Drosophila Notum Research Articles

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.

Protrusion-Mediated Signaling Regulates Patterning of the Developing Nervous System.

During brain development, the tissue pattern and specification are the foundation of neuronal circuit formation. Contact-mediated lateral inhibition is well known to play an important role in determining cell fate decisions in the nervous system by either regulating tissue boundary formation or the classical salt-and-pepper pattern of differentiation that results from direct neighboring cell contacts. In many systems, however, such as the Drosophila notum, Drosophila wing, zebrafish pigmented cells, and zebrafish spinal cord, the differentiation pattern occurs at multiple-cell diameter distances. In this review, we discuss the evidence and characteristics of long-distance patterning mechanisms mediated by cellular protrusions. In the nervous system, cellular protrusions deliver the Notch ligand Delta at long range to prevent cells from differentiating in their vicinity. By temporal control of protrusive activity, this mechanism can pattern differentiation in both space and time.

Expression of mammalian ASH1 and ASH4 in Drosophila reveals opposing functional roles in neurogenesis

To investigate whether the members of the mammalian Achaete-Scute Complex homologue (ASH) gene family have evolved functional differences, we used the patterning of bristles as a phenotypic marker. Drosophila uses a single genetic locus - the Achaete-Scute Complex - to demarcate the regions of the body where bristles can form. We found 4–5 Achaete-Scute Complex homologue genes (ASH) in the mammalian genome, which are homologous with scute in Drosophila. Although ASH2 and ASH3 have gained new functions during evolution, the function of ASH4 and its evolutionary changes are still unclear. In this study, we overexpressed mouse and human ASH1 and ASH4 in the Drosophila notum respectively. The results show that both the protein sequence and cis-regulatory elements of mammalian ASH1 have conserved an ancient proneural function during evolution. However, mouse ASH4 has lost proneural function partly due to truncation of a C-terminal amino acid domain. Interestingly, instead of a similar loss of proneural function, we found human ASH4 can actually inhibit Drosophila bristle development, implying that human ASH4 may be a potential factor relating to skin development in human being. Our results demonstrate gene duplication of the ASH family may have led to a novel function during evolution.

Jitterbug/Filamin and Myosin-II form a complex in tendon cells required to maintain epithelial shape and polarity during musculoskeletal system development

During musculoskeletal system development, mechanical tension is generated between muscles and tendon-cells. This tension is required for muscle differentiation and is counterbalanced by tendon-cells avoiding tissue deformation. Both, Jbug/Filamin, an actin-meshwork organizing protein, and non-muscle Myosin-II (Myo-II) are required to maintain the shape and cell orientation of the Drosophila notum epithelium during flight muscle attachment to tendon cells.Here we show that halving the genetic dose of Rho kinase (Drok), the main activator of Myosin-II, enhances the epithelial deformation and bristle orientation defects associated with jbug/Filamin knockdown. Drok and activated Myo-II localize at the apical cell junctions, tendon processes and are associated to the myotendinous junction. Further, we found that Jbug/Filamin co-distribute at tendon cells with activated Myo-II. Finally, we found that Jbug/Filamin and Myo-II are in the same molecular complex and that the actin-binding domain of Jbug/Filamin is necessary for this interaction.These data together suggest that Jbug/Filamin and Myo-II proteins may act together in tendon cells to balance the tension generated during development of muscles-tendon interaction, maintaining the shape and polarity of the Drosophila notum epithelium.

Transmission of cytokinesis forces via E-cadherin dilution and actomyosin flows.

During epithelial cytokinesis, the remodelling of adhesive cell-cell contacts between the dividing cell and its neighbours has profound implications for the integrity, arrangement and morphogenesis of proliferative tissues. In both vertebrates and invertebrates, this remodelling requires the activity of non-muscle myosin II (MyoII) in the interphasic cells neighbouring the dividing cell. However, the mechanisms that coordinate cytokinesis and MyoII activity in the neighbours are unknown. Here we show that in the Drosophila notum epithelium, each cell division is associated with a mechanosensing and transmission event that controls MyoII dynamics in neighbouring cells. We find that the ring pulling forces promote local junction elongation, which results in local E-cadherin dilution at the ingressing adherens junction. In turn, the reduction in E-cadherin concentration and the contractility of the neighbouring cells promote self-organized actomyosin flows, ultimately leading to accumulation of MyoII at the base of the ingressing junction. Although force transduction has been extensively studied in the context of adherens junction reinforcement to stabilize adhesive cell-cell contacts, we propose an alternative mechanosensing mechanism that coordinates actomyosin dynamics between epithelial cells and sustains the remodelling of the adherens junction in response to mechanical forces.

Notum deacylates Wnt proteins to suppress signalling activity.

Signalling by Wnts 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 Wnts from the cell surface. However, this view fails to explain specificity since 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 likely help Notum colocalise with Wnts. 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 Wnts and thus constitutes the first known extracellular protein deacylase.

Multiple feedback loops achieve robust localization of wingless expression in Drosophila notum development

Organ morphogenesis starts with the spatial patterning of different gene expressions in organ primordia, which is based on positional information provided by morphogens. To generate precise positional information, the robust localization of morphogen sources is needed. This can be realized by several different mechanisms, such as (i) by reducing the variations in spatial arrangement of morphogen sources, (ii) by reducing the variations in their source levels, and (iii) by increasing the degree of source localization with the sharp boundary that makes morphogen gradients steeper. Here we focus on the mechanism of localization of wingless expression, one of the important morphogens in Drosophila notum development. The mechanism of wingless-localization can be explained by a simple feed-forward loop network motif, but the real molecular network adopted by the organism is much more complex; it includes multiple feedback loops that function with the feed-forward loop in a coordinated manner. To clarify the functions of the molecular network, we decompose it into three sub-modules, each of which includes a single feedback loop, and examine their possible roles using a mathematical model. We demonstrate how the regulatory network for wingless expression realizes the conditions (i)–(iii) for its robust localization.

Intertissue Mechanical Stress Affects Frizzled-Mediated Planar Cell Polarity in the Drosophila Notum Epidermis

Frizzled/planar cell polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the anteroposterior axis of the Drosophila thorax (notum). A subset of the trichome-producing notum cells differentiate as "tendon cells," serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin, we show that maintenance of anteroposterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells, and its loss of function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat/dachsous systems. The chas phenotype arises during normal shortening of the IFMs and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechanoresponse of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by one or more interactions with other tissues during development and organogenesis influences the maintenance of its shape and PCP features.

Checking out the neighbourhood

Although we often like to think that we are smart and observant, the history of science is littered with seemingly obvious problems being overlooked. I was reminded of this when reading a recent paper on how patterns are formed within tissues. Incontrovertible genetic evidence had previously shown that signals involving Delta and Notch mediate the patterning of bristle cell fate in Drosophila species. This led to a widely accepted model of 'lateral inhibition', in which inhibitory signals between neighbouring cells lead to an alternating distribution of cell fates. So the problem was solved and researchers moved on. However, there was something amiss: a simple prediction of the lateral inhibition model is that any given cell should always contact another cell of an alternative fate, but examination of patterning in the Drosophila notum reveals that this is not the case. So what is going on?

Dynamic Filopodia Transmit Intermittent Delta-Notch Signaling to Drive Pattern Refinement during Lateral Inhibition

The organization of bristles on the Drosophila notum has long served as a popular model of robust tissue patterning. During this process, membrane-tethered Delta activates intracellular Notch signaling in neighboring epithelial cells, which inhibits Delta expression. This induces lateral inhibition, yielding a pattern in which each Delta-expressing mechanosensory organ precursor cell in the epithelium is surrounded on all sides by cells with active Notch signaling. Here, we show that conventional models of Delta-Notch signaling cannot account for bristle spacing or the gradual refinement of this pattern. Instead, the pattern refinement we observe using live imaging is dependent upon dynamic, basal actin-based filopodia and can be quantitatively reproduced by simulations of lateral inhibition incorporating Delta-Notch signaling by transient filopodial contacts between nonneighboring cells. Significantly, the intermittent signaling induced by these filopodial dynamics generates a type of structured noise that is uniquely suited to the generation of well-ordered, tissue-wide epithelial patterns.

Drosophila notal bristle as a novel assessment tool for pathogenic study of Tau toxicity and screening of therapeutic compounds

To elucidate the Tau gain-of-toxicity functional mechanism and to search for potential treatments, we overexpressed human Tau variants (hTau) in the dorsal mesothorax (notum) of Drosophila. Overexpression of Tau variants caused loss of notal bristles, and the phenotype was used for evaluating toxicity of ectopic Tau. The bristle loss phenotype was found to be highly associated with the toxicity of hyperphosphoryled Tau in flies. We have shown that the bristle loss phenotype can be rescued either by reducing Glycogen synthase kinase 3β (GSK3β)/Shaggy (Sgg) activity or overexpressing Bβ2 regulatory subunits of PP2A. Elevated expression of the Drosophila Bβ2 homolog, Twins (Tws), also alleviated neuritic dystrophy of the dorsal arborization (da) neuron caused by Tau aggregation. Additionally, lowering endogenous Tau dosage was beneficial as it ameliorated the bristle loss phenotype. Finally, the bristle loss phenotype was used to evaluate the efficacy of potential therapeutic compounds. The GSK3β inhibitor, alsterpaullone, was found to suppress toxicity of Tau in a concentration-dependent manner. The notum of Drosophila, thus, provides a new tool and insights into Tau-induced toxicity. It could also potentially assist in screening new drugs for possible therapeutic intervention.

Cdc42, Par6, and aPKC Regulate Arp2/3-Mediated Endocytosis to Control Local Adherens Junction Stability

By acting as a dynamic link between adjacent cells in a monolayer, adherens junctions (AJs) maintain the integrity of epithelial tissues while allowing for neighbor exchange. Although it is not currently understood how this combination of AJ stability and plasticity is achieved, junctionally associated actin filaments are likely to play a role, because actin-based structures have been implicated in AJ organization and in the regulation of junctional turnover. Here, through exploring the role of actin cytoskeletal regulators in the developing Drosophila notum, we have identified a critical role for Cdc42-aPKC-Par6 in the maintenance of AJ organization. In this system, the loss or inhibition of Cdc42-aPKC-Par6 leads to junctional discontinuities, the formation of ectopic junctional structures, and defects in apical actin cytoskeletal organization. Affected cells also undergo progressive apical constriction and, frequently, delamination. Surprisingly, this Cdc42-aPKC-Par6-dependent regulation of junctional stability was found to be independent of several well-known targets of Cdc42-aPKC-Par6: Baz, Lgl, Rac, and SCAR. However, similar AJ defects are observed in wasp, arp2/3, and dynamin mutant cells, suggesting a requirement for actin-mediated endocytosis in the maintenance of junctional stability downstream of Cdc42. This was confirmed in endocytosis assays, which revealed a requirement for Cdc42, Arp2/3, and Dynamin for normal rates of E-cadherin internalization. By focusing on the molecular mechanisms required to maintain an epithelium, this analysis reveals a novel role for the epithelial polarity machinery, Cdc42-Par6-aPKC, in local AJ remodeling through the control of Arp2/3-dependent endocytosis.

Human homolog of NOTUM, overexpressed in hepatocellular carcinoma, is regulated transcriptionally by β‐catenin/TCF

The Drosophila Notum gene, which is regulated by the Wingless pathway, encodes a secreted hydrolase that modifies heparan sulfate proteoglycans. In comparative analysis of the gene expression profiles in primary human hepatocellular carcinomas (HCC) and normal organs, we observed that the human ortholog of Drosophila Notum was overexpressed markedly in a subset of HCC, but expressed rarely in adult normal tissues. Immunoblotting confirmed the overexpression of NOTUM protein in 12 of 40 primary HCC cases (30%). High levels of NOTUM protein were significantly associated with intracellular (nuclear or cytoplasmic) accumulation of beta-catenin protein: all 10 HCC with high intracellular beta-catenin also had high NOTUM expression, whereas only 2 of 30 cases (6.7%) without intracellular beta-catenin had high NOTUM expression (P < 0.00001). NOTUM expression in HepG2 cells was downregulated significantly by induction of a dominant-negative mutant of TCF4, a beta-catenin partner. In vivo binding of the beta-catenin/TCF complex to the NOTUM promoter was demonstrated by chromatin immunoprecipitation in HepG2 and SW480 cells, where canonical Wnt signaling is activated constitutively. These findings provide evidence that NOTUM is a novel target of beta-catenin/TCF4 and is upregulated in Wnt/beta-catenin signaling-activated HCC.

Prepatterning the Drosophila notum: The three genes of the iroquois complex play intrinsically distinct roles

The Drosophila thorax exhibits 11 pairs of large sensory organs (macrochaetes) identified by their unique position. Remarkably precise, this pattern provides an excellent model system to study the genetic basis of pattern formation. In imaginal wing discs, the achaete–scute proneural genes are expressed in clusters of cells that prefigure the positions of each macrochaete. The activities of prepatterning genes provide positional cues controlling this expression pattern. The three homeobox genes clustered in the iroquois complex (araucan, caupolican and mirror) are such prepattern genes. mirror is generally characterized as performing functions predominantly different from the other iroquois genes. Conversely, araucan and caupolican are described in previous studies as performing redundant functions in most if not all processes in which they are involved. We have addressed the question of the specific role of each iroquois gene in the prepattern of the notum and we clearly demonstrate that they are intrinsically different in their contribution to this process: caupolican and mirror, but not araucan, are required for the neural patterning of the lateral notum. However, when caupolican and/or mirror expression is reduced, araucan loss of function has an effect on thoracic bristles development. Moreover, the overexpression of araucan is able to rescue caupolican loss of function. We conclude that, although retaining some common functionalities, the Drosophila iroquois genes are in the process of diversification. In addition, caupolican and mirror are required for stripe expression and, therefore, to specify the muscular attachment sites prepattern. Thus, caupolican and mirror may act as common prepattern genes for all structures in the lateral notum.

Tailup, a LIM-HD gene, and Iro-C cooperate inDrosophiladorsal mesothorax specification

The LIM-HD gene tailup (tup; also known as islet) has been categorised as a prepattern gene that antagonises the formation of sensory bristles on the notum of Drosophila by downregulating the expression of the proneural achaete-scute genes. Here we show that tup has an earlier function in the development of the imaginal wing disc; namely, the specification of the notum territory. Absence of tup function causes cells of this anlage to upregulate different wing-hinge genes and to lose expression of some notum genes. Consistently, these cells differentiate hinge structures or modified notum cuticle. The LIM-HD co-factors Chip and Ssdp are also necessary for notum specification. This suggests that Tup acts in this process in a complex with Chip and Ssdp. Overexpression of tup, together with araucan, a 'pronotum' gene of the iroquois complex (Iro-C), synergistically reinforces the weak capacity of either gene, when overexpressed singly, to induce ectopic notum-like development. Whereas the Iro-C genes are activated in the notum anlage by EGFR signalling, tup is positively regulated by Dpp signalling. Our data support a model in which the EGFR and Dpp signalling pathways, with their respective downstream Iro-C and tup genes, converge and cooperate to commit cells to the notum developmental fate.

Cytochrome c‐d regulates developmental apoptosis in the Drosophila retina

The role of cytochrome c (Cyt c) in caspase activation has largely been established from mammalian cell-culture studies, but much remains to be learned about its physiological relevance in situ. The role of Cyt c in invertebrates has been subject to considerable controversy. The Drosophila genome contains distinct cyt c genes: cyt c-p and cyt c-d. Loss of cyt c-p function causes embryonic lethality owing to a requirement of the gene for mitochondrial respiration. By contrast, cyt c-d mutants are viable but male sterile. Here, we show that cyt c-d regulates developmental apoptosis in the pupal eye. cyt c-d mutant retinas show a profound delay in the apoptosis of superfluous interommatidial cells and perimeter ommatidial cells. Furthermore, there is no apoptosis in mutant retinal tissues for the Drosophila homologues of apoptotic protease-activating factor 1 (Ark) and caspase 9 (Dronc). In addition, we found that cyt c-d--as with ark and dronc-regulates scutellar bristle number, which is known to depend on caspase activity. Collectively, our results indicate a role of Cyt c in caspase regulation of Drosophila somatic cells.

Asymmetric Rab11 Endosomes Regulate Delta Recycling and Specify Cell Fate in the Drosophila Nervous System

Drosophila sensory organ precursor (SOP) cells are a well-studied model system for asymmetric cell division. During SOP division, the determinants Numb and Neuralized segregate into the pIIb daughter cell and establish a distinct cell fate by regulating Notch/Delta signaling. Here, we describe a Numb- and Neuralized-independent mechanism that acts redundantly in cell-fate specification. We show that trafficking of the Notch ligand Delta is different in the two daughter cells. In pIIb, Delta passes through the recycling endosome which is marked by Rab 11. In pIIa, however, the recycling endosome does not form because the centrosome fails to recruit Nuclear fallout, a Rab 11 binding partner that is essential for recycling endosome formation. Using a mammalian cell culture system, we demonstrate that recycling endosomes are essential for Delta activity. Our results suggest that cells can regulate signaling pathways and influence their developmental fate by inhibiting the formation of individual endocytic compartments.

Larval Multidendrite Neurons Survive Metamorphosis and Participate in the Formation of Imaginal Sensory Axonal Pathways in the Notum of Drosophila

In each hemimesothorax of Drosophila, a cluster of five larval multidendrite neurons that survives metamorphosis is described. The cell bodies of these neurons, initially grouped together, spread out over the medial heminotum during early pupal stages and extend new dendrites. Growing axons from sensory bristle neurons first appear in a defined orientation specific for each macrochaete. They subsequently contact processes from the larval multidendrite neurons and then appear to follow the preestablished axon trajectories of the latter. Ablation of the multidendrite neurons during the larval stage causes bristle axons to adopt abnormal trajectories. We suggest that the persistent larval neurons participate in guiding axons of the bristles on the medial half of the notum to the posterior dorsal mesothoracic nerve leading to the central nervous system.

The Decapentaplegic morphogen gradient regulates the notal wingless expression through induction of pannier and u-shaped in Drosophila

The morphogen gradient of Wingless, a Wnt family member protein, provides positional information to cells in Drosophila imaginal discs. Elucidating the mechanism that precisely restricts the expression domain of wingless is important in understanding the two-dimensional patterning by secreted proteins in imaginal discs. In the pouch region of the wing disc, wingless is induced at the dorsal/ventral compartment boundary by Notch signaling in a compartment-dependent manner. In the notum region of the wing disc, wingless is also expressed across the dorsal/ventral axis, however, regulation of notal wingless expression is not fully understood. Here, we show that notal wingless expression is established through the function of Pannier, U-shaped and Wingless signaling itself. Initial wingless induction is regulated by two transcription factors, Pannier and U-shaped. At a later stage, wingless expression expands ventrally from the pannier expression domain by a Wingless signaling-dependent mechanism. Interestingly, expression of pannier and u-shaped is regulated by Decapentaplegic signaling that provides the positional information along the anterior/posterior axis, in a concentration-dependent manner. This suggests that the Decapentaplegic morphogen gradient is utilized not only for anterior/posterior patterning but also contributes to dorsal/ventral patterning through the induction of pannier, u-shaped and wingless during Drosophila notum development.

Involvement of pannier and u-shaped in regulation of Decapentaplegic-dependent wingless expression in developing Drosophila notum

In developing Drosophila notum, wingless expression is regulated by Decapentaplegic signaling positively and negatively so that only notal cells receiving optimal levels of Decapentaplegic signals express wingless (Sato et al., 1999b. Development 126, 1457–1466). Here, we show evidence that this Decapentaplegic-dependent regulation of notal wingless expression includes plural mechanisms, involving pannier and u-shaped. In the medial notum, Pannier and U-shaped form a complex (Haenlin et al., 1997. Genes Dev. 11, 3096–3108). The expression of pannier and u-shaped is positively regulated by Decapentaplegic signals emanating from the dorsal-most region. The Pannier/U-shaped complex serves as a repressor and a transcriptional activator, respectively, for wingless and u-shaped expression. In the more lateral region, wingless expression is up-regulated by U-shaped-unbound Pannier. wingless expression is also weakly regulated by its own signaling.