Synergistic activation by Glass and Pointed promotes neuronal identity in the Drosophila eye disc.

The integration of extrinsic signaling with cell-intrinsic transcription factors can direct progenitor cells to differentiate into distinct cell fates. In the developing Drosophila eye, differentiation of photoreceptors R1-R7 requires EGFR signaling mediated by the transcription factor Pointed, and our single-cell RNA-Seq analysis shows that the same photoreceptors require the eye-specific transcription factor Glass. We find that ectopic expression of Glass and activation of EGFR signaling synergistically induce neuronal gene expression in the wing disc in a Pointed-dependent manner. Targeted DamID reveals that Glass and Pointed share many binding sites in the genome of developing photoreceptors. Comparison with transcriptomic data shows that Pointed and Glass induce photoreceptor differentiation through intermediate transcription factors, including the redundant homologs Scratch and Scrape, as well as directly activating neuronal effector genes. Our data reveal synergistic activation of a multi-layered transcriptional network as the mechanism by which EGFR signaling induces neuronal identity in Glass-expressing cells.

Nuclear position and local acetyl-CoA production regulate chromatin state.

Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid ß-oxidation, which is also increased in the rim. Inhibition of fatty acid ß-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.

Epithelial monitoring through ligand-receptor segregation ensures malignant cell elimination.

Animals have evolved mechanisms, such as cell competition, to remove dangerous or nonfunctional cells from a tissue. Tumor necrosis factor signaling can eliminate clonal malignancies from Drosophila imaginal epithelia, but why this pathway is activated in tumor cells but not normal tissue is unknown. We show that the ligand that drives elimination is present in basolateral circulation but remains latent because it is spatially segregated from its apically localized receptor. Polarity defects associated with malignant transformation cause receptor mislocalization, allowing ligand binding and subsequent apoptotic signaling. This process occurs irrespective of the neighboring cells' genotype and is thus distinct from cell competition. Related phenomena at epithelial wound sites are required for efficient repair. This mechanism of polarized compartmentalization of ligand and receptor can generally monitor epithelial integrity to promote tissue homeostasis.

Multiview confocal super-resolution microscopy.

Confocal microscopy1 remains a major workhorse in biomedical optical microscopy owing to its reliability and flexibility in imaging various samples, but suffers from substantial point spread function anisotropy, diffraction-limited resolution, depth-dependent degradation in scattering samples and volumetric bleaching2. Here we address these problems, enhancing confocal microscopy performance from the sub-micrometre to millimetre spatial scale and the millisecond to hour temporal scale, improving both lateral and axial resolution more than twofold while simultaneously reducing phototoxicity. We achieve these gains using an integrated, four-pronged approach: (1) developing compact line scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; (2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; (3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labelled, thick samples; (4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than 20 distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae and adults; myoblasts in imaginal disks of Drosophila wings; and mouse renal, oesophageal, cardiac and brain tissues.

Drosophila TNFRs Grindelwald and Wengen bind Eiger with different affinities and promote distinct cellular functions.

The Drosophila tumour necrosis factor (TNF) ligand-receptor system consists of a unique ligand, Eiger (Egr), and two receptors, Grindelwald (Grnd) and Wengen (Wgn), and therefore provides a simple system for exploring the interplay between ligand and receptors, and the requirement for Grnd and Wgn in TNF/Egr-mediated processes. Here, we report the crystallographic structure of the extracellular domain (ECD) of Grnd in complex with Egr, a high-affinity hetero-hexameric assembly reminiscent of human TNF:TNFR complexes. We show that ectopic expression of Egr results in internalisation of Egr:Grnd complexes in vesicles, a step preceding and strictly required for Egr-induced apoptosis. We further demonstrate that Wgn binds Egr with much reduced affinity and is localised in intracellular vesicles that are distinct from those containing Egr:Grnd complexes. Altogether, our data provide insight into ligand-mediated activation of Grnd and suggest that distinct affinities of TNF ligands for their receptors promote different and non-redundant cellular functions.

Regulation of growth and cell fate during tissue regeneration by the two SWI/SNF chromatin-remodeling complexes of Drosophila.

To regenerate, damaged tissue must heal the wound, regrow to the proper size, replace the correct cell types, and return to the normal gene-expression program. However, the mechanisms that temporally and spatially control the activation or repression of important genes during regeneration are not fully understood. To determine the role that chromatin modifiers play in regulating gene expression after tissue damage, we induced ablation in Drosophila melanogaster imaginal wing discs, and screened for chromatin regulators that are required for epithelial tissue regeneration. Here, we show that many of these genes are indeed important for promoting or constraining regeneration. Specifically, the two SWI/SNF chromatin-remodeling complexes play distinct roles in regulating different aspects of regeneration. The PBAP complex regulates regenerative growth and developmental timing, and is required for the expression of JNK signaling targets and the growth promoter Myc. By contrast, the BAP complex ensures correct patterning and cell fate by stabilizing the expression of the posterior gene engrailed. Thus, both SWI/SNF complexes are essential for proper gene expression during tissue regeneration, but they play distinct roles in regulating growth and cell fate.

Exploiting Drosophila melanogaster Wing Imaginal Disc Eversion to Screen for New EMT Effectors.

In the early stages of Drosophila melanogaster (Drosophila) metamorphosis, a partial epithelial-mesenchymal transition (pEMT) takes place in the peripodial epithelium of wing imaginal discs. Blocking this pEMT results in adults with internalized wings and missing thoracic tissue. Using peripodial GAL4 drivers, GAL80ts temporal control, and UAS RNAi transgenes, one can use these phenotypes to screen for genes involved in the pEMT. Dominant modifier tests can then be employed to identify genetic enhancers and suppressors. To analyze a gene's role in the pEMT, one can then visualize peripodial cells in vivo at the time of eversion within the pupal case using live markers, and by dissecting, fixing, and immunostaining the prepupae. Alternatively, one can analyze the pEMT ex vivo by dissecting out wing discs and culturing them in the presence of ecdysone to induce eversion. This can provide a clearer view of the cellular processes involved and permit drug treatments to be easily applied.

Analysis of Epithelial Architecture and Planar Spindle Orientation in the Drosophila Wing Disc.

The Drosophila melanogaster wing imaginal disc is an epithelial sac that exhibits dramatic tissue growth during the larval stage. With its simple morphology and accessibility of genetic tools, studies using the wing disc have contributed to the understanding of the mechanisms of epithelial homeostasis including the control of mitotic spindle orientation. This chapter describes a detailed protocol for analyzing epithelial architecture and planar orientation of the mitotic spindle in the wing disc epithelium. The rapid dissection method, effective immunostaining, and mounting tips described here facilitate genetic and cell biological studies of the wing disc and can be applied to a wide array of studies using various Drosophila tissues.

Hippo signaling promotes Ets21c-dependent apical cell extrusion in the Drosophila wing disc.

Cell extrusion is a crucial regulator of epithelial tissue development and homeostasis. Epithelial cells undergoing apoptosis, bearing pathological mutations or possessing developmental defects are actively extruded toward elimination. However, the molecular mechanisms of Drosophila epithelial cell extrusion are not fully understood. Here, we report that activation of the conserved Hippo (Hpo) signaling pathway induces both apical and basal cell extrusion in the Drosophila wing disc epithelia. We show that canonical Yorkie targets Diap1, Myc and Cyclin E are not required for either apical or basal cell extrusion induced by activation of this pathway. Another target gene, bantam, is only involved in basal cell extrusion, suggesting novel Hpo-regulated apical cell extrusion mechanisms. Using RNA-seq analysis, we found that JNK signaling is activated in the extruding cells. We provide genetic evidence that JNK signaling activation is both sufficient and necessary for Hpo-regulated cell extrusion. Furthermore, we demonstrate that the ETS-domain transcription factor Ets21c, an ortholog of proto-oncogenes FLI1 and ERG, acts downstream of JNK signaling to mediate apical cell extrusion. Our findings reveal a novel molecular link between Hpo signaling and cell extrusion.

Hedgehog produced by the Drosophila wing imaginal disc induces distinct responses in three target tissues.

Hedgehog (Hh) is an evolutionarily conserved signaling protein that has essential roles in animal development and homeostasis. We investigated Hh signaling in the region of the Drosophila wing imaginal disc that produces Hh and is near the tracheal air sac primordium (ASP) and myoblasts. Hh distributes in concentration gradients in the anterior compartment of the wing disc, ASP and myoblasts, and activates genes in each tissue. Some targets of Hh signal transduction are common to the disc, ASP and myoblasts, whereas others are tissue-specific. Signaling in the three tissues is cytoneme-mediated and cytoneme-dependent. Some ASP cells project cytonemes that receive both Hh and Branchless (Bnl), and some targets regulated by Hh signaling in the ASP are also dependent on Bnl signal transduction. We conclude that the single source of Hh in the wing disc regulates cell type-specific responses in three discreet target tissues.

Determination of organ size: a need to focus on growth rate, not size.

The regulation of growth and the determination of organ-size in animals is an area of research that has received much attention during the past two and a half decades. Classic regeneration and cell-competition studies performed during the last century suggested that for size to be determined, organ-size is sensed and this sense of size feeds back into the growth control mechanism such that growth stops at the "correct" size. Recent work using Drosophila imaginal discs as a system has provided a particularly detailed cellular and molecular understanding of growth. Yet, a clear mechanistic basis for size-sensing has not emerged. I re-examine these studies from a different perspective and ask whether there is scope for alternate modes of size control in which size does not need to be sensed.

Drosophila as a model to understand autophagy deregulation in human disorders.

Autophagy has important functions in normal physiology to maintain homeostasis and protect against cellular stresses by the removal of harmful cargos such as dysfunctional organelles, protein aggregates and invading pathogens. The deregulation of autophagy is a hallmark of many diseases and therapeutic targeting of autophagy is highly topical. With the complex role of autophagy in disease it is essential to understand the genetic and molecular basis of the contribution of autophagy to pathogenesis. The model organism, Drosophila, provides a genetically amenable system to dissect out the contribution of autophagy to human disease models. Here we review the roles of autophagy in human disease and how autophagy studies in Drosophila have contributed to the understanding of pathophysiology.

FGF signaling promotes myoblast proliferation through activation of wingless signaling.

Indirect flight muscles (IFMs) are the largest muscles in Drosophila and are made up of hundreds of myonuclei. The generation of these giant muscles requires a large pool of wing disc associated adult muscle precursors (AMPs), however the factors that control proliferation to form this myoblast pool are incompletely known. Here, we examine the role of fibroblast growth factor (FGF) signaling in the proliferation of wing disc associated myoblasts. We find that the components of FGF signaling are expressed in myoblasts and surrounding epithelial cells of the wing disc. Next, we show that attenuation of FGF signaling results in a diminished myoblast pool. This reduction in the pool size is due to decreased myoblast proliferation. By contrast, activating the FGF signaling pathway increases the myoblast pool size and restores the proliferative capacity of FGF knockdown flies. Finally, our results demonstrate that the FGF receptor Heartless acts through up-regulating ß-catenin/Armadillo signaling to promote myoblast proliferation. Our studies identify a novel role for FGF signaling during IFM formation and uncover the mechanism through which FGF coordinates with Wingless signaling to promote myoblast proliferation.

Rif1 Functions in a Tissue-Specific Manner To Control Replication Timing Through Its PP1-Binding Motif.

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

An RNAi screen for secreted factors and cell-surface players in coordinating neuron and glia development in Drosophila.

The establishment of the functional nervous system requires coordinated development of neurons and glia in the embryo. Our understanding of underlying molecular and cellular mechanisms, however, remains limited. The developing Drosophila visual system is an excellent model for understanding the developmental control of the nervous system. By performing a systematic transgenic RNAi screen, we investigated the requirements of secreted proteins and cell-surface receptors for the development of photoreceptor neurons (R cells) and wrapping glia (WG) in the Drosophila visual system. From the screen, we identified seven genes whose knockdown disrupted the development of R cells and/or WG, including amalgam (ama), domeless (dome), epidermal growth factor receptor (EGFR), kuzbanian (kuz), N-Cadherin (CadN), neuroglian (nrg), and shotgun (shg). Cell-type-specific analysis revealed that ama is required in the developing eye disc for promoting cell proliferation and differentiation, which is essential for the migration of glia in the optic stalk. Our results also suggest that nrg functions in both eye disc and WG for coordinating R-cell and WG development.

Single cell transcriptomic landscapes of pattern formation, proliferation and growth in Drosophila wing imaginal discs.

Organ formation relies on the orchestration of pattern formation, proliferation and growth during development. How these processes are integrated at the individual cell level remains unclear. In the past decades, studies using Drosophila wing imaginal discs as a model system have provided valuable insights into pattern formation, growth control and regeneration. Here, we provide single cell transcriptomic landscapes of pattern formation, proliferation and growth of wing imaginal discs. We found that patterning information is robustly maintained in the single cell transcriptomic data and can provide reference matrices for computationally mapping single cells into discrete spatial domains. Assignment of wing disc single cells to spatial subregions facilitates examination of patterning refinement processes. We also clustered single cells into different proliferation and growth states and evaluated the correlation between cell proliferation/growth states and spatial patterning. Furthermore, single cell transcriptomic analyses allowed us to quantitatively examine disturbances of differentiation, proliferation and growth in a well-established tumor model. We provide a database to explore these datasets at http://drosophilayanlab-virtual-wingdisc.ust.hk:3838/v2/This article has an associated 'The people behind the papers' interview.

Dysfunction of LSD-1 induces JNK signaling pathway-dependent abnormal development of thorax and apoptosis cell death in Drosophila melanogaster.

Perilipins are evolutionarily conserved from insects to mammals. Lipid storage droplet-1 (LSD-1) is a member of the lipid droplet's surface-binding protein family and counterpart to mammalian perilipin 1. The role of LSD-1 has already been reported in lipid metabolism of Drosophila. However, the function of this gene during specific tissue development is still under investigation. Here, we found that LSD-1 is expressed in the notum of the wing imaginal disc, and notum-specific knockdown of Lsd-1 by pannir-GAL4 driver leads to split thorax phenotype in adults, suggesting an essential role of LSD-1 in development of Drosophila thorax. As overexpression of JNK homolog, bsk (basket) suppresses Lsd-1 knockdown phenotype, the role of LSD-1 in thorax development was proved to be dependent on the activity of the Drosophila c-Jun N-terminal kinase (JNK). The puckered (puc) expression led to significant decrease in the JNK activity in wing discs of Lsd-1 knockdown flies. In addition, we also detected that depletion of Lsd-1 enhances apoptotic cell death in the wing notum area. Taken together, these data demonstrated that LSD-1 functions in Drosophila thorax development by regulating JNK pathway.

Drosophila FGF cleavage is required for efficient intracellular sorting and intercellular dispersal.

How morphogenetic signals are prepared for intercellular dispersal and signaling is fundamental to the understanding of tissue morphogenesis. We discovered an intracellular mechanism that prepares Drosophila melanogaster FGF Branchless (Bnl) for cytoneme-mediated intercellular dispersal during the development of the larval Air-Sac-Primordium (ASP). Wing-disc cells express Bnl as a proprotein that is cleaved by Furin1 in the Golgi. Truncated Bnl sorts asymmetrically to the basal surface, where it is received by cytonemes that extend from the recipient ASP cells. Uncleavable mutant Bnl has signaling activity but is mistargeted to the apical side, reducing its bioavailability. Since Bnl signaling levels feedback control cytoneme production in the ASP, the reduced availability of mutant Bnl on the source basal surface decreases ASP cytoneme numbers, leading to a reduced range of signal/signaling gradient and impaired ASP growth. Thus, enzymatic cleavage ensures polarized intracellular sorting and availability of Bnl to its signaling site, thereby determining its tissue-specific intercellular dispersal and signaling range.

Developmental regulation of regenerative potential in Drosophila by ecdysone through a bistable loop of ZBTB transcription factors.

In many organisms, the regenerative capacity of tissues progressively decreases as development progresses. However, the developmental mechanisms that restrict regenerative potential remain unclear. In Drosophila, wing imaginal discs become unable to regenerate upon damage during the third larval stage (L3). Here, we show that production of ecdysone after larvae reach their critical weight (CW) terminates the window of regenerative potential by acting on a bistable loop composed of two antagonistic Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (ZBTB) genes: chinmo and broad (br). Around mid L3, ecdysone signaling silences chinmo and activates br to switch wing epithelial progenitors from a default self-renewing to a differentiation-prone state. Before mid L3, Chinmo promotes a strong regenerative response upon tissue damage. After mid L3, Br installs a nonpermissive state that represses regeneration. Transient down-regulation of ecdysone signaling or Br in late L3 larvae enhances chinmo expression in damaged cells that regain the capacity to regenerate. This work unveils a mechanism that ties the self-renewing and regenerative potential of epithelial progenitors to developmental progression.

An ADAMTS Sol narae is required for cell survival in Drosophila.

Cell survival is essential for all living organisms to cope against multiple environmental insults. Intercellular signaling between dying and surviving cells plays an important role to ensure compensatory proliferation, preventing tissue loss after environmental stresses. Here, we show that Sol narae (Sona), a Disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) in Drosophila is required for cell survival. sona exhibited a positive genetic interaction with Death-associated inhibitor of apoptosis 1 (Diap1), and a negative genetic interaction with reaper (rpr). Transcription patterns of sona, Diap1, and rpr genes in the pouch region of wing discs were coordinately changed after irradiation. Interestingly, there was a negative correlation in the expression levels of Sona and DIAP1, and both cell types, one with high Sona level and the other with high Diap1 level, were resistant to irradiation-induced cell death. The sona-expressing cells rarely entered into cell cycle themselves but promoted the nearby cells to proliferate in irradiation conditions. We found that these sona-expressing cells are able to upregulate Cyclin D (Cyc D) and increase tissue size. Furthermore, transient Sona overexpression increased survival rate and promoted development of flies in irradiation conditions. We propose that the two types of radiation-resistant cells, one with high Sona level and the other with high Diap1 level, communicate with dying cells and between each other for cell survival and proliferation in response to irradiation.