Wounding increases nuclear ploidy in wound-proximal epidermal cells of the Drosophila pupal notum.

After injury, tissues must replace cell mass and genome copy number. The mitotic cycle is one mechanism for replacement, but non-mitotic strategies have been observed in quiescent tissues to restore tissue ploidy after wounding. Here we report that nuclei of the mitotically capable Drosophila pupal notum enlarged following nearby laser ablation. Measuring DNA content, we determined that nuclei within 100 µm of a laser-wound increased their ploidy to ~8C, consistent with one extra S-phase. These data indicate non-mitotic repair strategies are not exclusively utilized by quiescent tissues and may be an underexplored wound repair strategy in mitotic tissues.

Live imaging basement membrane assembly under the pupal notum epithelium.

Basement membranes are sheet-like extracellular matrices containing Collagen IV, and they are conserved across the animal kingdom. Basement membranes usually line the basal surfaces of epithelia, where they contribute to structure, maintenance, and signaling. Although adult epithelia contact basement membranes, in early embryos the epithelia contact basement membranes only after basement membranes are assembled in embryogenesis. In Drosophila , the pupal notum epithelium is a useful model for live imaging epithelial cell behaviors, yet it is unclear when the basement membrane assembles in the pupa, as pupae are undergoing metamorphosis, similar to embryogenesis. To characterize the basement membrane in the pupal notum, we used spinning disk fluorescent microscopy to visualize Collagen IV subunit Vkg-GFP and adherens junction protein p120ctnRFP. Bright punctae of Vkg-GFP were observed in the X-Y plane, possibly representing Vkg-containing cells. We found that a thin continuous Vkg-containing basement membrane was evident at 14 h APF, which became more enriched with Vkg-GFP over the next 6 h, indicating the basement membrane is still assembling during that time. Live imaging of the pupal notum during this time could provide insight into formation, assembly, and repair of the basement membranes.

After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin ∼10 min after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension, we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin about 10 minutes after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

Peroxidasin is required for full viability in development and for maintenance of tissue mechanics in adults.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membranes that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved throughout the animal kingdom, indicating they are important, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a new CRISPR-generated catalytic null, and found that homozygotes were mostly lethal with 13 % viable escapers. Mouse mutants also show semi-lethality, with Mendelian analysis demonstrating ∼50 % lethality and ∼50 % escapers. Despite the strong mutations, the homozygous fly and mouse escapers had low but detectable levels of Col4 crosslinking, indicating the existence of inefficient alternative crosslinking mechanisms, probably responsible for the viable escapers. Fly mutant phenotypes are consistent with decreased basement membrane stiffness. Interestingly, we found that even after basement membranes are assembled and crosslinked in wild-type animals, continuing Peroxidasin activity is required in adults to maintain tissue stiffness over time. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

Wound-Induced Syncytia Outpace Mononucleate Neighbors during Drosophila Wound Repair.

All organisms have evolved to respond to injury. Cell behaviors like proliferation, migration, and invasion replace missing cells and close wounds. However, the role of other wound-induced cell behaviors is not understood, including the formation of syncytia (multinucleated cells). Wound-induced epithelial syncytia were first reported around puncture wounds in post-mitotic Drosophila epidermal tissues, but have more recently been reported in mitotically competent tissues such as the Drosophila pupal epidermis and zebrafish epicardium. The presence of wound-induced syncytia in mitotically active tissues suggests that syncytia offer adaptive benefits, but it is unknown what those benefits are. Here, we use in vivo live imaging to analyze wound-induced syncytia in mitotically competent Drosophila pupae. We find that almost half the epithelial cells near a wound fuse to form large syncytia. These syncytia use several routes to speed wound repair: they outpace diploid cells to complete wound closure; they reduce cell intercalation during wound closure; and they pool the resources of their component cells to concentrate them toward the wound. In addition to wound healing, these properties of syncytia are likely to contribute to their roles in development and pathology.

Analysis of Drosophila and mouse mutants reveals that Peroxidasin is required for tissue mechanics and full viability.

Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membrane that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved, indicating they are essential, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a newly generated catalytic null, and found that homozygotes were mostly lethal with 13% viable escapers. A Mendelian analysis of mouse mutants shows a similar pattern, with homozygotes displaying ~50% lethality and ~50% escapers. Despite the strong mutations, the homozygous escapers had low but detectable levels of Col4 crosslinking, indicating that inefficient alternative mechanisms exist and that are probably responsible for the viable escapers. Further, fly mutants have phenotypes consistent with a decrease in stiffness. Interestingly, we found that even after adult basement membranes are assembled and crosslinked, Peroxidasin is still required to maintain stiffness. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated.

A mathematical model of calcium signals around laser-induced epithelial wounds.

Cells around epithelial wounds must first become aware of the wound's presence in order to initiate the wound-healing process. An initial response to an epithelial wound is an increase in cytosolic calcium followed by complex calcium-signaling events. While these calcium signals are driven by both physical and chemical wound responses, cells around the wound will all be equipped with the same cellular components to produce and interact with the calcium signals. Here we have developed a mathematical model in the context of laser ablation of the Drosophila pupal notum that integrates tissue-level damage models with a cellular calcium-signaling toolkit. The model replicates experiments in the contexts of control wounds as well as knockdowns of specific cellular components, but it also provides new insights that are not easily accessible experimentally. The model suggests that cell-cell variability is necessary to produce calcium-signaling events observed in experiments; it quantifies calcium concentrations during wound-induced signaling events, and it shows that intercellular transfer of the molecule IP3 is required to coordinate calcium signals across distal cells around the wound. The mathematical model developed here serves as a framework for quantitative studies in both wound signaling and calcium signaling in the Drosophila system.

Piezo initiates transient production of collagen IV to repair damaged basement membranes.

Basement membranes are sheets of extracellular matrix separating tissue layers and providing mechanical support. Their mechanical properties are determined largely by their most abundant protein, Collagen IV (Col4). Although basement membranes are repaired after damage, little is known about how. To wit, since basement membrane is extracellular it is unknown how damage is detected, and since Col4 is long-lived it is unknown how it is regulated to avoid fibrosis. Using the basement membrane of the adult Drosophila midgut as a model, we show that repair is distinct from maintenance. In healthy conditions, midgut Col4 originates from the fat body, but after damage, a subpopulation of enteroblasts we term "matrix menders" transiently express Col4, and Col4 from these cells is required for repair. Activation of the mechanosensitive channel Piezo is required for matrix menders to upregulate Col4, and the signal to initiate repair is a reduction in basement membrane stiffness. Our data suggests that mechanical sensitivity may be a general property of Col4-producing cells.

Mounting Drosophila pupae for laser ablation and live imaging of the dorsal thorax.

This protocol describes the preparation of Drosophilamelanogaster pupae for laser ablation and live imaging of the notum (dorsal thorax). Because the pupa is stationary, it can be continuously live imaged for multiple days if desired, making it ideal for studying wound signaling and repair, from before laser ablation through wound closure. In this protocol, we demonstrate the processes of staging, partially dissecting, mounting, wounding, and live imaging the pupal notum, with the wounding occurring during the live imaging process. For complete details on the use and execution of this protocol, please refer to O'Connor et al. (2021b).

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum.

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue's dorsal or ventral side.

Regulation of Wnt distribution and function by Drosophila glypicans.

The extracellular distribution of secreted Wnt proteins is crucial for their ability to induce a response in target cells at short and long ranges to ensure proper development. Wnt proteins are evolutionarily conserved ligands that are lipid-modified, and their hydrophobic nature interferes with their solubility in the hydrophilic extracellular environment. This raises the question of how Wnt proteins spread extracellularly despite their lipid modifications, which are essential for both their secretion and function. Seminal studies on Drosophila Wingless (Wg), a prototypical Wnt, have discovered multiple mechanisms by which Wnt proteins spread. A central theme emerges from these studies: the Wnt lipid moiety is shielded from the aqueous environment, allowing the ligands to spread and remain viable for signaling. Wnt distribution in vivo is primarily facilitated by glypicans, which are cell-surface heparan sulfate proteoglycans, and recent studies have further provided mechanistic insight into how glypicans facilitate Wnt distribution. In this Review, we discuss the many diverse mechanisms of Wnt distribution, with a particular focus on glypican-mediated mechanisms.

Glypicans and cytonemes unite to distribute Wnt ligands.

Hu et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202009082) show that Glypican 4 participates in filopodia-mediated Wnt transport from endoderm to mesoderm in zebrafish embryos to facilitate intercellular communication between germ layers.

Zones of cellular damage around pulsed-laser wounds.

After a tissue is wounded, cells surrounding the wound adopt distinct wound-healing behaviors to repair the tissue. Considerable effort has been spent on understanding the signaling pathways that regulate immune and tissue-resident cells as they respond to wounds, but these signals must ultimately originate from the physical damage inflicted by the wound. Tissue wounds comprise several types of cellular damage, and recent work indicates that different types of cellular damage initiate different types of signaling. Hence to understand wound signaling, it is important to identify and localize the types of wound-induced cellular damage. Laser ablation is widely used by researchers to create reproducible, aseptic wounds in a tissue that can be live-imaged. Because laser wounding involves a combination of photochemical, photothermal and photomechanical mechanisms, each with distinct spatial dependencies, cells around a pulsed-laser wound will experience a gradient of damage. Here we exploit this gradient to create a map of wound-induced cellular damage. Using genetically-encoded fluorescent proteins, we monitor damaged cellular and sub-cellular components of epithelial cells in living Drosophila pupae in the seconds to minutes following wounding. We hypothesized that the regions of damage would be predictably arrayed around wounds of varying sizes, and subsequent analysis found that all damage radii are linearly related over a 3-fold range of wound size. Thus, around laser wounds, the distinct regions of damage can be estimated after measuring any one. This report identifies several different types of cellular damage within a wounded epithelial tissue in a living animal. By quantitatively mapping the size and placement of these different types of damage, we set the foundation for tracing wound-induced signaling back to the damage that initiates it.

Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection.

The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. Here, we show that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, we show that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. We present experimental and computational evidence that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium.

Extracellular spreading of Wingless is required for Drosophila oogenesis.

Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or whether they signal only in a juxtacrine manner to neighboring cells. The original evidence for long-range Wnt signaling arose from studies of Wg, a Drosophila Wnt protein, which patterns the wing disc over several cell diameters from a central source of Wg ligand. However, the requirement of long-range Wg for patterning was called into question when it was reported that replacing the secreted protein Wg with a membrane-tethered version, NRT-Wg, results in flies with normally patterned wings. We and others previously reported that Wg spreads in the ovary about 50 µm or 5 cell diameters, from the cap cells to the follicle stem cells (FSCs) and that Wg stimulates FSC proliferation. We used the NRT-wg flies to analyze the consequence of tethering Wg to the cap cells. NRT-wg homozygous flies are sickly, but we found that hemizygous NRT-wg/null flies, carrying only one copy of tethered Wingless, were significantly healthier. Despite their overall improved health, these hemizygous flies displayed dramatic reductions in fertility and in FSC proliferation. Further, FSC proliferation was nearly undetectable when the wg locus was converted to NRT-wg only in adults, and the resulting germarium phenotype was consistent with a previously reported wg loss-of-function phenotype. We conclude that Wg protein spreads from its source cells in the germarium to promote FSC proliferation.

Dally-like protein sequesters multiple Wnt ligands in the Drosophila germarium.

Cells in multicellular organisms rely on secreted ligands for development and morphogenesis. Several mechanisms modulate the availability and distribution of secreted ligands, determining their ability to signal locally and at long range from their source. One of these mechanisms is Dally-like protein (Dlp), a cell-surface glypican that exhibits biphasic functions in Drosophila wing discs, promoting Wg signaling at long-range from Wg source cells and inhibiting Wg signaling near source cells. In the germarium at the tip of the ovary, Dlp promotes long-range distribution of Wg from cap cells to follicle stem cells. However, the germarium also expresses other Wnts - Wnt2, Wnt4, and Wnt6 - that function locally in escort cells to promote oogenesis. Whether and how local functions of these Wnts are regulated remains unknown. Here we show that the dlp overexpression phenotype is multifaceted and phenocopies multiple Wnt loss-of-function phenotypes. Each aspect of dlp overexpression phenotype is suppressed by co-expression of individual Wnts, and the suppression pattern exhibited by each Wnt suggests that Wnts have functional specificity in the germarium. Further, dlp knockdown phenocopies Wnt gain-of-function phenotypes. Together these data show that Dlp inhibits the functions of each Wnt. All four Wnts co-immunoprecipitate with Dlp in S2R+ â€‹cells, suggesting that in the germarium, Dlp sequesters Wnts to inhibit local paracrine Wnt signaling. Our results indicate that Dlp modulates the availability of multiple extracellular Wnts for local paracrine Wnt signaling in the germarium.

DSS-induced damage to basement membranes is repaired by matrix replacement and crosslinking.

Basement membranes are an ancient form of animal extracellular matrix. As important structural and functional components of tissues, basement membranes are subject to environmental damage and must be repaired while maintaining functions. Little is known about how basement membranes get repaired. This paucity stems from a lack of suitable in vivo models for analyzing such repair. Here, we show that dextran sodium sulfate (DSS) directly damages the gut basement membrane when fed to adult Drosophila DSS becomes incorporated into the basement membrane, promoting its expansion while decreasing its stiffness, which causes morphological changes to the underlying muscles. Remarkably, two days after withdrawal of DSS, the basement membrane is repaired by all measures of analysis. We used this new damage model to determine that repair requires collagen crosslinking and replacement of damaged components. Genetic and biochemical evidence indicates that crosslinking is required to stabilize the newly incorporated repaired Collagen IV rather than to stabilize the damaged Collagen IV. These results suggest that basement membranes are surprisingly dynamic.

Basement membrane mechanics shape development: Lessons from the fly.

Basement membrane plays a foundational role in the structure and maintenance of many tissues throughout the animal kingdom. In addition to signaling to cells through cell-surface receptors, basement membrane directly influences the development and maintenance of organ shape via its mechanical properties. The mechanical properties of basement membrane are dictated by its composition, geometry, and crosslinking. Distinguishing between the ways the basement membrane influences morphology in vivo poses a major challenge. Drosophila melanogaster, already established as a powerful model for the analysis of cell signaling, has in recent years emerged as a tractable model for understanding the roles of basement membrane stiffness in vivo, in shaping and maintaining the morphology of tissues and organs. In addition to the plethora of genetic tools available in flies, the major proteins found in vertebrate basement membranes are all present in Drosophila. Furthermore, Drosophila has fewer copies of the genes encoding these proteins, making flies more amenable to genetic manipulation than vertebrate models. Because the development of Drosophila organs has been well-characterized, these different organ systems offer a variety of contexts for analyzing the role of basement membrane in development. The developing egg chamber and central nervous system, for example, have been important models for assessing the role of basement membrane stiffness in influencing organ shape. Studies in the nervous system have also shown how basement membrane stiffness can influence cellular migration in vivo. Finally, work in the imaginal wing disc has illuminated a distinct mechanism by which basement membrane can alter organ shape and size, by sequestering signaling ligands. This mini-review highlights the recent discoveries pertaining to basement membrane mechanics during Drosophila development.