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Lysosomes are digestive organelles that are crucial for nutrient sensing and metabolism. Lysosome impairment is linked to a broad spectrum of metabolic disorders, underscoring their importance to human health. Thus, lysosomes are an attractive target for metabolic disease therapies. In previous work, we discovered a novel class of tubular lysosomes that are morphologically and functionally distinct from traditionally described vesicular lysosomes. Tubular lysosomes are present in multiple tissues, are broadly conserved from invertebrates to mammals, are more proficient at degrading autophagic cargo than vesicular lysosomes, and delay signs of tissue aging when induced ectopically. Thus, triggering tubular lysosome formation presents one mechanism to increase lysosome activity and, notably, overproduction of the small lysosomal protein, SVIP, is a robust genetic strategy for triggering lysosomal tubulation on demand. In this study, we examine whether SVIP overexpression in the fly gut can suppress pathophysiological phenotypes associated with an obesogenic high-fat diet. Indeed, our results indicate that increasing SVIP expression in the fly gut reduces lipid accumulation, suppresses body mass increase, and improves survival in flies fed a high-fat diet. Collectively, these data hint that increasing lysosomal activity through induction of tubular lysosomal networks, could be one strategy to combat obesity-related pathologies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

Identifying the presence and strength of reproductive isolating barriers is necessary to understand how species form and then remain distinct in the face of ongoing gene flow. Here, we study reproductive isolation at two stages of the speciation process in the closely related mushroom-feeding species Drosophila recens and Drosophila subquinaria. We assess 3 isolating barriers that occur after mating, including the number of eggs laid, the proportion of eggs laid that hatched, and the number of adult offspring from a single mating. First, all 3 reproductive barriers are present between D. recens females and D. subquinaria males, which are at the late stages of speciation but still produce fertile daughters through which gene flow can occur. There is no evidence for geographic variation in any of these traits, concurrent with patterns of behavioural isolation. Second, all 3 of these reproductive barriers are strong between geographically distant conspecific populations of D. subquinaria, which are in the early stages of speciation and show genetic differentiation and asymmetric behavioural discrimination. The reduction in the number of eggs laid is asymmetric, consistent with patterns in behavioural isolation, and suggests the evolution of postmating prezygotic isolation due to cascade reinforcement against mating with D. recens. In summary, not only may postmating prezygotic reproductive barriers help maintain isolation between D. recens and D. subquinaria, but they may also drive the earliest stages of isolation within D. subquinaria., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Evolutionary Biology. 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.)

The discovery that homeotic genes in Drosophila are conserved and utilized for embryonic development throughout the animal kingdom, including humans, revolutionized the fields of developmental biology and evolutionary developmental biology (evo-devo). In a pair of back-to-back papers published in Cell in 1984, researchers at the Biozentrum in Basel, Switzerland, showed that the homeobox - previously identified as a sequence shared by homeotic genes in Drosophila - was also present in the genome of diverse animals. The first paper (McGinnis et al., 1984a) showed that genomes of both invertebrates and vertebrates contain sequences that cross-hybridized with Drosophila homeobox probes. The second paper (Carrasco et al., 1984) identified a cross-hybridizing sequence in the model system Xenopus laevis. They then isolated the first vertebrate homeobox-containing gene by cloning and sequencing of the corresponding genomic region. Finally, they showed that this gene (AC1, later renamed HoxC6) was expressed during embryonic development, the first evidence that developmentally expressed Drosophila genes could be used to isolate regulators of vertebrate embryonic development. These findings led to a flurry of activity in the evo-devo field, initially focused on isolating Hox genes across diverse species, and then expanding to isolation of other gene families based on Drosophila orthologs, an approach that continues today. This led to the notion of a conserved genetic toolkit for embryonic development, currently accepted, but unexpected at the time of its discovery. We attempt to provide some context for the sea-change in thinking that these discoveries brought about by referring to Jean Piaget's theories about the sequential acquisition of scientific knowledge., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Complex eukaryotes vary greatly in the mode and extent that their evolutionary histories have been shaped by the microbial communities that they host. A general understanding of the evolutionary consequences of host-microbe symbioses requires that we understand the relative importance of host phylogenetic divergence and other ecological processes in shaping variation in host-associated microbial communities. To contribute to this understanding, we described the bacterial communities hosted by several Drosophila species native to the Sonoran Desert of North America. Our sampling consisted of four species that span multiple dietary shifts to cactophily, as well as the dietary generalist D. melanogaster, allowing us to partition the influences of host phylogeny and extant ecology. We found that bacterial communities were compositionally indistinguishable when considering incidence only but varied when considering the relative abundances of bacterial taxa. Variation in community composition was not explained by host phylogenetic divergence but could be partially explained by dietary variation. In support of the important role of diet as a source of ecological selection, we found that specialist cactophilic Drosophila deviated more from neutral predictions than dietary generalists. Overall, our findings provide insight into the evolutionary and ecological factors that shape host-associated microbial communities in a natural context., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Evolutionary Biology.)

During the early stages of local adaptation and speciation, genetic differences tend to accumulate at certain regions of the genome leading to the formation of genomic islands of divergence (GIDs). This pattern may be due to selection and/or difference in the rate of recombination. Here, we investigate the possible causes of GIDs in Drosophila yakuba mayottensis, and reconfirm using field collection its association with toxic noni (Morinda citrifolia) fruits on the Mayotte island. Population genomics revealed lack of genetic structure on the island and identified 23 GIDs distinguishing D. y. mayottensis from generalist mainland populations of D. y. yakuba. The GIDs were enriched with gene families involved in the metabolism of lipids, sugars, peptides and xenobiotics, suggesting a role in host shift. We assembled a new genome for D. y. mayottensis and identified five novel chromosomal inversions. Twenty one GIDs (~99% of outlier windows) fell in low recombining regions or subspecies-specific inversions. However, only two GIDs were in collinear, normally recombining regions suggesting a signal of hard selective sweeps. Unlike D. y. mayottensis, D. sechellia, the only other noni-specialist, is known to be homosequential with its generalist relatives. Thus, whereas structural variation may disproportionally shape GIDs in some species, striking parallel adaptations can occur between species despite distinct genomic architectures., (© 2024 John Wiley & Sons Ltd.)

Drosophila suzukii is an invasive pest that affects many fresh, soft-skinned fruits such as strawberries and blueberries. To combat this pest, growers use various methods including insecticide sprays, biological control, and sanitation practices. However, these methods are becoming increasingly ineffective against D. suzukii due to increased resistance against insecticides and increasing labor costs. Sterile Insect Technique (SIT) has been used successfully to control many agricultural pests, but the use of irradiation, sex sorting, and the requirement to scale these insects make it costly to implement. pgSIT (precision guided Sterile Insect Technique) is a novel and efficient way to generate sterile males through genetic engineering and overcomes the drawbacks of traditional SIT. pgSIT has been implemented in multiple Dipteran insects, including D. suzukii, and has been shown to suppress wild insect populations. To further characterize sterile pgSIT males, we evaluated their fertility capacity, lack of mature sperm, and ability to induce a mating refractory period in D. suzukii wildtype females. In this study, we found that pgSIT sterile males do not produce mature sperm and can induce a refractory mating period in wildtype females. Furthermore, sperm DNA is not detected in the reproductive tracts of pgSIT-mated female reproductive tracts. These findings further support the penetrance of the pgSIT technology in D. suzukii and provide further supporting data to governing regulatory bodies in their evaluation of this technology., Competing Interests: Declarations. Competing interests: Authors affiliated with Agragene Inc. are employees of this company, which provided salary and other support for the project. Such employees may have shares or share options in Agragene Inc., (© 2025. The Author(s).)

In male animals, spermatogonia in testes differentiate into sperm, one of the most diverse cell types across species. Despite the evolutionary retention of key genes essential for spermatogenesis, the extent of their conservation remains unclear. To explore the genetic basis of spermatogenesis under strong selective pressure, we compare single-cell RNA sequencing (scRNA-seq) datasets from the testes of humans, mice, and fruit flies. Our analysis identifies conserved genes involved in key molecular programs, such as post-transcriptional regulation, meiosis, and energy metabolism. We perform gene knockout experiments of 20 candidate genes, three of which, when mutated in fruit flies, result in reduced male fertility, emphasizing the conservation of sperm centriole and steroid lipid processes across mammals and Drosophila. Additionally, deep-learning analysis uncovers potential transcriptional mechanisms driving gene-expression evolution. These findings establish a core genetic foundation for spermatogenesis, offering insights into sperm-phenotype evolution and the underlying mechanisms of male infertility., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

N 6 -methyladenosine (m 6 A) in eukaryotic RNA is an epigenetic modification that is critical for RNA metabolism, gene expression regulation, and the development of organisms. Aberrant expression of m 6 A components appears in a variety of human diseases. RNA m 6 A modification in Drosophila has proven to be involved in sex determination regulated by Sxl and may affect X chromosome expression through the MSL complex. The dosage-related effects under the condition of genomic imbalance (i.e. aneuploidy) are related to various epigenetic regulatory mechanisms. Here, we investigated the roles of RNA m 6 A modification in unbalanced genomes using aneuploid Drosophila . The results showed that the expression of m 6 A components changed significantly under genomic imbalance, and affected the abundance and genome-wide distribution of m 6 A, which may be related to the developmental abnormalities of aneuploids. The relationships between methylation status and classical dosage effect, dosage compensation, and inverse dosage effect were also studied. In addition, we demonstrated that RNA m 6 A methylation may affect dosage-dependent gene regulation through dosage-sensitive modifiers, alternative splicing, the MSL complex, and other processes. More interestingly, there seems to be a close relationship between MSL complex and RNA m 6 A modification. It is found that ectopically overexpressed MSL complex, especially the levels of H4K16Ac through MOF, could influence the expression levels of m 6 A modification and genomic imbalance may be involved in this interaction. We found that m 6 A could affect the levels of H4K16Ac through MOF, a component of the MSL complex, and that genomic imbalance may be involved in this interaction. Altogether, our work reveals the dynamic and regulatory role of RNA m 6 A modification in unbalanced genomes, and may shed new light on the mechanisms of aneuploidy-related developmental abnormalities and diseases., Competing Interests: SZ, RW, KL, SG, XL, JW, LZ, LS No competing interests declared, (© 2024, Zhang, Wang et al.)

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.)

Species that coexist in hybrid zones sexually isolate through reproductive character displacement, a mechanism that favours divergence between species. In Drosophila, behavioural and physiological traits discourage heterospecific mating between species. Recently, social network analysis revealed flies produce strain-specific and species-specific social structures. A gene, degrees of kevin bacon (dokb) has also been discovered that accounts for differences in social structures between flies. Why differences in social structures exist between drosophilids is currently unknown. Here we show through an experimental evolution study that six generations of selection in experimental sympatry led to the divergence of social structures measured in Drosophila pseudoobscura and Drosophila persimilis flies. We found that the frequency of hybrid offspring decreased within a few generations, suggesting social structures are associated with the sexual isolation of species. We also report increased species' differences in the concentration of the cuticular hydrocarbon 5, 9-pentacosadiene after six generations of selection. The mean concentration of this compound converged in female flies of both species and diverged in male flies of both species, suggesting a quantitative link between increased sexual dimorphism and sexual isolation. Our results suggest that chemical signals, together with social structures, increase the sexual isolation between species in hybrid zones., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Investigating the causes and consequences of niche partitioning in populations is a major goal in ecology and evolutionary biology. Previous studies have investigated genetic and environmentally induced variation in resource utility and their ecological implications. However, few studies have explored variability (non-genetic, stochastic variation) as a factor contributing to variation in resource utility. In this study, we studied the variability in foraging traits of Drosophila lutescens, a species of wild fruit fly. Using 70 iso-female lines from a single population, we observed two foraging traits, i.e., locomotive speed and resource preferences, in an "8"-shaped experimental arena containing different types of fruit juices. The mean locomotive speed and relative preference for orange juice over grape juice varied significantly among iso-female lines. Additionally, the degree of intraline variation (variability) was detected a fold-change of larger than 2-fold between the smallest line and the largest line. While the mean locomotive speed itself did not correlate with mean resource preferences, the variability of locomotive speed significantly correlated with that of resource preferences. These results suggest that the degree of variability within inbred lines for both locomotive activity and resource preference is potentially partly genetic and that a shared genetic basis may govern variability in these traits. The variability of a particular trait is considered to interact cooperatively with the variability of several other traits in creating phenotypic intraspecific variation within a population., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Hamamichi, Takahashi. 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.)

Anillin (Ani) is an evolutionarily conserved protein with a multi-domain structure that cross-links cytoskeletal proteins and plays an essential role in the formation of the contractile ring during cytokinesis. However, Ani is highly expressed in the human central nervous system (CNS), and it scaffolds myelin in the CNS of mice and modulates neuronal migration and growth in Caenorhabditis elegans. Although Ani is also highly expressed in the Drosophila CNS, its role remains unclear. In the present study, we showed that Ani is not only highly expressed in larval neuroblasts of the CNS, but also weakly expressed in the neuromuscular junction (NMJ) and axons. In addition, the ani knockdown in the nervous system led to pupal lethality, larval locomotor defects, and learning disability, along with abnormal morphology of the NMJ and distribution patterns of the mature neuropil in the CNS. These results show that Ani plays an important role also in the Drosophila nervous system., Competing Interests: Declaration of competing interest The authors declare no conflict of interests and competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The synaptonemal complex (SC) is a protein-rich structure essential for meiotic recombination and faithful chromosome segregation. Acting like a zipper to paired homologous chromosomes during early prophase I, the complex is a symmetrical structure where central elements are connected on two sides by the transverse filaments to the chromatin-anchoring lateral elements. Despite being found in most major eukaryotic taxa implying a deeply conserved evolutionary origin, several components of the complex exhibit unusually high rates of sequence turnover. This is puzzlingly exemplified by the SC of Drosophila, where the central elements and transverse filaments display no identifiable homologs outside of the genus. Here, we exhaustively examine the evolutionary history of the SC in Drosophila taking a comparative phylogenomic approach with high species density to circumvent obscured homology due to rapid sequence evolution. Contrasting starkly against other genes involved in meiotic chromosome pairing, SC genes show significantly elevated rates of coding evolution due to a combination of relaxed constraint and recurrent, widespread positive selection. In particular, the central element cona and transverse filament c(3)G have diversified through tandem and retro-duplications, repeatedly generating paralogs with novel germline activity. In a striking case of molecular convergence, c(3)G paralogs that independently arose in distant lineages evolved under positive selection to have convergent truncations to the protein termini and elevated testes expression. Surprisingly, the expression of SC genes in the germline is prone to change suggesting recurrent regulatory evolution which, in many species, resulted in high testes expression even though Drosophila males are achiasmic. Overall, our study recapitulates the poor conservation of SC components, and further uncovers that the lack of conservation extends to other modalities including copy number, genomic locale, and germline regulation. Considering the elevated testes expression in many Drosophila species and the common ancestor, we suggest that the activity of SC genes in the male germline, while still poorly understood, may be a prime target of constant evolutionary pressures driving repeated adaptations and innovations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Zakerzade 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.)

Enzyme-mediated modifications of tRNA, such as 5-methylcytosine (m5C) installed by nuclear-enriched NOP2/Sun RNA methyltransferase 2 (NSUN2), play a critical role in neuronal development and function. However, our understanding of these modifications' spatial installation and biological functions remains incomplete. In this study, we demonstrate that a nucleoplasm-localized G679R NSUN2 mutant, linked to intellectual disability, diminishes NSUN2-mediated tRNA m5C in human cell lines and Drosophila. Our findings indicate that inability of G679R-NSUN2 to install m5C is primarily attributed to its reduced binding to tRNA rather than its nucleoplasmic localization. Conversely, an NSUN2 variant lacking an internal intrinsically disordered region (ΔIDR-NSUN2) can install ∼80% m5C within the nucleoplasm. Furthermore, we show that tRNA m5C levels are positively correlated to cognitive performance in Drosophila, where expressing G679R-NSUN2 leads to the most severe social behavioral deficits while expressing ΔIDR-NSUN2 results in less pronounced deficits. This work illuminates the molecular mechanism underlying G679R disease mutation in cognitive function and offers valuable insights into the significance of the cellular localization of m5C installation on tRNA for neuronal function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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).)

Horizontal transfer of genetic material in eukaryotes has rarely been documented over short evolutionary timescales. Here, we show that two retrotransposons, Shellder and Spoink, invaded the genomes of multiple species of the melanogaster subgroup within the last 50 years. Through horizontal transfer, Spoink spread in D. melanogaster during the 1980s, while both Shellder and Spoink invaded D. simulans in the 1990s. Possibly following hybridization, D. simulans infected the island endemic species D. mauritiana (Mauritius) and D. sechellia (Seychelles) with both TEs after 1995. In the same approximate time-frame, Shellder also invaded D. teissieri, a species confined to sub-Saharan Africa. We find that the donors of Shellder and Spoink are likely American Drosophila species from the willistoni, cardini, and repleta groups. Thus, the described cascade of TE invasions could only become feasible after D. melanogaster and D. simulans extended their distributions into the Americas 200 years ago, likely aided by human activity. Our work reveals that cascades of TE invasions, likely initiated by human-mediated range expansions, could have an impact on the genomic and phenotypic evolution of geographically dispersed species. Within a few decades, TEs could invade many species, including island endemics, with distributions very distant from the donor of the TE., 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.)

The primary antiviral immunity in insects is mediated by the RNA interference (RNAi) pathway. To counteract this antiviral RNAi response, viruses employ virulence factors known as viral suppressors of RNAi (VSR). The question of whether host factors can activate a counter-counter-defense mechanism to cope with VSR-mediated RNA silencing suppression remains unanswered. In this study, cyclin-dependent kinase 12 (CDK12) was identified to interact with B2, a VSR of Flock House virus (FHV), and the critical amino acids responsible for dsRNA binding and dimerization in B2 were essential for this interaction. Silencing of CDK12 facilitated FHV RNA accumulation only in the context of B2, not for FHVΔB2. Notably, CDK12 abrogated the RNAi suppression exerted by B2. Furthermore, the knockdown of CDK12 inhibited the production of vsiRNAs in FHV-infected Drosophila cells. This study revealed that CDK12 mediated a counter-counter-defense strategy against VSR, thereby enhancing antiviral RNAi immunity in Drosophila .IMPORTANCEThe arms race between virus and host immunity is never-ending. This study enhances our understanding of antiviral defenses in insects by uncovering a novel counter-counter-defense mechanism against viral suppressors of RNA interference (VSRs). The RNA interference (RNAi) pathway serves as a primary antiviral response in insects, but viruses, such as Flock House virus (FHV), have evolved VSRs like B2 to disrupt this defense. Our research identifies cyclin-dependent kinase 12 (CDK12) as a critical host factor that interacts with the VSR B2. The discovery that CDK12 can counteract B2-mediated RNAi suppression and stimulate the production of viral small interfering RNAs (vsiRNAs) in FHV-infected Drosophila cells highlights its pivotal role in enhancing antiviral RNAi immunity. This study not only reveals a new dimension of host-virus interactions but also opens avenues for developing strategies to strengthen RNAi-based antiviral defenses., Competing Interests: The authors declare no conflict of interest.

Many biological systems operate near the physical limits to their performance, suggesting that aspects of their behavior and underlying mechanisms could be derived from optimization principles. However, such principles have often been applied only in simplified models. Here, we explore a detailed mechanistic model of the gap gene network in the Drosophila embryo, optimizing its 50+ parameters to maximize the information that gene expression levels provide about nuclear positions. This optimization is conducted under realistic constraints, such as limits on the number of available molecules. Remarkably, the optimal networks we derive closely match the architecture and spatial gene expression profiles observed in the real organism. Our framework quantifies the tradeoffs involved in maximizing functional performance and allows for the exploration of alternative network configurations, addressing the question of which features are necessary and which are contingent. Our results suggest that multiple solutions to the optimization problem might exist across closely related organisms, offering insights into the evolution of gene regulatory networks., Competing Interests: Competing interests statement:The authors declare no competing interest.

Spatial omics technologies have enabled the creation of intricate spatial maps that capture molecular features and tissue morphology, providing valuable insights into the spatial associations and functional organization of tissues. Accurate annotation of spot or domain types is essential for downstream spatial omics analyses, but this remains challenging. Therefore, this study aimed to develop a manually curated spatial omics database (SpatialRef, https://bio.liclab.net/spatialref/), to provide comprehensive and high-quality spatial omics data with known spot labels across multiple species. The current version of SpatialRef aggregates >9 million manually annotated spots across 17 Human, Mouse and Drosophila tissue types through extensive review and strict quality control, covering multiple spatial sequencing technologies and >400 spot/domain types from original studies. Furthermore, SpatialRef supports various spatial omics analyses about known spot types, including differentially expressed genes, spatially variable genes, Gene Ontology (GO)/KEGG annotation, spatial communication and spatial trajectories. With a user-friendly interface, SpatialRef facilitates querying, browsing and visualizing, thereby aiding in elucidating the functional relevance of spatial domains within the tissue and uncovering potential biological effects., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The design, analysis and mining of large-scale 'omics studies with the goal of advancing biological and biomedical understanding require use of a range of bioinformatics tools, including approaches tailored to needs specific to a given species and/or technology. The FlyRNAi database at the Drosophila RNAi Screening Center and Transgenic RNAi Project (DRSC/TRiP) Functional Genomics Resources (https://fgr.hms.harvard.edu/tools) supports an increasingly broad group of technologies and species. Recently, for example, we expanded the database to include additional new data-centric resources that facilitate mining and analysis of single-cell transcriptomics. In addition, we have applied our approaches to CRISPR reagent and gene-centric bioinformatics approaches in Drosophila to arthropod vectors of infectious diseases. Building on our previous comprehensive reports on the FlyRNAi database, here we focus on new and updated resources with a primary focus on data-centric tools. Altogether, our suite of online resources supports various stages of functional genomics studies for Drosophila and other arthropods, and facilitate a wide range of reagent design, analysis, data mining and analysis approaches by biologists and biomedical experts studying Drosophila, other common genetic model species, arthropod vectors and/or human biology., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 .

Chromatin immunoprecipitation (ChIP) is a common approach for studying the binding pattern of proteins on DNA sequences or the landscape of histones with different marks throughout the genome. ChIP is used on various organisms, including Drosophila This protocol provides a detailed overview of the immunoprecipitation portion of a ChIP procedure from samples of Drosophila nervous systems, specifically antennae and brains, that have already been fixed and sheared. These methods can be applied to other tissues of interest after optimizing for sample size and other relevant parameters., (© 2025 Cold Spring Harbor Laboratory Press.)

Chromatin immunoprecipitation (ChIP) is a technique used to study specific protein-DNA interaction. Briefly, in this technique, antibodies to proteins of interest are used to isolate regions of DNA where these proteins bind. ChIP samples can be processed and analyzed in different ways. One of the approaches for assessing the results of ChIP experiments is quantitative PCR (qPCR). qPCR is used to quantitatively measure the amount of DNA fragments that have been isolated, reflecting the signal of specific proteins interacting with these fragments. This protocol describes both the "percent input" method and the "fold enrichment" method for ChIP-qPCR data analysis using Drosophila tissues as an example. The "percent input" method measures signals of DNA fragments against the input measurement. In contrast, the "fold enrichment" method quantifies the amplified signal strength relative to a background control. Because the quality of primers is critical for the reliability of ChIP-qPCR results, this protocol also describes how to measure primer amplification efficiency using Drosophila genomic DNA., (© 2025 Cold Spring Harbor Laboratory Press.)

Inborn errors of selenoprotein expression arise from deleterious variants in genes encoding selenoproteins or selenoprotein biosynthetic factors, some of which are associated with neurodegenerative disorders. This study shows that bi-allelic selenocysteine tRNA-specific eukaryotic elongation factor (EEFSEC) variants cause selenoprotein deficiency, leading to progressive neurodegeneration. EEFSEC deficiency, an autosomal recessive disorder, manifests with global developmental delay, progressive spasticity, ataxia, and seizures. Cerebral MRI primarily demonstrated a cerebellar pathology, including hypoplasia and progressive atrophy. Exome or genome sequencing identified six different bi-allelic EEFSEC variants in nine individuals from eight unrelated families. These variants showed reduced EEFSEC function in vitro, leading to lower levels of selenoproteins in fibroblasts. In line with the clinical phenotype, an eEFSec-RNAi Drosophila model displays progressive impairment of motor function, which is reflected in the synaptic defects in this model organisms. This study identifies EEFSEC deficiency as an inborn error of selenocysteine metabolism. It reveals the pathophysiological mechanisms of neurodegeneration linked to selenoprotein metabolism, suggesting potential targeted therapies., Competing Interests: Declaration of interests B.B. is a co-founder, shareholder, and CTO of AIRAmed GmbH., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The chromatin state plays an important role in regulating gene expression, which affects organismal development and plasticity. Proteins, including transcription factors, chromatin modulatory proteins, and histone proteins, usually with modifications, interact with gene loci involved in cellular differentiation, function, and modulation. One molecular method used to characterize protein-DNA interactions is chromatin immunoprecipitation (ChIP). ChIP uses antibodies to immunoprecipitate specific proteins cross-linked to DNA fragments. This approach, in combination with quantitative PCR (qPCR) or high-throughput DNA sequencing, can determine the enrichment of a certain protein or histone modification around specific gene loci or across the whole genome. ChIP has been used in Drosophila to characterize the binding pattern of transcription factors and to elucidate the roles of regulatory proteins in gene expression during development and in response to environment stimuli. This review outlines ChIP procedures using tissues from the Drosophila nervous system as an example and discusses all steps and the necessary optimization., (© 2025 Cold Spring Harbor Laboratory Press.)

Chromatin immunoprecipitation (ChIP) is a well-characterized procedure used to reveal specific patterns of protein-DNA interactions and identify the binding sites of proteins on DNA. ChIP has been used to study many aspects of Drosophila biology, including neurobiology. This protocol describes in detail how to prepare cross-linked chromatin from Drosophila antennae and brains followed by immunoprecipitation (X-ChIP). We first describe tissue dissection, chromatin cross-linking with formaldehyde, quenching of the cross-linking, homogenization of tissues, and sonication for shearing the chromatin. Additionally, we describe how to optimize the sonication efficiency and fixation time and concentration using Drosophila brain samples as an example. These parameters are crucial for successful ChIP., (© 2025 Cold Spring Harbor Laboratory Press.)

After injury, severed axons separated from their somas activate programmed axon degeneration, a conserved pathway to initiate their degeneration within a day. Conversely, severed projections deficient in programmed axon degeneration remain morphologically preserved with functional synapses for weeks to months after axotomy. How this synaptic function is sustained remains currently unknown. Here, we show that dNmnat overexpression attenuates programmed axon degeneration in distinct neuronal populations. Severed projections remain morphologically preserved for weeks. When evoked, they elicit a postsynaptic behavior, a readout for preserved synaptic function. We used ribosomal pulldown to isolate the translatome from these projections 1 week after axotomy. Translatome candidates of enriched biological classes identified by transcriptional profiling are validated in a screen using a novel automated system to detect evoked antennal grooming as a proxy for preserved synaptic function. RNAi-mediated knockdown reveals that transcripts of the mTORC1 pathway, a mediator of protein synthesis, and of candidate genes involved in protein ubiquitination and Ca 2+ homeostasis are required for preserved synaptic function. Our translatome dataset also uncovers several uncharacterized Drosophila genes associated with human disease. It may offer insights into novel avenues for therapeutic treatments., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

In the Drosophila brain, neuronal diversity originates from approximately 100 neural stem cells, each dividing asymmetrically. Precise mapping of cell lineages at the single-cell resolution is crucial for understanding the mechanisms that direct neuronal specification. However, existing methods for high-resolution lineage tracing are notably time-consuming and labor-intensive. Here, we outline the best practices for lineage tracing using CLADES (cell lineage access driven by an edition sequence), a revolutionary approach to neuronal lineage tracing that addresses the limitations of previous methods. CLADES effectively traces the birth order of neurons using approximately 100 samples. The technique relies on a genetic cascade of reporter activations and deactivations that delineate lineage progression through color-coded markers. This system not only facilitates the detailed mapping of neuronal lineages but also holds the potential to be applied to tracking biological events and producing cell types for therapeutic purposes., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

Purpose: This study details a novel syndromic form of autosomal recessive intellectual disability resulting from recessive variants in GTF3C3, encoding a key component of the DNA-binding transcription factor IIIC, which has a conserved role in RNA polymerase III-mediated transcription., Methods: Exome sequencing, minigene analysis, molecular modeling, RNA polymerase III reporter gene assays, and Drosophila knockdown models were utilized to characterize GTF3C3 variants., Results: Twelve affected individuals from 7 unrelated families were identified with homozygous or compound heterozygous missense variants in GTF3C3 including c.503C>T p.(Ala168Val), c.1268T>C p.(Leu423Pro), c.1436A>G p.(Tyr479Cys), c.2419C>T p.(Arg807Cys), and c.2420G>A p.(Arg807His). The cohort presented with intellectual disability, variable nonfamilial facial features, motor impairments, seizures, and cerebellar/corpus callosum malformations. Consistent with disruptions in intra- and intermolecular interactions observed in molecular modeling, RNA polymerase III reporter assays confirmed that the majority of missense variants resulted in a loss of function. Minigene analysis of the recurrent c.503C>T p.(Ala168Val) variant confirmed the introduction of a cryptic donor site into exon 4, resulting in mRNA missplicing. Consistent with the clinical features of this cohort, neuronal loss of Gtf3c3 in Drosophila induced seizure-like behavior, motor impairment, and learning deficits., Conclusion: These findings confirm that GTF3C3 variants result in an autosomal recessive form of syndromic intellectual disability., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Single-cell Hi-C (scHi-C) is a collection of protocols for studying genomic interactions within individual cells. Although data analysis for scHi-C resembles data analysis for bulk Hi-C, the unique challenges of scHi-C, such as high noise and protocol-specific biases, require specialized data processing strategies. In this tutorial chapter, we focus on using pairtools, a suite of tools optimized for scHi-C data, demonstrating its application on a Drosophila snHi-C dataset. While centered on pairtools for snHi-C data, the principles outlined are applicable across scHi-C variants with minor adjustments. This educational chapter aims to guide researchers in using open-source tools for scHi-C analysis, emphasizing critical steps of contact pair extraction, detection of ligation junctions, filtration, and deduplication., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mitotic exit is a necessary step for highly specialized cells to terminally differentiate and acquire unique functions. The FUCCI system can be used to visualize mitotic and post-mitotic cells during development and regeneration in both live organisms and fixed tissues. Here we describe a Fly-FUCCI protocol for assaying mitotic exit in Drosophila embryos., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

G protein-coupled receptors (GPCRs) are essential cell surface proteins involved in transducing extracellular signals into intracellular responses, regulating various physiological processes. This study validated the use of the Tango assay, a sensitive method for detecting GPCR activation, in Drosophila Schneider 2 (S2) cells, focusing on the human Dopamine Receptor D4 (DRD4). Plasmids encoding the LexA-tagged human DRD4 receptor and a luciferase reporter were co-transfected into Drosophila S2 cells and stimulated with dopamine. Receptor activation was measured by quantifying the luciferase activity. The system showed high specificity for dopamine, with no activation in response to octopamine, a non-ligand for DRD4. Furthermore, the system effectively detects activation by a novel compound. These results demonstrate that Drosophila S2 cells, coupled with the Tango assay, provide a viable model for studying human GPCR function and ligand specificity. This system enables the rapid screening of potential GPCR ligands in a cost-effective cellular model.

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

A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf , that bind to N 6 methylated adenosines (m 6 A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR-Cas9 genome editing advance the efficient use of this technique for safer applications in humans., (© 2024 Haussmann et al.; Published by Cold Spring Harbor Laboratory Press.)

Visualizing RNA expression in the Drosophila epidermis during pupal development is challenging because the tissue is fragile during early pupal stages and increasingly impermeable at later stages. Here, we present a protocol for tissue dissection and detection of RNA in situ. We describe steps for using the hybridization chain reaction (HCR) in early and late Drosophila pupal stages (and larval imaginal discs). This protocol facilitates the study of dynamically changing patterns in gene expression during pupal development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genomic insulators are DNA elements that prevent transcriptional activation of a promoter by an enhancer when interposed. We present a protocol for insulator-seq that enables high-throughput screening of genomic insulators using a plasmid-based massively parallel reporter assay in Drosophila cultured cells. We describe steps for insulator reporter plasmid library generation, transient transfection into cultured cells, and sequencing library preparation and provide a pipeline for data analysis. For complete details on the use and execution of this protocol, please refer to Tonelli et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Drosophila establishes social clusters in groups, yet the underlying principles remain poorly understood. Here, we performed a systemic analysis of social network behavior (SNB) that quantifies individual social distance (SD) in a group over time. The SNB assessment in 175 inbred strains from the Drosophila Genetics Reference Panel showed a tight association of short SD with long developmental time, low food intake, and hypoactivity. The developmental inferiority in short-SD individuals was compensated by their group culturing. By contrast, developmental isolation silenced the beneficial effects of social interactions in adults and blunted the plasticity of SNB under physiological challenges. Transcriptome analyses revealed genetic diversity for SD traits, whereas social isolation reprogrammed select genetic pathways, regardless of SD phenotypes. In particular, social deprivation suppressed the expression of the neuropeptide Drosulfakinin ( Dsk ) in three pairs of adult brain neurons. Male-specific DSK signaling to cholecystokinin-like receptor 17D1 mediated the SNB plasticity. In fact, transgenic manipulations of the DSK neuron activity were sufficient to imitate the state of social experience. Given the functional conservation of mammalian Dsk homologs, we propose that animals may have evolved a dedicated neural mechanism to encode early-life experience and transform group properties adaptively., Competing Interests: JJ, KK, TG, JL, TK, CL No competing interests declared, (© 2024, Jeong, Kwon et al.)

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.

Background/objectives: Neural differentiation requires a multifaceted program to alter gene expression along the proliferation to the differentiation axis. While critical changes occur at the level of transcription, post-transcriptional mechanisms allow fine-tuning of protein output. We investigated the role of tRNAs in regulating gene expression during neural differentiation in Drosophila larval brains., Methods: We quantified tRNA abundance in neural progenitor-biased and neuron-biased brains using the hydrotRNA-seq method. These tRNA data were combined with cell type-specific mRNA decay measurements and transcriptome profiles in order to model how tRNA abundance affects mRNA stability and translation efficiency., Results: We found that (1) tRNA abundance is largely constant between neural progenitors and neurons but significant variation exists for 10 nuclear tRNA genes and 8 corresponding anticodon groups, (2) tRNA abundance correlates with codon-mediated mRNA decay in neuroblasts and neurons, but does not completely explain the different stabilizing or destabilizing effects of certain codons, and (3) changes in tRNA levels support a shift in translation optimization from a program supporting proliferation to a program supporting differentiation., Conclusions: These findings reveal coordination between tRNA expression and codon usage in transcripts that regulate neural development.

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.)

More than 40 years ago, studies of the Drosophila engrailed and Hox genes led to major discoveries that shaped the history of developmental biology. We learned that these genes define the state of determination of cells that populate particular spatially defined regions: the identity of segmental domains by Hox genes, and the identity of posterior developmental compartments by engrailed. Hence, the boundaries that delimit spatial domains depend on engrailed. Here, we review the engrailed field, which now includes orthologs in Drosophila and mouse, as well as many other animals. We focus on fly and mouse and highlight additional functions that span early stages of embryogenesis and neural development., 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.

Satellite DNAs are highly repetitive, tandemly arranged sequences, typically making up large portions (> 20%) of the eukaryotic genome. Most satDNAs are fast evolving and changes in their abundance and nucleotide composition may be related to genetic incompatibilities between species. Here, we used Illumina paired-end sequencing raw data and graph-based read-clustering with the TAREAN bioinformatic tool to study the satDNAs in two cactophilic neotropical cryptic species of Drosophila from the buzzatii cluster (repleta group), D. serido and D. antonietae, from five localities in Brazil. Both species share the same four families of satDNAs: pBuM, DBC-150, CDSTR138 and CDSTR230. They represent less than 4% of the genomic DNA and there are no large differences in the abundance of each satDNA between species. Despite not being the most abundant satDNA, CDSTR138 was found to be associated with most centromeres. All four satDNAs showed instances where repeats are more homogeneous within than between species, a phenomenon known as concerted evolution. On the other hand, there was no evidence for concerted evolution at the population level. Thus, these satDNAs may also be useful as potential markers for species identification. The low levels of satDNA differentiation (both quantitatively as qualitatively) between the two species might be among the reasons that allowed the establishment of a hybrid zone between the two species in the southern coast of Brazil., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethical approval: Not applicable., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Types A and B Niemann-Pick disease (NPD) are inherited multisystem lysosomal storage disorders due to mutations in the SMPD1 gene. Respiratory dysfunction is a key hallmark of NPD, yet the mechanism for this is underexplored. SMPD1 encodes acid sphingomyelinase (ASM), which hydrolyses sphingomyelin to ceramide and phosphocholine. Here, we present a Drosophila model of ASM loss-of-function, lacking the fly orthologue of SMPD1, dASM, modelling several aspects of the respiratory pathology of NPD. dASM is expressed in the late-embryonic fly respiratory network, the trachea, and is secreted into the tracheal lumen. Loss of dASM results in embryonic lethality, and the tracheal lumen fails to fill normally with gas prior to eclosion. We demonstrate that the endocytic clearance of luminal constituents prior to gas-filling is defective in dASM mutants, and is coincident with autophagic, but not lysosomal defects, in late stage embryonic trachea. Finally, we show that although bulk sphingolipids are unchanged, dietary loss of lipids in combination with genetic and pharmacological block of ceramide synthesis rescues the airway gas-filling defects. We highlight myriocin as a potential therapeutic drug for the treatment of the developmental respiratory defects associated with ASM deficiency, and present a new NPD model amenable to genetic and pharmacological screens., (© The Author(s) 2024. Published by Oxford University Press.)