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In most Eukaryota, telomeres are protected by the CST complex, composed of CTC1, STN1 and TEN1. In Drosophila, instead, another complex is present, composed of Modigliani, Tea and Verrocchio. We performed a search for STN1 orthologs in Arthropoda, in order to verify if Verrocchio can be considered as such. We found that STN1 in Arthropoda is shorter than in other Metazoa and shares the same architecture with Verrocchio. Despite high sequence divergence between human and Drosophila, we have discovered that Verrocchio is an ortholog of STN1., Competing Interests: Declarations. Ethical approval: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The quick-to-court (qtc) gene is expressed in both males and females but affects only the mating behavior of males, probably due to the different composition of isoforms between the sexes. We tested this hypothesis and examined the sex-specific expression of qtc transcripts in the tissues of male and female Oregon-R flies. It was found that some qtc transcripts, such as qtc-RM and qtc-RN, are testis-specific, while others like qtc-RH are found in ovaries but absent in testes. No sex-specific transcripts were identified in the brain, suggesting further investigation into specific brain structures may be needed. There is likely a complex regulation of qtc gene expression, which is potentially influenced by various factors in different tissues., 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.)

Balanced self-renewal and differentiation of stem cells are crucial for maintaining tissue homeostasis, but the underlying mechanisms of this process remain poorly understood. Here, from an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), we identify a factor, Pax, which is orthologous to mammalian PXN, coordinates the proliferation and differentiation of ISCs during both normal homeostasis and injury-induced midgut regeneration in Drosophila. Loss of Pax promotes ISC proliferation while suppressing its differentiation into absorptive enterocytes (ECs). Mechanistically, our findings demonstrate that Pax is a conserved target gene of the Hippo signaling pathway in both Drosophila and mammals. Subsequent investigations have revealed Pax interacts with Yki and enhances its cytoplasmic localization, thereby establishing a feedback regulatory mechanism that attenuates Yki activity and ultimately inhibits ISCs proliferation. Additionally, Pax induces the differentiation of ISCs into ECs by activating Notch expression, thus facilitating the differentiation process. Overall, our study highlights Pax as a pivotal component of the Hippo and Notch pathways in regulating midgut homeostasis, shedding light on this growth-related pathway in tissue maintenance and intestinal function., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

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.)

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.

Unlike most species that use telomerase for telomere maintenance, many dipterans, including Drosophila , rely on three telomere-specific retrotransposons (TRs)- HeT-A , TART , and TAHRE -to form tandem repeats at chromosome ends. Although TR transcription is crucial in their life cycle, its regulation remains poorly understood. This study identifies the Mediator complex, E2F1-Dp, and Scalloped/dTEAD as key regulators of TR transcription. Reducing the activity of the Mediator or Sd/dTEAD increases TR expression and telomere length, while overexpressing E2F1-Dp or depleting Rbf1 stimulates TR transcription. The Mediator and Sd/dTEAD regulate this process through E2F1-Dp. CUT&RUN (Cleavage under targets and release using nuclease) analysis shows direct binding of CDK8, Dp, and Sd/dTEAD to telomeric repeats, with motif enrichment revealing E2F- and TEAD-binding sites. These findings uncover the Mediator complex's role in controlling TR transcription and telomere length through E2F1-Dp and Sd, coupling the transcriptional regulation of the TR life cycle with host cell-cycle machinery to protect chromosome ends in Drosophila .

We modelled and calibrated the distributions of the seven-stripe patterns of Even-skipped (Eve) and Fushi-tarazu (Ftz) pair-rule proteins along the anteroposterior axis of the Drosphila embryo, established during early development. We have identified the putative repressive combinations for five Eve enhancers, and we have explored the relationship between Eve and Ftz for complementary patterns. The regulators of Eve and Ftz are stripe-specific DNA enhancers with embryo position-dependent activation rates and are regulated by the gap family of proteins. We achieved remarkable data matching of the Eve stripe pattern, and the calibrated model reproduces gap gene mutation experiments. Extended work inferring the Wingless (Wg) fourteen stripe pattern from Eve and Ftz enhancers have been proposed, clarifying the hierarchical structure of Drosphila's genetic expression network during early development., Competing Interests: Declaration of competing interest The authors declare no conflict or competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. 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.)

Small Open Reading Frames (smORFs) of less than 100 codons remain mostly uncharacterised. About a thousand smORFs per genome encode peptides and microproteins about 70-80 aa long, often containing recognisable protein structures and markers of translation, and these are referred to as short Coding Sequences (sCDSs). The characterisation of individual sCDSs has provided examples of smORFs' function and conservation, but we cannot infer the functionality of all other metazoan smORFs from these. sCDS function has been characterised at a genome-wide scale in yeast and bacteria, showing that hundreds can produce a phenotype, but attempts in metazoans have been less successful. Either most sCDSs are not functional, or classic experimental techniques do not work with smORFs due to their shortness. Here, we combine extensive proteomics with bioinformatics and genetics in order to detect and corroborate sCDS function in Drosophila . Our studies nearly double the number of sCDSs with detected peptides and microproteins and an experimentally corroborated function. Finally, we observe a correlation between proven sCDS protein function and bioinformatic markers such as conservation and GC content. Our results support that sCDSs peptides and microproteins act as membrane-related regulators of canonical proteins, regulators whose functions are best understood at the cellular level, and whose mutants produce little, if any, overt morphological phenotypes.

Live imaging of transcription in the Drosophila embryo using the MS2 or PP7 systems is transforming our understanding of transcriptional regulation. However, insertion of MS2/PP7 stem-loops into endogenous genes requires laborious CRISPR genome editing. Here, we exploit the previously described Minos-mediated integration cassette (MiMIC) transposon system in Drosophila to establish a method for simply and rapidly inserting MS2/PP7 cassettes into any of the thousands of genes carrying a MiMIC insertion. In addition to generating a variety of stem-loop donor fly stocks, we have made new stocks expressing the complementary coat proteins fused to different fluorescent proteins. We show the utility of this MiMIC-based approach by MS2/PP7 tagging of endogenous genes and the long non-coding RNA roX1, then imaging their transcription in living embryos. We also present live transcription data from larval brains, the wing disc and ovary, thereby extending the tissues that can be studied using the MS2/PP7 system. Overall, this first high-throughput method for tagging mRNAs in Drosophila will facilitate the study of transcription dynamics of thousands of endogenous genes in a range of Drosophila tissues., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The ribosomal RNA (rRNA) is one of the most heavily modified RNA species in nature. Although we have advanced knowledge of the sites, functions, and the enzymology of many of the rRNA modifications from all kingdoms of life, we lack basic understanding of many of those that are not universally present. A single N 3 modified uridine base (m 3 U) was identified to be present on the 28 S rRNA from humans and frogs but absent in bacteria or yeast. Here, we show that the equivalent m 3 U is present in Drosophila and that the Ptch/CG12128 enzyme and its human homolog SPOUT1 are both necessary and sufficient for carrying out the modification. The Ptch-modified U is at a functional center of the large ribosomal subunit, and, consistently, ptch -mutant cells suffer loss of ribosomal functions. SPOUT1, suggested to be the most druggable RNA methyltransferases in humans, represents a unique target where ribosomal functions could be specifically compromised in cancer cells.

Identifying a suitable mating partner is an ancient and critical biological problem. How a fruit fly distinguishes a fly of the same species from flies of innumerable related species remains unclear. We analyze the Ir52 receptors, expressed in taste neurons on the fly legs and encoded by a cluster of genes. We find that the cluster shows dynamic evolution, rapidly expanding and contracting over evolutionary time. We develop a novel in vivo expression system and find that Ir52 receptors respond differently to pheromone extracts of different fly species. The receptors are activated by some compounds and inhibited by others, with different receptors showing distinct response profiles. Circuit mapping shows that Ir52 neurons are pre-synaptic to sexually dimorphic neurons that overlap with neurons acting in courtship behavior. Our results support a model in which Ir52 receptors detect information about the species of a potential mating partner., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

To identify genes required for brain growth, we took an RNAi knockdown reverse genetic approach in Drosophila . One potential candidate isolated from this effort is the anti-lipogenic gene adipose ( adp ). Adp has an established role in the negative regulation of lipogenesis in the fat body of the fly and adipose tissue in mammals. While fat is key to proper development in general, adp has not been investigated during brain development. Here, we found that RNAi knockdown of adp in neuronal stem cells and neurons results in reduced brain lobe volume and sought to replicate this with a mutant fly. We generated a novel adp mutant that acts as a loss-of-function mutant based on buoyancy assay results. We found that despite a change in fat content in the body overall and a decrease in the number of larger (>5 µm) brain lipid droplets, there was no change in the brain lobe volume of mutant larvae. Overall, our work describes a novel adp mutant that can functionally replace the long-standing adp 60 mutant and shows that the adp gene has no obvious involvement in brain growth.

Although A-to-I RNA editing leads to similar effects to A-to-G DNA mutation, nonsynonymous RNA editing (recoding) is believed to confer its adaptiveness by 'epigenetically' regulating proteomic diversity in a temporospatial manner, avoiding the pleiotropic effect of genomic mutations. Recent discoveries on the evolutionary trajectory of Ser>Gly auto-editing site in insect Adar gene demonstrated a selective advantage to having an editable codon compared to uneditable ones. However, apart from pure observations, quantitative approaches for justifying the adaptiveness of individual RNA editing sites are still lacking. We performed a comparative genomic analysis on 113 Diptera species, focusing on the Adar Ser>Gly auto-recoding site in Drosophila . We only found one species having a derived Gly at the corresponding site, and this occurrence was significantly lower than genome-wide random expectation. This suggests that the Adar Ser>Gly site is unlikely to be genomically replaced with G during evolution, and thus indicating the advantage of editable status over hardwired genomic alleles. Similar trends were observed for the conserved Ile>Met recoding in gene Syt1 . In the light of evolution, we established a comparative genomic approach for quantitatively justifying the adaptiveness of individual editing sites. Priority should be given to such adaptive editing sites in future functional studies.

The Mediator complex plays a pivotal role in facilitating RNA polymerase II-dependent transcription in eukaryotes. Within this complex, the CDK8 kinase module (CKM), comprising CDK8, Cyclin C (CycC), Med12 and Med13, serves as a dissociable subcomplex that modulates the activity of the small Mediator complex. Genetic studies in Drosophila have revealed distinct phenotypes associated with mutations in CKM subunits, but the underlying mechanisms have remained unclear. Using Drosophila as a model, we generated transgenic strains to deplete individually or simultaneously the four CKM subunits in all possible combinations, uncovering unique phenotypes in the eyes and wings. Depletion of CDK8-CycC enhanced E2F1 target gene expression and promoted cell-cycle progression, whereas Med12-Med13 depletion had no significant impact on these processes. Instead, depleting Med12-Med13 altered the expression of ribosomal protein genes and fibrillarin, and reduced nascent protein synthesis, indicating a severe reduction in ribosome biogenesis and cellular growth compared to the loss of CDK8-CycC. These findings reveal distinct in vivo roles for CKM subunits, with Med12-Med13 disruption having a more pronounced effect on ribosome biogenesis and protein synthesis than CDK8-CycC loss., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Muscle development and diversity require a large number of spatially and temporally regulated events controlled by transcription factors (TFs). Drosophila has long stood as a model to study myogenesis due to the highly conserved key TFs involved at all stages of muscle development. While many studies focused on the diversification of Drosophila larval musculature, how distinct adult muscle types are generated is much less characterized. Here, we identify an essential regulator of Drosophila thoracic flight muscle development, the Hox TF Antennapedia (Antp). Correcting a long-standing belief that flight muscle development occurs without the input of Hox TFs, we show that Antp intervenes at several stages of flight muscle development, from the establishment of the progenitor pool in the embryo to myoblast differentiation in the early pupa. Furthermore, the precisely regulated clearance of Hox in the developing flight muscle fibers is required to allow for fibrillar muscle fate diversification, setting these muscles apart from all other adult tubular muscle types.

SAYP and Bap170, subunits of the SWI/SNF remodeling complex, have the ability to support enhancer-dependent transcription when artificially recruited to the promoter on a transgene. We found that the phenomenon critically depends on two subunits of the Mediator kinase module, Med12 and Med13 but does not require the two other subunits of the module (Cdk8 and CycC) or other subunits of the core part of the complex. A cooperation of the above proteins in active transcription was also observed at endogenous loci, but the contribution of the subunits to the activity of a particular gene differed in different loci. The factors SAYP/Bap170 and Med12/Med13 did not form sufficiently stable interactions in the extract, and their cooperation was apparently local at regulatory elements, the presence of SAYP and Bap170 in a locus being necessary for stable recruitment of Med12 and Med13 to the locus. In addition to the above factors, the Nelf-A protein was found to participate in the process. The cooperation of the factors, independent of enzymatic activities of the complexes they are part of, appears to be a novel mechanism that maintains promoter activity and may be used in many loci of the genome. Extended intrinsically disordered regions of the factors were assumed to sustain the mechanism.

R-loops are involved in many biological processes in cells, yet the regulatory principles for R-loops in vivo and their impact on development remain to be explored. Here, we modified the CUT&Tag strategy to profile R-loops in Drosophila at multiple developmental stages. While high GC content promotes R-loop formation in mammalian cells, it is not required in Drosophila. In contrast, RNAPII abundance appears to be a universal inducing factor for R-loop formation, including active promoters and enhancers, and H3K27me3 decorated repressive regions and intergenic repeat sequences. Importantly, such a regulatory relationship is dynamically maintained throughout development, and development-related transcription factors may regulate RNAPII activation and R-loop dynamics. By ablating Spt6, we further showed the global R-loop induction coupled with RNAPII pausing. Importantly, depending on the gene length, genes underwent up- or down-regulation, both of which were largely reversed by rnh1 overexpression, suggesting that R-loops play a significant role in the divergent regulation of transcription by Spt6 ablation. DNA damage, defects in survival, and cuticle development were similarly alleviated by rnh1 overexpression. Altogether, our findings indicate that dynamic R-loop regulation is dictated by RNAPII pausing and transcription activity, and plays a feedback role in gene regulation, genome stability maintenance, and Drosophila development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Identifying the molecular origins by which new morphological structures evolve is one of the long-standing problems in evolutionary biology. To date, vanishingly few examples provide a compelling account of how new morphologies were initially formed, thereby limiting our understanding of how diverse forms of life derived their complex features. Here, we provide evidence that the large projections on the Drosophila eugracilis phallus that are implicated in sexual conflict have evolved through the partial co-option of the trichome genetic network. These unicellular apical projections on the phallus postgonal sheath are reminiscent of trichomes that cover the Drosophila body but are up to 20-fold larger in size. During their development, they express the transcription factor Shavenbaby, the master regulator of the trichome network. Consistent with the co-option of the Shavenbaby network during the evolution of the D. eugracilis projections, somatic mosaic CRISPR-Cas9 mutagenesis shows that shavenbaby is necessary for their proper length. Moreover, misexpression of Shavenbaby in the sheath of D. melanogaster, a naive species that lacks these projections, is sufficient to induce small trichomes. These induced projections rely on a genetic network that is shared to a large extent with the D. eugracilis projections, indicating its partial co-option but also some genetic rewiring. Thus, by leveraging a genetically tractable evolutionary novelty, our work shows that the trichome-forming network is flexible enough that it can be partially co-opted in a new context and subsequently refined to produce unique apical projections that are barely recognizable compared with their simpler ancestral beginnings., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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.)

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.

Understanding the principles underlying the design of robust, yet flexible patterning systems is a key problem in developmental biology. In the Drosophila wing, Hedgehog (Hh) signaling determines patterning outputs using dynamical properties of the Hh gradient. In particular, the pattern of collier ( col ) is established by the steady-state Hh gradient, whereas the pattern of decapentaplegic ( dpp ), is established by a transient gradient of Hh known as the Hh overshoot. Here, we use mathematical modeling to suggest that this dynamical interpretation of the Hh gradient results in specific robustness and precision properties. For instance, the location of the anterior border of col , which is subject to self-enhanced ligand degradation is more robustly specified than that of dpp to changes in morphogen dosage, and we provide experimental evidence of this prediction. However, the anterior border of dpp expression pattern, which is established by the overshoot gradient is much more precise to what would be expected by the steady-state gradient. Therefore, the dynamical interpretation of Hh signaling offers tradeoffs between robustness and precision to establish tunable patterning properties in a target-specific manner., Competing Interests: RR, AL No competing interests declared, MN Reviewing editor, eLife, (© 2024, Reyes et al.)

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.)

Many organisms, including cosmopolitan drosophilids, show circadian plasticity, varying their activity with changing dawn-dusk intervals 1 . How this behaviour evolves is unclear. Here we compare Drosophila melanogaster with Drosophila sechellia, an equatorial, ecological specialist that experiences minimal photoperiod variation, to investigate the mechanistic basis of circadian plasticity evolution 2 . D. sechellia has lost the ability to delay its evening activity peak time under long photoperiods. Screening of circadian mutants in D. melanogaster/D. sechellia hybrids identifies a contribution of the neuropeptide pigment-dispersing factor (Pdf) to this loss. Pdf exhibits species-specific temporal expression, due in part to cis-regulatory divergence. RNA interference and rescue experiments in D. melanogaster using species-specific Pdf regulatory sequences demonstrate that modulation of this neuropeptide's expression affects the degree of behavioural plasticity. The Pdf regulatory region exhibits signals of selection in D. sechellia and across populations of D. melanogaster from different latitudes. We provide evidence that plasticity confers a selective advantage for D. melanogaster at elevated latitude, whereas D. sechellia probably suffers fitness costs through reduced copulation success outside its range. Our findings highlight this neuropeptide gene as a hotspot locus for circadian plasticity evolution that might have contributed to both D. melanogaster's global distribution and D. sechellia's specialization., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Rabex-5 (also called RabGEF1), a protein originally characterized for its Rab5 GEF function, also has an A20-like E3 ubiquitin ligase domain. We and others reported that Rabex-5 E3 activity promotes Ras mono- and di-ubiquitination to inhibit Ras signaling in Drosophila and mammals. Subsequently, we reported that Rabex-5 inhibits Notch signaling in the Drosophila hematopoietic system. Here we report genetic interactions using Rabex-5 transgenes encoding domain-specific mutations that show that Rabex-5 requires an intact E3 domain to inhibit Notch signaling in the epithelial tissue of the developing wing. Surprisingly, we discovered that Rabex-5 with an impaired E3 domain but active Rab5 GEF domain suppresses Notch loss-of-function phenotypes and enhances both Notch duplication phenotypes and activated Ras phenotypes consistent with a model that the Rab5 GEF activity of Rabex-5 might positively regulate Ras and Notch. Positive and negative regulation of developmental signaling by its different catalytic domains could allow Rabex-5 to precisely coordinate developmental signaling to fine-tune patterning. Finally, we report that Rabex-5 also inhibits the overgrowth due to loss of PTEN or activation of PI3K but not activation of AKT. Inhibition of Ras, Notch, and PI3K signaling may explain why Rabex-5 is deleted in some cancers. Paradoxically, Rabex-5 is reported to be an oncogene in other cancers. We propose that Rabex-5 acts as a tumor suppressor via its E3 activity to inhibit Ras, Notch, and PI3K signaling and as an oncogene via its Rab5 GEF activity to enhance Ras and Notch signaling., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Reimels 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.)

The translational repressor Nanos (Nos) regulates a single target, maternal hunchback (hb) mRNA, to govern abdominal segmentation in the early Drosophila embryo. Nos is recruited to sites in the 3' UTR of hb mRNA in collaboration with the sequence-specific RNA-binding protein Pumilio (Pum); on its own, Nos has no binding specificity. Nos is expressed at other stages of development, but very few mRNA targets that might mediate its action at these stages have been described. Nor has it been clear whether Nos is targeted to other mRNAs in concert with Pum or via other mechanisms. In this report, we identify mRNAs targeted by Nos via 2 approaches. First, we identify mRNAs depleted upon expression of a chimera bearing Nos fused to the nonsense mediated decay (NMD) factor Upf1. We find that, in addition to hb, Upf1-Nos depletes approximately 2,600 mRNAs from the maternal transcriptome in early embryos. Virtually all of these appear to be targeted in a canonical, hb-like manner in concert with Pum. In a second, more conventional approach, we identify mRNAs that are stabilized during the maternal zygotic transition (MZT) in embryos from nos- females. Most (86%) of the 1,185 mRNAs regulated by Nos are also targeted by Upf1-Nos, validating use of the chimera. Previous work has shown that 60% of the maternal transcriptome is degraded in early embryos. We find that maternal mRNAs targeted by Upf1-Nos are hypoadenylated and inefficiently translated at the ovary-embryo transition; they are subsequently degraded in the early embryo, accounting for 59% of all destabilized maternal mRNAs. We suggest that the late ovarian burst of Nos represses a large fraction of the maternal transcriptome, priming it for later degradation by other factors in the embryo., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Marhabaie 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.)

Tissue-specific gene knockout by CRISPR/Cas9 is a powerful approach for characterizing gene functions during development. However, this approach has not been successfully applied to most Drosophila tissues, including the Drosophila neuromuscular junction (NMJ). To expand tissue-specific CRISPR to this powerful model system, here we present a CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) toolkit for knocking out genes in motoneurons, muscles, and glial cells. We validated the efficacy of CRISPR-TRiM by knocking out multiple genes in each tissue, demonstrated its orthogonal use with the Gal4/UAS binary expression system, and showed simultaneous knockout of multiple redundant genes. We used CRISPR-TRiM to discover an essential role for SNARE components in NMJ maintenance. Furthermore, we demonstrate that the canonical ESCRT pathway suppresses NMJ bouton growth by downregulating retrograde Gbb signaling. Lastly, we found that axon termini of motoneurons rely on ESCRT-mediated intra-axonal membrane trafficking to release extracellular vesicles at the NMJ. Thus, we have successfully developed an NMJ CRISPR mutagenesis approach which we used to reveal genes important for NMJ structural plasticity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen 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.)

Yorkie (Yki) is a key effector of the Hippo pathway that activates the expression of targets by associating with the transcription factor Scalloped. Various upstream signals, such as cell polarity and mechanical cues, control transcriptional programs by regulating Yki activity. Searching for Yki regulatory factors has far-reaching significance for studying the Hippo pathway in development and human diseases. In this study, we identify Calpain-A (CalpA) and Calpain-B (CalpB), two calcium (Ca 2+ )-dependent modulatory proteases of the calpain family, as critical regulators of Yki in Drosophila that interact with Yki, respectively. Ca 2+ induces Yki cleavage in a CalpA/CalpB-dependent manner, and the protease activity of CalpA/CalpB is pivotal for the cleavage. Furthermore, overexpression of CalpA or CalpB in Drosophila partially restores the large wing phenotype caused by Yki overexpression, and F98 of Yki is an important cleavage site by the Ca 2+ -calpains axis. Our study uncovers a unique mechanism whereby the Ca 2+ -calpain axis modulates Yki activity through protein cleavage., 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.)

Notch signaling is a highly conserved pathway between mammals and Drosophila and plays a key role in various biological processes. Drosophila has emerged as a powerful model for studying hematopoiesis and leukemia. In exception to crystal cells, the strength of Notch signaling in Drosophila lymph gland cortical zone (CZ)/intermediate zone (IZ) cells is weak. However, the influence of Notch activation in the lymph gland CZ/IZ cells and circulating hemocytes on hematopoietic homeostasis maintenance is unclear. Here, we showed that Notch activation in lymph gland CZ/IZ cells induced overdifferentiation of progenitors. Moreover, Notch activation promoted lamellocyte generation via NFκB/Toll signaling activation and increased reactive oxygen species (ROS). In addition, we found that Notch activation in lymph gland CZ/IZ cells and circulating hemocytes caused caspase-independent and nonautophagic cell death. However, crystal cell autophagy was activated by upregulation of the expression of the target gene of the Hippo/Yki pathway Diap1. Moreover, we showed that Notch activation could alleviate cytokine storms and improve the survival of Ras v12 leukemia model flies. Our study revealed the various mechanisms of hematopoietic dysregulation induced by Notch activation in healthy flies and the therapeutic effect of Notch activation on leukemia model flies., 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 Ltd. All rights reserved.)

Selective elimination of cancer cells without causing deleterious effects on normal cells is an ideal anti-cancer strategy. Here, using Drosophila cancer model, we performed an in vivo RNAi screen for anti-cancer targets that selectively eliminate tumors without affecting normal tissue growth. In Drosophila imaginal epithelium, clones of cells expressing oncogenic Ras with simultaneous mutations in the cell polarity gene scribble (Ras V12 /scrib -/- ) develop into malignant tumors. We found that knockdown of Crk, the Drosophila ortholog of human CRK (CT10 regulatory kinase) and CRKL (Crk-like) adapter proteins, significantly suppresses growth of Ras V12 /scrib -/- tumors by inducing c-Jun N-terminal kinase (JNK)-mediated apoptosis, while it does not affect growth of normal epithelium. Mechanistically, Crk inhibition blocks Yorkie (Yki)/YAP activity by impairing F-actin accumulation, an upstream event of Yki/YAP activation in tumors. Inhibition of Yki/YAP in tumors causes intracellular JNK signaling to be used for apoptosis induction. Given that molecules and signaling pathways identified in Drosophila are highly conserved and activated in human cancers, our findings would provide a novel, to the best of our knowledge, anti-cancer strategy against YAP-activated cancers., (© 2024. The Author(s).)

Here, we present a protocol to quantify interactions among difficult-to-express proteins from Drosophila cells using the select western blot-free tagged-protein interaction (SWFTI) assay. We describe steps for plasmid design, cell plating, protein expression, and immunoprecipitation preparation. We then detail procedures for protein labeling, gel purification, and protein quantification. This protocol offers a fluorescence-based technique for rapid quantification of ectopically expressed proteins that are fused to SNAP and CLIP tags without the need for membrane transfer. For complete details on the use and execution of this protocol, please refer to Lin et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Most RNA-protein condensates are composed of heterogeneous immiscible phases. However, how this multiphase organization contributes to their biological functions remains largely unexplored. Drosophila germ granules, a class of RNA-protein condensates, are the site of mRNA storage and translational activation. Here, using super-resolution microscopy and single-molecule imaging approaches, we show that germ granules have a biphasic organization and that translation occurs in the outer phase and at the surface of the granules. The localization, directionality, and compaction of mRNAs within the granule depend on their translation status, translated mRNAs being enriched in the outer phase with their 5'end oriented towards the surface. Translation is strongly reduced when germ granule biphasic organization is lost. These findings reveal the intimate links between the architecture of RNA-protein condensates and the organization of their different functions, highlighting the functional compartmentalization of these condensates., (© 2024. The Author(s).)

RNA interference (RNAi) drives powerful antiviral immunity in plants and animals so that many viruses must express viral suppressor of RNAi (VSR) to establish virulent infection. However, little is known about the immune responses conferring resistance against viruses that have evolved the counter-defensive strategy to suppress antiviral RNAi. In this study, we discover that Drosophila cells infected with Drosophila C virus (DCV), a natural viral pathogen of Drosophila known to harbor a potent VSR, exhibit heightened expression of circular RNA circZfh1. circZfh1 confers virus resistance in the presence of viral suppression of antiviral RNAi. Furthermore, we validate that circZfh1 encodes a 274-amino acid protein, CRAV, essential for its antiviral activity. Notably, CRAV differs from its parental Zfh1 gene in a different reading frame, with the C-terminal 69 amino acids unique to CRAV. Our analysis also reveals the presence of CRAV in species within the melanogaster subgroup, with the C-terminal unique fragment undergoing accelerated evolution. Expression of CRAV upregulates the expression of the cytokine Upd3, which binds to its receptor, stimulating the JAK-STAT pathway and enhancing the immune response to DCV infection. Notably, CRISPR/Cas9 knockout of circZfh1 significantly enhances DCV replication in vitro and in vivo , with circZfh1-knockout adult flies displaying heightened disease susceptibility to DCV. In summary, our findings unveil a Drosophila protein-coding circular RNA that activates an innate immune signaling pathway crucial for virus resistance following the suppression of antiviral RNAi by viruses, thereby elucidating a novel counter-defensive strategy.IMPORTANCEEukaryotic hosts possess a complex, multilayered immune system that guards against pathogen invasion. In fruit flies, RNA interference (RNAi) drives robust antiviral immunity, prompting many viruses to express viral suppressors of RNAi (VSRs) to establish virulent infections. However, little is known about immune responses that confer resistance against viruses with potent VSRs. In this study, we discovered that Drosophila cells infected with Drosophila C virus (DCV), a natural viral pathogen possessing a potent VSR, upregulated the expression of circular RNA circZfh1. circZfh1 exhibits DCV-specific antiviral activity, encoding a 274-amino acid protein, CRAV, crucial for its antiviral effects. As a different reading frame from its parental Zfh1 gene, the C-terminal 69 amino acids are unique to CRAV, undergoing faster evolution. CRAV activates the JAK-STAT pathway, enhancing the immune response to DCV infection. Therefore, our work uncovers a new strategy for suppressing viral counter-defense through protein-coding circular RNA in fruit flies., Competing Interests: The authors declare no conflict of interest.

Post-larval hematopoiesis in Drosophila largely depends upon the stockpile of progenitors present in the blood-forming organ/lymph gland of the larvae. During larval stages, the lymph gland progenitors gradually accumulate reactive oxygen species (ROS), which is essential to prime them for differentiation. Studies have shown that ROS triggers the activation of JNK (c-Jun Kinase), which upregulates fatty acid oxidation (FAO) to facilitate progenitor differentiation. Intriguingly, despite having ROS, the entire progenitor pool does not differentiate simultaneously in the late larval stages. Using expression analyses, genetic manipulation and pharmacological approaches, we found that the Drosophila NF-κB transcription factor Relish (Rel) shields the progenitor pool from the metabolic pathway that inducts them into the differentiation program by curtailing the activation of JNK. Although ROS serves as the metabolic signal for progenitor differentiation, the input from ROS is monitored by the developmental signal TAK1, which is regulated by Relish. This developmental circuit ensures that the stockpile of ROS-primed progenitors is not exhausted entirely. Our study sheds light on how, during development, integrating NF-κB-like factors with metabolic pathways seem crucial to regulating cell fate transition during development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ramesh 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.)

Despite increasing in mass approximately 100-fold during larval life, the Drosophila CNS maintains its characteristic form. Dynamic interactions between the overlying basement membrane and underlying surface glia are known to regulate CNS structure in Drosophila, but the genes and pathways that establish and maintain CNS morphology during development remain poorly characterized. To identify genes that regulate CNS shape in Drosophila, we conducted an EMS-based, forward genetic screen of the second chromosome, uncovered 50 mutations that disrupt CNS structure, and mapped these alleles to 17 genes. Analysis of whole genome sequencing data wedded to genetic studies uncovered the affected gene for all but 1 mutation. Identified genes include well-characterized regulators of tissue shape, like LanB1, viking, and Collagen type IV alpha1, and previously characterized genes, such as Toll-2 and Rme-8, with no known role in regulating CNS structure. We also uncovered that papilin and C1GalTA likely act in the same pathway to regulate CNS structure and found that the fly homolog of a glucuronosyltransferase, B4GAT1/LARGE1, that regulates Dystroglycan function in mammals is required to maintain CNS shape in Drosophila. Finally, we show that the senseless-2 transcription factor is expressed and functions specifically in surface glia found on peripheral nerves but not in the CNS to govern CNS structure, identifying a gene that functionally subdivides a glial subtype along the peripheral-central axis. Future work on these genes should clarify the genetic mechanisms that ensure the homeostasis of CNS form during development., 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 initially homogeneous epithelium of the early Drosophila embryo differentiates into regional subpopulations with different behaviours and physical properties that are needed for morphogenesis. The factors at top of the genetic hierarchy that control these behaviours are known, but many of their targets are not. To understand how proteins work together to mediate differential cellular activities, we studied in an unbiased manner the proteomes and phosphoproteomes of the three main cell populations along the dorso-ventral axis during gastrulation using mutant embryos that represent the different populations. We detected 6111 protein groups and 6259 phosphosites of which 3398 and 3433 were differentially regulated, respectively. The changes in phosphosite abundance did not correlate with changes in host protein abundance, showing phosphorylation to be a regulatory step during gastrulation. Hierarchical clustering of protein groups and phosphosites identified clusters that contain known fate determinants such as Doc1, Sog, Snail, and Twist. The recovery of the appropriate known marker proteins in each of the different mutants we used validated the approach, but also revealed that two mutations that both interfere with the dorsal fate pathway, Toll 10B and serpin27a ex do this in very different manners. Diffused network analyses within each cluster point to microtubule components as one of the main groups of regulated proteins. Functional studies on the role of microtubules provide the proof of principle that microtubules have different functions in different domains along the DV axis of the embryo., Competing Interests: JG, HN, EV, BD, MT, GG, MF, TH, AC, RZ, MK, EP, YW, ML No competing interests declared, (© 2024, Gomez et al.)

Deubiquitinases (DUBs) are essential for the maintenance of protein homeostasis and assembly of proteins into functional complexes. Despite growing interest in DUBs biological functions, the roles of DUBs in regulating intestinal stem cells (ISCs) and gut homeostasis remain largely unknown. Here, we perform an in vivo RNAi screen through induced knock-down of DUBs expression in adult midgut ISCs and enteroblasts (EBs) to identify DUB regulators of intestinal homeostasis in Drosophila. We screen 43 DUBs and identify 8 DUBs that are required for ISCs homeostasis. Knocking-down of usp1, CG7857, usp5, rpn8, usp10 and csn5 decreases the number of ISCs/EBs, while knocking-down of CG4968 and usp8 increases the number of ISCs/EBs. Moreover, knock-down of usp1, CG4968, CG7857, or rpn8 in ISCs/EBs disrupts the intestinal barrier integrity and shortens the lifespan, indicating the requirement of these DUBs for the maintenance of gut homeostasis. Furthermore, we provide evidences that USP1 mediates ISC lineage differentiation via modulating the Notch signaling activity. Our study identifies, for the first time, the deubiquitinases required for the maintenance of intestinal homeostasis in Drosophila, and provide new insights into the functional links between the DUBs and intestinal homeostasis., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The translocator protein TSPO is an evolutionary conserved mitochondrial protein overexpressed in various contexts of neurodegeneration. Friedreich Ataxia (FA) is a neurodegenerative disease due to GAA expansions in the FXN gene leading to decreased expression of frataxin, a mitochondrial protein involved in the biosynthesis of iron-sulfur clusters. We previously reported that Tspo was overexpressed in a Drosophila model of this disease generated by CRISPR/Cas9 insertion of approximately 200 GAA in the intron of fh, the fly frataxin gene. Here, we describe a new Drosophila model of FA with 42 GAA repeats, called fh-GAAs. The smaller expansion size allowed to obtain adults exhibiting hallmarks of the FA disease, including short lifespan, locomotory defects and hypersensitivity to oxidative stress. The reduced lifespan was fully rescued by ubiquitous expression of human FXN, confirming that both frataxins share conserved functions. We observed that Tspo was overexpressed in heads and decreased in intestines of these fh-GAAs flies. Then, we further overexpressed Tspo specifically in glial cells and observed improved survival. Finally, we investigated the effects of Tspo overexpression in healthy flies. Increased longevity was conferred by glial-specific overexpression, with opposite effects in neurons. Overall, this study highlights protective effects of glial TSPO in Drosophila both in a neurodegenerative and a healthy context., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The Drosophila compound eye is an attractive system for unraveling how tissues are specified and patterned. Puli et al. recently demonstrated that eye size and spacing are controlled by the defective proventriculus (dve) gene. This impacts our understanding of hypertelorism, a disorder associated with mutations in special AT-rich binding protein 1 (SATB1), the human ortholog of Dve., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase ( OPA1 ) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 ( hOPA1 ) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1 . This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies., Competing Interests: YN, JO, RM, SH, ES, SU, TS, AS No competing interests declared, (© 2023, Nitta, Osaka et al.)

Obesity remains one of the largest health problems in the world, arising from the excess storage of triglycerides (TAGs). However, the full complement of genes that are important for regulating TAG storage is not known. The Glut1 gene encodes a Drosophila glucose transporter that has been identified as a potential obesity gene through genetic screening. Yet, the tissue-specific metabolic functions of Glut1 are not fully understood. Here, we characterized the role of Glut1 in the fly brain by decreasing neuronal Glut1 levels with RNAi and measuring glycogen and TAGs. Glut1RNAi flies had decreased TAG and glycogen levels, suggesting a nonautonomous role of Glut1 in the fly brain to regulate nutrient storage. A group of hormones that regulate metabolism and are expressed in the fly brain are Drosophila insulin-like peptides (Ilps) 2, 3, and 5. Interestingly, we observed blunted Ilp3 and Ilp5 expression in neuronal Glut1RNAi flies, suggesting Glut1 functions in insulin-producing neurons (IPCs) to regulate whole-organism TAG and glycogen storage. Consistent with this hypothesis, we also saw fewer TAGs and glycogens and decreased expression of Ilp3 and Ilp5 in flies with IPC-specific Glut1RNAi . Together, these data suggest Glut1 functions as a nutrient sensor in IPCs, controlling TAG and glycogen storage and regulating systemic energy homeostasis.

The Drosophila Id gene extramacrochaetae (emc) is required during Drosophila eye development for proper cell fate specification within the R7 equivalence group. Without emc, R7 cells develop like R1/6 cells, and there are delays and deficits in differentiation of non-neuronal cone cells. Although emc encodes an Inhibitor of DNA-binding (Id) protein that is known to antagonize proneural bHLH protein function, no proneural gene is known for R7 or cone cell fates. These fates are also independent of daughterless (da), which encodes the ubiquitous E protein heterodimer partner of proneural bHLH proteins. We report here that the effects of emc mutations disappear in the absence of da, and are partially mimicked by forced expression of Da dimers, indicating that emc normally restrains da from interfering with R7 and cone cell specification, as occurs in emc mutants. emc, and da, regulate three known contributors to R7 fate, which are Notch signaling, Rap1, and Sevenless. R7 specification is partially restored to emc mutant cells by mutation of RapGap1, confirming that Rap1 activity, in addition to Notch activity, is a critical target of emc. These findings exemplify how mutations of an Id protein gene can affect processes that do not require any bHLH protein, by restraining Da activity within physiological bounds., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here, we address the function of Drosophila Spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, the cells of which undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of Spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of Spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelope., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl - /H + antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells., (© 2024. The Author(s).)

The nuclear factor binding the κ light chain in B-cells (NFκB) is involved in a wide range of cellular processes including development, growth, innate immunity, and sleep. However, genetic studies of the role of specific NFκB transcription factors in sleep have been limited. Drosophila fruit flies carry three genes encoding NFκB transcription factors, Dorsal, Dorsal Immunity Factor (Dif), and Relish. We previously found that loss of the Relish gene from fat body suppressed daily nighttime sleep, and abolished infection-induced sleep. Here we show that Dif regulates daily sleep and recovery sleep following prolonged wakefulness. Mutants of Dif showed reduced daily sleep and suppressed recovery in response to sleep deprivation. Pan-neuronal knockdown of Dif strongly suppressed daily sleep, indicating that in contrast to Relish, Dif functions from the central nervous system to regulate sleep. Based on the unique expression pattern of a Dif- GAL4 driver, we hypothesized that its effects on sleep were mediated by the pars intercerebralis (PI). While RNAi knock-down of Dif in the PI reduced daily sleep, it had no effect on the recovery response to sleep deprivation. However, recovery sleep was suppressed when RNAi knock-down of Dif was distributed across a wider range of neurons. Induction of the nemuri (nur) antimicrobial peptide by sleep deprivation was reduced in Dif mutants and pan-neuronal overexpression of nur also suppressed the Dif mutant phenotype by significantly increasing sleep and reducing nighttime arousability. Together, these findings indicate that Dif functions from brain to target nemuri and to promote deep sleep., (© The Author(s) 2024. Published by Oxford University Press on behalf of Sleep Research Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research., (© 2024. The Author(s).)

The chromosomes in multicellular eukaryotes are organized into a series of topologically independent loops called TADs. In flies, TADs are formed by physical interactions between neighboring boundaries. Fly boundaries exhibit distinct partner preferences, and pairing interactions between boundaries are typically orientation-dependent. Pairing can be head-to-tail or head-to-head. The former generates a stem-loop TAD, while the latter gives a circle-loop TAD. The TAD that encompasses the Drosophila even skipped ( eve ) gene is formed by the head-to-tail pairing of the nhomie and homie boundaries. To explore the relationship between loop topology and the physical and regulatory landscape, we flanked the nhomie boundary region with two attP sites. The attP sites were then used to generate four boundary replacements: λ DNA , nhomie forward (WT orientation), nhomie reverse (opposite of WT orientation), and homie forward (same orientation as WT homie ). The nhomie forward replacement restores the WT physical and regulatory landscape: in MicroC experiments, the eve TAD is a 'volcano' triangle topped by a plume, and the eve gene and its regulatory elements are sequestered from interactions with neighbors. The λ DNA replacement lacks boundary function: the endpoint of the 'new' eve TAD on the nhomie side is ill-defined, and eve stripe enhancers activate a nearby gene, eIF3j . While nhomie reverse and homie forward restore the eve TAD, the topology is a circle-loop, and this changes the local physical and regulatory landscape. In MicroC experiments, the eve TAD interacts with its neighbors, and the plume at the top of the eve triangle peak is converted to a pair of 'clouds' of contacts with the next-door TADs. Consistent with the loss of isolation afforded by the stem-loop topology, the eve enhancers weakly activate genes in the neighboring TADs. Conversely, eve function is partially disrupted., Competing Interests: WK, MF, PS, JJ No competing interests declared, (© 2024, Ke, Fujioka et al.)

The T-box (Tbx) proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. They are highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. Here, we report posttranscriptional and translational regulation of midline (mid), a Tbx member in Drosophila. We found that the 3'UTR of mid has mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid-mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla luciferase reporter significantly reduced the activity of the Luciferase, whereas deleting only the degradation elements from the 3'UTR resulted in reduced activity, but not as much. Overexpression of mid in MP2, an embryonic neuroblast, showed no significant difference in the levels of mid-mRNA between the 2 transgenes, with and without the 3'UTR, indicating the absence of posttranscriptional regulation of mid in MP2. Moreover, while elevated mid-RNA was detected in MP2 in nearly all hemisegments, only a fifth of those hemisegments had elevated levels of the protein. Overexpression of the 2 transgenes resulted in MP2-lineage defects at about the same frequency. These results indicate a translational/posttranslational regulation of mid in MP2. The regulation of ectopically expressed mid in the wing imaginal disc was complex. In the wing disc, where mid is not expressed, the ectopic expression of the transgene lacking the 3'UTR had a higher level of mid-RNA and the protein had a stronger phenotypic effect. These results indicate that the 3'UTR can subject mid-mRNA to degradation in a cell- and tissue-specific manner. We further report a balancer-mediated transgenerational modifier effect on the expression and gain of function effects of the 2 transgenes., 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. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Accurate chromosome segregation during meiosis requires the maintenance of sister chromatid cohesion, initially established during premeiotic S phase. In human oocytes, DNA replication and cohesion establishment occur decades before chromosome segregation and deterioration of meiotic cohesion is one factor that leads to increased segregation errors as women age. Our previous work led us to propose that a cohesion rejuvenation program operates to establish new cohesive linkages during meiotic prophase in Drosophila oocytes and depends on the cohesin loader Nipped-B and the cohesion establishment factor Eco. In support of this model, we recently demonstrated that chromosome-associated cohesin turns over extensively during meiotic prophase and failure to load cohesin onto chromosomes after premeiotic S phase results in arm cohesion defects in Drosophila oocytes. To identify proteins required for prophase cohesion rejuvenation but not S phase establishment, we conducted a Gal4-UAS inducible RNAi screen that utilized two distinct germline drivers. Using this strategy, we identified 29 gene products for which hairpin expression during meiotic prophase, but not premeiotic S phase, significantly increased segregation errors. Prophase knockdown of Brahma or Pumilio, two positives with functional links to the cohesin loader, caused a significant elevation in the missegregation of recombinant homologs, a phenotype consistent with premature loss of arm cohesion. Moreover, fluorescence in situ hybridization confirmed that Brahma, Pumilio, and Nipped-B are required during meiotic prophase for the maintenance of arm cohesion. Our data support the model that Brahma and Pumilio regulate Nipped-B-dependent cohesin loading during rejuvenation. Future analyses will better define the mechanism(s) that govern meiotic cohesion rejuvenation and whether additional prophase-specific positives function in this process., Competing Interests: Conflict of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)