type sensilla, suggesting its perception as a bitter compound. Additionally, we identify painless as crucial for both ribose aversion and the neuronal response to ribose., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Wingless (Wg)/Wnt signaling plays a critical role in both development and adult tissue homeostasis. In the Drosophila larval wing disc epithelium, the orderly delivery of Wg/Wnt to the apical and basal cell surfaces is essential for wing development. Here, we identified Ehbp1 as the switch that dictates the direction of Wg/Wnt polarized intracellular transport: the Adaptor Protein complex 1 (AP-1) delivers Wg/Wnt to the basolateral cell surface, and its sequestration by Ehbp1 redirects Wg/Wnt for apical delivery. Genetic analyses showed that Ehbp1 specifically regulates the polarized distribution of Wg/Wnt, a process that depends on the dedicated Wg/Wnt cargo receptor Wntless. Mechanistically, Ehbp1 competes with Wntless for AP-1 binding, thereby preventing the unregulated basolateral Wg/Wnt transport. Reducing Ehbp1 expression, or removing the coiled-coil motifs within its bMERB domain, leads to basolateral Wg/Wnt accumulation. Importantly, the regulation of polarized Wnt delivery by EHBP1 is conserved in vertebrates. The generality of this switch mechanism for regulating intracellular transport remains to be determined in future studies., (© 2024. The Author(s).)

Defining the time of action for morphogens requires tools capable of temporally controlled perturbations. To study how the transcription factor Dorsal affects patterning of the Drosophila embryonic dorsal-ventral axis, we used two light-inducible tags that trigger either nuclear export or degradation of Dorsal under blue light. Nuclear export of Dorsal leads to loss of the high-threshold, ventrally expressed target gene snail (sna), while the low-threshold, laterally expressed target gene short-gastrulation (sog) is retained. In contrast, degradation of Dorsal results in retention of sna, loss of sog, and lower nuclear levels compared to when Dorsal is exported from the nucleus. To understand why nuclear export causes loss of sna but degradation does not, we investigated Dorsal kinetics using photobleaching and found that it rapidly re-enters the nucleus even under blue-light conditions favoring export. The associated kinetics of Dorsal being rapidly imported and exported continuously are likely responsible for loss of sna but, alternatively, can support sog. Collectively, our results indicate that this dynamic patterning process is influenced by both Dorsal concentration and nuclear retention., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Drosophila NF-κB transcription factor Dif has been well known for its function in innate immunity, and recent study also reveals its role in neuronal cells. However, the underlying mechanisms of Dif in the brain remain elusive. In this study, we aim to investigate the function of Dif in Drosophila brain development and how Dif regulates structure and plasticity of the brain to affect aging and behaviors. Based on the analysis of differentially expressed genes, we identified key genes associated with cell division, development and aging in the brain of Dif 1 loss of function mutant. In Dif 1 larvae, we found that the metamorphosis and brain development were delayed, and cell division was decreased. In Dif 1 adults, the number of neuron cells was reduced in the brain, the lifespan and locomotor activity were decreased, protein markers associated with aging-related neurodegenerative diseases in the brain were altered in abundance or activity. Our results indicated that Dif plays a crucial role in brain plasticity and neurogenesis, dysfunction of Dif delays larval brain development and impacts proliferation of neuronal cells, resulting in aging adult brain by regulating expression of key genes in multiple signaling pathways involved in cell division, neurogenesis and aging., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homologue of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Because MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process., Competing Interests: Conflicts of interest: The authors declare no financial conflict of interest.

Lateral inhibition mediates alternative cell fate decision and produces regular cell fate patterns with fate symmetry breaking (SB) relying on the amplification of small stochastic differences in Notch activity via an intercellular negative-feedback loop. Here, we used quantitative live imaging of endogenous Scute (Sc), a proneural factor, and of a Notch activity reporter to study the emergence of sensory organ precursor cells in the pupal abdomen of Drosophila. SB was observed at low Sc levels and was not preceded by a phase of intermediate Sc expression and Notch activity. Thus, mutual inhibition may only be transient in this context. In support of the intercellular feedback loop model, cell-to-cell variations in Sc levels promoted fate divergence. The size of the apical area of competing cells did not detectably bias this fate choice. Surprisingly, cells that were in direct contact at the time of SB could adopt the sensory organ precursor cell fate, albeit at low frequency (10%). These lateral inhibition defects were corrected by cellular rearrangements, not cell fate change, highlighting the role of cell-cell intercalation in pattern refinement., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Ribonucleoprotein (RNP) granules are membraneless condensates that organize the intracellular space by compartmentalization of specific RNAs and proteins. Studies have shown that RNA tunes the phase behaviour of RNA-binding proteins, but the role of intermolecular RNA-RNA interactions in RNP granules in vivo remains less explored. Here we determine the role of a sequence-specific RNA-RNA kissing-loop interaction in assembly of mesoscale oskar RNP granules in the female Drosophila germline. We show that a two-nucleotide mutation that disrupts kissing-loop-mediated oskar messenger RNA dimerization impairs condensate formation in vitro and oskar granule assembly in the developing oocyte, leading to defective posterior localization of the RNA and abrogation of oskar-associated processing bodies upon nutritional stress. This specific trans RNA-RNA interaction acts synergistically with the scaffold RNA-binding protein, Bruno, in driving condensate assembly. Our study highlights the architectural contribution of an mRNA and its specific secondary structure and tertiary interactions to the formation of an RNP granule that is essential for embryonic development., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Sex pheromones play a crucial role in species recognition and reproductive isolation. Despite being largely species-specific in drosophilids, the mechanisms underlying pheromone detection, production, and their influence on mating behavior remain poorly understood. Here, we compare the chemical profiles of Drosophila bipectinata and D. melanogaster, the mating behaviors in both species, as well as the tuning properties of Or67d receptors, which are expressed by neurons in antennal trichoid sensilla at1. Through single sensillum recordings, we demonstrate that the D. bipectinata Or67d-ortholog exhibits similar sensitivity to cis-vaccenyl acetate (cVA) as compared to D. melanogaster but in addition also responds uniquely to (Z)-11-eicosen-1-yl-acetate (Z11-20:Ac), a compound exclusively produced by D. bipectinata males. Through courtship behavior assays we found that, surprisingly, perfuming the flies with Z11-20:Ac did not reveal any aphrodisiacal or anti-aphrodisiacal effects in mating assays. The behavioral relevance of at1 neuron channels in D. bipectinata compared to D. melanogaster seems to be restricted to its formerly shown function as an aggregation pheromone. Moreover, the non-specific compound cVA affected copulation negatively in D. bipectinata and could potentially act as a premating isolation barrier. As both ligands of Or67d seem to govern different behaviors in D. bipectinata, additional neurons detecting at least one of those compounds might be involved. These results underscore the complexity of chemical signaling in species recognition and raise intriguing questions about the evolutionary implications of pheromone detection pathways in Drosophila species., (© 2024. The Author(s).)

Heart failure (HF) is highly prevalent. Mechanisms underlying HF remain incompletely understood. Splicing factors (SF), which control pre-mRNA alternative splicing, regulate cardiac structure and function. This study investigated regulation of the splicing factor heterogeneous nuclear ribonucleoprotein-L (hnRNPL) in the failing heart. hnRNPL protein increased in left ventricular tissue from mice with transaortic constriction-induced HF and from HF patients. In left ventricular tissue, hnRNPL was detected predominantly in nuclei. Knockdown of the hnRNPL homolog Smooth in Drosophila induced cardiomyopathy. Computational analysis of predicted mouse and human hnRNPL binding sites suggested hnRNPL-mediated alternative splicing of tropomyosin, which was confirmed in C2C12 myoblasts. These findings identify hnRNPL as a sensor of cardiac dysfunction and suggest that disturbances of hnRNPL affect alternative splicing in HF., (© 2024 Federation of European Biochemical Societies.)

Objectives: The mitochondrial enzyme L-2-hydroxyglutarate dehydrogenase (L2HGDH) regulates the abundance of L-2-hydroxyglutarate (L-2HG), a potent signaling metabolite capable of influencing chromatin architecture, mitochondrial metabolism, and cell fate decisions. Loss of L2hgdh activity in humans induces ectopic L-2HG accumulation, resulting in neurodevelopmental defects, altered immune cell function, and enhanced growth of clear cell renal cell carcinomas. To better understand the molecular mechanisms that underlie these disease pathologies, we used the fruit fly Drosophila melanogaster to investigate the endogenous functions of L2hgdh., Methods: L2hgdh mutant adult male flies were analyzed under normoxic and hypoxic conditions using a combination of semi-targeted metabolomics and RNA-seq. These multi-omic analyses were complemented by tissue-specific genetic studies that examined the effects of L2hgdh mutations on the Drosophila renal system (Malpighian tubules; MTs)., Results: Our studies revealed that while L2hgdh is not essential for growth or viability under standard culture conditions, L2hgdh mutants are hypersensitive to hypoxia and expire during the reoxygenation phase with severe disruptions of mitochondrial metabolism. Moreover, we find that the fly renal system is a key site of L2hgdh activity, as L2hgdh mutants that express a rescuing transgene within the MTs survive hypoxia treatment and exhibit normal levels of mitochondrial metabolites. We also demonstrate that even under normoxic conditions, L2hgdh mutant MTs experience significant metabolic stress and are sensitized to aberrant growth upon Egfr activation., Conclusions: These findings present a model in which renal L2hgdh activity limits systemic L-2HG accumulation, thus indirectly regulating the balance between glycolytic and mitochondrial metabolism, enabling successful recovery from hypoxia exposure, and ensuring renal tissue integrity., 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 Author(s). Published by Elsevier GmbH.. All rights reserved.)

ɣ-aminobutyric acid (GABA) is a four‑carbon amino acid acting as the main inhibitory transmitter in the invertebrate and vertebrate nervous systems. The metabolism of GABA is well compartmentalized in the cell and the uptake of cytosolic GABA into the mitochondrial matrix is required for its degradation. A previous study carried out in the fruit fly Drosophila melanogaster indicated that the mitochondrial aspartate/glutamate carrier (AGC) is responsible for mitochondrial GABA accumulation. Here, we investigated the transport of GABA catalysed by the human and D. melanogaster AGC proteins through a well-established method for the study of the substrate specificity and the kinetic parameters of the mitochondrial carriers. In this experimental system, the D. melanogaster spliced AGC isoforms (Aralar1-PA and Aralar1-PE) and the human AGC isoforms (AGC1/aralar1 and AGC2/citrin) are unable to transport GABA both in homo- and in hetero-exchange with either glutamate or aspartate, i.e. the canonical substrates of AGC. Moreover, GABA has no inhibitory effect on the exchange activities catalysed by the investigated AGCs. Our data demonstrate that AGC does not transport GABA and the molecular identity of the GABA transporter in human and D. melanogaster mitochondria remains unknown., 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 B.V. All rights reserved.)

We have analyzed the organization of the microtubule system in photoreceptor cells and pigment cells within the adult Drosophila compound eye. Immunofluorescence localization of tubulin and of Short stop, a spectraplakin that has been reported to be involved in the anchorage of microtubule minus ends at the membrane, suggests the presence of non-centrosomal microtubule-organizing centers at the distal tip of the visual cells. Ultrastructural analyses confirm that microtubules emanate from membrane-associated plaques at the site of contact with cone cells and that all microtubules are aligned in distal-proximal direction within the photoreceptor cells. Determination of microtubule polarities demonstrated that about 95% of the microtubules in photoreceptor cells are oriented with their plus end in the direction of the synapse. Pigment cells in the eye contain only microtubules aligned in distal-proximal direction, with their plus end pointing towards the retinal floor. There, two populations of microtubules can be distinguished, single microtubules and bundled microtubules, the latter associated with actin filaments. Whereas microtubules in both photoreceptor cells and pigment cells are acetylated and mono/bi-glutamylated on α-tubulin, bundled microtubules in pigment cells are apparently also mono/bi-glutamylated on β-tubulin, providing the possibility of binding different microtubule-associated proteins., (© 2024. The Author(s).)

Germ cells mutant for bam or bgcn are locked in a germline stem cell (GSC)-like state, leading to tumor-like overgrowth in Drosophila ovaries. Our previous studies have demonstrated that germline overgrowth in bam mutants can be suppressed by defects in the miRNA pathway but enhanced by a null mutation in hippo. However, the genetic epistasis between the miRNA and Hippo pathways still remains unknown. Here, we determined that the miRNA pathway acts downstream of the Hippo pathway in regulating this process. Germ cells mutant for bam or bgcn and defective in both pathways divide very slowly, phenocopying those defective only in the miRNA pathway. In addition, we found that Yki, a key oncoprotein in the Hippo pathway, promotes the growth of both wild-type germ cells and bam mutant GSC-like cells. Like wild-type GSCs, bam mutant GSC-like cells predominantly stay in the G2 phase. Remarkably, many of those defective in the miRNA pathway are arrested before entering this phase. Furthermore, our studies identified bantam as a critical miRNA promoting germline overgrowth in bam or bgcn mutants. Taken together, these findings establish a genetic circuitry controlling Drosophila female germline overgrowth., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Stem cell loss in aging and disease is associated with nuclear deformation. Yet, how nuclear shape influences stem cell homeostasis is poorly understood. We investigated this connection using Drosophila germline stem cells, as survival of these stem cells is compromised by dysfunction of the nuclear lamina, the extensive protein network that lines the inner nuclear membrane and gives shape to the nucleus. To induce nuclear distortion in germline stem cells, we used the GAL4-UAS system to increase expression of the permanently farnesylated nuclear lamina protein, Kugelkern, a rate limiting factor for nuclear growth. We show that elevated Kugelkern levels cause severe nuclear distortion in germline stem cells, including extensive thickening and lobulation of the nuclear envelope and nuclear lamina, as well as alteration of internal nuclear compartments. Despite these changes, germline stem cell number, proliferation, and female fertility are preserved, even as females age. Collectively, these data demonstrate that disruption of nuclear architecture does not cause a failure of germline stem cell survival or homeostasis, revealing that nuclear deformation does not invariably promote stem cell loss., Competing Interests: Declaration of competing interest The authors have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The minute wasp Habrobracon hebetor venom (HH venom) is a potent cocktail of toxins that paralyzes the victim's muscles and suppresses humoral and cellular immunity. This study examined the effect of HH venom on specific biochemical, physiological, and ultrastructural characteristics of the thoracic and nervous (CNS) tissues of Drosophila melanogaster under in vitro conditions. Venom treatment modulated the activities of superoxide dismutase (SOD) and catalase (CAT), endogenous Drome-AKH level, and affected the relative viability of the cells. Additionally, it reduced the expression of genes related to the immune system in the CNS, including Keap1, Relish, Nox, Eiger, Gadd45, and Domeless, as well as in the thoracic muscles, except for Nox. Besides, venom treatment led to deteriorative changes in the ultrastructure of muscle cells, particularly affecting the mitochondria. When venom and Drosophila melanogaster-adipokinetic hormone (Drome-AKH) were applied together, the effects of the venom alone were often modulated. The harmful effect of the venom on SOD activity was relatively reduced and the activity returned to a level similar to that of the control. In the CNS, the simultaneous application of venom and hormones abolished the suppression of previously reported immune-related genes (except for Gadd45), whereas in the muscles, this was only true for Eiger. Additionally, Drome-AKH restored cell structure to a level comparable to that of the control and lessened the harmful effects of HH venom on muscle mitochondria. These findings suggest a general body response of D. melanogaster to HH venom and a partial defensive role of Drome-AKH in this process., 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 Inc. All rights reserved.)

Maternal nutrition, including the availability of micronutrients such as zinc, influences the health of the offspring. Using Drosophila melanogaster, we studied the impact of zinc deficiency on development and reproduction, as well as the effects of maternal zinc status on the offspring's expression of zinc transporters across F1 to F3 generations. Zinc deficiency was induced by adding N,N,N',N'-Tetrakis (2-pyridylmethyl)-ethylenediamine (TPEN) to the diet on which the eggs representing the F0 generation flies were laid. Then, virgin F0 females were mated with control males to produce F1, and subsequently thereafter to generate F2 and F3. Offspring from F1 to F3 were analyzed for body zinc status and zinc transporter mRNA levels. We found that zinc deficiency significantly (p < 0.05) impaired the development of flies, as evidenced by a reduced eclosion rate of zinc-deficient flies. Similarly, zinc deficiency significantly (p < 0.05) reduced the egg-laying rate in F0 flies, highlighting its impact on reproductive functions. Also, zinc levels were consistently lower in the F0 and persisted in subsequent generations for both male and female offspring, indicating transgenerational alterations in zinc status. Furthermore, gene expression analysis revealed significant (p < 0.05) variations in the mRNA levels of dZip42C.1, dZnT63C, dZip71B, and dZnT35C genes across different generations and between male and female offspring. These findings indicate gender-specific dynamics of gene expression in response to zinc deficiency, suggesting potential regulatory mechanisms involved in maintaining zinc homeostasis. Our study emphasizes the detrimental effects of zinc deficiency on development and reproduction in Drosophila and highlights potential implications for offspring and human health., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Insect prophenoloxidases (PPOs) are important immunity proteins for defending against the invading pathogens and parasites. As a Type-Ⅲ copper-containing proteins, unlike Homo sapiens tyrosinases, the insect PPOs and most bacterial tyrosinases contain no signal peptides for unknown reason, however they can still be released. To this end, we fused different signal peptides to Drosophila melanogaster PPOs for in vitro and in vivo expression, respectively. We demonstrate that an artificial signal peptide can help PPO secretion in vitro. The secreted PPO appeared larger than wild-type PPO on molecular weight sizes due to glycosylation when expressed in S2 cells. Two asparagine residues for potential glycosylation in PPO1 were identified when a signal peptide was fused. After purification, the glycosylated PPO1 lost zymogen activity. When PPO1 containing a signal peptide was over-expressed in Drosophila larvae, the glycosylation and secretion of PPO1 was detected in vivo. Unlike insect PPO, human tyrosinase needs a signal peptide for protein expression and maintaining enzyme activity. An artificial signal peptide fused to bacterial tyrosinase had no influence on the protein expression and enzyme activity. These Type-Ⅲ copper-containing proteins from different organisms may evolve to perform their specific functions. Intriguingly, our study revealed that the addition of calcium inhibits PPO secretion from the transiently cultured larval hindguts in vitro, indicating that the calcium concentration may regulate PPO secretion. Taken together, insect PPOs can maintain enzyme activities without any signal peptide., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The Snail superfamily of transcription factors plays a crucial role in metazoan development; one of the most important vertebrate members of this family is Snai1 which is orthologous to the Drosophila melanogaster esg gene. This review offers a comprehensive examination of the roles of the esg gene in Drosophila development, covering its expression pattern and downstream targets, and draws parallels between the vertebrate Snai1 family proteins on controlling the epithelial-to-mesenchymal transition and esg. This gene regulates stemness, ploidy, and pluripontency. esg is expressed in various tissues during development, including the gut, imaginal discs, and neuroblasts. The functions of the esg include the suppression of differentiation in intestinal stem cells and the preservation of diploidy in imaginal cells. In the nervous system development, esg expression also inhibits neuroblast differentiation, thus regulating the number of neurons and the moment in development of neuronal differentiation. Loss of esg function results in diverse developmental defects, including defects in intestinal stem cell maintenance and differentiation, and alters imaginal disc and nervous system development. Expression levels of esg also play a role in regulating longevity and metabolism in adult stages. This review provides an overview of the current understanding of esg's developmental role, emphasizing cellular and tissue effects that arise from its loss of function. The insights gained may contribute to a better understanding of evolutionary conserved developmental mechanisms and certain metabolic diseases., (© 2024 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Switching from mitotic spermatogonia to meiotic spermatocytes is critical to producing haploid sperms during male germ cell differentiation. However, the underlying mechanisms of this switch remain largely unexplored. In Drosophila melanogaster, the gene RpL38 encodes the ribosomal protein L38, one component of the 60S subunit of ribosomes. We found that its depletion in spermatogonia severely diminished the production of mature sperms and thus led to the infertility of male flies. By examining the germ cell differentiation in testes, we found that RpL38-knockdown blocked the transition from spermatogonia to spermatocytes and accumulated spermatogonia in the testis. To understand the intrinsic reason for this blockage, we conducted proteomic analysis for these spermatogonia populations. Differing from the control spermatogonia, the accumulated spermatogonia in RpL38-knockdown testes already expressed many spermatocyte markers but lacked many meiosis-related proteins, suggesting that spermatogonia need to prepare some important proteins for meiosis to complete their switch into spermatocytes. Mechanistically, we found that the expression of bag of marbles (bam), a crucial determinant in the transition from spermatogonia to spermatocytes, was inhibited at both the mRNA and protein levels upon RpL38 depletion. We also confirmed that the bam loss phenocopied RpL38 RNAi in the testis phenotype and transcriptomic profiling. Strikingly, overexpressing bam was able to fully rescue the testis abnormality and infertility of RpL38-knockdown flies, indicating that bam is the key effector downstream of RpL38 to regulate spermatogonia differentiation. Overall, our data suggested that germ cells start to prepare meiosis-related proteins as early as the spermatogonial stage, and RpL38 in spermatogonia is required to regulate their transition toward spermatocytes in a bam-dependent manner, providing new knowledge for our understanding of the transition process from spermatogonia to spermatocytes in Drosophila spermatogenesis., (© 2024. Science China Press.)