Is Drosophila Dpp/BMP morphogen spreading required for wing patterning and growth?

Secreted signaling molecules act as morphogens to control patterning and growth in many developing tissues. Since locally produced morphogens spread to form a concentration gradient in the surrounding tissue, spreading is generally thought to be the key step in the non-autonomous actions. Here, we review recent advances in tool development to investigate morphogen function using the role of decapentaplegic (Dpp)/bone morphogenetic protein (BMP)-type ligand in the Drosophila wing disc as an example. By applying protein binder tools to distinguish between the roles of Dpp spreading and local Dpp signaling, we found that Dpp signaling in the source cells is important for wing patterning and growth but Dpp spreading from this source cells is not as strictly required as previously thought. Given recent studies showing unexpected requirements of long-range action of different morphogens, manipulating endogenous morphogen gradients by synthetic protein binder tools could shed more light on how morphogens act in developing tissues.

Ecdysone and gene expressions for chromatin remodeling, histone modification, and Broad Complex in relation to pupal commitment in Bombyx mori.

In the present study, we tried to clarify when and how pupal commitment (PT) better to use PC occurs and what is involved in the PT of Bombyx mori. To clarify this, we examined the responsiveness of a wing disc to ecdysone, referring to metamorphosis-related BR-C, development-related Myc and Wnt, and chromatin remodeling-related genes at around the predicted PT stage of the Bombyx wing disc. Wing disc responsiveness to juvenile hormone (JH) and ecdysone was examined using Methoprene and 20-hydroxyecdysone (20E) in vitro. The body weight of B. mori increased after the last larval ecdysis, peaked at Day 5 of the fifth larval instar (D5L5), and then decreased. The responsiveness of the wing disc to JH decreased after the last larval ecdysis up to D3L5. Bmbr-c (the Broad Complex of B. mori) showed enhanced expression in D4L5 wing discs with 20E treatment. Some chromatin remodeler and histone modifier genes (Bmsnr1, Bmutx, and Bmtip60) showed upregulation after being cultured with 20E in D4L5 wing discs. A low concentration of 20E is suggested to induce responsiveness to 20E in D4L5 wing discs. Bmbr-c, Bmsnr1, Bmutx, and Bmtip60 were upregulated after being cultured with a low concentration of 20E in D4L5 wing discs. The expression of Bmmyc and Bmwnt1 did not show a change after being cultured with or without 20E in D4L5 wing discs, while enhanced expression was observed with 20E in D5L5 wing discs. From the present results, we concluded that PT of the wing disc of B. mori occurred beginning on D4L5 with the secretion of low concentrations of ecdysteroids. Bmsnr1, Bmutx, Bmtip60, and BR-C are also involved.

Chromatin remodelling and retrotransposons activities during regeneration in Drosophila.

Regeneration is a response mechanism aiming to reconstruct lost or damaged structures. To achieve this, the cells repopulating the lost tissue often have to change their original identity, a process that involves chromatin remodelling.We have analysed the issue of chromatin remodelling during regeneration in the wing disc of Drosophila . In this disc the ablation of the central region (the pouch) induces the regenerative response of the cells from the lateral region (the hinge), which reconstitute the wing pouch. We have examined euchromatin and heterochromatin histone marks during the process and find that heterochromatin marks disappear but are recovered when regeneration is complete. Euchromatin marks are not modified. We also describe the transcription of two retrotransposons, Roo and F-element in the regenerating cells. We have established a temporal correlation between the alterations of heterochromatin marks and the levels of transcription of two retrotransposons, Roo and F-element, both during embryonic development and in the regeneration process.

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.

Increased lateral tension is sufficient for epithelial folding in Drosophila.

The folding of epithelial sheets is important for tissues, organs and embryos to attain their proper shapes. Epithelial folding requires subcellular modulations of mechanical forces in cells. Fold formation has mainly been attributed to mechanical force generation at apical cell sides, but several studies indicate a role of mechanical tension at lateral cell sides in this process. However, whether lateral tension increase is sufficient to drive epithelial folding remains unclear. Here, we have used optogenetics to locally increase mechanical force generation at apical, lateral or basal sides of epithelial Drosophila wing disc cells, an important model for studying morphogenesis. We show that optogenetic recruitment of RhoGEF2 to apical, lateral or basal cell sides leads to local accumulation of F-actin and increase in mechanical tension. Increased lateral tension, but not increased apical or basal tension, results in sizeable fold formation. Our results stress the diversification of folding mechanisms between different tissues and highlight the importance of lateral tension increase for epithelial folding.

Wingless counteracts epithelial folding by increasing mechanical tension at basal cell edges in Drosophila.

The modulation of mechanical tension is important for sculpturing tissues during animal development, yet how mechanical tension is controlled remains poorly understood. In Drosophila wing discs, the local reduction of mechanical tension at basal cell edges results in basal relaxation and the formation of an epithelial fold. Here, we show that Wingless, which is expressed next to this fold, promotes basal cell edge tension to suppress the formation of this fold. Ectopic expression of Wingless blocks fold formation, whereas the depletion of Wingless increases fold depth. Moreover, local depletion of Wingless in a region where Wingless signal transduction is normally high results in ectopic fold formation. The depletion of Wingless also results in decreased basal cell edge tension and basal cell area relaxation. Conversely, the activation of Wingless signal transduction leads to increased basal cell edge tension and basal cell area constriction. Our results identify the Wingless signal transduction pathway as a crucial modulator of mechanical tension that is important for proper wing disc morphogenesis.

JAK/STAT signaling in stem cells and regeneration: from Drosophila to vertebrates.

The JAK/STAT pathway is a conserved metazoan signaling system that transduces cues from extracellular cytokines into transcriptional changes in the nucleus. JAK/STAT signaling is best known for its roles in immunity. However, recent work has demonstrated that it also regulates critical homeostatic processes in germline and somatic stem cells, as well as regenerative processes in several tissues, including the gonad, intestine and appendages. Here, we provide an overview of JAK/STAT signaling in stem cells and regeneration, focusing on Drosophila and highlighting JAK/STAT pathway functions in proliferation, survival and cell competition that are conserved between Drosophila and vertebrates.

Coupling cell proliferation rates to the duration of recruitment controls final size of the Drosophila wing.

Organ growth driven by cell proliferation is an exponential process. As a result, even small variations in proliferation rates, when integrated over a relatively long developmental time, will lead to large differences in size. How organs robustly control their final size despite perturbations in cell proliferation rates throughout development is a long-standing question in biology. Using a mathematical model, we show that in the developing wing of the fruit fly, Drosophila melanogaster, variations in proliferation rates of wing-committed cells are inversely proportional to the duration of cell recruitment, a differentiation process in which a population of undifferentiated cells adopt the wing fate by expressing the selector gene, vestigial. A time-course experiment shows that vestigial-expressing cells increase exponentially while recruitment takes place, but slows down when recruitable cells start to vanish, suggesting that undifferentiated cells may be driving proliferation of wing-committed cells. When this observation is incorporated in our model, we show that the duration of cell recruitment robustly determines a final wing size even when cell proliferation rates of wing-committed cells are perturbed. Finally, we show that this control mechanism fails when perturbations in proliferation rates affect both wing-committed and recruitable cells, providing an experimentally testable hypothesis of our model.

Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability.

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.

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.

A dynamic cell recruitment process drives growth of the Drosophila wing by overscaling the vestigial expression pattern.

Organs mainly attain their size by cell growth and proliferation, but sometimes also grow through recruitment of undifferentiated cells. Here we investigate the participation of cell recruitment in establishing the pattern of Vestigial (Vg), the product of the wing selector gene in Drosophila. We find that the Vg pattern overscales along the dorsal-ventral (DV) axis of the wing imaginal disc, i.e., it expands faster than the DV length of the pouch. The overscaling of the Vg pattern cannot be explained by differential proliferation, apoptosis, or oriented-cell divisions, but can be recapitulated by a mathematical model that explicitly considers cell recruitment. When impairing cell recruitment genetically, we find that the Vg pattern almost perfectly scales and adult wings are approximately 20% smaller. Conversely, impairing cell proliferation results in very small wings, suggesting that cell recruitment and cell proliferation additively contribute to organ growth in this system. Furthermore, using fluorescent reporter tools, we provide direct evidence that cell recruitment is initiated between early and mid third-instar larval development. Altogether, our work quantitatively shows when, how, and by how much cell recruitment shapes the Vg pattern and drives growth of the Drosophila wing.

Nano-CT imaging of larvae in the ant Pheidole hyatti reveals coordinated growth of a rudimentary organ necessary for soldier development.

The growth of imaginal discs in holometabolous insects is coordinated with larval growth to ensure the symmetrical and proportional development of the adult appendages. In ants, the differential growth of these discs generates distinct castes-the winged male and queen castes and the wingless worker caste. In the hyperdiverse ant genus Pheidole, the worker caste is composed of two morphologically distinct subcastes: small-headed minor workers and larger, big-headed, soldiers. Although these worker subcastes are completely wingless, soldier larvae develop rudimentary forewing discs that function in generating the disproportionate head-to-body scaling and size of soldiers. It remains unclear, however, how rudimentary forewing discs in soldier larvae are coordinated with other imaginal discs. Here we show, using quantitative nano-CT imaging and three-dimensional analyses, that the increase in the volume of the soldier rudimentary forewing discs is coordinated with larval size as well as with the increase in the volume of the leg and eye-antennal (head) discs. However, relative to larval size, we found that when the rudimentary forewing discs appear during the last larval instar, they are relatively smaller but increase in volume faster than that of the head (eye-antennal) and leg discs. These findings show that the rudimentary wing disc in soldier larvae has evolved novel patterns of inter-organ coordination as compared with other insects to generate the big-headed soldier caste in Pheidole. More generally, our study raises the possibility that novel patterns of inter-organ coordination are a general feature of rudimentary organs that acquire novel regulatory functions during development and evolution.

Distinct regenerative potential of trunk and appendages of Drosophila mediated by JNK signalling.

The Drosophila body comprises a central part, the trunk, and outgrowths of the trunk, the appendages. Much is known about appendage regeneration, but little about the trunk. As the wing imaginal disc contains a trunk component, the notum, and a wing appendage, we have investigated the response to ablation of these two components. We find that, in contrast with the strong regenerative response of the wing, the notum does not regenerate. Nevertheless, the elimination of the wing primordium elicits a proliferative response of notum cells, but they do not regenerate wing; they form a notum duplicate. Conversely, the wing cells cannot regenerate an ablated notum; they overproliferate and generate a hinge overgrowth. These results suggest that trunk and appendages cannot be reprogrammed to generate each other. Our experiments demonstrate that the proliferative response is mediated by JNK signalling from dying cells, but JNK functions differently in the trunk and the appendages, which may explain their distinct regenerative potential.

COP9 signalosome subunits protect Capicua from MAPK-dependent and -independent mechanisms of degradation.

The COP9 signalosome removes Nedd8 modifications from the Cullin subunits of ubiquitin ligase complexes, reducing their activity. Here, we show that mutations in the Drosophila COP9 signalosome subunit 1b (CSN1b) gene increase the activity of ubiquitin ligases that contain Cullin 1. Analysis of CSN1b mutant phenotypes revealed a requirement for the COP9 signalosome to prevent ectopic expression of Epidermal growth factor receptor (EGFR) target genes. It does so by protecting Capicua, a transcriptional repressor of EGFR target genes, from EGFR pathway-dependent ubiquitylation by a Cullin 1/SKP1-related A/Archipelago E3 ligase and subsequent proteasomal degradation. The CSN1b subunit also maintains basal Capicua levels by protecting it from a separate mechanism of degradation that is independent of EGFR signaling. As a suppressor of tumor growth and metastasis, Capicua may be an important target of the COP9 signalosome in cancer.

The formation of the Thickveins (Tkv) gradient in Drosophila wing discs: A theoretical study.

The development of the wing imaginal disc (wing disc) is commonly adopted for the studies of patterning and growth which are two fundamental problems in developmental biology. Decapentaplegic (Dpp) signaling regulates several aspects of wing development, such as the anterior (A)-posterior (P) patterning, cellular growth rate, and cell adhesion. The distribution and activity of Dpp signaling are controlled in part by the expression level of its major type I receptor, Thickveins (Tkv). In this paper, we focus on theoretically investigating mechanisms by which the highly asymmetric pattern of Tkv is established in Drosophila wing discs. To the end, a mathematical model of Hh signaling and Dpp signaling is proposed and validated by comparisons with experimental observations. Our model provides a comprehensive view of the formation of Tkv gradients in wing discs. We found that engrailed (En), Hedgehog (Hh) signaling, and Dpp signaling cooperate to establish the asymmetric gradients of Tkv and pMad in the wing disc. Moreover, our model suggests a Brinker-mediated mechanism of Dpp-dependent repression of Tkv. Based on this mechanism, a couple of predicted experimental observations have been provided for further lab confirmation.

Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs.

Endocytosis of ligand-receptor complexes regulates signal transduction during development. In particular, clathrin and dynamin-dependent endocytosis has been well studied in the context of patterning of the Drosophila wing disc, wherein apically secreted Wingless (Wg) encounters its receptor, DFrizzled2 (DFz2), resulting in a distinctive dorso-ventral pattern of signaling outputs. Here, we directly track the endocytosis of Wg and DFz2 in the wing disc and demonstrate that Wg is endocytosed from the apical surface devoid of DFz2 via a dynamin-independent CLIC/GEEC pathway, regulated by Arf1, Garz, and class I PI3K. Subsequently, Wg containing CLIC/GEEC endosomes fuse with DFz2-containing vesicles derived from the clathrin and dynamin-dependent endocytic pathway, which results in a low pH-dependent transfer of Wg to DFz2 within the merged and acidified endosome to initiate Wg signaling. The employment of two distinct endocytic pathways exemplifies a mechanism wherein cells in tissues leverage multiple endocytic pathways to spatially regulate signaling.

miR-8 modulates cytoskeletal regulators to influence cell survival and epithelial organization in Drosophila wings.

The miR-200 microRNA family plays important tumor suppressive roles. The sole Drosophila miR-200 ortholog, miR-8 plays conserved roles in Wingless, Notch and Insulin signaling - pathways linked to tumorigenesis, yet homozygous null animals are viable and often appear morphologically normal. We observed that wing tissues mosaic for miR-8 levels by genetic loss or gain of function exhibited patterns of cell death consistent with a role for miR-8 in modulating cell survival in vivo. Here we show that miR-8 levels impact several actin cytoskeletal regulators that can affect cell survival and epithelial organization. We show that loss of miR-8 can confer resistance to apoptosis independent of an epithelial to mesenchymal transition while the persistence of cells expressing high levels of miR-8 in the wing epithelium leads to increased JNK signaling, aberrant expression of extracellular matrix remodeling proteins and disruption of proper wing epithelial organization. Altogether our results suggest that very low as well as very high levels of miR-8 can contribute to hallmarks associated with cancer, suggesting approaches to increase miR-200 microRNAs in cancer treatment should be moderate.

Complementary expression of optomotor-blind and the Iroquois complex promotes fold formation to separate wing notum and hinge territories.

Animal morphogenesis requires folds or clefts to separate populations of cells which are often associated with different cell affinities. In the Drosophila wing imaginal disc, the regional expression of the Iroquois complex (Iro-C) in the notum leads to the formation of the hinge/notum (H/N) fold that separates the wing hinge and notum territories. Although Decapentaplegic (Dpp) signaling has been revealed as essential for the hinge/notum subdivision through the restriction of Iro-C toward the notum region, the mechanism by which the H/N border develops into a fold is unknown. Here, we report that a Dpp target gene, optomotor-blind (omb), mediates the role of Dpp signaling in Iro-C inhibition. omb is complementarily expressed on the dorsal hinge side, abutting the Iro-C domain along the H/N border. Ectopic omb expression inhibits Iro-C in the notum territory, independent of known Iro-C regulators Msh and Stat92E. Uniform manipulation of either omb or Iro-C genes spanning the presumptive H/N border significantly suppresses H/N fold formation via inhibition of the apical microtubule enrichment. Ectopically sharp border or discontinuity in level of Iro-C or Omb is enough to generate ectopic fold formation. These results reveal that omb and Iro-C not only are complementarily expressed but also cooperate to promote H/N fold formation. Our data help to understand how Dpp signaling is interpreted region-specifically during tissue subdivision.

Scaling morphogen gradients during tissue growth by a cell division rule.

Morphogen gradients guide the patterning of tissues and organs during the development of multicellular organisms. In many cases, morphogen signaling is also required for tissue growth. The consequences of this interplay between growth and patterning are not well understood. In the Drosophila wing imaginal disc, the morphogen Dpp guides patterning and is also required for tissue growth. In particular, it was recently reported that cell division in the disc correlates with the temporal increase in Dpp signaling. Here we mathematically model morphogen gradient formation in a growing tissue, accounting also for morphogen advection and dilution. Our analysis defines a new scaling mechanism, which we term the morphogen-dependent division rule (MDDR): when cell division depends on the temporal increase in morphogen signaling, the morphogen gradient scales with the growing tissue size, tissue growth becomes spatially uniform and the tissue naturally attains a finite size. This model is consistent with many properties of the wing disc. However, we find that the MDDR is not consistent with the phenotype of scaling-defective mutants, supporting the view that temporal increase in Dpp signaling is not the driver of cell division during late phases of disc development. More generally, our results show that local coupling of cell division with morphogen signaling can lead to gradient scaling and uniform growth even in the absence of global feedbacks. The MDDR scaling mechanism might be particularly beneficial during rapid proliferation, when global feedbacks are hard to implement.

Improving Parameter Inference from FRAP Data: an Analysis Motivated by Pattern Formation in the Drosophila Wing Disc.

Fluorescence recovery after photobleaching (FRAP) is used to obtain quantitative information about molecular diffusion and binding kinetics at both cell and tissue levels of organization. FRAP models have been proposed to estimate the diffusion coefficients and binding kinetic parameters of species for a variety of biological systems and experimental settings. However, it is not clear what the connection among the diverse parameter estimates from different models of the same system is, whether the assumptions made in the model are appropriate, and what the qualities of the estimates are. Here we propose a new approach to investigate the discrepancies between parameters estimated from different models. We use a theoretical model to simulate the dynamics of a FRAP experiment and generate the data that are used in various recovery models to estimate the corresponding parameters. By postulating a recovery model identical to the theoretical model, we first establish that the appropriate choice of observation time can significantly improve the quality of estimates, especially when the diffusion and binding kinetics are not well balanced, in a sense made precise later. Secondly, we find that changing the balance between diffusion and binding kinetics by changing the size of the bleaching region, which gives rise to different FRAP curves, provides a priori knowledge of diffusion and binding kinetics, which is important for model formulation. We also show that the use of the spatial information in FRAP provides better parameter estimation. By varying the recovery model from a fixed theoretical model, we show that a simplified recovery model can adequately describe the FRAP process in some circumstances and establish the relationship between parameters in the theoretical model and those in the recovery model. We then analyze an example in which the data are generated with a model of intermediate complexity and the parameters are estimated using models of greater or less complexity, and show how sensitivity analysis can be used to improve FRAP model formulation. Lastly, we show how sophisticated global sensitivity analysis can be used to detect over-fitting when using a model that is too complex.