Drosophila Development Research Articles

Global coupling of R-loopdynamics with RNA polymerase II modulates gene expression and early development of Drosophila.

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.

SUMOylation of Warts kinase promotes neural stem cell reactivation

A delicate balance between neural stem cell (NSC) quiescence and proliferation is important for adult neurogenesis and homeostasis. Small ubiquitin-related modifier (SUMO)-dependent post-translational modifications cause rapid and reversible changes in protein functions. However, the role of the SUMO pathway during NSC reactivation and brain development is not established. Here, we show that the key components of the SUMO pathway play an important role in NSC reactivation and brain development in Drosophila. Depletion of SUMO/Smt3 or SUMO conjugating enzyme Ubc9 results in notable defects in NSC reactivation and brain development, while their overexpression leads to premature NSC reactivation. Smt3 protein levels increase with NSC reactivation, which is promoted by the Ser/Thr kinase Akt. Warts/Lats, the core protein kinase of the Hippo pathway, can undergo SUMO- and Ubc9-dependent SUMOylation at Lys766. This modification attenuates Wts phosphorylation by Hippo, leading to the inhibition of the Hippo pathway, and consequently, initiation of NSC reactivation. Moreover, inhibiting Hippo pathway effectively restores the NSC reactivation defects induced by SUMO pathway inhibition. Overall, our study uncovered an important role for the SUMO-Hippo pathway during Drosophila NSC reactivation and brain development.

Affinity hierarchies and amphiphilic proteins underlie the co-assembly of nucleolar and heterochromatin condensates.

Nucleoli are surrounded by Pericentromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. Nucleoli are the sites of ribosome synthesis, while the repeat-rich PCH is essential for chromosome segregation, genome stability, and transcriptional silencing. How and why these two distinct condensates co-assemble is unclear. Here, using high-resolution live imaging of Drosophila embryogenesis, we find that de novo establishment of PCH around the nucleolus is highly dynamic, transitioning from the nuclear edge to surrounding the nucleolus. Eliminating the nucleolus by removing the ribosomal RNA genes (rDNA) resulted in increased PCH compaction and subsequent reorganization into a toroidal structure. In addition, in embryos lacking rDNA, some nucleolar proteins were redistributed into new bodies or 'neocondensates', including enrichment in the PCH toroidal hole. Combining these observations with physical modeling revealed that nucleolar-PCH associations can be mediated by a hierarchy of interaction strengths between PCH, nucleoli, and 'amphiphilic' protein(s) that have affinities for both nucleolar and PCH components. We validated this model by identifying a candidate amphiphile, a DEAD-Box RNA Helicase called Pitchoune, whose depletion or mutation of its PCH interaction motif disrupted PCH-nucleolar associations. Together, this study unveils a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrates that nucleoli are required for normal PCH organization, and identifies Pitchoune as an amphiphilic molecular link required for PCH-nucleolar associations.

Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs.

Retinoblastoma tumor suppressor proteins (Rb) are highly conserved metazoan transcriptional corepressors involved in regulating the expression of thousands of genes. The vertebrate lineage and the Drosophila genus independently experienced an Rb gene duplication event, leading to the expression of several Rb paralogs whose unique and redundant roles in gene regulation remain to be fully explored. Here, we used a novel CRISPRi system in Drosophila to identify the significance of paralogy in the Rb family. We engineered dCas9 fusions to the fly Rbf1 and Rbf2 paralogs and deployed them to gene promoters in vivo, studying them in their native chromatin context. By directly querying the in vivo response of dozens of genes to Rbf1 and Rbf2 targeting, using both transcriptional as well as sensitive developmental readouts, we find that Rb paralogs function as "soft repressors" and have highly context-specific activities. Our comparison of targeting endogenous genes to reporter genes in cell culture identified striking differences in activity, underlining the importance of using CRISPRi effectors in a physiologically relevant context to identify paralog-specific activities. Our study uncovers the complexity of Rb-mediated transcriptional regulation in a living organism, and serves as a stepping stone for future CRISPRi development in Drosophila.

OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO’s role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5’-TAACNGT-3’ OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

OVO positively regulates essential maternal pathways by binding near the transcriptional start sites in the Drosophila female germline.

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO's role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5'-TAACNGT-3' OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

Muscle-fiber specific genetic manipulation of Drosophila sallimus severely impacts neuromuscular development, morphology, and physiology.

The ability of skeletal muscles to contract is derived from the unique genes and proteins expressed within muscles, most notably myofilaments and elastic proteins. Here we investigated the role of the sallimus (sls) gene, which encodes a structural homologue of titin, in regulating development, structure, and function of Drosophila melanogaster. Knockdown of sls using RNA interference (RNAi) in all body-wall muscle fibers resulted in embryonic lethality. A screen for muscle-specific drivers revealed a Gal4 line that expresses in a single larval body wall muscle in each abdominal hemisegment. Disrupting sls expression in single muscle fibers did not impact egg or larval viability nor gross larval morphology but did significantly alter the morphology of individual muscle fibers. Ultrastructural analysis of individual muscles revealed significant changes in organization. Surprisingly, muscle-cell specific disruption of sls also severely impacted neuromuscular junction (NMJ) formation. The extent of motor-neuron (MN) innervation along disrupted muscles was significantly reduced along with the number of glutamatergic boutons, in MN-Is and MN-Ib. Electrophysiological recordings revealed a 40% reduction in excitatory junctional potentials correlating with the extent of motor neuron loss. Analysis of active zone (AZ) composition revealed changes in presynaptic scaffolding protein (brp) abundance, but no changes in postsynaptic glutamate receptors. Ultrastructural changes in muscle and NMJ development at these single muscle fibers were sufficient to lead to observable changes in neuromuscular transduction and ultimately, locomotory behavior. Collectively, the data demonstrate that sls mediates critical aspects of muscle and NMJ development and function, illuminating greater roles for sls/titin.

In-vivo toxicological study of cysteamine modified carbon dots derived from Ruellia simplex on fruit fly for potential bioimaging

Modern medicine must shift its paradigm from conventional treatment strategies to theranostics to meet individuals' needs. Carbon dots, as a class of fluorescent materials, provide biocompatible and multifunctional solutions for a wide range of applications including clinical sensing, imaging, and drug delivery. Various studies have focused on their synthesis, photophysical properties, and innoxious nature but their potential biomedical applications for the in-vivo toxicity are yet limited. In this work, previously synthesized C-Dots (CDs) were utilized to determine their effect on the development of Drosophila melanogaster. Simultaneously, the genotoxic potential of CDs was evaluated on specific larval cell types that play important roles in immunological defence as well as growth and development. The gut organ toxicity of both CDs was studied using DAPI and DCFH-DA dyes wherein RS-CDs didn't show significant toxicity to the concentration 500 μg/mL whereas RS-Cys-CDs showed nuclear fragmentation and modest ROS (reactive oxygen species) production. Subsequently, trypan-blue assay, larvae crawling assay, touch sensitivity, adult phenotype, and survivability assay were performed. The trypan-blue assay shows the non-toxic nature of both CDs even at the concentration of 500 μg/mL. The high concentrations of RS-Cys-CD (500 μg/mL) were further associated with the alteration in touch behaviour and decrease in pupa hatching. In-vivo and ex-vivo fluorescence assessment of both the CDs exhibit bright fluorescence in green and red channels upon excitation at 485 and 577 nm respectively. The prominent imaging results from RS-Cys-CDs highlight the positive impact of surface modification.

The Hox Gene, abdominal A controls timely mitotic entry of neural stem cell and their growth during CNS development in Drosophila.

The size of a cell is important for its function and physiology. Interestingly, size variation can be easily observed in clonally derived embryonic and hematopoietic stem cells. Here, we investigated the regulation of stem cell growth and its association with cell fate. We observed heterogeneous sizes of neuroblasts or neural stem cells (NSCs) in the Drosophila ventral nerve cord (VNC). Specifically, thoracic NSCs were larger than those in the abdominal region of the VNC. Our research uncovered a significant role of the Hox gene abdominal A (abdA) in the regulation of abdominal NSC growth. Developmental expression of AbdA retards their growth and delays mitotic entry compared to thoracic NSCs. The targeted loss of abdA enhanced their growth and caused an earlier entry into mitosis with a faster cycling rate. Furthermore, ectopic expression of abdA reduced the size of thoracic NSCs and delayed their entry into mitosis. We suggest that abdA plays an instructive role in regulating NSC size and exit from quiescence. This study demonstrates for the first time the involvement of abdA in NSC fate determination by regulating their growth, entry into mitosis and proliferation rate, and thus their potential to make appropriate number of progeny for CNS patterning.

Genetic characterisation of candidate ecdysteroid kinases in Drosophila melanogaster.

Ecdysteroids are major hormones in insects and control moulting, growth, reproduction, physiology, and behaviour. The biosynthesis of ecdysteroids such as 20-hydroxyecdysone (20E) from dietary sterols is well characterised, but ecdysteroid catabolism is poorly understood. Ecdysteroid kinases (EcKs) mediate the reversible phosphorylation of ecdysteroids, which has been implicated in ecdysteroid recycling during embryogenesis and reproduction in various insects. However, to date only two EcK-encoding genes have been identified, in the silkworm Bombyx mori and the mosquito Anopheles gambiae. Previously, we identified two ecdysteroid kinase-like (EcKL) genes-Wallflower (Wall) and Pinkman (pkm)-in the model fruit fly Drosophila melanogaster that are orthologs of the ecdysteroid 22-kinase gene BmEc22K. Here, using gene knockdown, knockout and misexpression, we explore Wall and pkm's possible functions and genetically test the hypothesis that they encode EcKs. Wall and pkm null mutants are viable and fertile, suggesting they are not essential for development or reproduction, whereas phenotypes arising from RNAi and somatic CRISPR appear to derive from off-target effects or other artefacts. However, misexpression of Wall results in dramatic phenotypes, including developmental arrest, and defects in trachea, cuticle and pigmentation. Wall misexpression fails to phenocopy irreversible ecdysteroid catabolism through misexpression of Cyp18a1, suggesting Wall does not directly inactivate 20E. Additionally, Wall misexpression phenotypes are not attenuated in Cyp18a1 mutants, strongly suggesting Wall is not an ecdysteroid 26-kinase. We hypothesise that the substrate of Wall in this misexpression experiment and possibly generally is an unknown, atypical ecdysteroid that plays essential roles in Drosophila development, and may highlight aspects of insect endocrinology that are as-yet uncharacterised. We also provide preliminary evidence that CG5644 encodes an ecdysteroid 22-kinase conserved across Diptera.

A PRE loop at the dac locus acts as a topological chromatin structure that restricts and specifies enhancer-promoter communication.

Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function; it restricts enhancer-promoter communication and contributes to enhancer specificity.

The Enigmas of Tissue Closure: Inspiration from Drosophila.

Hollow structures are essential for development and physiological activity. The construction and maintenance of hollow structures never cease throughout the lives of multicellular animals. Epithelial tissue closure is the main strategy used by living organisms to build hollow structures. The high diversity of hollow structures and the simplicity of their development in Drosophila make it an excellent model for the study of hollow structure morphogenesis. In this review, we summarize the tissue closure processes in Drosophila that give rise to or maintain hollow structures and highlight the molecular mechanisms and distinct cell biology involved in these processes.

Day-night gene expression reveals circadian gene disco as a candidate for diel-niche evolution in moths.

Temporal ecological niche partitioning is an underappreciated driver of speciation. While insects have long been models for circadian biology, the genes and circuits that allow adaptive changes in diel-niches remain poorly understood. We compared gene expression in closely related day- and night-active non-model wild silk moths, with otherwise similar ecologies. Using an ortholog-based pipeline to compare RNA-Seq patterns across two moth species, we find over 25 pairs of gene orthologs showing differential expression. Notably, the gene disco, involved in circadian control, optic lobe and clock neuron development in Drosophila, shows robust adult circadian mRNA cycling in moth heads. Disco is highly conserved in moths and has additional zinc-finger domains with specific nocturnal and diurnal mutations. We propose disco as a candidate gene for the diversification of temporal diel-niche in moths.

Glycogen homeostasis and mtDNA expression require motor neuron to muscle TGFβ/Activin Signaling in Drosophila.

Maintaining metabolic homeostasis requires coordinated nutrient utilization between intracellular organelles and across multiple organ systems. Many organs rely heavily on mitochondria to generate (ATP) from glucose, or stored glycogen. Proteins required for ATP generation are encoded in both nuclear and mitochondrial DNA (mtDNA). We show that motoneuron to muscle signaling by the TGFβ/Activin family member Actβ positively regulates glycogen levels during Drosophila development. Remarkably, we find that levels of stored glycogen are unaffected by altering cytoplasmic glucose catabolism. Instead, Actβ loss reduces levels of mtDNA and nuclearly encoded genes required for mtDNA replication, transcription and translation. Direct RNAi mediated knockdown of these same nuclearly encoded mtDNA expression factors also results in decreased glycogen stores. Lastly, we find that expressing an activated form of the type I receptor Baboon in muscle restores both glycogen and mtDNA levels in actβ mutants, thereby confirming a direct link between Actβ signaling, glycogen homeostasis and mtDNA expression.

Exploring the expression of CG18269 under Zelda's regulation in early embryonic development of Drosophila melanogaster

Exploring the expression of CG18269 under Zelda's regulation in early embryonic development of Drosophila melanogaster

Redesigning the Drosophila histone gene cluster: an improved genetic platform for spatiotemporal manipulation of histone function.

Mutating replication-dependent (RD) histone genes is an important tool for understanding chromatin-based epigenetic regulation. Deploying this tool in metazoans is particularly challenging because RD histones in these organisms are typically encoded by many genes, often located at multiple loci. Such gene arrangements make the ability to generate homogenous histone mutant genotypes by site-specific gene editing quite difficult. Drosophila melanogaster provides a solution to this problem because the RD histone genes are organized into a single large tandem array that can be deleted and replaced with transgenes containing mutant histone genes. In the last ∼15 years several different RD histone gene replacement platforms were developed using this simple strategy. However, each platform contains weaknesses that preclude full use of the powerful developmental genetic capabilities available to Drosophila researchers. Here we describe the development of a newly engineered platform that rectifies many of these weaknesses. We used CRISPR to precisely delete the RD histone gene array (HisC), replacing it with a multifunctional cassette that permits site-specific insertion of either one or two synthetic gene arrays using selectable markers. We designed this cassette with the ability to selectively delete each of the integrated gene arrays in specific tissues using site-specific recombinases. We also present a method for rapidly synthesizing histone gene arrays of any genotype using Golden Gate cloning technologies. These improvements facilitate the generation of histone mutant cells in various tissues at different stages of Drosophila development and provide an opportunity to apply forward genetic strategies to interrogate chromatin structure and gene regulation.

Body plan evolvability: The role of variability in gene regulatory networks.

Recent computational modeling of early fruit fly (Drosophila) development has characterized the degree to which gene regulation networks can be robust to natural variability. In the first few hours of development, broad spatial gradients of maternally derived transcription factors activate embryonic gap genes. These gap patterns determine the subsequent segmented insect body plan through pair-rule gene expression. Gap genes are expressed with greater spatial precision than the maternal patterns. Computational modeling of the gap-gap regulatory interactions provides a mechanistic understanding for this robustness to maternal variability in wild-type (WT) patterning. A long-standing question in evolutionary biology has been how a system which is robust, such as the developmental program creating any particular species' body plan, is also evolvable, i.e. how can a system evolve or speciate, if the WT form is strongly buffered and protected? In the present work, we use the WT model to explore the breakdown of such Waddington-type 'canalization'. What levels of variability will push the system out of the WT form; are there particular pathways in the gene regulatory mechanism which are more susceptible to losing the WT form; and when robustness is lost, what types of forms are most likely to occur (i.e. what forms lie near the WT)? Manipulating maternal effects in several different pathways, we find a common gap 'peak-to-step' pattern transition in the loss of WT.We discuss these results in terms of the evolvability of insect segmentation, and in terms of experimental perturbations and mutations which could test the model predictions. We conclude by discussing the prospects for using continuum models of pattern dynamics to investigate a wider range of evo-devo problems.

Fat body-derived juvenile hormone acid methyltransferase functions to maintain iron homeostasis in Drosophila melanogaster.

Iron homeostasis is of critical importance to living organisms. Drosophila melanogaster has emerged as an excellent model to study iron homeostasis, while the regulatory mechanism of iron metabolism remains poorly understood. Herein, we accidently found that knockdown of juvenile hormone (JH) acid methyltransferase (Jhamt) specifically in the fat body, a key rate-limiting enzyme for JH synthesis, led to iron accumulation locally, resulting in serious loss and dysfunction of fat body. Jhamt knockdown-induced phenotypes were mitigated by iron deprivation, antioxidant and Ferrostatin-1, a well-known inhibitor of ferroptosis, suggesting ferroptosis was involved in Jhamt knockdown-induced defects in the fat body. Further study demonstrated that upregulation of Tsf1 and Malvolio (Mvl, homolog of mammalian DMT1), two iron importers, accounted for Jhamt knockdown-induced iron accumulation and dysfunction of the fat body. Mechanistically, Kr-h1, a key transcription factor of JH, acts downstream of Jhamt inhibiting Tsf1 and Mvl transcriptionally. In summary, the findings indicated that fat body-derived Jhamt is required for the development of Drosophila by maintaining iron homeostasis in the fat body, providing unique insight into the regulatory mechanisms of iron metabolism in Drosophila.

Evolution and development of Drosophila melanogaster under different thermal conditions affected cell sizes and sensitivity to paralyzing hypoxia

Environmental gradients cause evolutionary and developmental changes in the cellular composition of organisms, but the physiological consequences of these effects are not well understood. Here, we studied experimental populations of Drosophila melanogaster that had evolved in one of three selective regimes: constant 16 °C, constant 25 °C, or intergenerational shifts between 16 °C and 25 °C. Genotypes from each population were reared at three developmental temperatures (16 °C, 20.5 °C, and 25 °C). As adults, we measured thorax length and cell sizes in the Malpighian tubules and wing epithelia of flies from each combination of evolutionary and developmental temperatures. We also exposed flies from these treatments to a short period of nearly complete oxygen deprivation to measure hypoxia tolerance. For genotypes from any selective regime, development at a higher temperature resulted in smaller flies with smaller cells, regardless of the tissue. At every developmental temperature, genotypes from the warm selective regime had smaller bodies and smaller wing cells but had larger tubule cells than did genotypes from the cold selective regime. Genotypes from the fluctuating selective regime were similar in size to those from the cold selective regime, but their cells of either tissue were the smallest among the three regimes. Evolutionary and developmental treatments interactively affected a fly’s sensitivity to short-term paralyzing hypoxia. Genotypes from the cold selective regime were less sensitive to hypoxia after developing at a higher temperature. Genotypes from the other selective regimes were more sensitive to hypoxia after developing at a higher temperature. Our results show that thermal conditions can trigger evolutionary and developmental shifts in cell size, coupled with changes in body size and hypoxia tolerance. These patterns suggest links between the cellular composition of the body, levels of hypoxia within cells, and the energetic cost of tissue maintenance. However, the patterns can be only partially explained by existing theories about the role of cell size in tissue oxygenation and metabolic performance.

Long non-coding RNAs involved in Drosophila development and regeneration.

The discovery of functional long non-coding RNAs (lncRNAs) changed their initial concept as transcriptional noise. LncRNAs have been identified as regulators of multiple biological processes, including chromatin structure, gene expression, splicing, mRNA degradation, and translation. However, functional studies of lncRNAs are hindered by the usual lack of phenotypes upon deletion or inhibition. Here, we used Drosophila imaginal discs as a model system to identify lncRNAs involved in development and regeneration. We examined a subset of lncRNAs expressed in the wing, leg, and eye disc development. Additionally, we analyzed transcriptomic data from regenerating wing discs to profile the expression pattern of lncRNAs during tissue repair. We focused on the lncRNA CR40469, which is upregulated during regeneration. We generated CR40469 mutant flies that developed normally but showed impaired wing regeneration upon cell death induction. The ability of these mutants to regenerate was restored by the ectopic expression of CR40469. Furthermore, we found that the lncRNA CR34335 has a high degree of sequence similarity with CR40469 and can partially compensate for its function during regeneration in the absence of CR40469. Our findings point to a potential role of the lncRNA CR40469 in trans during the response to damage in the wing imaginal disc.