Drosophila Imaginal Discs Research Articles

Enhanced expression of the myogenic factor Myocyte enhancer factor-2 in imaginal disc myoblasts activates a partial, but incomplete, muscle development program

The Myocyte enhancer factor-2 (MEF2) transcription factor plays a vital role in orchestrating muscle differentiation. While MEF2 cannot effectively induce myogenesis in naïve cells, it can potently accelerate myogenesis in mesodermal cells. This includes in Drosophila melanogaster imaginal disc myoblasts, where triggering premature muscle gene expression in these adult muscle progenitors has become a paradigm for understanding the regulation of the myogenic program. Here, we investigated the global consequences of MEF2 overexpression in the imaginal wing disc myoblasts, by combining RNA-sequencing with RT-qPCR and immunofluorescence. We observed the formation of sarcomere-like structures that contained both muscle and cytoplasmic myosin, and significant upregulation of muscle gene expression, especially genes essential for myofibril formation and function. These transcripts were functional since numerous myofibrillar proteins were detected in discs using immunofluorescence. Interestingly, muscle genes whose expression is restricted to the adult stages were not activated in these adult myoblasts. These studies confirm a broad activation of the myogenic program in response to MEF2 expression and suggest that additional regulatory factors are required for promoting the adult muscle-specific program. Our findings contribute to understanding the regulatory mechanisms governing muscle development and highlight the multifaceted role of MEF2 in orchestrating this intricate process.

The pleiotropic functions of Pri smORF peptides synchronize leg development regulators

The last decade witnesses the emergence of the abundant family of smORF peptides, encoded by small ORF (<100 codons), whose biological functions remain largely unexplored. Bioinformatic analyses here identify hundreds of putative smORF peptides expressed in Drosophila imaginal leg discs. Thanks to a functional screen in leg, we found smORF peptides involved in morphogenesis, including the pioneer smORF peptides Pri. Since we identified its target Ubr3 in the epidermis and pri was known to control leg development through poorly understood mechanisms, we investigated the role of Ubr3 in mediating pri function in leg. We found that pri plays several roles during leg development both in patterning and in cell survival. During larval stage, pri activates independently of Ubr3 tarsal transcriptional programs and Notch and EGFR signaling pathways, whereas at larval pupal transition, Pri peptides cooperate with Ubr3 to insure cell survival and leg morphogenesis. Our results highlight Ubr3 dependent and independent functions of Pri peptides and their pleiotropy. Moreover, we reveal that the smORF peptide family is a reservoir of overlooked developmental regulators, displaying distinct molecular functions and orchestrating leg development.

Dilp8 and its candidate receptor, Drl, are involved in the transdetermination of the Drosophila imaginal disc.

Drosophila imaginal disc cells can change their identity under stress conditions through transdetermination (TD). Research on TD can help elucidate the in vivo process of cell fate conversion. We previously showed that the overexpression of winged eye (wge) induces eye-to-wing TD in the eye disc and that an insulin-like peptide, Dilp8, is then highly expressed in the disc. Although Dilp8 is known to mediate systemic developmental delay via the Lgr3 receptor, its role in TD remains unknown. This study showed that Dilp8 is expressed in specific cells that do not express eye or wing fate markers during Wge-mediated TD and that the loss of Dilp8 impairs the process of eye-to-wing transition. Thus, Dilp8 plays a pivotal role in the cell fate conversion under wge overexpression. Furthermore, we found that instead of Lgr3, another candidate receptor, Drl, is involved in Wge-mediated TD and acts locally in the eye disc cells. We propose a model in which Dilp8-Drl signaling organizes cell fate conversion in the imaginal disc during TD.

Polycomb safeguards imaginal disc specification through control of the Vestigial-Scalloped complex.

A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are misregulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex, which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high-throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate.

Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration.

Epithelial repair relies on the activation of stress signaling pathways to coordinate tissue repair. Their deregulation is implicated in chronic wound and cancer pathologies. Using TNF-α/Eiger-mediated inflammatory damage to Drosophila imaginal discs, we investigate how spatial patterns of signaling pathways and repair behaviors arise. We find that Eiger expression, which drives JNK/AP-1 signaling, transiently arrests proliferation of cells in the wound center and is associated with activation of a senescence program. This includes production of the mitogenic ligands of the Upd family, which allows JNK/AP-1-signaling cells to act as paracrine organizers of regeneration. Surprisingly, JNK/AP-1 cell-autonomously suppress activation of Upd signaling via Ptp61F and Socs36E, both negative regulators of JAK/STAT signaling. As mitogenic JAK/STAT signaling is suppressed in JNK/AP-1-signaling cells at the center of tissue damage, compensatory proliferation occurs by paracrine activation of JAK/STAT in the wound periphery. Mathematical modelling suggests that cell-autonomous mutual repression between JNK/AP-1 and JAK/STAT is at the core of a regulatory network essential to spatially separate JNK/AP-1 and JAK/STAT signaling into bistable spatial domains associated with distinct cellular tasks. Such spatial stratification is essential for proper tissue repair, as coactivation of JNK/AP-1 and JAK/STAT in the same cells creates conflicting signals for cell cycle progression, leading to excess apoptosis of senescently stalled JNK/AP-1-signaling cells that organize the spatial field. Finally, we demonstrate that bistable separation of JNK/AP-1 and JAK/STAT drives bistable separation of senescent signaling and proliferative behaviors not only upon tissue damage, but also in RasV12, scrib tumors. Revealing this previously uncharacterized regulatory network between JNK/AP-1, JAK/STAT, and associated cell behaviors has important implications for our conceptual understanding of tissue repair, chronic wound pathologies, and tumor microenvironments.

RNAi screen in the Drosophila wing of genes encoding proteins related to cytoskeleton organization and cell division

Cell division and cytoskeleton organization are fundamental processes participating in the development of Drosophila imaginal discs. In this manuscript we describe the phenotypes in the adult fly wing generated by knockdowns of 85% of Drosophila genes encoding proteins likely related to the regulation of cell division and cytoskeleton organization. We also compile a molecular classification of these proteins into classes that describe their expected or known main biochemical characteristics, as well as mRNA expression in the wing disc and likely protein subcellular localization for a subset of these genes. Finally, we analyze in more detail one protein family of cytoskeleton genes (Arp2/3 complex), and define the consequences of interfering with cell division for wing growth and patterning.

Bilateral JNK activation is a hallmark of interface surveillance and promotes elimination of aberrant cells.

Tissue-intrinsic defense mechanisms eliminate aberrant cells from epithelia and thereby maintain the health of developing tissues or adult organisms. 'Interface surveillance' comprises one such distinct mechanism that specifically guards against aberrant cells which undergo inappropriate cell fate and differentiation programs. The cellular mechanisms which facilitate detection and elimination of these aberrant cells are currently unknown. We find that in Drosophila imaginal discs, clones of cells with inappropriate activation of cell fate programs induce bilateral JNK activation at clonal interfaces, where wild type and aberrant cells make contact. JNK activation is required to drive apoptotic elimination of interface cells. Importantly, JNK activity and apoptosis are highest in interface cells within small aberrant clones, which likely supports the successful elimination of aberrant cells when they arise. Our findings are consistent with a model where clone size affects the topology of interface contacts and thereby the strength of JNK activation in wild type and aberrant interface cells. Bilateral JNK activation is unique to 'interface surveillance' and is not observed in other tissue-intrinsic defense mechanisms, such as classical 'cell-cell competition'. Thus, bilateral JNK interface signaling provides an independent tissue-level mechanism to eliminate cells with inappropriate developmental fate but normal cellular fitness. Finally, oncogenic Ras-expressing clones activate 'interface surveillance' but evade elimination by bilateral JNK activation. Combined, our work establishes bilateral JNK interface signaling and interface apoptosis as a new hallmark of interface surveillance and highlights how oncogenic mutations evade tumor suppressor function encoded by this tissue-intrinsic surveillance system.

Distinct signaling signatures drive compensatory proliferation via S-phase acceleration.

Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, we investigate models of compensatory proliferation in Drosophila imaginal discs. We find that compensatory proliferation is associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models.

PTP10D-mediated cell competition is not obligately required for elimination of polarity-deficient clones.

Animal organs maintain tissue integrity and ensure removal of aberrant cells through several types of surveillance mechanisms. One prominent example is the elimination of polarity-deficient mutant cells within developing Drosophila imaginal discs. This has been proposed to require heterotypic cell competition dependent on the receptor tyrosine phosphatase PTP10D within the mutant cells. We report here experiments to test this requirement in various contexts and find that PTP10D is not obligately required for the removal of scribble (scrib) mutant and similar polarity-deficient cells. Our experiments used identical stocks with which another group can detect the PTP10D requirement, and our results do not vary under several husbandry conditions including high and low protein food diets. Although we are unable to identify the source of the discrepant results, we suggest that the role of PTP10D in polarity-deficient cell elimination may not be absolute.

Chromatin remodeler Dmp18 regulates apoptosis by controlling H2Av incorporation in Drosophila imaginal disc development.

Programmed Cell Death (PCD) or apoptosis is a highly conserved biological process and plays essential roles both in the development and stress context. In Drosophila, expression of pro-apoptotic genes, including reaper (rpr), head involution defective (hid), grim, and sickle (skl), is sufficient to induce cell death. Here, we demonstrate that the chromatin remodeler Dmp18, the homolog of mammalian Znhit1, plays a crucial role in regulating apoptosis in eye and wing development. We showed that loss of Dmp18 disrupted eye and wing development, up-regulated transcription of pro-apoptotic genes, and induced apoptosis. Inhibition of apoptosis suppressed the eye defects caused by Dmp18 deletion. Furthermore, loss of Dmp18 disrupted H2Av incorporation into chromatin, promoted H3K4me3, but reduced H3K27me3 modifications on the TSS regions of pro-apoptotic genes. These results indicate that Dmp18 negatively regulates apoptosis by mediating H2Av incorporation and histone H3 modifications at pro-apoptotic gene loci for transcriptional regulation. Our study uncovers the role of Dmp18 in regulating apoptosis in Drosophila eye and wing development and provides insights into chromatin remodeling regulating apoptosis at the epigenetic levels.

Elucidating the Role of Chmp1 Overexpression in the Transport of Polyamines in Drosophila melanogaster.

Polyamines are small organic cations that are essential for many biological processes such as cell proliferation and cell cycle progression. While the metabolism of polyamines has been well studied, the mechanisms by which polyamines are transported into and out of cells are poorly understood. Here, we describe a novel role of Chmp1, a vesicular trafficking protein, in the transport of polyamines using a well-defined leg imaginal disc assay in Drosophila melanogaster larvae. We show that Chmp1 overexpression had no effect on leg development in Drosophila, but does attenuate the negative impact on leg development of Ant44, a cytotoxic drug known to enter cells through the polyamine transport system (PTS), suggesting that the overexpression of Chmp1 downregulated the PTS. Moreover, we showed that the addition of spermine did not rescue the leg development in Chmp1-overexpressing leg discs treated with difluoromethylornithine (DFMO), an inhibitor of polyamine metabolism, while putrescine and spermidine did, suggesting that there may be unique mechanisms of import for individual polyamines. Thus, our data provide novel insight into the underlying mechanisms that are involved in polyamine transport and highlight the utility of the Drosophila imaginal disc assay as a fast and easy way to study potential players involved in the PTS.

The Gr64 cluster of gustatory receptors promotes survival and proteostasis of epithelial cells in Drosophila.

Gustatory Receptor 64 (Gr64) genes are a cluster of 6 neuronally expressed receptors involved in sweet taste sensation in Drosophila melanogaster. Gr64s modulate calcium signalling and excitatory responses to several different sugars. Here, we discover an unexpected nonneuronal function of Gr64 receptors and show that they promote proteostasis in epithelial cells affected by proteotoxic stress. Using heterozygous mutations in ribosome proteins (Rp), which have recently been shown to induce proteotoxic stress and protein aggregates in cells, we show that Rp/+ cells in Drosophila imaginal discs up-regulate expression of the entire Gr64 cluster and depend on these receptors for survival. We further show that loss of Gr64 in Rp/+ cells exacerbates stress pathway activation and proteotoxic stress by negatively affecting autophagy and proteasome function. This work identifies a noncanonical role in proteostasis maintenance for a family of gustatory receptors known for their function in neuronal sensation.

Single-Cell Atlas of the Drosophila Leg Disc Identifies a Long Non-Coding RNA in Late Development.

The Drosophila imaginal disc has been an excellent model for the study of developmental gene regulation. In particular, long non-coding RNAs (lncRNAs) have gained widespread attention in recent years due to their important role in gene regulation. Their specific spatiotemporal expressions further support their role in developmental processes and diseases. In this study, we explored the role of a novel lncRNA in Drosophila leg development by dissecting and dissociating w1118 third-instar larval third leg (L3) discs into single cells and single nuclei, and performing single-cell RNA-sequencing (scRNA-seq) and single-cell assays for transposase-accessible chromatin (scATAC-seq). Single-cell transcriptomics analysis of the L3 discs across three developmental timepoints revealed different cell types and identified lncRNA:CR33938 as a distal specific gene with high expression in late development. This was further validated by fluorescence in-situ hybridization (FISH). The scATAC-seq results reproduced the single-cell transcriptomics landscape and elucidated the distal cell functions at different timepoints. Furthermore, overexpression of lncRNA:CR33938 in the S2 cell line increased the expression of leg development genes, further elucidating its potential role in development.

Glial regenerative response in the imaginal discs of Drosophila melanogaster.

Glial regenerative response in the imaginal discs of Drosophila melanogaster.

Fluorescent labeling of genomic loci in Drosophila imaginal discs with heterologous DNA-binding proteins

SummaryUsing the Drosophila melanogaster Hox gene Ultrabithorax (Ubx) as an example, we demonstrate the use of three heterologous DNA-binding protein systems—LacI/LacO, ParB1/ParS1, and ParB2/ParS2—to label genomic loci in imaginal discs with the insertion of a small DNA tag. We compare each system, considering the impact of labeling in genomic regions (1) inside versus outside of a transcribed gene body and (2) with varying chromatin accessibility. We demonstrate the value of this system by interrogating the relationship between gene expression level and enhancer-promoter distance, as well as inter-allelic distance at the Ubx locus. We find that the distance between an essential intronic cis-regulatory element, anterobithorax (abx), and the promoter does not vary with expression level. In contrast, inter-allelic distance correlates with Ubx expression level.

Preparation of a Single-cell Suspension from Drosophila Wing Imaginal Discs.

The wing imaginal discs in Drosophila larvae are a pair of sac-like structures that later form the wings of the adult fly. During the past decades, wing discs have been used as a simple and accessible model system, for identifying genes and deciphering signaling cascades that play crucial roles in many aspects of development. In this protocol, we describe a simple method for preparing a cell suspension from wing discs (see Graphical abstract). This method can also be applied to the preparation of single-cell suspensions from other types of Drosophila tissues. When combined with genetic labeling, the dissociated cells are suitable for downstream analysis, such as flow cytometry. This method was recently used to isolate different populations of cells from Drosophila imaginal discs ( Yang et al., 2022 ). Graphical abstract: Procedures to prepare a single-cell suspension from Drosophila imaginal discs. Illustration of the main steps to dissect, temporarily store, and dissociate imaginal discs from Drosophila larvae. Refer to the Procedure section for detailed description of each step.

Cultivation and Live Imaging of Drosophila Imaginal Discs.

In this chapter, I present a method for the ex vivo cultivation and live imaging of Drosophila imaginal disc explants using low concentrations of the steroid hormone 20-hydroxyecdysone (20E). This method has been optimized for analyzing cellular dynamics during wing disc growth and leverages recent insights from in vivo experiments demonstrating that 20E is required for growth and patterning of the imaginal tissues. Using this protocol, we directly observe wing disc proliferation at a rapid rate for at least 13h during live imaging. The orientation of tissue growth is also consistent with that inferred from indirect in vivo techniques. Thus, this method provides an improved way of studying dynamic cellular processes and tissue movements during imaginal disc development. I first describe the preparation of the growthmedium and the dissection, and then I include a protocol for mounting and live imaging of the explants.

BTG1 Mutation Promotes Aggressive Lymphoma Development By Lowering the Threshold to MYC Activation and Generating “Super-Competitor” B Cells

BTG1 Mutation Promotes Aggressive Lymphoma Development By Lowering the Threshold to MYC Activation and Generating “Super-Competitor” B Cells

Ecdysone regulates the Drosophila imaginal disc epithelial barrier, determining the length of regeneration checkpoint delay.

Regeneration of Drosophila imaginal discs, larval precursors to adult tissues, activates a regeneration checkpoint that coordinates regenerative growth with developmental progression. This regeneration checkpoint results from the release of the relaxin-family peptide Dilp8 from regenerating imaginal tissues. Secreted Dilp8 protein is detected within the imaginal disc lumen, in which it is separated from its receptor target Lgr3, which is expressed in the brain and prothoracic gland, by the disc epithelial barrier. Here, we demonstrate that following damage the imaginal disc epithelial barrier limits Dilp8 signaling and the duration of regeneration checkpoint delay. We also find that the barrier becomes increasingly impermeable to the transepithelial diffusion of labeled dextran during the second half of the third instar. This change in barrier permeability is driven by the steroid hormone ecdysone and correlates with changes in localization of Coracle, a component of the septate junctions that is required for the late-larval impermeable epithelial barrier. Based on these observations, we propose that the imaginal disc epithelial barrier regulates the duration of the regenerative checkpoint, providing a mechanism by which tissue function can signal the completion of regeneration.

Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants.

Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, we analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, we found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. Our analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. Our findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted.