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The aryl hydrocarbon receptor (AHR) plays important roles in intestinal homeostasis, limiting tumour growth and promoting differentiation in the intestinal epithelium. Spineless, theDrosophilahomolog of AHR, has only been studied in the context of development but not in the adult intestine. Here, we show thatspinelessis upregulated in the adult intestinal epithelium after infection withPseudomonas entomophila(P.e.). Spineless knockdown increased stem cell proliferation following infection-induced injury. Spineless overexpression limited intestinal stem cell proliferation and reduced survival after infection. In two tumour models, using eitherNotchRNAi or constitutively active Yorkie, Spineless suppressed tumour growth and doubled the lifespan of tumour-bearing flies. At the transcriptional level it reversed the gene expression changes induced in Yorkie tumours, counteracting cell proliferation and altered metabolism. These findings demonstrate a new role for Spineless in the adultDrosophilamidgut and highlight the evolutionarily conserved functions of AHR/Spineless in the control of proliferation and differentiation of the intestinal epithelium.

SUMMARYHow biological form emerges from cell fate decisions and tissue remodelling is a fundamental question in development biology. However, an understanding of how these processes operate side-by-side to set precise and robust patterns is largely missing. Here, we investigate this interplay during the process of vein refinement in theDrosophilapupal wing. By following reporters of signalling activity dynamically, together with tissue flows, we show that longitudinal vein refinement arises from a combination of local tissue deformation and cell fate adjustments controlled by a signalling network involving Notch, Dpp, and EGFR. Perturbing large-scale convergence and extension tissue flows does not affect vein refinement, showing that pre-patterned vein domains are able to intrinsically refine to the correct width. A minimal biophysical description taking into account key signalling interactions recapitulates the intrinsic tissue ability to establish a thin, regular vein independently of large-scale tissue flows. Supporting this prediction, artificial proveins optogenetically generated orthogonal to the axis of wing elongation refine against large-scale flows. Overall, we find that signalling-mediated updating of cell fate is a key contributor to reproducible patterning.

Steroid hormones control various cellular activities in a context-dependent manner. For example, ecdysone, which acts through a type II nuclear receptor, has seemingly opposite effects in Drosophila wing precursors, promoting proliferation during larval stages, and triggering proliferation arrest at pupariation. We find that wing precursors proliferate normally in the complete absence of the ecdysone receptor (EcR), whether ecdysone is present or not, suggesting that ecdysone overrides a default antiproliferative activity of the receptor. By contrast, termination of proliferation by high concentration of 20E at the end of larval life involves conventional gene regulation by the ligand-receptor complex. The switch from one mode of regulation to the other is determined by ligand level, as measured with a calibrated EcR transcriptional reporter andex vivoproliferation assays. Accordingly, RNA Seq analysis uncovers distinct transcriptional responses to different doses of ecdysone. Some genes are only activated at high doses (high threshold targets) and likely to comprise genes that stop proliferation at pupariation, when ecdysone titres are high. We find that other target genes respond to all physiological concentrations of ecdysone. Some of these genes are known to promote proliferation and could therefore contribute to the pro-proliferation activity of low-level ecdysone. Finally, we show mathematically and with synthetic reporters that relatively simple combinations of regulatory elements can recapitulate the behaviour of both types of target genes.

Regulation of the Wnt signaling pathway is critically important for a number of cellular processes in both development and adult mammalian biology. This Perspective will provide a summary of current and emerging therapeutic opportunities in modulating Wnt signaling, especially through inhibition of Notum carboxylesterase activity. Notum was recently shown to act as a negative regulator of Wnt signaling through the removal of an essential palmitoleate group. Inhibition of Notum activity may represent a new approach to treat disease where aberrant Notum activity has been identified as the underlying cause. Reliable screening technologies are available to identify inhibitors of Notum, and structural studies are accelerating the discovery of new inhibitors. A selection of these hits have been optimized to give fit-for-purpose small molecule inhibitors of Notum. Three noteworthy examples are LP-922056 (26), ABC99 (27), and ARUK3001185 (28), which are complementary chemical tools for exploring the role of Notum in Wnt signaling.

Cells within developing tissues rely on morphogens to assess positional information. Passive diffusion is the most parsimonious transport model for long-range morphogen gradient formation but does not, on its own, readily explain scaling, robustness and planar transport. Here, we argue that diffusion is sufficient to ensure robust morphogen gradient formation in a variety of tissues if the interactions between morphogens and their extracellular binders are considered. A current challenge is to assess how the affinity for extracellular binders, as well as other biophysical and cell biological parameters, determines gradient dynamics and shape in a diffusion-based transport system. Technological advances in genome editing, tissue engineering, live imaging and in vivo biophysics are now facilitating measurement of these parameters, paving the way for mathematical modelling and a quantitative understanding of morphogen gradient formation and modulation.

Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human disease such as colorectal cancer and Alzheimer’s disease supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we describe the discovery and profile of 8l (ARUK3001185) as a potent, selective and brain pentrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identifed 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases and drug targets.

Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of the role Notum plays in disease, supporting the need to discover new small-molecule inhibitors. A crystallographic X-ray fragment screen was performed, which identified fragment hit 1,2,3-triazole 7 as an attractive starting point for a structure-based drug design hit-to-lead program. Optimization of 7 identified oxadiazol-2-one 23dd as a preferred example with properties consistent with drug-like chemical space. Screening 23dd in a cell-based TCF/LEF reporter gene assay restored the activation of Wnt signaling in the presence of Notum. Mouse pharmacokinetic studies with oral administration of 23dd demonstrated good plasma exposure and partial blood-brain barrier penetration. Significant progress was made in developing fragment hit 7 into lead 23dd (>600-fold increase in activity), making it suitable as a new chemical tool for exploring the role of Notum-mediated regulation of Wnt signaling.

Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer's disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.

As organs and tissues approach their normal size during development or regeneration, growth slows down, and cell proliferation progressively comes to a halt. Among the various processes suggested to contribute to growth termination,1-10 mechanical feedback, perhaps via adherens junctions, has been suggested to play a role.11-14 However, since adherens junctions are only present in a narrow plane of the subapical region, other structures are likely needed to sense mechanical stresses along the apical-basal (A-B) axis, especially in a thick pseudostratified epithelium. This could be achieved by nuclei, which have been implicated in mechanotransduction in tissue culture.15 In addition, mechanical constraints imposed by nuclear crowding and spatial confinement could affect interkinetic nuclear migration (IKNM),16 which allows G2 nuclei to reach the apical surface, where they normally undergo mitosis.17-25 To explore how mechanical constraints affect IKNM, we devised an individual-based model that treats nuclei as deformable objects constrained by the cell cortex and the presence of other nuclei. The model predicts changes in the proportion of cell-cycle phases during growth, which we validate with the cell-cycle phase reporter FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator).26 However, this model does not preclude indefinite growth, leading us to postulate that nuclei must migrate basally to access a putative basal signal required for S phase entry. With this refinement, our updated model accounts for the observed progressive slowing down of growth and explains how pseudostratified epithelia reach a stereotypical thickness upon completion of growth.

The Wnt signaling suppressor Notum is a promising target for osteoporosis, Alzheimer's disease, and colorectal cancers. To develop novel Notum inhibitors, we used an X-ray crystallographic fragment screen with the Diamond-SGC Poised Library (DSPL) and identified 59 fragment hits from the analysis of 768 data sets. Fifty-eight of the hits were found bound at the enzyme catalytic pocket with potencies ranging from 0.5 to >1000 μM. Analysis of the fragments' diverse binding modes, enzymatic inhibitory activities, and chemical properties led to the selection of six hits for optimization, and five of these resulted in improved Notum inhibitory potencies. One hit, 1-phenyl-1,2,3-triazole 7, and its related cluster members, have shown promising lead-like properties. These became the focus of our fragment development activities, resulting in compound 7d with IC50 0.0067 μM. The large number of Notum fragment structures and their initial optimization provided an important basis for further Notum inhibitor development.

Summary Deciphering gene function requires the ability to control gene expression in space and time. Binary systems such as the Gal4/UAS provide a powerful means to modulate gene expression and to induce loss or gain of function. This is best exemplified in Drosophila, where the Gal4/UAS system has been critical to discover conserved mechanisms in development, physiology, neurobiology, and metabolism, to cite a few. Here we describe a transgenic light-inducible Gal4/UAS system (ShineGal4/UAS) based on Magnet photoswitches. We show that it allows efficient, rapid, and robust activation of UAS-driven transgenes in different tissues and at various developmental stages in Drosophila. Furthermore, we illustrate how ShineGal4 enables the generation of gain and loss-of-function phenotypes at animal, organ, and cellular levels. Thanks to the large repertoire of UAS-driven transgenes, ShineGal4 enriches the Drosophila genetic toolkit by allowing in vivo control of gene expression with high temporal and spatial resolutions., Graphical abstract, Highlights • ShineGal4 is a Drosophila transgenic optogenetic Gal4 system • It allows rapid and robust light activation of UAS reporters in various tissues • It can be actuated in given regions of interest or enhancer-defined patterns • ShineGal4 enables the exploration of gene function with high spatiotemporal resolution, di Pietro et al. develop a GAL4/UAS optogenetic system that allows fast and efficient control of gene expression. This enables the investigation of gene function with high spatiotemporal resolution in Drosophilain vivo.

Summary This one-step method generates, for any locus, a conditional knockout allele in Drosophila. The allele carries a bright fluorescent marker for easy screening and an attP landing site for subsequent genetic manipulations. After removing the selectable marker with Cre, the attP site can be used to insert DNA fragments expressing tagged or mutant alleles to determine protein localization and function in a tissue- and stage-specific manner. Only a single round of CRISPR-Cas9-mediated editing is required. For complete details on the use and execution of this protocol, please refer to the DWnt4[cKO] example in Yu et al. (2020)., Graphical abstract, Highlights • One-step CRISPR-Cas9 protocol to generate conditional knockout alleles in Drosophila • Facile screening with a removeable fluorescent marker • Integrase site included to enable subsequent “plug&play” manipulations, This one-step method generates, for any locus, a conditional knockout allele in Drosophila. The allele carries a bright fluorescent marker for easy screening and an attP landing site for subsequent genetic manipulations. After removing the selectable marker with Cre, the attP site can be used to insert DNA fragments expressing tagged or mutant alleles to determine protein localization and function in a tissue- and stage-specific manner. Only a single round of CRISPR-Cas9-mediated editing is required.

Funding Information: Acknowledgements We thank the Core Services and Advanced Technologies at the Cancer Research UK Beatson Institute (C596/A17196 and A31287), and particularly the Biological Services Unit, Histology Service and Molecular Technologies; members of the Sansom and Katajisto laboratories for discussions of the data and manuscript; and BRC Oxford for supplying patient material. O.J.S. and his laboratory members were supported by Cancer Research UK (A28223, A21139, A12481 and A17196). D.J.F. and M.C.H. were supported by the UK Medical Research Council (MR/R017247/1 and MR/J50032X/1, respectively). SpecifiCancer CRUK Grand Challenge (C7932/A29055) is funded by Cancer Research UK and the Mark Foundation for Cancer Research. P.K. and his laboratory members were supported by the Academy of Finland Centre of Excellence MetaStem (266869, 304591 and 320185), the ERC Starting Grant 677809, the Swedish Research Council 2018-03078, the Cancerfonden 190634, the Jane and Aatos Erkko Foundation and the Cancer Foundation Finland. N.P. was supported by the Finnish Cultural Foundation, the Biomedicum Helsinki Foundation, the Orion Research Foundation sr and The Paulo Foundation. P.V.F. was supported by Alzheimer’s Research UK and The Francis Crick Institute. The ARUK UCL Drug Discovery Institute receives its core funding from Alzheimer’s Research UK (520909). The Francis Crick Institute receives its core funding from Cancer Research UK (FC001002), the UK Medical Research Council (FC001002) and the Wellcome Trust (FC001002). Publisher Copyright: © 2021, The Author(s), under exclusive licence to Springer Nature Limited. The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling(1), but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)(2). Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.

Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting β-catenin degradation downstream of Wnt receptors. Unexpectedly, we find that blocking Wnt receptor activity in APC-deficient cells inhibits Wnt signaling independently of Wnt ligand. We also show that inducible loss of APC is rapidly followed by Wnt receptor activation and increased β-catenin levels. In contrast, APC2 loss does not promote receptor activation. We show that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, we demonstrate conservation of this mechanism in Drosophila intestinal stem cells. We propose a model in which APC and APC2 function to promote β-catenin degradation, and APC also acts as a molecular “gatekeeper” to block receptor activation via the clathrin pathway.

Carboxylesterase Notum is a negative regulator of the Wnt signaling pathway. There is an emerging understanding of the role Notum plays in disease, supporting the need to discover new small-molecule inhibitors. A crystallographic X-ray fragment screen was performed, which identified fragment hit 1,2,3-triazole

A relatively small number of proteins have been suggested to act as morphogens—signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1–3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4–6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors. Genetic studies in Drosophila combined with structural analyses show that glypicans bind palmitoylate moieties in Wnt ligands, and thus shield Wnt ligands from their aqueous environments to enable them to signal to their distant receptors.

SummaryPlanar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. Here we dissect the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for study of PCP. We found that PCP was normal in quintuple mutant wings that rely solely on membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. We then used a nanobody-based approach to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. We conclude that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization.

Morphogen gradients provide positional information during development. To uncover the minimal requirements for morphogen gradient formation, we have engineered a synthetic morphogen in Drosophila wing primordia. We show that an inert protein, green fluorescent protein (GFP), can form a detectable diffusion-based gradient in the presence of surface-associated anti-GFP nanobodies, which modulate the gradient by trapping the ligand and limiting leakage from the tissue. We next fused anti-GFP nanobodies to the receptors of Dpp, a natural morphogen, to render them responsive to extracellular GFP. In the presence of these engineered receptors, GFP could replace Dpp to organize patterning and growth in vivo. Concomitant expression of glycosylphosphatidylinositol (GPI)–anchored nonsignaling receptors further improved patterning, to near–wild-type quality. Theoretical arguments suggest that GPI anchorage could be important for these receptors to expand the gradient length scale while at the same time reducing leakage.

In developing tissues, cells estimate their spatial position by sensing graded concentrations of diffusible signaling proteins called morphogens. Morphogen-sensing pathways exhibit diverse molecular architectures, whose roles in controlling patterning dynamics and precision remain unclear. Here, combining cell-based in vitro gradient reconstitution, genetic re-wiring, and mathematical modeling, we systematically analyzed the unique architectural features of the Sonic Hedgehog pathway. The combination of double-negative regulatory logic and negative feedback through the PTCH receptor accelerates gradient formation and improves robustness to variation in the morphogen production rate compared to alternative designs. The ability to isolate morphogen patterning from concurrent developmental processes, and to compare the patterning behaviors of alternative, re-wired, pathway architectures offers a powerful way to understand and engineer multicellular patterning.

Ribosomes are multicomponent molecular machines that synthesize all of the proteins of living cells. Most of the genes that encode the protein components of ribosomes are therefore essential. A reduction in gene dosage is often viable albeit deleterious and is associated with human syndromes, which are collectively known as ribosomopathies1–3. The cell biological basis of these pathologies has remained unclear. Here, we model human ribosomopathies in Drosophila and find widespread apoptosis and cellular stress in the resulting animals. This is not caused by insufficient protein synthesis, as reasonably expected. Instead, ribosomal protein deficiency elicits proteotoxic stress, which we suggest is caused by the accumulation of misfolded proteins that overwhelm the protein degradation machinery. We find that dampening the integrated stress response4 or autophagy increases the harm inflicted by ribosomal protein deficiency, suggesting that these activities could be cytoprotective. Inhibition of TOR activity—which decreases ribosomal protein production, slows down protein synthesis and stimulates autophagy5—reduces proteotoxic stress in our ribosomopathy model. Interventions that stimulate autophagy, combined with means of boosting protein quality control, could form the basis of a therapeutic strategy for this class of diseases. Recasens-Alvarez et al. model human ribosomopathies and find that apoptosis and cellular stress result from proteotoxic stress that overwhelms the degradation machinery.

CRISPR/Cas technology allows the creation of double strand breaks and hence loss of function mutations at any location in the genome. This technology is now routine for many organisms and cell lines. Here we describe how CRISPR/Cas can be combined with other DNA manipulation techniques (e.g. homology-based repair, site-specific integration and Cre or FLP-mediated recombination) to create sophisticated tools to measure and manipulate gene activity. In one class of applications, a single site-specific insertion generates a transcriptional reporter, a loss-of function allele, and a tagged allele. In a second class of modifications, essential sequences are deleted and replaced with an integrase site, which serves as a platform for the creation of custom reporters, transcriptional drivers, conditional alleles and regulatory mutations. We describe how these tools and protocols can be implemented easily and efficiently. Importantly, we also highlight unanticipated failures, which serve as cautionary tales, and suggest mitigating measures. Our tools are designed for use inDrosophilabut the lessons we draw are likely to be widely relevant.AUTHOR SUMMARYThe genome contains all the information that an organism needs to develop and function throughout its life. One of the goal of genetics is to decipher the role of all the genes (typically several thousands for an animal) present in the genome. One approach is to delete each gene and assay the consequences. Deletion of individual genes is now readily achieved with a technique called CRISPR/Cas9. However, simple genetic deletion provides limited information. Here we describe strains and DNA vectors that streamline the generation of more sophisticated genetic tools. We describe general means of creating alleles (genetic variants) that enable gene activity to be measured and experimentally modulated in space and time. Although the tools we describe are universally applicable, each gene requires special consideration. Based on our experience of successes and failures, we suggest measures to maximise the chances that engineered alleles serve their intended purpose. Although our methods are designed for use in Drosophila, they could be adapted to any organism that is amenable to CRISPR/Cas9 genome modification.

Wnt proteins activate a conserved signalling pathway that controls development and tissue homeostasis in all metazoans. The intensity of Wnt signalling must be tightly controlled to avoid diseases caused by excess or ectopic signalling. Over the years, many proteins dedicated to Wnt function have been identified, including Porcupine, which appends a palmitoleate moiety that is essential for signalling activity. This lipid inevitably affects subcellular trafficking and solubility, as well as providing a target for post-translational modulation. We review here the life history of Wnts, starting with progression through the secretory pathway, continuing with release and spread in the extracellular space, and finishing with the various proteins that dampen or inactivate Wnts in the extracellular space.

The apical and basolateral membranes of epithelia are insulated from each other, preventing the transfer of extracellular proteins from one side to the other1. Thus, a signalling protein produced apically is not expected to reach basolateral receptors. Evidence suggests that Wingless, the main Drosophila Wnt is secreted apically in the embryonic epidermis2, 3. However, in the wing imaginal disc epithelium, Wingless is mostly seen on the basolateral membrane where it spreads from secreting to receiving cells 4, 5. Here we examine the apico-basal movement of Wingless in Wingless-producing cells of wing imaginal discs. We find that it is presented first on the apical surface before making its way to the basolateral surface, where it is released and allowed to interact with signalling receptors. We show that Wingless transcytosis involves Dynamin-dependent endocytosis from the apical surface. Subsequent trafficking from early apical endosomes to the basolateral surface requires Godzilla, a member of the RNF family of membrane-anchored E3 ubiquitin ligases. Without such transport, Wingless signalling is strongly reduced in this tissue.

Decapentaplegic has long been thought to be a morphogen that controls patterning and growth in Drosophila wings, but hard evidence for the requisite long-range action has only now come from two new studies.

Summary Planar cell polarity (PCP) organizes the orientation of cellular protrusions and migratory activity within the tissue plane. PCP establishment involves the subcellular polarization of core PCP components. It has been suggested that Wnt gradients could provide a global cue that coordinates local PCP with tissue axes. Here, we dissect the role of Wnt ligands in the orientation of hairs of Drosophila wings, an established system for the study of PCP. We found that PCP was normal in quintuple mutant wings that rely solely on the membrane-tethered Wingless for Wnt signaling, suggesting that a Wnt gradient is not required. We then used a nanobody-based approach to trap Wntless in the endoplasmic reticulum, and hence prevent all Wnt secretion, specifically during the period of PCP establishment. PCP was still established. We conclude that, even though Wnt ligands could contribute to PCP, they are not essential, and another global cue must exist for tissue-wide polarization., Graphical Abstract, Highlights • Reporter knockin alleles identify all the Wnt genes expressed in the Drosophila wing • A quintuple mutant removes all diffusible DWnts from the wing but leaves PCP unperturbed • The secretion and release of Wnts is dispensable for Drosophila wing PCP establishment • The wing margin does not provide a global cue for PCP, Planar cell polarity (PCP) requires Frizzled, which transduces Wnt signaling. Yu et al show that the secretion and release of Wnts is dispensable for proper PCP establishment in the Drosophila wing. The global cue organizing PCP in this tissue remains unknown but does not originate from the wing margin.

Extensive apoptosis is often seen in patterning mutants, suggesting that tissues can detect and eliminate potentially harmful mis-specified cells. Here, we show that the pattern of apoptosis in the embryonic epidermis of Drosophila is not a response to fate mis-specification but can instead be explained by the limiting availability of prosurvival signaling molecules released from locations determined by patterning information. In wild-type embryos, the segmentation cascade elicits the segmental production of several epidermal growth factor receptor (EGFR) ligands, including the transforming growth factor Spitz (TGFα), and the neuregulin, Vein. This leads to an undulating pattern of signaling activity, which prevents expression of the proapoptotic gene head involution defective (hid) throughout the epidermis. In segmentation mutants, where specific peaks of EGFR ligands fail to form, gaps in signaling activity appear, leading to coincident hid up-regulation and subsequent cell death. These data provide a mechanistic understanding of how cell survival, and thus appropriate tissue size, is made contingent on correct patterning., Cells lacking essential patterning determinants are eliminated from developing tissues by apoptosis; this study shows that cell death in fruit fly segmentation mutants results from disruption of the pattern of EGFR signalling, which in normal embryos coordinates segment size with patterning information., Author summary In many tissues, defective cells are eliminated by a process called apoptosis. This process prevents the emergence of rogue cells, which could be detrimental to normal physiology. Apoptosis is particularly apparent in developing embryos that lack appropriate positional information, and it has been suggested that in the absence of clear positional instructions, cells are unable to acquire a defined fate and commit suicide as a result. Here, we have used mutant fruit fly embryos lacking essential segmental determinants to identify the molecular signals that trigger apoptosis in response to mispatterning. We found that cells do not trigger apoptosis in response to conflicting fate determinants. Instead, mispatterning disrupts a tissue size control system that removes excess cells in oversized segments. Specifically, correct patterning information leads to the segmentally repeated production of survival signals, which activate the epidermal growth factor receptor, and this system is disrupted in patterning mutants leading to reproducible patterns of apoptosis. We propose that a similar, though less obvious, process also occurs in normal embryos. In such embryos, each segment would initially comprise a slight excess of cells and would then be trimmed down to a size specified by the pattern of survival signal production and the range of these signals. We suggest that a similar regulatory logic could ensure the coordination of tissue patterning and size in a variety of developing tissues.

The caspase-mediated regulation of many cellular processes, including apoptosis, justifies the substantial interest in understanding all of the biological features of these enzymes. To complement functional assays, it is critical to identify caspase-activating cells in live tissues. Our work describes new caspase-reporters that, for the first time, provide direct information concerning the initial steps of the caspase activation cascade in Drosophila tissues. One of our caspase-sensors has capitalized on the rapid subcellular localization change of a fluorescent marker to uncover novel cellular apoptotic events. These refer to the actin-mediated positioning of the nucleus before cell delamination. The other construct has benefited from a caspase-induced nuclear translocation of a QF transcription factor. This feature enables the genetic manipulation of caspase-activating cells, whilst showing the spatio-temporal patterns of apical caspase activation. Collectively, our sensors offer new experimental opportunities that are already illuminating unknown aspects of caspase-dependent processes in apoptotic and non-apoptotic cellular scenarios.Summary statementWe describe a novel set of caspase sensors that directly detect early caspase activation. The exclusive features of our reporters uncovered unknown stages of apoptosis and properties of caspase-activating cells.

Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.

Dpp, a member of the BMP family, is a morphogen that specifies positional information in Drosophila wing precursors. In this tissue, Dpp expressed along the anterior-posterior boundary forms a concentration gradient that controls the expression domains of target genes, which in turn specify the position of wing veins. Dpp also promotes growth in this tissue. The relationship between the spatio-temporal profile of Dpp signalling and growth has been the subject of debate, which has intensified recently with the suggestion that the stripe of Dpp is dispensable for growth. With two independent conditional alleles of dpp, we find that the stripe of Dpp is essential for wing growth. We then show that this requirement, but not patterning, can be fulfilled by uniform, low level, Dpp expression. Thus, the stripe of Dpp ensures that signalling remains above a pro-growth threshold, while at the same time generating a gradient that patterns cell fates. DOI: http://dx.doi.org/10.7554/eLife.22546.001, eLife digest From the wings of a butterfly to the fingers of a human hand, living tissues often have complex and intricate patterns. Developmental biologists have long been fascinated by the signals – called morphogens – that guide how these kinds of pattern develop. Morphogens are substances that are produced by groups of cells and spread to the rest of the tissue to form a gradient. Depending on where they sit along this gradient, cells in the tissue activate different sets of genes, and the resulting pattern of gene activity ultimately defines the position of the different parts of the tissue. Decades worth of studies into how limbs develop in animals from mice to fruit flies have revealed common principles of morphogen gradients that regulate the development of tissue patterns. Morphogens have been shown to help regulate the growth of tissues in a number of different animals as well. However, how the morphogens regulate tissue size and what role their gradients play in this process remain topics of intense debate in the field of developmental biology. In the developing wing of a fruit fly, a morphogen called Dpp is expressed in a thin stripe located in the centre and spreads to the rest of the tissue to form a gradient. Bosch, Ziukaite, Alexandre et al. have now characterised where and when the Dpp morphogen must be produced to regulate both the final size of the fly’s wing and the number of cells the wing eventually contains. The experiments involved preventing the production of Dpp in the developing wing in specific cells and at specific stages of development. This approach confirmed that Dpp must be produced in the central stripe for the wing to grow. Matsuda and Affolter and, independently, Barrio and Milán report the same findings in two related studies. Moreover, Bosch et al. and Barrio and Milán also conclude that the gradient of Dpp throughout the wing is not required for growth. Further work will be needed to explain how the Dpp signal regulates the growth of the wing. The answer to this question will contribute to a better understanding of the role of morphogens in regulating the size of human organs and how a failure to do so might cause developmental disorders. DOI: http://dx.doi.org/10.7554/eLife.22546.002

Summary Jun N-terminal kinase (JNK) often mediates apoptosis in response to cellular stress. However, during normal development, JNK signaling controls a variety of live cell behaviors, such as during dorsal closure in Drosophila embryos. During this process, the latent proapoptotic activity of JNK becomes apparent following Dpp signaling suppression, which leads to JNK-dependent transcriptional activation of the proapoptotic gene reaper. Dpp signaling also protects cells from JNK-dependent apoptosis caused by epithelial disruption. We find that repression of reaper transcription by Dpp is mediated by Schnurri. Moreover, reporter gene analysis shows that a transcriptional regulatory module comprising AP-1 and Schnurri binding sites located upstream of reaper integrate the activities of JNK and Dpp. This arrangement allows JNK to control a migratory behavior without triggering apoptosis. Dpp plays a dual role during dorsal closure. It cooperates with JNK in stimulating cell migration and also prevents JNK from inducing apoptosis., Graphical Abstract, Highlights • Dpp/BMP signaling prevents JNK from activating expression of proapoptotic reaper • The transcriptional repressor Schnurri mediates the antiapoptotic effect of Dpp • AP-1 and Schnurri binding sites integrate the inputs of JNK and Dpp on reaper • Dpp signaling ensures the survival of dorsal edge cells during dorsal closure, JNK often mediates apoptosis in response to cellular stress, yet JNK signaling controls a variety of live cell behaviors during normal development. In Drosophila embryos, JNK can direct dorsal closure because concurrent Dpp signaling, mediated by the repressor Schnurri, prevents activation of the proapoptotic gene reaper by JNK.

Wnts are evolutionarily conserved secreted signaling proteins that, in various developmental contexts, spread from their site of synthesis to form a gradient and activate target gene expression at a distance. However, the requirement for Wnts to spread has never been directly tested. Here we used genome engineering to replace the endogenous wingless gene, which encodes the main Drosophila Wnt, with one that expresses a membrane-tethered form of the protein. Surprisingly, the resulting flies were viable and produced normally patterned appendages of nearly the right size, albeit with a delay. We show that, in the prospective wing, prolonged wingless transcription followed by memory of earlier signaling allow persistent expression of relevant target genes, thus explaining why Wingless release is dispensable. We suggest that patterning and growth can proceed without an instructive Wingless gradient, even though the spread of Wingless likely increases the rate of proliferation.

Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless-expressing Drosophila S2 cells, Wingless is present on exosome-like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless-containing exosome-like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome-like vesicles. Using these exosome markers and a cell-based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless-expressing cells.

Summary Wnt signaling is a key regulator of development that is often associated with cancer. Wingless, a Drosophila Wnt homolog, has been reported to be a survival factor in wing imaginal discs. However, we found that prospective wing cells survive in the absence of Wingless as long as they are not surrounded by Wingless-responding cells. Moreover, local autonomous overactivation of Wg signaling (as a result of a mutation in APC or axin) leads to the elimination of surrounding normal cells. Therefore, relative differences in Wingless signaling lead to competitive cell interactions. This process does not involve Myc, a well-established cell competition factor. It does, however, require Notum, a conserved secreted feedback inhibitor of Wnt signaling. We suggest that Notum could amplify local differences in Wingless signaling, thus serving as an early trigger of Wg signaling-dependent competition., Graphical Abstract Highlights ► Wingless-blind cells survive within large groups but die as part of small clones ► Patches of APC or axin mutant cells trigger apoptosis in surrounding normal cells ► The competitive nature of axin mutant cells is not mediated by Myc ► Tissue takeover by axin cells requires Notum, a secreted inhibitor of Wg signaling

Epithelial homeostasis and the avoidance of diseases such as cancer require the elimination of defective cells by apoptosis. Here, we investigate how loss of apical determinants triggers apoptosis in the embryonic epidermis of Drosophila. Transcriptional profiling and in situ hybridisation show that JNK signalling is upregulated in mutants lacking Crumbs or other apical determinants. This leads to transcriptional activation of the pro-apoptotic gene reaper and to apoptosis. Suppression of JNK signalling by overexpression of Puckered, a feedback inhibitor of the pathway, prevents reaper upregulation and apoptosis. Moreover, removal of endogenous Puckered leads to ectopic reaper expression. Importantly, disruption of the basolateral domain in the embryonic epidermis does not trigger JNK signalling or apoptosis. We suggest that apical, not basolateral, integrity could be intrinsically required for the survival of epithelial cells. In apically deficient embryos, JNK signalling is activated throughout the epidermis. Yet, in the dorsal region, reaper expression is not activated and cells survive. One characteristic of these surviving cells is that they retain discernible adherens junctions despite the apical deficit. We suggest that junctional integrity could restrain the pro-apoptotic influence of JNK signalling.

Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome-wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts.

Tolerance verification permits to check the product conformity and to verify assumptions made by the designer. For conformity assessment, the uncertainty associated with the values of the measurands must be known. The Guide to the Expression of Uncertainty in Measurement (ISO-GUM) describes the measurement uncertainty evaluation, and in the ISO TS 17450 part 2, the notion of the uncertainty is generalized to the specification and the verification. The uncertainty is divided into correlation uncertainty, specification uncertainty and measurement uncertainty. Correlation uncertainty characterizes the fact that the intended functionality and the controlled characteristics may not be perfectly correlated. Therefore, we propose a new specified characteristics based on the statistical tolerancing approach which are in direct relationship with the design intent: the probability distribution of maximum range of the transmission error (the transmission error is the main source of vibratory and acoustic nuisances), and the evaluation of this characteristic based on 3D acquisition by Monte Carlo simulation and tooth contact analysis. Moreover, the measurement uncertainty of the evaluation of this characteristic is estimated by Monte Carlo simulation.