dissecting-fixing-and-visualizing-the-drosophila-pupal-notum

Dissecting, Fixing, and Visualizing the <em>Drosophila</em> 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 ...

Vanderbilt University

The coordination of cell-cycle events with developmental processes is essential for the reproductive success of organisms. In Drosophila melanogaster, meiosis is tightly coupled to oocyte development, and early embryos undergo specialized S-M mitoses that are supported by maternal products. We previously showed that the small phosphoprotein α-endosulfine (Endos) is required for normal oocyte meiotic maturation and early embryonic mitoses in Drosophila. In this study, we performed a genetic screen for dominant enhancers of endos(00003) and identified several genomic regions that, when deleted, lead to impaired fertility of endos(00003)/+ heterozygous females. We uncovered matrimony (mtrm), which encodes a Polo kinase inhibitor, as a strong dominant enhancer of endos. mtrm(126) +/+ endos(00003) females are sterile because of defects in early embryonic mitoses, and this phenotype is reverted by removal of one copy of polo. These results provide compelling genetic evidence that excessive Polo activity underlies the strong functional interaction between endos(00003) and mtrm(126). Moreover, we show that endos is required for the increased expression of Mtrm in mature oocytes, which is presumably loaded into early embryos. These data are consistent with the model that maternal endos antagonizes Polo function in the early embryo to ensure normal mitoses through its effects on Mtrm expression during late oogenesis. Finally, we also identified genomic deletions that lead to loss of viability of endos(00003)/+ heterozygotes, consistent with recently published studies showing that endos is required zygotically to regulate the cell cycle during development.

A Genetic Screen for Dominant Enhancers of the Cell-Cycle Regulator α-Endosulfine Identifies Matrimony as a Strong Functional Interactor in Drosophila.

Matrix metalloproteinases (MMPs) are extracellular proteases that can cleave extracellular matrix and alter signaling pathways. They have been implicated in many disease states, but it has been difficult to understand the contribution of individual MMPs, as there are over 20 MMPs in vertebrates. The vertebrate MMPs have overlapping substrates, they exhibit genetic redundancy and compensation, and pharmacological inhibitors are non-specific. In contrast, there are only two MMP genes in Drosophila, DmMmp1 and DmMmp2, which makes Drosophila an attractive system to analyze the basis of MMP specificity. Previously, Drosophila MMPs have been categorized by their pericellular localization, as Mmp1 appeared to be secreted and Mmp2 appeared to be membrane-anchored, suggesting that protein localization was the critical distinction in this small MMP family. We report here that products of both genes are found at the cell surface and released into media. Additionally, we show that products of both genes contain GPI-anchors, and unexpectedly, that GPI-anchored MMPs promote cell adhesion when they are rendered inactive. Finally, by using new reagents and assays, we show that the two MMPs cleave different substrates, suggesting that this is the important distinction within this smallest MMP family.

Both Drosophila matrix metalloproteinases have released and membrane-tethered forms but have different substrates.

Basement membrane is a highly conserved sheet-like extracellular matrix in animals, underlying simple and complex epithelia, and wrapping around tissues like muscles and nerves. Like the tissues they support, basement membranes become damaged by environmental insults. Although it is clear that basement membranes are repaired after damage, virtually nothing is known about this process. For example, it is not known how repaired basement membranes compare to undamaged ones, whether basement membrane components are necessary for epithelial wound closure, or whether there is a hierarchy of assembly that repairing basement membranes follow, similar to the hierarchy of assembly of embryonic basement membranes. In this report, we address these questions using the basement membrane of the Drosophila larval epidermis as a model system. By analyzing the four main basement membrane proteins - laminin, collagen IV, perlecan, and nidogen - we find that although basement membranes are repaired within a day after mechanical damage in vivo, thickened and disorganized matrix scars are evident with all four protein components. The new matrix proteins that repair damaged basement membranes are provided by distant adipose and muscle tissues rather than by the local epithelium, the same distant tissues that provide matrix proteins for growth of unwounded epithelial basement membranes. To identify a hierarchy of repair, we tested the dependency of each of the basement membrane proteins on the others for incorporation after damage. For proper incorporation after damage, nidogen requires laminin, and perlecan requires collagen IV, but surprisingly collagen IV does not to depend on laminin. Thus, the rules of basement membrane repair are subtly different than those of de novo assembly.

A scar-like lesion is apparent in basement membrane after wound repair in vivo.

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  models for analyzing such repair. Here, we show that dextran sodium sulfate (DSS) directly damages the gut basement membrane when fed to adult  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.

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

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.

Extracellular spreading of Wingless is required for Drosophila oogenesis.

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.

Kimberly S. LaFever

Program in Developmental Biology

Vanderbilt University

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

Kimberly S. LaFever

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Program in Developmental Biology

Vanderbilt University

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

Program in Developmental Biology