InteractiveFly: GeneBrief

Matrix metalloproteinase 1 and Matrix metalloproteinase 2: Biological Overview | References

BIOLOGICAL OVERVIEW

Matrix metalloproteinases (MMPs) are a large conserved family of extracellular proteases, a number of which are expressed during neuronal development and upregulated in nervous system diseases. Primarily on the basis of studies using pharmaceutical inhibitors, MMPs have been proposed to degrade the extracellular matrix to allow growth cone advance during development and hence play largely permissive roles in axon extension. This study shows that MMPs are not required for axon extension in the Drosophila embryo, but rather are specifically required for the execution of several stereotyped motor axon pathfinding decisions. The Drosophila genome contains only two MMP homologs, Mmp1 and Mmp2. Mmp1 was isolated in a misexpression screen to identify molecules required for motoneuron development. Misexpression of either MMP inhibits the regulated separation/defasciculation of motor axons at defined choice points. Conversely, motor nerves in Mmp1 and Mmp2 single mutants and Mmp1 Mmp2 double mutant embryos are loosely bundled/fasciculated, with ectopic axonal projections. Quantification of these phenotypes reveals that the genetic requirement for Mmp1 and Mmp2 is distinct in different nerve branches, although generally Mmp2 plays the predominant role in pathfinding. Using both an endogenous MMP inhibitor and MMP dominant-negative constructs, it was demonstrated that MMP catalytic activity is required for motor axon fasciculation. In support of the model that MMPs promote fasciculation, it was found that the defasciculation observed when MMP activity is compromised is suppressed by otherwise elevating interaxonal adhesion - either by overexpressing Fas2 or by reducing Sema-1a dosage. These data demonstrate that MMP activity is essential for embryonic motor axon fasciculation (Miller, 2008).

Motor axons navigate an extracellular environment rich with potentially competing attractive and repulsive cues. Remarkably, motor axon growth cones are able to both interpret and integrate the signals present in this complex environment en route to their individual muscle targets. The particular axonal trajectory taken by any given motoneuron depends on the nature of the extracellular cues encountered by the extending axon as well as the complement of receptor or adhesion molecules expressed on its growth cone. In addition, several molecules required for either the activation or distribution of extracellular guidance molecules have recently been implicated in axon guidance (Miller, 2008).

The number and diversity of molecules implicated in motor axon pathfinding suggest that work in genetic model systems will continue to be essential to identify and tease apart the relative contributions of proteins involved in this process. In particular, the Drosophila embryo provides an important model for the study of motor axon pathfinding as a result of the small number of motoneurons, their defined trajectories and invariant muscle targets. Work by a number of groups has led to the identification and characterization of molecules critical for pathfinding and target recognition by Drosophila motor axons. An underlying principle to emerge from these studies is that in order for axons to reach their muscle targets, the activity of adhesion molecules that promote the fasciculation and/or bundling of motor axons must be precisely balanced with repulsive signals that trigger the defasciculation and/or separation of the extending axons (Miller, 2008).

Although the mechanisms responsible for limiting defasciculation to defined choice points in the periphery are not clear, a number of molecules necessary for proper defasciculation have been identified. In particular, repulsive signaling mediated by the Semaphorin-Plexin (Sema-Plex) pathway is essential for motor axon defasciculation. In wild-type embryos, axons of the intersegmental nerve branch b (ISNb) defasciculate from the primary ISN pathway and innervate the ventrolateral muscle (VLM) field. In embryos with reduced Sema-Plex pathway activity, however, ISNb axons fail to reach their targets and often remain bundled with the primary ISN branch - a phenotype consistent with diminished interaxonal repulsion. Furthermore, embryos with loss-of-function (LOF) mutations in nervy and protein kinase A RII, two genes that have been proposed to antagonize Sema-Plex signaling, exhibit premature and excessive motor axon defasciculation. By contrast, LOF mutations in the genes for cell adhesion molecules Fasciclin II (FasII) or Connectin (Con) suppress LOF mutations in Sema-1a and plexA, arguing that Sema-1a and PlexA stimulate defasciculation by overcoming axon-axon adhesion maintained by FasII and Con. These genetic interaction studies demonstrate the importance of balancing attractive and repulsive forces to enable correct fasciculation and pathfinding (Miller, 2008).

To understand how the precise balance of attraction and repulsion is achieved, the roles of additional molecules capable of modulating fasciculation of extending motor axons must be characterized. A number of studies have investigated the roles of metalloproteinases in axon extension and guidance. The metzincin metalloproteinases are zinc-dependent extracellular proteases that are subdivided into four subfamilies based on structure: astacins, serralysins, matrix metalloproteinases (MMPs) and adamlysins - a subfamily that includes the ADAMs (a disintegrin and a metalloproteinase) (Sternlicht, 2001). Classic models of metalloproteinase function in neuronal development proposed that they acted to degrade extracellular matrix (ECM) in order to clear a path for advancing axons. Recently, the roles of metalloproteinases in axonogenesis have been revisited in a number of experimental systems. These studies indicate that relevant neuronal metalloproteinase substrates include molecules directly involved in mediating axon pathfinding, including guidance receptors and their ligands. Among the metalloproteinases, the ADAM family is most strongly implicated in the regulation of axon guidance. For instance, ADAM10 terminates the interaction between ephrin A2 and EphA by cleaving ephrin A2, thereby facilitating axon retraction in vitro (Hattori, 2000). Analyses of Drosophila embryos mutant for the ADAM family homolog kuzbanian (kuz) further support the idea that ADAMs regulate particular guidance events; kuz mutations display genetic interactions with mutations in the repulsive midline factor slit. Interestingly, independent work from several groups has recently provided evidence that tolloid-related 1 (tlr1; also known as tolkin - FlyBase), a Drosophila astacin-family metalloproteinase, acts through its TGFβ ligand Dawdle to regulate motor axon guidance in the embryo (Miller, 2008).

As a family, MMPs are able to cleave nearly every component of the ECM, as well as numerous signaling molecules and cell surface receptors (Sternlicht, 2001). In the CNS, investigations of MMP function have largely centered on the roles of these proteases in nervous system disease, as MMPs are known to be dramatically upregulated in a host of CNS diseases, as well as following nervous system injury (Yong, 2005; Yong, 2001). However, in large part due to issues of redundancy and compensation among the twenty-four vertebrate MMP family members, the normal physiological roles of MMPs in the nervous system have remained largely elusive. Notably, a number of vertebrate MMPs display neuronal expression patterns in the embryo, suggesting that they may be involved in normal nervous system development. In support of this model, studies of Xenopus retinal ganglion cell axon guidance using MMP pharmaceutical inhibitors suggest that MMPs are required for specific pathfinding decisions (Hehr, 2005). Drosophila affords an attractive genetic model system in which to study MMP function since there are only two MMP family members in the fly, Mmp1 and Mmp2 (Llano, 2002; Llano, 2000; Page-McCaw, 2003). Whereas Mmp1 is a secreted protein, Mmp2 contains a GPI-anchor sequence and has been shown to be membrane-bound in tissue culture cells (Miller, 2008).

This work presents an analysis of MMP function during Drosophila embryonic neuronal development. Both LOF and gain-of-function (GOF) analyses support the model that MMP activity promotes motor axon fasciculation in the embryo. Misexpression of either Mmp1 or Mmp2 drives excessive motor axon fasciculation. By contrast, aberrant defasciculation was found in MMP LOF mutants. Although Mmp1 mutants display relatively mild pathfinding defects, many motor axons separate prematurely and aberrantly in Mmp2 single mutants and Mmp1 Mmp2 double mutants, indicating that Mmp2 plays a primary role in motor axon fasciculation. The embryonic expression of both MMPs was analyzed, and it was found that whereas Mmp1 exhibits a limited embryonic expression profile, Mmp2 is expressed in neurons and glia - supporting a primary role for Mmp2 in embryonic neuronal development. Importantly, aberrant motor axon defasciculation was found in embryos misexpressing the endogenous MMP inhibitor Timp and in embryos misexpressing MMP dominant-negative constructs, indicating that MMP catalytic activity is essential for pathfinding. Finally, it was shown that the defasciculation phenotype exhibited by MMP LOF mutants are dominantly suppressed by LOF mutations in Sema-1a, arguing that MMP activity normally acts to promote fasciculation by antagonizing Sema-1a function. Together, these results indicate that MMPs are not required for motor axon extension per se, but instead may modulate the responses of the axons of defined neuronal populations to specific guidance cues (Miller, 2008). To further investigate the possibility that MMP activity plays a role in neuronal development, the embryonic expression patterns of Mmp1 and Mmp2 were characterized. Previous studies have established that both genes are embryonically expressed (Llano, 2002; Llano, 2000; Page-McCaw, 2003). Using anti-Mmp1 antibodies, Mmp1 protein was found to be expressed in essentially the same spatiotemporal expression profile as has been described for Mmp1 RNA. The most prominent embryonic expression of Mmp1 is in the proventriculus and hindgut. Consistent with previous studies (Llano, 2000; Page-McCaw, 2003), Mmp1 CNS expression was found to be restricted to small clusters of segmentally repeating cells at the CNS midline. Mmp1 expression was also detected in the chordotonal organs of the peripheral nervous system and in two cells situated in the ventral mesodermal region. This expression is undetectable in Mmp1-null mutant embryos, (Mmp1²/Mmp1^Q112*), confirming antibody specificity (Miller, 2008).

The expression pattern of Mmp2 was characterized via whole-mount RNA in situ hybridization. In contrast to Mmp1, Mmp2 is widely expressed in the embryonic CNS. To identify the neuronal cells, wild-type embryos were double labeled with Mmp2 RNA and markers for specific neural and glial populations. It was found that Mmp2 is expressed in midline glia as Mmp2 RNA is co-expressed with Wrapper in these cells. Next whether Mmp2 is expressed in additional glial populations by was ested by co-labeling embryos with Mmp2 RNA and the glial marker anti-Repo. At stage 15, Mmp2 and Repo are co-expressed in approximately three glial cells per hemisegment situated at the base of motor nerve roots. The position and morphology of these cells suggest they correspond to exit glia, a group of peripheral glia originating within the CNS before migrating into the periphery during embryogenesis along extending motor axons. To confirm that these Mmp2-expressing cells are glia, it was asked whether they are absent in embryos mutant for glial cells missing (gcm), in which the number of glial cells is greatly reduced. In support of this conclusion, gcm mutant embryos specifically lack the Mmp2-expressing cells situated at the boundary between the CNS and periphery (Miller, 2008).

The observation that Mmp2-positive cells within the CNS do not co-express Repo suggested that they are probably neurons. To determine whether they correspond to well-characterized subsets of motoneurons or interneurons, embryos were double labeled with Mmp2 RNA and antibodies specific for particular neuronal populations. Co-expression between Mmp2 and Islet, a marker for distinct motoneuron and interneuron populations, was detected in three neurons per hemisegment in the lateral CNS. It was next asked whether these Mmp2-expressing neurons are Hb9-positive motoneurons. No co-expression was detected between Hb9 and Mmp2 RNA, suggesting that the Mmp2-positive neurons in the lateral CNS are Islet-positive interneurons. In sum, whereas Mmp1 exhibits a limited neuronal expression pattern, Mmp2 is expressed in stereotyped populations of neurons and glia, consistent with a role for Mmp2 in neuronal development (Miller, 2008).

This work demonstrates that the level of MMP catalytic activity dictates the degree of motor axon fasciculation in the Drosophila embryo. MMP misexpression is sufficient to inhibit separation of motor axons during outgrowth, but both of the primary embryonic motor nerve branches display striking defasciculation in MMP LOF mutants. The opposing axonal phenotypes observed in MMP LOF and GOF embryos indicates that the level of MMP activity is critical for pathfinding and further suggests that the relevant MMP substrate(s) plays an instructive role in motor axon guidance. In support of the hypothesis that MMPs influence axon outgrowth by modulating the activity of established guidance cues, Mmp2 LOF mutants were shown to be dominantly suppressed by a null mutation in Sema-1a, arguing that MMP function is tightly coupled to guidance decisions. Possible substrates for Mmp2 in motor axon pathfinding are considered and these findings are put in the context of proposed neural functions for metalloproteinases in vertebrates and invertebrates (Miller, 2008).

Both fly MMPs were previously shown to be expressed in the embryonic CNS, suggesting that they regulate aspects of neuronal development. However, the finding that both MMP single mutants and the Mmp1 Mmp2 double mutant survived embryogenesis called into question the extent of any possible roles for the MMPs in embryogenesis. This work presents genetic evidence that MMP catalytic activity is essential for motor axon fasciculation. Whereas Mmp1 mutants display subtle fasciculation errors, it was found that motor axons in Mmp2 mutants are markedly defasciculated, with many embryonic nerves appearing frayed and poorly organized. Consistent with this phenotypic analysis, the CNS expression profile of Mmp2 is considerably broader than that of Mmp1: Mmp2 is expressed in midline glia, in clusters of interneurons and in peripheral/exit glia but CNS expression of Mmp1 is limited to the midline. The prominent expression of Mmp1 and Mmp2 at the CNS midline prompted an examination of whether either MMP might be required for proper guidance there. However, no alteration was found in the behavior of axons at the midline in either MMP LOF or GOF mutant backgrounds, and no genetic interactions were found between Mmp2 and Slit or Mmp1 and Robo. These data indicate that MMPs do not contribute significantly to embryonic midline guidance in the fly (Miller, 2008).

Although the Mmp1 and Mmp2 LOF phenotypes are distinct, several pieces of evidence suggest that they have overlapping substrate specificities and can cleave the same guidance cue(s). First, misexpression of either Mmp1 or Mmp2 yields qualitatively indistinguishable guidance phenotypes with many motor axons remaining inappropriately bundled together. Second, misexpression of an Mmp1 dominant-negative transgene gives phenotypes nearly identical to those observed with a dominant negative Mmp2. Furthermore, the phenotypes observed with these constructs are stronger and more penetrant than the phenotypes of Mmp1 LOF mutants, suggesting that the Mmp1 dominant-negative transgene affects motor axon pathfinding by interfering with Mmp2 function by binding to the relevant Mmp2 substrate(s). Lastly, if Mmp1 and Mmp2 cleave the same substrate(s), they might be expected to be genetically redundant, since removal of one would be compensated for by the presence of the other. In fact, Mmp1 and Mmp2 show partially redundant roles in SNa pathfinding; the double mutant phenotype is significantly stronger than the phenotype observed in either single mutant. These results are in agreement with analyses of enzymatic activity of vertebrate MMPs that suggest that there is overlap between the substrates cleaved by individual MMPs (Page-McCaw, 2007; Miller, 2008).

Mmp2 contains a predicted GPI anchor and is membrane associated in Drosophila tissue culture cells (Llano, 2002). Thus, the expression pattern of Mmp2 in the embryo would be expected to reflect the locations of Mmp2-dependent proteolysis. Mmp2 RNA was found to be expressed in restricted populations of interneurons and peripheral glia, but not in motoneurons. Peripheral glia originate at the lateral edge of the CNS and migrate into the periphery along elongating motor axons. By the end of embryogenesis, they extend cytoplasmic processes and wrap axon bundles in a manner similar to vertebrate non-myelinating Schwann cells. It is proposed that peripheral glial-derived Mmp2 modulates the activity of factors required for pathfinding. This model implies that peripheral glia play a significant role in regulating motor axon fasciculation. This finding contrasts slightly with the results of (Sepp, 2001) who found more subtle errors in the motor axon projection pattern when peripheral glia were genetically ablated. One possible explanation for the weaker phenotypes in the peripheral glia-ablated embryos relative to Mmp2 LOF mutants is that peripheral glia express several factors that influence axon pathfinding in opposing directions - for example, proteins that both inhibit and stimulate fasciculation. In this way, peripheral glia would somewhat resemble midline glia which express both an axonal attractant (Netrin) and repellent (Slit). Therefore, ablation of the entire cellular population would be expected to yield different phenotypes than mutating individual molecules. Another possibility is that although Mmp2 is likely to act locally, its substrate might be secreted and could regulate motor axon guidance at a distance. In this case, Mmp2 need not be expressed at the site of fasciculation decisions, and either midline or interneuron-derived Mmp2 might provide the relevant proteolytic activity (Miller, 2008).

In principle, since MMP cleavage might either activate or inhibit the function of a molecule required for axon guidance, the motor axon phenotypes observed in MMP mutants could be expected to be identical to or opposite that of the phenotypes displayed by substrate mutations. Based solely on phenotypic considerations, several guidance molecules could be considered candidate MMP substrates. For example, LOF mutations in a number of genes give hyperfasciculation and/or stalled motor axon phenotypes. These include beaten path (beat) and sidestep (side), two immunoglobulin superfamily proteins required for proper defasciculation of both ISNb and SNa. There are also five CNS-expressed receptor protein tyrosine phosphatases (RPTPs) that have combinatorial roles in the regulation of motor axon pathfinding. A number of these RPTPs, in particular LAR, are involved in ISNb defasciculation decisions. Additionally, Plexin proteins and their receptors, the semaphorins, are critical regulators of motor axon fasciculation. Sema-Plex pathway activity promotes inter-axonal repulsion so that LOF mutations in Sema-Plex pathway components result in ISNb stall phenotypes. Importantly, it has also been shown that for axons to remain tightly bundled during normal axon outgrowth, Sema-Plex signaling must be actively antagonized, as LOF mutations in two downstream inhibitors, nervy and Protein kinase A, give aberrant defasciculation phenotypes similar to that observed in MMP mutations. Hence, levels of Sema-plex activity must be tightly controlled to ensure that defasciculation occurs properly at guidance choice points. And similar to what is described in this study for MMPs, reciprocal GOF and LOF mutations in the pathway can result in opposing hyper- and hypo-fasciculation phenotypes (Miller, 2008).

The MMP family as a whole does not cleave a conserved amino acid sequence in their targets, meaning that Drosophila substrates must be determined empirically, not computationally. One identified Mmp1 substrate, Ninjurin A (NijA), represented an appealing candidate in motor axon guidance as it is a signaling protein that regulates cell adhesion whose vertebrate homologs are upregulated in response to nerve injury (Zhang, 2006). However, no aberrations to motor axon pathfinding were found in either NijA LOF or GOF mutants, indicating that NijA is unlikely to be a relevant substrate in this context. Although few other Drosophila MMP substrates have been identified, the Drosophila homologs of several putative vertebrate MMP substrates make appealing candidates for MMP targets in embryonic CNS development. For instance, vertebrate membrane type MMP1 (MT1-MMP), has been shown to interact with the transmembrane heparan sulfate proteoglycan Syndecan 1 and trigger Syndecan 1 ectodomain shedding (Endo, 2003). Syndecan 1 processing stimulated cell migration on collagen, suggesting that this cleavage has functional consequences in vivo. Interestingly, Fox (2005) identified Drosophila Syndecan (Sdc) as a ligand for the LAR RPTP. Accordingly, genetic interaction studies indicate that Sdc and LAR act in concert to regulate ISNb pathfinding. As it is currently unknown whether LAR binds membrane-bound or soluble Sdc, MMP activity could potentially regulate the LAR/Sdc interaction. In addition, MT1-MMP has also recently been shown to be required for ectodomain shedding of Semaphorin 4D in a model of tumor-induced angiogenesis - a processing event required for the induction of blood vessel growth in vivo (Basile, 2007). Semaphorin signaling plays a well-documented role in regulating motor axon behavior. Furthermore, since Sema-1a mutations display strong genetic interactions with Mmp2 mutations in this system, it is conceivable that MMPs directly modulate Sema-Plex signaling activity (Miller, 2008).

MMP expression levels are highly elevated in a number of neuronal pathologies and after nervous system injury. MMP upregulation in CNS disease states raises the issue of whether MMP induction has an overall positive or negative effect on disease outcome. There is substantial evidence that the net effect of high MMP expression in some diseases is detrimental (Yong, 2005; Yong, 2001). For example, treatment with broad-spectrum metalloproteinase inhibitors is able to alleviate or prevent experimental autoimmune encephalomyelitis (EAE), a mouse multiple sclerosis model. There is also, however, growing recognition of beneficial functions for MMPs following CNS injury. The diverse functions for MMPs in disease states have become increasingly apparent as investigators have moved beyond the use of general metalloproteinase inhibitors to the study of particular MMPs. For example, increased expression of individual MMPs has been shown to correlate with periods of regeneration and repair following nervous system injury (Ahmed, 2005; Demestre, 2004; Shubayev, 2004). The functional significance of elevated MMP expression on regenerating axons has not been established, though in some regeneration models treatment with active MMPs promotes axon outgrowth (Heine, 2004; Siebert, 2001). In regeneration, it is thought that MMPs influence axon growth by degrading chondroitin sulphate proteoglycans (CSPGs), which normally inhibit regrowth beyond the glial scar (Miller, 2008).

In the context of neuronal development, there is substantial support for the idea that metalloproteinases, and in particular the ADAM subfamily, regulate axon outgrowth and pathfinding (McFarlane, 2003). Early work in the field suggested that metalloproteinases play a largely permissive role in axon outgrowth - by degrading the ECM in order to clear a path for extending axons. In support of a role for MMPs in outgrowth, it has been shown that a number of MMPs are expressed on the growth cones of vertebrate neurites extending in vitro. More recent work has demonstrated that in vitro, metalloproteinases are capable of modulating the interactions between guidance cues and their receptors (Galko, 2000; Hattori, 2000). For example, the interaction between ephrin A2 and Eph receptor is terminated by ephrin A2 cleavage via ADAM10 (also known as Kuzbanian-like - FlyBase) and/or Kuz. Functionally, this cleavage allows growth cone withdrawal of hippocampal neurons in culture, as a cleavage-inhibiting mutation delays axon retraction (Hattori, 2000). Metalloproteinases have also been implicated in DCC (deleted in colorectal carcinoma) receptor activity as broad-spectrum metalloproteinase inhibitors inhibit ectodomain shedding of DCC and potentiate netrin-mediated axon outgrowth (Galko, 2000). In vivo support for the role of ADAM proteases in axon outgrowth and pathfinding comes from work in Drosophila (Fambrough, 1996; Schimmelpfeng, 2001). kuz mutant embryos display ectopic axon crossing at the midline suggesting that kuz is required for repulsive signaling mediated by Slit-Roundabout (Robo). Supporting this idea, kuz and slit mutations genetically interact, and Kuz appears to be required for the clearance of the Robo receptor from commissural axons (Miller, 2008).

Although a number of vertebrate MMPs display neuronal expression patterns in the embryo, until relatively recently there was little direct evidence supporting a role for this metalloproteinase subclass in axon pathfinding. Studies of retinal ganglion cell (RGC) pathfinding in frogs argue that MMP activity is required for axon guidance at several defined choice points. Hehr (2005) used an MMP-specific inhibitor to demonstrate that MMPs are required for RGC guidance decisions both at the optic chiasm and tectum. Hehr work suggested that MMPs are normally required for axon guidance during vertebrate development, though the particular MMPs involved in RGC pathfinding remain to be identified. Exploiting the relative simplicity of the Drosophila model system, this study has established that individual MMPs play critical and distinct roles in well-defined axon pathfinding decisions during development. To extend this work to more complex vertebrate systems, it will be critical to analyze axon outgrowth and pathfinding in MMP single and compound mutant mice (Miller, 2008).

Tumor-derived MMPs regulate cachexia in a Drosophila cancer model

Cachexia, the wasting syndrome commonly observed in advanced cancer patients, accounts for up to one-third of cancer-related mortalities. A Drosophila larval model of organ wasting was established whereby epithelial overgrowth in eye-antennal discs leads to wasting of the adipose tissue and muscles. The wasting is associated with fat-body remodeling and muscle detachment and is dependent on tumor-secreted matrix metalloproteinase 1 (Mmp1). Mmp1 can both modulate TGF β signaling in the fat body and disrupt basement membrane (BM)/extracellular matrix (ECM) protein localization in both the fat body and the muscle. Inhibition of TGFβ signaling or Mmps in the fat body/muscle using a QF2-QUAS binary expression system rescues muscle wasting in the presence of tumor. Altogether, this study proposes that tumor-derived Mmps are central mediators of organ wasting in cancer cachexia (Lodge, 2021).

Interorgan communication ensures that organs respond in synchrony with alterations in the physiological state of the organism. One disease context where interorgan communication is relevant is cancer cachexia, where tumors cause loss of body fat and skeletal muscle, anorexia, metabolic deregulation, and systemic inflammation. Cachexia is present in up to 80% of advanced-stage cancers, and up to one-third of patients die from complications associated with cachexia. Early detection of the syndrome is further impeded by the lack of effective biomarkers, and it therefore remains a major unmet clinical need. Thus far, cancer cachexia has been studied mainly in rodent models where organ-specific genetic manipulations and large-scale, systematic in vivo functional studies (genetic screens) are challenging. Important parallels exist in how physiology is regulated between the vinegar fly Drosophila melanogaster and humans, whereby evolutionarily conserved enzymes and equivalent organs regulate carbohydrate and lipid metabolism. This makes the fly a valuable model organism for the study of cachexia. The fly fat body stores excess fat as triglycerides, thus functioning as the adipose tissues, while a group of specialized cells, called oenocytes, can function as hepatocytes by mobilizing lipids during periods of starvation. The fly muscles are also critical mediators of systemic energy homeostasis that coordinate energy supply and utilization, and are capable of inducing metabolic changes in distant tissues (Lodge, 2021).

In the last few years, models of cachexia have emerged in both Drosophila and zebrafish, in which several tumor-derived factors have been shown to modulate host physiology. The insulin/insulin growth factor (IGF) antagonist imaginal morphogenesis protein-late 2 (ImpL2) released by neoplastic cancer cells has been shown to suppress insulin signaling by binding to circulating Drosophila insulin-like peptides to cause insulin resistance in peripheral tissues. Gut-derived tumors, induced by the overexpression of the co-transcriptional factor Yorkie (Yki), also produce the PDGF- and VEGF-related factor 1 (Pvf1) ligand to nonautonomously activate host Pvr/MEK signaling and induce tissue wasting. Recently, it was shown that high-sugar-diet (HSD)-enhanced tumorigenesis upregulates Branchless (Bnl), a Drosophila fibroblast growth factor (FGF) ligand, to induce muscle wasting, which in turn releases proline, which fuels tumor growth. However, the signaling regulation that occurs in the tumor to drive tissue remodeling is so far unclear, and whether the remodeled host tissues communicate with each other is poorly understood (Lodge, 2021).

In this study, a larval cachexia model was created, enabling visualization of onset and progression of the disease, including the associated lipid mobilization, muscle detachment, and tissue remodeling within an intact animal. Among several cachectic factors, it was found that tumor-secreted Mmp1 alters the availability of Gbb in the tumor and triggers TGFβ activation in the fat body, resulting in the disruption of basement membrane (BM)/extracellular matrix (ECM) proteins at the adipocyte cell-cell adhesion site. In addition, Mmp1 and Mmp2 also act directly on the fat body and the muscle to trigger their disruption. Interestingly, the disassembly of fat-body cell-cell adhesion in the absence of tumor is sufficient to induce muscle detachment, suggesting that adipocyte breakdown releases signals that culminate in muscle detachment. Together, this study has gained valuable insights into the interplay between tumor, muscle, and adipose tissue during cachexia (Lodge, 2021).

The Gyc76C receptor Guanylyl cyclase and the Foraging cGMP-dependent kinase regulate extracellular matrix organization and BMP signaling in the developing wing of Drosophila melanogaster

The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, this study has identified Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. This study identified a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases and shows that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. Parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues are discussed (Schleede, 2015).

The vein cells that develop from the ectodermal epithelia of the Drosophila melanogaster wing are positioned, elaborated and maintained by a series of well-characterized intercellular signaling pathways. The wing is easily visualized, and specific mutant vein phenotypes have been linked to changes in specific signals, making the wing an ideal tissue for examining signaling mechanisms, for identifying intracellular and extracellular crosstalk between different pathways, and for isolating new pathway components (Schleede, 2015).

The venation defect, the loss of the posterior crossvein (PCV), is used to identify and characterize participants in Bone Morphogenetic Protein (BMP) signaling. The PCV is formed during the end of the first day of pupal wing development, well after the formation of the longitudinal veins (LVs, numbered L1-L6), and requires localized BMP signaling in the PCV region between L4 and L5. Many of the homozygous viable crossveinless mutants identified in early genetic screens have now been shown to disrupt direct regulators of BMP signaling, especially those that bind BMPs and regulate BMP movement and activity in the extracellular space. The PCV is especially sensitive to loss of these regulators because of the long range over which signaling must take place, and the role many of these BMP regulators play in the assembly or disassembly of a BMP-carrying 'shuttle' (Schleede, 2015).

The BMP Decapentaplegic (Dpp) is secreted by the pupal LVs, possibly as a heterodimer with the BMP Glass bottom boat (Gbb). This stimulates autocrine and short-range BMP signaling in the LVs that is relatively insensitive to extracellular BMP regulators. However, Dpp and Gbb also signal over a long range by moving into the intervein tissues where the PCV forms. In order for this to occur, the secreted BMPs must bind the D. melanogaster Chordin homolog Short gastrulation (Sog) and the Twisted gastrulation family member Crossveinless (Cv, termed here Cv-Tsg2 to avoid confusion with other 'Cv' gene names). The Sog/Cv-Tsg2 complex facilitates the movement of BMPs from the LVs through the extracellular space, likely by protecting BMPs from binding to cell bound molecules such as their receptors. In order to stimulate signaling in the PCV, BMPs must also be freed from the complex. The Tolloid-related protease (Tlr, also known as Tolkin) cleaves Sog, lowering its affinity for BMPs, and Tsg family proteins help stimulate this cleavage. Signaling is further aided in the PCV region by a positive feedback loop, as BMP signaling increases localized expression of the BMP-binding protein Crossveinless 2 (Cv-2, recently renamed BMPER in vertebrates). Cv-2 also binds Sog, cell surface glypicans and the BMP receptor complex, and likely acts as a co-receptor and a transfer protein that frees BMPs from Sog. The lipoprotein Crossveinless-d (Cv-d) also binds BMPs and glypicans and helps signaling by an unknown mechanism (Schleede, 2015).

PCV development takes place in a complex and changing extracellular environment, but while there is some evidence that PCV-specific BMP signaling can be influenced by changes in tissue morphology or loss of the cell-bound glypican heparan sulfate proteoglycans, other aspects of the environment have not been greatly investigated. During the initial stages of BMP signaling in the PCV, at 15-18 hours after pupariation (AP), the dorsal and ventral wing epithelia form a sack that retains only a few dorsal to ventral connections from earlier stages; the inner, basal side of the sack is filled with extracellular matrix (ECM) proteins, both diffusely and in laminar aggregates. As BMP signaling in the PCV is maintained and refined, from 18-30 hours AP, increasing numbers of dorsal and ventral epithelial cells adhere, basal to basal, flattening the sack. The veins form as ECM-filled channels between the two epithelia, while in intervein regions scattered pockets of ECM are retained basolaterally between the cells within each epithelium; a small amount of ECM is also retained at the sites of basal-to-basal contact. This changing ECM environment could potentially alter BMP movement, assembly of BMP-containing complexes, and signal reception, as has been demonstrated in other developmental contexts in Drosophila (Schleede, 2015).

This study demonstrates the strong influence of the pupal ECM on PCV-specific long-range BMP signaling, through the identification of a previously unknown ECM-regulating pathway in the wing. In a screen conducted for novel crossveinless mutations on the third chromosome, a mutation was found in the guanylyl cyclase at 76C (gyc76C) locus, which encodes one of five transmembrane, receptor class guanylyl cyclases in D. melanogaster. Gyc76C has been previously characterized for its role in Semaphorin-mediated axon guidance; Malpighian tubule physiology, and the development of embryonic muscles and salivary glands. Like the similar mammalian natriuretic peptide receptors NPR1 and NPR2, the guanylyl cyclase activity of Gyc76C is likely regulated by secreted peptides, and can act via a variety of downstream cGMP sensors (Schleede, 2015).

The evidence suggests that Gyc76C influences BMP signaling in the pupal wing by changing the activity of the cGMP-dependent kinase Foraging (For; also known as Dg2 or Pkg24A), also a novel role for this kinase. But rather than controlling BMP signal transduction in a cell-autonomous manner, evidence is provided that Gyc76C and Foraging regulate BMP signaling non-autonomously by dramatically altering the wing ECM during the period of BMP signaling in the PCV. This effect is largely mediated by changing the levels and activity of matrix metalloproteinases (Mmps), especially Drosophila Mmp2. Genetic interactions suggest that the ECM alterations affect the extracellular mobility and activity of the BMP-binding protein Sog (Schleede, 2015).

This provides the first demonstration of Gyc76C and For activity in the developing wing, and the first evidence these proteins can act by affecting Mmp activity. Moreover, the demonstration of in vivo link from a guanylyl cyclase to Mmps and the ECM, and from there to long-range BMP signaling, may have parallels with findings in mammalian cells and tissues. NPR and NO-mediated changes in cGMP activity can on the one hand change matrix metalloproteinase expression secretion and activity, and on the other change in BMP and TGFβ signaling (Schleede, 2015).

Mutation 3L043, uncovered by a genetic screen to identify homozygous lethal mutations required for PCV development, is a novel allele of gyc76C, a transmembrane peptide receptor that, like vertebrate NPRs, acts as a guanylyl cyclase. gyc76C is likely linked by cGMP production to the activity of the cGMP-dependent kinase For, and that Gyc76C and For define a new pathway for the regulation of wing ECM. This pathway appears to act largely through changes in the activity of ECM-remodeling Mmp enzymes. Loss of gyc76C or For alter both the organization of the wing ECM and the levels of the two D. melanogaster Mmps, and the gyc76C knockdown phenotype can be largely reversed by knockdown of Mmp2. This is the first indication of a role for cGMP, Gyc76C and For function in the developing wing, and their effects on the ECM provides a novel molecular output for each (Schleede, 2015).

Gyc76C and For are necessary for the normal refinement and maintenance of long-range BMP signaling in the posterior crossvein region of the pupal wing; in fact, crossvein loss is the most prominent aspect of the adult gyc76C knockdown phenotype. The evidence suggests that this effect is also mediated by changes in Mmp activity, and most likely the Mmp-dependent reorganization of the ECM. In fact, analysis using genetic mosaics finds no evidence for a reliable, cell autonomous effect of cGMP activity on BMP signal transduction in the wing. Thus, this apparent crosstalk between receptor guanylyl cyclase activity and BMP signaling in the wing is mediated by extracellular effects (Schleede, 2015).

It is noteworthy that the cGMP activity mediated by NPR or nitric oxide signaling can change also Mmp gene expression, secretion or activation in many different mammalian cells and tissues. Both positive and negative effects have been noted, depending on the cells, the context, and the specific Mmp. Given the strong role of the ECM in cell-cell signaling, the contribution of cGMP-mediated changes in Mmp activity to extracellular signaling may be significant. There is also precedent for cGMP activity specifically affecting BMP and TGFβ signaling in mammals. cGMP-dependent kinase activity increases BMP signaling in C2C12 cells, and this effect has been suggested to underlie some of the effects of nitric oxide-induced cGMP on BMP-dependent pulmonary arterial hypertension. Conversely, atrial natriuretic peptide stimulates the guanylyl cyclase activities of NPR1 and NPR2 and can inhibit TGFβ activity in myofibroblasts; this inhibition has been suggested to underlie the opposing roles of atrial natriuretic peptide and TGFβ during hypoxia-induced remodeling of the pulmonary vasculature. However, unlike the pathway observed in the fly wing, these mammalian effects are thought to be mediated by the intracellular modulation of signal transduction, with cGMP-dependent kinases altering BMP receptor activity or the phosphorylation and nuclear accumulation of receptor-activated Smads. Nonetheless, it remains possible that there are additional layers of regulation mediated through extracellular effects, underscoring the importance of testing cell autonomy (Schleede, 2015).

Aside from its role in adult Malpighian tubule physiology, Gyc76C was previously shown to have three developmental effects: in the embryo it regulates the repulsive axon guidance mediated by Semaphorin 1A and Plexin A, the proper formation and arrangement of somatic muscles, and lumen formation in the salivary gland. All these may have links to the ECM. Loss of gyc76C from embryonic muscles affects the distribution and vesicular accumulation of the βintegrin Mys, and reduces laminins and the integrin regulator Talin in the salivary gland. The axon defects likely involve a physical interaction between Gyc76C and semaphorin receptors that affects cGMP levels; nonetheless, gyc76C mutant axon defects are very similar to those caused by loss of the perlecan Trol (Schleede, 2015).

The parallels between the different contexts of Gyc76C action are not exact, however. First, only the wing phenotype has been linked to a change in Mmp activity. Second, unlike the muscle phenotype, the wing phenotype is not accompanied by any obvious changes in integrin levels or distribution, beyond those caused by altered venation. Finally, most gyc76C mutant phenotypes are reproduced by loss of the Pkg21D (Dg1) cytoplasmic cGMP-dependent kinase, instead of For (Dg2, Pkg24A) as found in the wing, and thus may be mediated by different kinase targets (Schleede, 2015).

For has been largely analyzed for behavioral mutant phenotypes, and the overlap between Pkg21D and For targets is unknown. While many targets have been identified for the two mammalian cGMP-dependent kinases, PRKG1 (which exists in alpha and beta isoforms) and PRKG2, it is not clear if either of these is functionally equivalent to For. One of the protein isoforms generated by the for locus has a putative protein interaction/dimerization motif with slight similarity to the N-terminal binding/dimerization domains of alpha and beta PRKG1, but all three For isoforms have long N-terminal regions that are lacking from PRKG1 and PRKG2. In fact, a recent study suggested that For is instead functionally equivalent to PRKG2: Like PRKG2, For can stimulate phosphorylation of FOXO, and is localized to cell membranes in vitro. But For apparently lacks the canonical myristoylation site that is thought to account for the membrane localization and thus much of the target specificity of PRKG2. FOXO remains the only identified For target, and foxo null mutants are viable with normal wings (Schleede, 2015).

The loss of long range BMP signaling in the PCV region caused by knockdown of gyc76C can, like the ECM, be largely rescued by knockdown of Mmp2. Two results suggest that it is the alteration to the ECM that affects long-range BMP signaling, rather than some independent effect of Mmp2. First, the BMP signaling defects caused by gyc76C knockdown were rescued by directly manipulating the ECM through the overexpression of the perlecan Trol. Second, when Mmp activity is inhibited by overexpression of the diffusible Mmp inhibitor TIMP, this not only rescued the PCV BMP signaling defects caused by gyc76C knockdown, but also led to ectopic BMP signaling, not throughout the region of TIMP expression, but only in those regions with abnormal accumulation of ECM (Schleede, 2015).

The Mmp2-mediated changes in the ECM likely affect long-range BMP signaling by altering the activity of extracellular BMP-binding proteins, particularly Sog. The BMPs Dpp and Gbb produced in the LVs bind Sog and Cv-Tsg2, shuttle into the PCV region, and are released there by Tlr-mediated cleavage of Sog and transfer to Cv-2 and the receptors. Genetic interaction experiments suggest that knockdown of gyc76C both increases Sog's affinity for BMPs and reduces the movement of the Sog/Cv-Tsg2/BMP complex into the crossvein region (Schleede, 2015).

Collagen IV provides the best-studied example for how the ECM might affect Sog activity. The two D. melanogaster collagen IV chains regulate BMP signaling in other contexts, and they bind both Sog and the BMP Dpp. Results suggest that collagen IV helps assemble and release a Dpp/Sog/Tsg shuttling complex, and also recruits the Tld protease that cleaves Sog cleavage and releases Dpp for signaling. D. melanogaster Mmp1 can cleave vertebrate Collagen IV. Since reduced Gyc76C and For activity increases abnormal Collagen IV aggregates throughout the wing and diffuse Collagen IV in the veins, it ishypothesized that these Collagen IV changes both foster the assembly or stability of Sog/Cv-Tsg2/BMP complexes and tether them to the ECM, favoring the sequestration of BMPs in the complex and reducing thelong-range movement of the complex into the region of the PCV (Schleede, 2015).

While few other D. melanogaster Mmp targets have been identified, it is likely that Mmp1 and Mmp2 share the broad specificity of their mammalian counterparts, so other ECM components, known or unknown, might be involved. For instance, vertebrate Perlecan and can be cleaved by Mmps. Trol regulates BMP signaling in other D. melanogaster contexts, and Trol overexpression rescue gyc76C knockdown's effects on BMP signaling. But while null trol alleles are lethal before pupal stages, normal PCVs were formed in viable and even adult lethal alleles like trol^G0023, and actin-Gal 4-driven expression of trol-RNAi using any of four different trol-RNAi lines did not alter adult wing venation. Loss of the D. melanogaster laminin B chain shared by all laminin trimers strongly disrupts wing venation, and a zebrafish laminin mutation can reduce BMP signaling (Schleede, 2015).

Finally, it was recently shown that Dlp, one of the two D. melanogaster glypicans, can be removed from the cell surface by Mmp2. While gyc76C knockdown did not detectably alter anti-Dlp staining in the pupal wing, it is noteworthy that Dlp and the second glypican Dally are required non-autonomously for BMP signaling in the PCV and that they bind BMPs and other BMP-binding proteins (Schleede, 2015).

Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation

Tissue remodeling is a crucial process in animal development and disease progression. Coordinately controlled by the two main insect hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), tissues are remodeled context-specifically during insect metamorphosis. Previous work has discovered that two matrix metalloproteinases (Mmps) cooperatively induce fat body cell dissociation in Drosophila. However, the molecular events involved in this Mmps-mediated dissociation are unclear. This study reports that JH and 20E coordinately and precisely control the developmental timing of Mmps-induced fat body cell dissociation. During the larval-prepupal transition, the anti-metamorphic factor Kr-h1 was found to transduce JH signaling, which directly inhibited Mmps expression and activated expression of tissue inhibitor of metalloproteinases (timp), and thereby suppressed Mmps-induced fat body cell dissociation. It is also noted that upon a decline in the JH titer, a prepupal peak of 20E suppresses Mmps-induced fat body cell dissociation through the 20E primary-response genes, E75 and Blimp-1, which inhibited expression of the nuclear receptor and competence factor βftz-F1. Moreover, upon a decline in the 20E titer, βftz-F1 expression was induced by the 20E early-late response gene DHR3, and then βftz-F1 directly activated Mmps expression and inhibited timp expression, causing Mmps-induced fat body cell dissociation during 6-12 hrs after puparium formation. In conclusion, coordinated signaling via JH and 20E finely tunes the developmental timing of Mmps-induced fat body cell dissociation. These findings shed critical light on hormonal regulation of insect metamorphosis (Jia, 2017).

MMPs and tissue inhibitor of metalloproteinases (TIMPs) play crucial roles in regulating tissue remodeling in both vertebrates and Drosophila. Previous work has demonstrated the collaborative functions of Mmp1 and Mmp2 in inducing fat body cell dissociation in Drosophila. timp mutant adults show autolyzed tissue in the abdominal cavity and inflated wings, a phenotype consistent with the role of timp in BM integrity and remodeling. The current study clarified the role of timp in inhibiting the enzymatic activity of Mmps and thus, Mmp-induced fat body cell dissociation. In mammals, Mmps activity in vivo is controlled at different levels, including the regulation by gene expression, the zymogens activation, and the inhibition of active enzymes by TIMPs. These studies unify the important inhibitory roles of timp/TIMP in regulating tissue remodeling in both Drosophila and mammals. In addition to regulating Mmp expression, JH and 20E signals differentially regulate timp expression, with the stimulatory role of Kr-h1 and the inhibitory role of βftz-F1. Because timp inhibits the enzymatic activity of Mmps in the Drosophila fat body, it is concluded that JH and 20E coordinately control Mmps activity at both the mRNA and enzymatic levels (Jia, 2017).

Previously work has show the requirement of both JH and its receptors to inhibit fat body cell dissociation in Drosophila. This study demonstrated the ability of Kr-h1 to transduce JH signaling to decrease Mmp expression and to induce timp expression during larval-prepupal transition. Moreover, a Kr-h1-binding sites (KBS) was identified in the Mmp1 promoter, indicating that Kr-h1 directly represses Mmp1 expression. Interestingly, Kr-h1 expression gradually increases from initiation of wandering (IW) to 3 h APF when induced by JH and 20E in an overlapping manner, thus inhibiting the enzymatic activity of Mmps and Mmp-induced fat body cell dissociation during the larval-prepupal transition. Moreover, Kr-h1 acts as an anti-metamorphic factor by inhibiting 20E signaling. It is proposed, in addition to directly affecting the expression of Mmps and timp, that Kr-h1 might also indirectly regulate their expression by inhibiting 20E signaling (Jia, 2017).

Two consecutive 20E pulses control timely metamorphosis in Drosophila. Together with previous findings, the current results show that the conserved 20E transcriptional cascade precisely controls the timing of Mmp-induced fat body cell dissociation. In general, the first 20E signal pulse plays an inhibitory role during the larval-prepupal transition; however, it is a prerequisite for the expression of βftz-F1, which induces the second 20E signal pulse during the prepupal-pupal transition and the expression of Mmps. Because of the requirement for the first 20E signal pulse, blockade of the 20E receptor prevents fat body cell dissociation. When JH titer declines, the prepupal peak of 20E activates expression of two 20E primary-response genes, E75 and Blimp-1, to inhibit fat body cell dissociation: E75 represses DHR3 transactivation of βftz-F1 expression, and Blimp-1 directly represses βftz-F1 expression. During the prepupal-pupal transition, DHR3 directly induces βftz-F1 expression from 6 h APF to 12 APF. Before pupation, βftz-F1 induces Mmp expression and represses timp expression. Moreover, an FBS was identified in the Mmp2 promoter, demonstrating that βftz-F1 directly induces Mmp2 expression. Finally, within 6 h before pupation, Mmp1 and Mmp2 cooperatively induce fat body cell dissociation, with each assuming a distinct role (Jia, 2017).

Insect metamorphosis is coordinately controlled by JH and 20E, whereas the hormonal control of tissue remodeling is strictly context-specific. Different larval tissues and adult organs might have distinct, yet precise, developmental fates and timing. Knowledge regarding this question is poor. Based on previous preliminary information, this study clarified the detailed molecular mechanisms by which JH and 20E precisely control the developmental timing of Mmp-induced fat body cell dissociation at both mRNA and enzymatic levels in Drosophila, and a working model is provided of hormonal control of tissue remodeling in animals (see Model showing developmental timing of Mmp-induced fat body cell dissociation is coordinately and precisely controlled by JH and 20E in Drosophila). In summary, at first, Kr-h1 transduces JH signaling to inhibit Mmp-induced fat body cell dissociation during larval-prepupal transition. Then when JH titer declines, the prepupal peak of 20E suppresses Mmp-induced fat body cell dissociation through E75 and Blimp-1, which inhibit βftz-F1 expression. Finally, until 20E titer declines, DHR3 induces βftz-F1 expression, and βftz-F1 covers the 20E-triggered transcriptional cascade to activate Mmp-induced fat body cell dissociation within 6 h before pupation. This study provides an excellent sample for better understanding the hormonal regulation of insect metamorphosis (Jia, 2017).

NADPH oxidase-generated reactive oxygen species in mature follicles are essential for Drosophila ovulation

Ovarian reactive oxygen species (ROS) are believed to regulate ovulation in mammals, but the details of ROS production in follicles and the role of ROS in ovulation in other species remain underexplored. In Drosophila ovulation, matrix metalloproteinase 2 (MMP2) is required for follicle rupture by degradation of posterior follicle cells surrounding a mature oocyte. MMP2 activation and follicle rupture are regulated by the neuronal hormone octopamine (OA) and the octopamine receptor in mushroom body (OAMB). This study investigated the role of the superoxide-generating enzyme NADPH oxidase (NOX) in Drosophila ovulation. Nox is highly enriched in mature follicle cells, and Nox knockdown in these cells leads to a reduction in superoxide and to defective ovulation. Similar to MMP2 activation, NOX enzymatic activity is also controlled by the OA/OAMB-Ca(2+) signaling pathway. In addition, this study reports that extracellular superoxide dismutase 3 (SOD3) is required to convert superoxide to hydrogen peroxide, which acts as the key signaling molecule for follicle rupture, independent of MMP2 activation. Given that Nox homologs are expressed in mammalian follicles, the NOX-dependent hydrogen peroxide signaling pathway that is described in this study could play a conserved role in regulating ovulation in other species (Li, 2018).

Ovulation is a key step in animal reproduction and involves multiple endocrine, paracrine, and autocrine signaling molecules, such as progesterone, epidermal growth factors, and prostaglandins. These molecules ultimately activate proteinases that break down the ovarian follicle wall, releasing a fertilizable oocyte. Several lines of evidence indicate that reactive oxygen species (ROS) also play indispensable roles in mammalian ovulation. However, there is no genetic evidence to support an in vivo role of ROS in ovulation, and the enzymes responsible for ROS production during ovulation are still unknown (Li, 2018).

ROS are oxygen-derived, chemically reactive small molecules and include superoxide anion (O2*-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH*). The physiological generation of ROS can occur as a byproduct of aerobic metabolism or as the primary function of the family of NADPH oxidases (NOXs). NOX enzymes transfer an electron across the cell membrane from NADPH in the cytosol to oxygen (O2) in the luminal or extracellular space. This movement of an electron generates O2*-, which can be rapidly converted into H2O2 by superoxide dismutases (SODs) (Li, 2018).

The mammalian NOX family comprises seven members (NOX1-5 and DUOX1-2), which have marked differences in tissue distribution and play a variety of physiological roles. Members of this family are also expressed in mammalian ovaries. Nox4 and Nox5, for example, are expressed in human granulosa cells. NOX4 and its accessory proteins in human granulosa cells show age-dependent reductions in protein expression, which correlates with low fertility. Importantly, pharmacological inhibition of NOX enzymes blocks follicle-stimulating hormone-induced oocyte maturation in mouse cumulus-oocyte complex in vitro. Despite these observations, a role for NOX in mammalian ovulation has not been demonstrated (Li, 2018).

The NOX family of enzymes is evolutionarily conserved across species. The Drosophila genome contains one Nox gene encoding NOX and one Duox gene encoding DUOX. DUOX has an additional peroxidase domain and has been well studied in gut-microbe interaction, wing formation, and wound healing. Much less is known about Nox. Earlier work reported that Nox regulates ovarian muscle contraction, which somehow influences ovulation. However, the mechanism of NOX regulation of ovulation and the cellular localization of NOX in Drosophila remain unclear (Li, 2018).

Recent work challenges the concept that ovulation is controlled by ovarian muscle contraction in Drosophila. Instead, Drosophila ovulation involves active proteolytic degradation of the follicle wall and follicle rupture and shares much in common with mammalian ovulation. Like in mammals, each oocyte in Drosophila is encapsulated in a layer of somatic follicle cells to form an egg chamber, which develops through 14 distinct stages to become a mature follicle (stage-14 egg chamber) in ovarioles. In mature follicles, the zinc finger transcription factor Hindsight (HNT) induces the expression of matrix metalloproteinase 2 (MMP2) in posterior follicle cells and octopamine receptor in mushroom body (OAMB) in all follicle cells. During ovulation, octopamine (OA) is released from neuron terminals in the ovary and binds to its receptor OAMB in stage-14 follicle cells. OAMB receptor activation causes an increase in intracellular calcium that activates MMP2 enzymatic activity, which breaks down posterior follicle cells and induces follicle rupture. Strikingly, the entire process of follicle rupture can be recapitulated ex vivo by culturing isolated mature follicles with OA in the absence of ovarian muscles and oviducts. This work casts doubt on the proposed involvement of ovarian muscles in follicle rupture/ovulation (Li, 2018).

This study investigated the role of Nox in Drosophila ovulation. Surprisingly, it was found that ovarian muscle Nox does not play a major role in ovulation but rather that Nox is enriched in mature follicle cells and is essential for follicle rupture/ovulation. OA/OAMB-Ca2+ signaling activates NOX enzymatic activity to produce extracellular O2*-, which is converted into H2O2 by an extracellular SOD3. These results suggest that NOX-produced ROS in mature follicles play a conserved role in regulating follicle rupture/ovulation across species (Li, 2018).

Ovarian ROS are indispensable for ovulation in mice. However, the site of production of ROS is unknown and it is unclear whether ROS play a conserved role in ovulation across species. This study provides genetic evidence that follicular ROS are required for ovulation in Drosophila. NOX, whose activity is regulated by follicular adrenergic signaling, regulates follicle rupture and ovulation by producing O2*- in the extracellular space of mature follicle cells. In addition, the data suggest that an extracellular SOD3 converts this O2*- into H2O2, which is the key signaling molecule responsible for regulating follicle rupture. H2O2 can partially mimic LH in regulating cumulus expansion and gene expression in mammalian follicles. It is thus plausible that H2O2 plays a conserved role in regulating follicle rupture/ovulation from insects to mammals (Li, 2018).

Members of the NOX family are also expressed in mouse and human granulosa cells and are functional in producing ROS. Norepinephrine, the mammalian counterpart of OA, is highly enriched in human follicular fluid and causes ROS generation in human granulosa cells. It will be interesting to determine whether norepinephrine plays a similar role as OA in generating ROS through regulating NOX activity during follicle rupture/ovulation in mammals (Li, 2018).

Why would Drosophila mature follicles use NOX to generate ROS during follicle rupture? ROS can be generated through the mitochondrial respiratory chain and membrane-bound NOX family enzymes, as well as by a host of intracellular enzymes, such as xanthine oxidase, cyclooxygenases, cytochrome p450 enzymes, and lipoxygenases that produce ROS as part of their normal enzymatic function. As high-level cytoplasmic ROS are detrimental to cell function and viability, limiting O2*-/H2O2 production in the extracellular environment may be essential for cell viability and function. This is consistent with the finding that overexpression of Sod1, which presumably produces extra-cytoplasmic H2O2, led to a disruption in follicle rupture and egg laying. Interestingly, Nox-knockdown follicles overexpressing Sod1 had normal follicle rupture, likely due to compensation of NOX-generated H2O2 by intracellularly produced H2O2, whereas bathing Nox-knockdown follicles in H2O2 did not rescue the defect in OA-induced follicle rupture. These findings suggest that local ROS production is essential for cellular physiology, while global ROS may be detrimental (Li, 2018).

Interestingly, Sod3 knockdown alone was sufficient to cause follicle rupture defects in Drosophila, yet mice lacking SOD3 are healthy and fertile. It is possible that SOD1 can compensate for the loss of SOD3 in mouse follicles, as mice lacking SOD1 or both SOD1 and SOD3 are subfertile or infertile, respectively (Li, 2018).

This study solved a conundrum in Drosophila ovulation. Previous work demonstrated that follicle rupture requires OA/OAMB induction of MMP2 activity in posterior follicle cells. However, OA/OAMB induces a rise in intracellular Ca2+ in all mature follicle cells. What is the role of OA/OAMB-Ca2+ in nonposterior follicle cells? This work demonstrated that OA/OAMB-Ca2+ signaling activates NOX in all follicle cells to produce O2.*- and H2O2, which are important for follicle rupture. NOX-generated ROS had a minimal effect on MMP2 activity, implying that these ROS regulate an independent pathway that is required for follicle rupture. Further studies should test whether region-specific Nox knockdown, such as only in nonposterior follicle cells, causes a follicle rupture defect (Li, 2018).

The targets of H2O2 in regulating follicle rupture are still unknown. Biological redox reactions catalyzed by H2O2 typically affect protein function by promoting the oxidation of cysteine residues. The best-characterized examples of H2O2-mediated signal transduction include several protein tyrosine phosphatases in growth factor signaling pathways, such as platelet-derived growth factor, epidermal growth factor (EGF), insulin, and B cell receptor signaling. Oxidation of the cysteine residue in the active-site motif of these phosphatases reversibly inactivates phosphatase activity and promotes growth factor signaling. The timing of H2O2 production and follicle rupture makes it unlikely that H2O2 promotes follicle rupture in Drosophila follicle cells by regulating growth factor signaling. The peak production of O2*- (and presumably of H2O2) is ~30-40 min after OA stimulation, which coincides with the beginning of follicle rupture. There is not enough time to allow growth factor signaling-mediated transcription and translation to occur before rupture happens. Alternatively, H2O2 is also involved in the activation of the ADAM (a disintegrin and metalloprotease) family of metalloproteases, possibly through direct oxidation of a cysteine residue that prevents the inhibition of catalytic domain by the prodomain of the enzyme. The idea is favored that NOX-generated H2O2 activates ADAM or other proteinases to regulate follicle rupture in addition to MMP2 activation. Microarray and RNA-sequencing analysis identified multiple proteinases that are up-regulated in Drosophila follicle cells during ovulation, and at least six different proteinases have been suggested to be involved in mammalian ovulation. Recent bioinformatics and large-scale proteomic analyses have predicted >500 proteins containing redox-active cysteine residues, some of which could serve as the downstream effectors of H2O2 for follicle rupture (Li, 2018).

Drosophila MMP2 regulates the matrix molecule faulty attraction (Frac) to promote motor axon targeting in Drosophila

Matrix metalloproteinases (MMPs) are widely hypothesized to regulate signaling events through processing of extracellular matrix (ECM) molecules. It has been shown that membrane-associated Mmp2 is expressed in exit glia and contributes to motor axon targeting (Miller, 2008). To identify possible substrates, a yeast interaction screen was undertaken for Mmp2-binding proteins, and the novel ECM protein faulty attraction (Frac: CG7526) was identified. Frac encodes a multidomain extracellular protein rich in epidermal growth factor (EGF) and calcium-binding EGF domains, related to the vertebrate Fibrillin and Fibulin gene families. It is expressed in mesodermal domains flanking Mmp2-positive glia. The juxtaposition of Mmp2 and Frac proteins raises the possibility that Frac is a proteolytic target of Mmp2. Consistent with this hypothesis, levels of full-length Frac are increased in Mmp2 loss-of-function (LOF) and decreased in Mmp2 gain-of-function (GOF) embryos, indicating that Frac cleavage is Mmp2 dependent. To test whether frac is necessary for axon targeting, axon guidance was characterized in frac LOF mutants. Motor axons in frac LOF embryos are loosely associated and project ectopically, a phenotype essentially equivalent to that of Mmp2 LOF. The phenotypic similarity between enzyme and substrate mutants argues that Mmp2 activates Frac. In addition, Mmp2 overexpression pathfinding phenotypes depend on frac activity, indicating that Mmp2 is genetically upstream of frac. Last, overexpression experiments suggest that Frac is unlikely to have intrinsic signaling activity, raising the possibility that an Mmp2-generated Frac fragment acts as a guidance cue cofactor. Indeed, genetic evidence is presented that Frac regulates a non-canonical LIM kinase 1-dependent bone morphogenetic protein signaling pathway in motoneurons necessary for axon pathfinding during embryogenesis (Miller, 2011).

Frac is expressed in the embryonic mesoderm concurrent with axon pathfinding and directly adjacent to Mmp2-expressing exit glia. It was also shown that Frac processing in embryos is Mmp2 dependent. frac LOF alleles display marked defects in axon pathfinding, which are tantamount to those displayed by Mmp2 LOF mutants, providing evidence that Mmp2 cleaves and activates Frac. Genetic interaction analyses was undertaken to elucidate the mechanism of Mmp2-Frac signaling. These studies argue that (1) the Frac fragment generated by Mmp2 does not have inherent signaling activity, and (2) frac contributes to the activation of a noncanonical BMP signaling pathway in motoneurons. These data are the first to demonstrate that proteolysis of an ECM molecule is involved in regulating the distribution or activation of a signaling cue during axon guidance (Miller, 2011).

Motor axons in Drosophila selectively defasciculate at guidance choice points as they follow individual routes to their synaptic targets. Repulsive signaling driven by the Semaphorin1a-PlexinA pathway acts in motor axons to promote interaxonal repulsion essential for axon separation. However, the mechanism by which defasciculation is normally confined to axon choice points is unknown. This study has presented evidence that an Mmp2-Frac pathway is necessary to limit axon defasciculation. Together, these data argue that a Frac cleavage product acts in the mesodermal ECM to signal axons to remain bundled. Because Mmp2 regulates Frac processing, signal activation may be precisely modulated both spatially and temporally. It is hypothesized that the Mmp2-Frac pathway provides a cue to axons to remain bundled during outgrowth and overcomes the interaxonal repulsion driven by the Sema1a-PlexA pathway. This antagonistic relationship receives strong support from the finding that both Mmp2 and Frac are dominantly suppressed by Sema1a mutations. It is speculated that Mmp2 is inactive at choice points, allowing the Sema1a-PlexA pathway to promote motor axon separation at these locations (Miller, 2011).

Among the metalloproteinases, the ADAM (a disintegrin and metalloproteinase domain) family has been most intimately linked to the regulation of axon targeting to date. In particular, the transmembrane protein ADAM10/Kuzbanian (Kuz) regulates the ectodomain shedding of a number of neural substrates, including the Roundabout receptor, Notch, and GPI-linked Ephrin A2. Given the central role that proteolysis is likely to play in sculpting nervous system connectivity and function, why are there few examples of MMP activity in this process? Analysis of the MMP family in neuronal development in vertebrates has likely been primarily obscured by the functional redundancy among the 24 MMP family members. Broad-spectrum MMP inhibitors and compound mouse mutants are beginning to resolve their functions. For example, MMP inhibitors have been used in an analysis of retinal ganglion cell guidance in Xenopus, and evidence was found for MMP function at two distinct choice points. There is also emerging evidence that MMP-9 elicits stable modifications of spine structure in long-term potentiation via ECM remodeling, arguing that synaptic plasticity requires MMPs (Miller, 2011).

In Drosophila, Mmp1 is secreted whereas Mmp2 is anchored to the membrane via a GPI link. The membrane association of Mmp2 may position it well to regulate defined aspects of neuronal development. In addition to the work presented in this study, Mmp2 has been shown to be required for dendritic reshaping of sensory neurons in the adult. A pulse of Mmp2 expression in epithelial cells directly apposed to a class of adult sensory neurons is coincident with the dendritic remodeling. Furthermore, dendritic reshaping is blocked in Mmp2 mutants, and clonal analysis indicates that Mmp2 acts locally in dendritic morphogenesis and reshapes only those dendrites in direct contact with Mmp2-positive epithelia. Hence, this work provides a second example of a local function of Mmp2 defining neuronal morphology in development. Given the specialized microenvironments likely present in the neuronal ECM, the spatiotemporal control of proteolysis provided by a membrane-associated MMP is predicted to be of fundamental importance (Miller, 2011).

This study has uncovered an Mmp2-Frac signaling module that promotes proper motor axon targeting. Motor axons have a precisely modulated attraction for their mesodermal ECM substrate. They must be sufficiently attracted to the mesodermal ECM to leave the ventral nerve cord and initiate migration on it, yet they need to limit exploration on this substrate and maintain strong interaxonal adhesion. The data argue that an Mmp2-dependent Frac cleavage fragment keeps motor axons on track. Because Mmp2 is expressed by motor-axon-associated exit glia, this processing event is positioned only in the vicinity of extending axons. In general terms, the ECM is positioned to regulate the distribution, activation, and presentation of growth factors, raising a number of possibilities for the role of Frac in axon guidance (Miller, 2011).

A relatively straightforward model based on the role of the related Fibrillin family in vertebrates is that Frac acts as a reservoir or sink for a guidance cue, which is released to signal via Mmp2-dependent cleavage. Vertebrate Fibrillins play major functional roles in limiting the bioavailability of TGFβs. TGFβs are targeted to Fibrillin scaffolds by latent TGFβ-binding proteins and are released from this latent Fibrillin-associated complex via proteolysis and integrin-mediated activation. Support for the inhibitory role for Fibrillins in the TGFβ pathway comes from work on human MFS, which is a connective tissue disorder caused by mutations in human Fibrillin-1. Remarkably, the aortic root dilation associated with MFS can be attenuated or reversed with losartan, a drug with anti-TGFβ signaling activity. The implication from these studies is that Fibrillin normally sequesters TGFβ, so that a decrease in Fibrillin dosage results in excessive TGFβ signaling. If a related mechanism is at play in Mmp2-Frac signaling, it would be predicted that (1) frac LOF embryos would phenotypically resemble Mmp2 GOF because in both scenarios too much signal is released, and that (2) frac LOF embryos would exhibit the opposite phenotype of Mmp2 LOF because in the first case excess signal is released, whereas in Mmp2 LOF mutants, too little signal is generated. The finding that frac LOF and Mmp2 LOF mutants display essentially identical phenotypes, which are opposite to those displayed by Mmp2 GOF, suggests that Frac does not simply act to sequester or store a guidance cue (Miller, 2011).

A second model is that the Frac cleavage product has intrinsic signaling activity. Until recently, there have been few compelling examples of the direct action of matrix molecules in signaling in neuronal development. However, it has been demonstrated that Thrombospondin (TSP), another EGF-domain-containing matrix protein, is secreted by astrocytes to promote synaptogenesis. It was further demonstrated that EGF domains in TSP directly bind to the neuronal α2δ-1 calcium channel subunit to increase synapse formation. If Frac were solely responsible for providing the pro-fasciculation signal, the simplest prediction is that increasing Frac levels would increase axon bundling. In fact, it was found that motor axons separate prematurely and inappropriately in 24B>frac embryos, arguing against this model. The pathfinding phenotypes in these embryos is interpreted to result from a 'dominant negative-like' effect of elevated Frac. If the addition of excess Frac to the signaling system effectively dilutes out the guidance cue (BMP), then decreased fasciculation is expected to result (Miller, 2011).

Frac is structurally related to the vertebrate Fibrillin family, making the interaction between Fibrillins and TGFβs potentially relevant. Furthermore, in Drosophila, BMPs have been shown to be under complex regulation by type IV Collagens, underscoring the essential role(s) matrix molecules play in this pathway. In addition, pMad is present in motoneuron nuclei as motor axons leave the ventral nerve cord, demonstrating that the pathway is active during guidance. In light of these data, genetic interactions between frac and the BMP pathway were analyzed. Strikingly, activation of BMP signaling in motoneurons via overexpression of an activated type I receptor (sax^act) strongly suppresses the guidance defects associated with both frac LOF and GOF, arguing that frac regulates a BMP cue. This hypothesis is supported by motor axon guidance phenotypes displayed by LIMK1 mutants and genetic interactions between frac and LIMK1. In conclusion, this study has described a novel pathway controlling motor axon targeting. It is proposed that Mmp2 expressed on the surface of exit glia controls the processing of the matrix molecule Frac, which sends a pro-fasciculation signal to extending axons. These findings highlight the complex interactions between motor axons, glia, and the mesodermal matrix during motor axon targeting and lay the groundwork for future investigations into Mmp2-Frac pathway function (Miller, 2011).

Dendrite reshaping of adult Drosophila sensory neurons requires Matrix Metalloproteinase-mediated modification of the basement membranes

In response to changes in the environment, dendrites from certain neurons change their shape, yet the mechanism remains largely unknown. This study shows that dendritic arbors of adult Drosophila sensory neurons are rapidly reshaped from a radial shape to a lattice-like shape within 24 hr after eclosion. This radial-to-lattice reshaping arises from rearrangement of the existing radial branches into the lattice-like pattern, rather than extensive dendrite pruning followed by regrowth of the lattice-shaped arbors over the period. It was also found that the dendrite reshaping is completely blocked in mutants for the matrix metalloproteinase (Mmp) 2. Further genetic analysis indicates that Mmp2 promotes the dendrite reshaping through local degradation of the basement membrane upon which dendrites of the sensory neurons innervate. These findings suggest that regulated proteolytic alteration of the extracellular matrix microenvironment might be a fundamental mechanism to drive a large-scale change of dendritic structures during reorganization of neuronal circuits (Yasunaga, 2010).

The Drosophila dendrite arborization (da) sensory neurons provide a suitable system for systematic analysis of dendritic morphogenesis. Recent studies have demonstrated that the subtype-specific dendritic patterns of class IV da (C4 da) neurons are determined by intrinsic factors, such as transcription factors, as well as extrinsic cues, such as repulsive interactions between neighboring dendrites. The C4 da neurons are born by mid-embryogenesis, and extend their two-dimensional dendrites between the epidermis and the underlying musculature during the late-embryonic and larval stages. Following a period of growth and development in larval stages, the larval dendritic arbors are completely replaced with adult-specific processes as a result of extensive pruning and subsequent regeneration of dendrite arbors during metamorphosis (Kuo, 2005; Shimono, 2009; Williams, 2005). In contrast to the significant progress being made to understand embryonic/larval dendrite morphogenesis in C4 da neurons, much less is known about how C4 da neurons remodel and regenerate their dendritic arbors in the pupal/adult stages (Yasunaga, 2010).

This study investigated the arbor dynamics of C4 da neurons following elaboration of the adult-specific dendrite arbors; a rapid, highly stereotyped reshaping of these dendrite arbors was found following eclosion. Similar to their larval counterparts, dendrites of adult C4 da neurons initially elaborated dendritic trees in a radial fashion, and covered the whole body wall prior to eclosion. However, in contrast to what is observed during larval development, this radial arrangement of the dendritic arbor was rapidly rearranged to a lattice-like shape within 24 hr after eclosion. Time-lapse imaging revealed that this radial-to-lattice reshaping was largely due to rearrangement of the existing radial processes into a lattice-like pattern, rather than extensive pruning of the radially arranged dendrites followed by regrowth of new arbors into a lattice pattern. Mutations in Mmp2, which encodes a GPI-anchored MMP, blocked this radial-to-lattice reshaping of C4 da dendrites without affecting other aspects of dendrite growth or development, and Mmp2 expression in epithelial cells adjacent to C4 da dendrites was transiently increased at exactly the time when C4 da dendrites undergo the radial-to-lattice reshaping. Therefore, Mmp2 is a critical regulator of the dendrite arbor reshaping. Furthermore, it was found that epithelial Mmp2 promotes the dendrite reshaping through local modification of the basement membrane (BM) upon which C4 da dendrites grow, suggesting that alteration of the ECM microenvironment might be a general mechanism for driving the structural plasticity of dendritic arbors in vivo (Yasunaga, 2010).

Large-scale reshaping of dendritic arbors is observed in the CNS and PNS including RGCs, olfactory neurons, and cortical neurons. In all cases, the dendrite reshaping is thought to be achieved mostly by elimination of existing dendrite branches and/or growth of new dendrite branches, although detailed analysis of these reshaping events has been hampered by the difficulty in visualizing the entire dendrite arbor of throughout the reshaping process. This report shows that large-scale reshaping of the entire dendrite arbor occurs in adult Drosophila sensory neurons over a 24 hr period following eclosion. Surprisingly, this dendrite reshaping is achieved by the rearrangement of the existing branches, rather than by extensive pruning of the existing dendrites and elongation of new branches. Indeed, time-lapse imaging indicates that major branches were rearranged, moving laterally toward the groove between the lateral tergosternal muscles (LTM) fibers just after eclosion. This dendrite rearrangement requires the activity of the Mmp2, as the rearrangement is completely blocked in Mmp2 mutants. Further genetic analyses indicate that the Mmp2-mediated degradation of the BM drives the dendrite rearrangement. Finally, Mmp2 expression is upregulated at the precise location (epithelial cells) and time (just following eclosion) at which it is required for this dendrite rearrangement, suggesting that the precise spatiotemporal regulation of Mmp2 expression is one mechanism to achieve this large-scale dendrite arbor reshaping (Yasunaga, 2010).

In addition to the rearrangement of existing dendrite branches, it was found that Mmp2 also regulates elongation of terminal branches along the DV axis. Notably, time-lapse imaging indicates that nascent protrusions often sprouted both inside and outside of the grooves; however, the latter ones immediately disappeared, suggesting that new protrusions sprouting inside of the grooves are selectively maintained. In addition, although some growing terminals extended toward the outside of the grooves, they immediately retracted back to the groove, and then restarted elongation inside the grooves. These branch growth behaviors suggest that the terminal elongation is promoted by the microenvironment inside the grooves and/or inhibited by the microenvironment outside the grooves. Most likely, Mmp2-mediated modification of the ECM provides such a microenvironment, since the DV-directed elongation of terminal branches was inhibited in Mmp2 mutants (Yasunaga, 2010).

Mammalian MMPs have been implicated in the structural plasticity of spines; however, the existence of more than 20 MMPs in mammals constrains genetic dissection of in vivo functions. In contrast, Drosophila has only two MMPs, Mmp1 and Mmp2, thus providing opportunity to analyze the neural functions of MMPs (Llano, 2000: Llano, 2002; Page-McCaw, 2003). This study provides genetic evidence that Mmp2 is essential for dendrite reshaping through modification of the BM upon which the C4 da dendrites innervate. The mammalian MMPs are known to cleave multiple ECM components, including laminins and collagens (Page-McCaw, 2007). Although less information is available for the substrate of the Drosophila Mmps, several biochemical studies suggest that Mmp1 and Mmp2 are able to cleave multiple ECM components in vitro as well (Llano, 2000; Llano, 2002). Indeed, Mmp1 and Mmp2 are both involved in the BM degradation in imaginal disc eversion and in epithelial tumor invasion in vivo (Srivastava, 2007; Uhlirova, 2006). However, despite their shared roles in many systems, Mmp2, but not Mmp1, is required for the BM degradation during dendrite reshaping. This specific function of Mmp2 in the dendrite reshaping presumably arises from the transient upregulation of Mmp2 in the epithelial cells during 0-24 hr after eclosion. In addition, Mmp2 is a GPI-anchored protein, whereas Mmp1 is a secreted proteinase, suggesting that the epithelial-derived Mmp2 is likely tethered to the epithelial cell surface. It is thus possible that the localized Mmp2 activity ensures the local degradation of the BM at the interface between the epidermis and the LTMs. Collectively, the spatiotemporally regulated expression of Mmp2 provides one molecular explanation for how the BM upon which C4 da dendrites grow is modulated in a specific pattern during the particular time window (Yasunaga, 2010).

It is proposed that the Mmp2-mediated BM degradation drives the dendrite reshaping in adult C4 da neurons. This idea is supported by the following lines of evidence. First, the reshaping dendrites are in direct contact with the BM, as revealed by EM experiments. Second, Mmp2 expression is transiently elevated in the epithelial cells at the exact time that C4 da neurons undergo dendrite reshaping. Third, Mmp2 acts locally, since Mmp2 mutant epithelial clones affect the dendrite reshaping only in dendrites situated directly below the Mmp2 mutant epithelial cells. Lastly, dendrite reshaping, but not dendrite growth/branching, is completely blocked in Mmp2 mutants. How does the BM degradation promote the dendrite reshaping? One possible scenario is that Mmp2 may loosen the dendrite-BM interaction, thereby facilitating the branch dynamics. The remnant BM might also function as a 'template' for the dendrite reshaping, since the reshaped branches appeared to be in contact with the remnant BM in the grooves between the LTMs. In regard to this possibility, it is notable that the ECM plays a critical role for dendrite morphogenesis through the direct interaction with the cell adhesion molecules, such as integrins. It is thus feasible that the dynamic changes of the ECM distribution upon which C4 da neurons grow may impact the dendrite patterning. In support of this model, the reshaped dendrites extended new terminals. Alternatively, Mmp2 might release molecules required for the dendrite reshaping from bound stores. Recent reports demonstrate that several axon guidance molecules, such as Semaphorins, Netrin, and Robos, also function as guidance factors for dendrites. For example, Semaphorin 3A acts as an attractive signal for the apical dendrites of pyramidal neurons in the mouse cortex. Similarly, dendrites of motoneurons in Drosophila CNS are attracted by Netrin and/or Slit. Since the secreted guidance molecules are often associated with the ECM, it is possible that the Mmp2-mediated BM degradation may cause a redistribution of the dendrite guidance factors on the ECM, thereby leading to reshaping of dendritic patterns. Finally, it is also possible that Mmp2 modulates the activity of important molecules for the dendrite reshaping by direct cleavage (Yasunaga, 2010).

MMP expression levels are elevated in a number of neuronal pathologies and after nervous system injury. For example, MMP9 expression is elevated shortly after ischemia. Likewise, multiple MMPs are immediately induced within 24 hr of an acute insult, such as spinal cord compression injury. Interestingly, increased dendrite dynamics have been observed in animal models of pathological conditions, such as the dentate gyrus following temporal lobe epilepsy and in CA1 hippocampal neurons following global ischemia. It is thus possible that upregulation of MMPs might contribute to the deregulation of dendrite dynamics under these pathological conditions. In the context of neuronal development, extensive dendrite reshaping occurs in the central and peripheral nervous systems during the critical period, and the reshaping is likely induced by afferent activity. Although intrinsic factors, including calcium signaling, have been implicated in this dendrite reshaping, the role of ECM dynamics in the critical period remains largely unknown. Recent studies indicate that MMP9 is significantly induced in the hippocampus and the cortex by elevated neural activity. Therefore, it would be of great interest to examine whether regulated proteolysis of the local ECM by MMPs might be a conserved mechanism to drive the reshaping of dendrite arbors in both developmental and pathological conditions (Yasunaga, 2010).

Spatial restriction of FGF signaling by a matrix metalloprotease controls branching morphogenesis

FGF signaling is a central regulator of branching morphogenesis processes, such as angiogenesis or the development of branched organs including lung, kidney, and mammary gland. The formation of the air sac during the development of the Drosophila tracheal system is a powerful genetic model to investigate how FGF signaling patterns such emerging structures. This article describes the characterization of the Drosophila matrix metalloprotease Mmp2 as an extracellular inhibitor of FGF morphogenetic function. Mmp2 expression in the developing air sac is controlled by the Drosophila FGF homolog Branchless and then participates in a negative feedback and lateral inhibition mechanism that defines the precise pattern of FGF signaling. The signaling function for MMPs described here may not be limited to branching morphogenesis processes (Wang, 2010).

To explore their potential role in morphogenesis, the expression patterns of Drosophila mmp1 and mmp2 were studied using GFP reporter strains. Consistent with previously published in situ hybridization data (Page-McCaw, 2003), it was found that both Drosophila mmp genes are active in the larval air sac primordium (ASP) as this structure forms and migrates across the wing imaginal disc, invading larval tissues. In the course of air sac outgrowth, mmp1 is evenly expressed throughout the tubular structure, whereasmmp2 levels become progressively more prominent in the distal end of the air sac and the tip cells (Wang, 2010).

To investigate whether the striking expression pattern of the Drosophila mmp genes in the ASP might point to a role in air sac formation, Mmp1 or Mmp2 function in the tracheal system of Drosophila third instar larvae was disrupted. This was achieved by the expression of specific RNAi constructs for either gene (UAS-mmp1RNAi and UAS-mmp2RNAi, respectively) under the spatial control of the trachea-specific btlGal4 driver and the temporal control of a temperature-sensitive Gal80ts suppressor. The efficacy of the RNAi-mediated knockdown was validated by PCR and antibody staining. Whereas knockdown of mmp1 had only a subtle effect, the phenotype caused by loss of mmp2 was dramatic: air sac extension was impaired, resulting in a severely deformed structure. The characteristic elongated and pointed shape of the air sac was lost. Instead of a single well-defined tip, mmp2- deficient air sacs displayed multiple tips and sometimes had a multilobed appearance. Proliferation appeared unaffected upon knockdown of mmp2 in the ASP as shown by anti-phospho H3 staining, indicating that the mmp2 loss-of-function (LOF) phenotype is not caused by an insufficient supply of tracheoblasts for air sac development. Expression of Drosophila Timp, a specific MMP inhibitor, under btlGal4/Gal80ts control resulted in a multitip phenotype indistinguishable from the one elicited by mmp2RNAi. Coexpression of Mmp2, but not Mmp1, largely reverted the ASP defect caused by TIMP. Moreover, the function of Mmp2 is continuously required to maintain the ordered outgrowth of the ASP (Wang, 2010).

In order for outgrowth to proceed normally, the ASP has to be patterned into stalk and tip cells. Under wild-type conditions, characteristic actin-rich filopodia emanate from the migrating ASP tip and extend toward the source of Bnl/FGF signaling. The multitipped, migration-deficient ASP caused by mmp2 LOF, however, are characterized by the widespread appearance of such filopodia. It is concluded that the patterning into stalk and tip cells might be disturbed under mmp2 LOF conditions, resulting in an expansion of tip territory. To confirm this interpretation, the expression of the tip cell marker esg was monitored in mmp2 LOF air sacs using a GFP reporter under the control of an esgGal4 driver or a straight esg-LacZ reporter. Expression of mmp2RNAi either in the tip cell domain (using the esgGal4 driver) or throughout the tracheal system (using btlGal4) causes a significant expansion of esg expression (Wang, 2010).

It is concluded that Mmp2 function is required to spatially constrain the tip cell region. It is plausible that the failure to migrate and the multitip phenotype are direct consequences of an expansion of the tip cell domain at the expense of the stalk cells (Wang, 2010).

The esg transcription factor controls tip-cell-specific functions in the developing tracheal system of the Drosophila embryo. It is reasonable to assume that esg would similarly confer tip cell specification in the larval ASP. If that were the case, one might expect that the expanded domain of esg expression that was have seen under conditions of reduced mmp2 expression might be causal for the multitip phenotype. To test this possibility, esg was overexpressed throughout the third instar ASP under the control of btlGal4/tubGal80ts, thereby expanding the expression of esg beyond the prospective tip domain. Interestingly, this manipulation caused a multitip phenotype resembling that of mmp2 LOF conditions. It is concluded that the expansion of the esg expression domain is sufficient to mediate the mmp2 LOF phenotype. This finding supports the idea that the mmp2 mutant phenotype is a consequence of a patterning defect caused by an expansion of tip cell fate (Wang, 2010).

Tip cell fate is specified by FGF signaling. Therefore, the expansion of the tip cell domain under mmp2 LOF conditions might be caused by a spread of FGF signaling activity. Such FGF signaling activity in the tip region can be visualized by staining with a phospho-specific (dpERK) antibody, which recognizes the doubly phosphorylated, active form of Drosophila ERK. Consistent with previous reports, dpERK staining was found to be restricted to the tip area of the wild-type ASP. However, upon suppression of Mmp2 activity, either by expression of mmp2RNAi or of timp, dpERK staining was expanded broadly throughout the air sac. This result suggests that the mmp2 LOF phenotype is caused by an expansion of FGF signaling. Consequently, tracheoblasts that would otherwise become part of the stalk are misspecified and adopt ectopic tip cell fates. Consistent with this interpretation, deliberate activation of FGF signaling by overexpression of FGF receptor throughout the air sac can phenocopy the Mmp2 LOF phenotype. These data suggest that Mmp2 can prevent FGF signaling in prospective stalk cells and thereby restrict FGF activity to the tip cell domain (Wang, 2010).

To directly confirm that Mmp2 can suppress FGF signaling, experiments were conducted in Drosophila S2 cells. Transient coexpression of Bnl and Btl potently activates ERK phosphorylation, as monitored by immunoblotting with the dpERK antibody. This ERK response can be abrogated by coexpression with Mmp2. A catalytically inactive mutant, Mmp2E258A, however, has no effect. In agreement with the in vivo data, this result suggests that Mmp2 can interfere with Bnl/Btl signaling. To investigate whether this effect is specific for the FGF pathway or whether other receptor tyrosine kinases might also be affected, a similar experiment was conducted in which ERK was activated by expression of Drosophila EGF receptor and a soluble form of its ligand, Keren. Expression of EGF-R and sKrn causes ERK phosphorylation to a similar degree as Bnl/Btl expression. Significantly, however, this activation is insensitive to the presence of Mmp2. It is concluded that the regulatory function of Mmp2 on air sac development is selective for the FGF pathway. Mmp2 signaling might therefore control the balance between EGF-regulated cell proliferation and FGF-mediated patterning and cell migration (Wang, 2010).

Many examples of branching morphogenesis require a lateral inhibition process that serves to spatially restrict a tip domain in the outgrowing organ. Lateral inhibition is mediated by an inhibitory signal that is released by distal cells once they have adopted tip cell identity, to stop their neighbors from doing the same. In this manner, spreading of tip fate into the adjacent stalk cell area is prevented, assuring the correct patterning and structure of the forming organ. The data presented so far are compatible with the idea that Mmp2 is part of a tip-cell-specific lateral inhibition mechanism. Consistent with this model, FGF signaling itself can induce Mmp2 expression in the ASP. This conclusion was further confirmed by real-time RT-PCR and western blotting (Wang, 2010).

The lateral inhibition model predicts that Mmp2 expression is required in the tip cells themselves, to restrict expansion of tip cell territory. To test this notion, a clonal analysis strategy was adopted. Using MARCM technology, random clones of GFP-marked cells were generated that were homozygous for themmp2 LOF allele mmp2G535R or the mmp1 LOF allele mmp1Q273*. In parallel, mmp2RNAi or Timp was clonally expressed using the flp-out Gal4 driver system in developing larvae. All strategies resulted in the generation of GFP-labeled clones that lacked the capacity to express Mmp2 activity. The location of these clones within the mRFP-labeled air sac was recorded. Both strategies showed that the mmp2-deficient cells rarely contributed to tip territory, whereas control clones that lack Mmp1 function or express GFP only were randomly distributed across the whole area of the air sac, including the tip. This is interpretated to mean that mmp2-deficient cells, even if they were the first to receive an FGF signal, would not be able to maintain tip fate, as they could not inhibit FGF signaling in their wild-type neighbors. Those neighbor cells expressing Mmp2 normally would then exert a lateral inhibition effect preventing mmp2-deficient clonal cells from receiving FGF signaling. In other words, cells of the ASP compete with each other to contribute to the tip. Cells that lack Mmp2 activity are at a disadvantage and will likely lose the ability to respond to FGF signals, and assume a subsidiary stalk cell role (Wang, 2010).

Finally, an experiment wss designed to directly visualize the paracrine effect of Mmp2 on FGF signaling in the ASP. To this end, a small number of clones expressing Mmp2 were induced in the ASP. The strain used here also carried the hs-bnl transgene. Thereby, Bnl expression can be ubiquitously activated by exposing wandering third instar larvae to a mild heat treatment. The resulting elevated levels of FGF throughout the ASP made it easier to observe inhibitory functions of Mmp2. Strikingly, areas were found of diminished ERK phosphorylation adjacent to Mmp2-expressing clones, as monitored by dpERK staining. Control clones that expressed only GFP never caused such an effect (Wang, 2010).

Several observations in this experiment are noteworthy. First, the inhibitory effect of Mmp2 expression on ERK signaling is strictly nonautonomous. Only cells adjacent to the Mmp2- expressing clones showed decreased ERK activity. The clonal cells themselves are impervious to the inhibitory activity of Mmp2. This finding explains the persistent FGF activity in the ASP tip cells even after Mmp2 expression is induced, and supports the concept that tip cells act as classical organizers that secrete signals to which neighboring cells respond but to which they themselves are insensitive (Wang, 2010).

Second, the paracrine inhibitory effect that Mmp2-expressing cells exert on FGF signaling in their neighbors is not gradual. The affected cells adjacent to the Mmp2-expressing clones have either normal or dramatically decreased ERK signaling activity, but none show intermediary levels. This suggests that Mmp2 activity influences a yes/no decision. Such a mechanism would be consistent with the proposed function of the FGF-Mmp2 signaling circuit to distinguish between two distinct cell fate choices: tip or stalk (Wang, 2010).

Third, not all cells touching Mmp2-expressing clones show decreased ERK activity. The basis for this anisotropic effect of Mmp2 is not clear, but it might be related to the previous point: cells can adopt either an ERK on (tip cell) or an ERK off (stalk cell) state, a decision that is influenced by the interplay between FGF, FGF receptor, and Mmp2. Stochastic variations in signaling might tip the balance one way or the other, especially in the experimental setting employed here, in which high ubiquitous levels of FGF are present (Wang, 2010).

Matrix metalloproteases have long been implicated in invasion and branching morphogenesis. Whereas many studies focus on MMP-dependent extracellular matrix (ECM) remodeling in this context, a different role for Mmp2 was documented in this study: controlling the spatial pattern of FGF signaling. It should be noted that the signaling function of Mmp2 documented here by in vivo and cell-culture evidence does not rule out a mechanical contribution of MMP to air sac outgrowth and invasion. Interestingly, Guha (2009) has very recently reported that Mmp2 clears ECM components around the outgrowing ASP, which may facilitate the movement of the structure (Wang, 2010).

The function of Mmp2 as a modulator of FGF signaling and as part of a lateral inhibition mechanism can be explained by the following model: tracheal cells that receive the FGF signal first will activate ERK to induce gene expression programs that direct budding and air sac formation. Among the activated transcription units is the mmp2 gene, which is required for the release of an inhibitory signal that nonautonomously prevents further FGF responses in adjacent cells. This Mmp2-mediated lateral inhibition mechanism would thereby restrict the spreading tip cell fate through the prospective air sac. The nature of the inhibitory signal that is delivered by the Mmp2-expressing tip cells is still unknown (Wang, 2010).

It is likely that the mechanisms described here for the Drosophila air sac are also employed by other species and developmental processes. For example, it has been shown that cells with high levels of FGF activity have a competitive advantage in populating the tips or 'terminal end buds' of invading ducts during murine mammary development (Lu, 2008), a finding that is indicative of a lateral inhibition process. Interestingly, it is well established that both MMPs and FGF signaling make critical contributions to mammary development. It is therefore tempting to speculate that the regulatory interplay between MMPs and FGFs operates broadly in invasive growth and branching morphogenesis (Wang, 2010).

Regulation of Drosophila matrix metalloprotease Mmp2 is essential for wing imaginal disc:trachea association and air sac tubulogenesis

The Drosophila Dorsal Air Sac Primordium (ASP) is a tracheal tube that grows toward Branchless FGF-expressing cells in the wing imaginal disc. This study shows that the ASP arises from a tracheal branch that invades the basal lamina of the disc to juxtapose directly with disc cells. The role of matrix metalloproteases (Mmps) was examined; reducing Mmp2 activity perturbed disc-trachea association, altered peritracheal distributions of collagen IV and Perlecan, misregulated ASP growth, and abrogated development of the dorsal air sacs. Whereas the function of the membrane-tethered Mmp2 in the ASP is non-cell autonomous, it was found that Mmp2 may have distinct tissue-specific roles in the ASP and disc. These findings demonstrate a critical role for Mmp2 in tubulogenesis post-induction, and implicate Mmp2 in regulating dynamic and essential changes to the extracellular matrix (Guha, 2009).

The invasive coupling of the wing disc with the Tr2 transverse connective and the ECM remodeling that accompanies ASP growth led to an investigation of how the presence of a Basal lamina (BL) impacts FGF signaling and the roles of MMPs. Based on the capacity of Bnl/FGF to signal through the disc and tracheal BL, it is concluded that these BL are functionally transparent to FGF. Based on the effects of btl-TIMP, ap-TIMP, btl-MMP2 RNAi and ap-MMP2RNAi, and on the phenotypes of Mmp2 mutants, it is concluded that Dm-MMP2 has essential roles sculpting the disc-trachea association and remodeling the ECM during ASP induction and growth. This discussion considers mechanisms underlying disc-trachea association, disc to trachea FGF signaling and the role of Dm-Mmp2 in tissue contact, ECM remodeling and organ morphogenesis (Guha, 2009).

Invasive coupling of the wing disc and trachea must involve several distinct processes. First, progenitor disc and trachea cells, which originate independently in the embryo, must establish contact. Disc cells migrate towards the trachea during embryogenesis, leading to direct juxtaposition. Collagen IV is not detected at this stage, so it seems unlikely that the BL had fully formed. The current experiments did not address whether Mmps are required for the early association of disc and trachea. However, it was found that L3 discs remain associated with trachea in Mmp2^w307 and Mmp1^Q273 mutants. Since the Mmp2^w307 allele harbors a nonsense mutation and is a genetic null, these findings suggest that Mmp2 is not required to join these tissues (Guha, 2009).

Second, the arrangement of BL over the invasively coupled tracheal segment requires precise position-specific synthesis as well as continuous remodeling as the disc and trachea grow. Since core proteins of the ECM (e.g. collagen IV) are expressed by only a few disc cells, most BL components are presumably recruited from circulating stores in the hemolymph; little is known of the processes that bring these components to appropriate locations or regulate their assembly. It is not known, for instance, whether the absence of a distinct tracheal BL where the transverse connective contacts the disc is a consequence of insufficient levels of components. Alternatively, if availability of BL components is not limiting, it is not known whether the enzymes that synthesize BL are absent from these locations, or whether proteases that degrade the core components might be activated there. The changes in levels of Mmp2 that were engineered had significant effects on the ECM and its components. The presence of ectopic BL around disc-associated trachea in ap-TIMP animals suggests that neither components nor synthetic enzymes are limiting. Moreover, the accumulation of collagen IV and Perlecan in Mmp2 mutants revealed that proteolysis of ECM components is dependent upon Mmp2 and regulates BL assembly around disc-associated tracheae. collagen IV and Perlecan are either substrates of Mmp2 or their levels are dependent on another component that is. The distinct effects of reducing Mmp2 activity in the trachea (increased collagen IV and stunted ASP growth) or in the disc (hypertrophic ASP growth) show that location and level of Mmp2 activity is critically important for normal association and growth of the ASP. They also imply that the location and level of Mmp2 expression must be precisely regulated and that Mmp2, a membrane-tethered enzyme, might have different substrates in the disc and trachea extracellular milieu. A possible explanation is that despite the absence of a lamina densa separating the ASP and disc, distinct collagen-containing layers overly each tissue (Guha, 2009).

Third, the invasively coupled transverse connective and ASP nestle within the plane of the disc such that the overlying ECM forms a relatively flat sheet. However, reducing Mmp2 activity led to the partial extrusion of the disc-associated trachea. This phenotype revealed that the character of the disc:trachea association is sensitive to the composition of the ECM, and is impaired if the system's capacity to remodel the ECM is reduced (Guha, 2009).

The BL is rich with proteins that bind and sequester growth factors, and it therefore has the potential to block movement of proteins such as FGF. However, ectopic expression of Bnl/FGF in the disc induces invasive outgrowths from regions of the trachea that do not contact the disc and are separated from disc cells by two layers of BL. Invasive coupling and direct contact are not therefore prerequisites for signaling and growth. The ectopic expression assay is a qualitative measure of FGF signaling and does not ascertain whether direct apposition facilitates signaling; however, the results suggest that the BL is functionally transparent to FGF signaling. Tunneling may be needed for other purposes, for example, for the ASP to interact with the disc and to develop together with other thoracic structures during pupal development. It is speculated that the functional transparency of the BL is likely to be general property, that the BL may be generally transparent to signaling proteins and growth factors. Such transparency would be relevant to the mechanisms that distribute signaling proteins, since constraining signaling proteins to restrict their influence to only their intended targets would seem to be an essential feature (Guha, 2009).

Although tube formation is essential to generate many vertebrate organs, Drosophila offers few relevant models. Strategies for making tubes have been classified according to the apical-basal polarity of the founding cells. Some, such as the vertebrate mammary gland, hair follicle and early pancreas, form from clusters of cells that initially lack polarity but acquire apical-basal polarity as they coalesce around a central lumen. Others, such as the vertebrate liver, lung and neural tube and the Drosophila salivary glands, form directly from morphogenetic movements of polarized epithelial sheets. The progenitors of these tubes retain their apical-basal polarity as they generate tubular extrusions (Guha, 2009).

The ASP is an example of the latter type of tubulogenesis. The cells of the ASP retain the apical-basal polarity of the tracheal epithelium from which they emerge. Many of the cells in the ASP are mitotically active, distinguishing the ASP from the Drosophila salivary gland, whose cells invaginate from an epithelial sheet but do not divide. The process of ASP tubulogenesis is therefore more like that of the vertebrate liver, lung and neural tube, which also grow by coupling cell division to invagination and morphogenesis (Guha, 2009).

Mmps have been implicated in organ morphogenesis in a variety of contexts. A relevant example is HGF-induced tubulogenesis by MDCK cells cultured in 3D-matricies. Initial stages of tube morphogenesis required ERK activation, after which tube growth was dependent on Mmps but independent of ERK (O'Brien, 2004). Since the Drosophila ASP was induced but its growth was stunted in genetic backgrounds that reduced Mmp function, Mmps also appear to have a stage-specific role in ASP morphogenesis (Guha, 2009).

A matrix metalloproteinase mediates airway remodeling in Drosophila

Organ size typically increases dramatically during juvenile growth. This growth presents a fundamental tension, as organs need resiliency to resist stresses while still maintaining plasticity to accommodate growth. The extracellular matrix (ECM) is central to providing resiliency, but how ECM is remodeled to accommodate growth is poorly understood. This study investigated remodeling of Drosophila respiratory tubes (tracheae) that elongate continually during larval growth, despite being lined with a rigid cuticular ECM. Cuticle is initially deposited with a characteristic pattern of repeating ridges and valleys known as taenidia. For tubes to elongate, this study found that the extracellular protease Mmp1 is required for expansion of ECM between the taenidial ridges during each intermolt period. Mmp1 protein localizes in periodically spaced puncta that are in register with the taenidial spacing. Mmp1 also degrades old cuticle at molts, promotes apical membrane expansion in larval tracheae, and promotes tube elongation in embryonic tracheae. Whereas work in other developmental systems has demonstrated that MMPs are required for axial elongation occurring in localized growth zones, this study demonstrates that MMPs can also mediate interstitial matrix remodeling during growth of an organ system (Glasheen, 2010).

This study found that larval tracheal tubes elongate their apical matrix at discrete sites along the long axis of the tube. Immediately after a molt in wild-type animals, the new apical matrix (cuticle) is constructed with taenidial ridges at a fixed interval of ~ 0.8 µm. During intermolt tube elongation, this matrix expands the taenidial interval to ~ 1.6 µm. At molting, this fully expanded matrix is discarded and replaced with a new matrix with a taenidial interval again at 0.8 µm, which will again expand about two-fold. This precise remodeling of the taenidia is accomplished by the extracellular protease Mmp1, which is localized in discrete apical puncta, each associated with an individual taenidium. In Mmp1 mutants, the taenidial ridges do not expand, the taenidial interval remains fixed, and larvae cannot elongate their tracheae as their bodies elongate, causing stretched tubes that eventually break. In normal tube elongation, ECM expansion is coupled with cellular apical membrane expansion, and Mmp1 is required for the coordination of ECM and cellular expansion; Mmp1 is able to promote both aspects of tube expansion when overepressed in embryos. Mmp1 is also required for the other important tracheal cuticle remodeling event in larvae: degrading cuticle into pieces that can be discarded at each molt. Thus, Mmp1 tracheal tubes cannot elongate because they cannot remodel apical extracellular matrix either to expand it or to discard it (Glasheen, 2010).

On first inspection, it is difficult to account for the progressively deteriorating phenotype of Mmp1 mutants. Although the tracheal system appears morphologically normal at hatching in Mmp1 null mutants, within several hours, they develop taut stretched tracheal systems. As has been previously reported, this phenotype worsens throughout larval life: by second instar, many animals have broken dorsal trunks and some death occurs; by third instar, nearly all animals have tracheal breaks and all eventually die (Page-McCaw, 2003). This presents an apparent paradox as early third instar mutants, with shortened tracheal systems, have normal spacing of their taenidia. This paradox is resolved, however, when one considers that in each instar taenidial expansion is a requirement for tube elongation. Thus, at the start of second instar, although the taenidia are correctly spaced, they comprise a tube that is already shorter than wild-type and is virtually unexpandable, and thus, tubes frequently break as the animal grows. By third instar, although the taenidia are again deposited with normal spacing, the collective failures of elongation in the previous instars make the entire tracheal system very short and highly abnormal. Taken alone, the taenidial expansion data might suggest that the Mmp1 tubes fail to elongate at all. If Mmp1 mutant tubes were unable to achieve any elongation, then the tracheal system of third instar larvae would be ~ 1/8 the length of wild-type controls; it was observed that an Mmp1 tracheal segment is about 1/4 the length of wild-type. Thus, despite the lack of taenidial expansion, there appears to be some kind of other elongation at work in these mutant tubes. Possibilities include an aberrant brute-force stretching of unremodeled cuticle, or a burst of tube elongation during molts when the tube releases cuticle (Glasheen, 2010).

In electron micrographs of late Mmp1 mutants, the cuticle appears to separate from the epithelial layer in late Mmp1 samples, but not in wild-type; the explanation of artefactual sample fracturing is unlikely, as cell membranes appeared intact in TEMs from both genotypes. Two models were envisioned to explain this separation. One possibility is that matrix components are still secreted by the mutant cells, but they require matrix remodeling to become incorporated into cuticle, and so they accumulate between the cells and the unremodeled cuticle, creating a gap. They would have to be electron-transparent to be consistent with the images, and there are reports of electron-transparent cuticle layers in insects; indeed, the taenidial cores of the TEMs can appear electron-transparent. Alternatively, it is possible that Mmp1 is required for processing adhesion molecules that hold the cuticle to the cell layer, so that adhesion is lost in the absence of the Mmp1. The first model is favored since it accounts for the fate of the matrix components that should have been deposited in the cuticle in the absence of remodeling (Glasheen, 2010).

An important question arising from this study is how MMP localization and activation is controlled to produce uniform tube elongation. One simple model is that the cells can sense tension and respond by secreting or activating Mmp1. However, this model is not supported by the observation that Tubby (Tb) mutants, with slack in the tracheal tubes, still increase the intertaenidial distance during the intermolt period. Thus, the hypothesis is favored that tube elongation is under developmental regulation. The Tb mutant phenotype indicates that such a tube elongation program is independent of body elongation. One possible mechanism is that the developmental program could trigger Mmp1 secretion from cytoplasmic vesicles to the apical ECM; how MMP secretion is controlled is an open question. The protease appears to be localized in extracellular puncta precisely coordinated with the taenidia. This localization pattern may be ultimately directed by the actin cytoskeleton, which appears to pattern the taenidia during new cuticle secretion. Consistent with the possibility of actin localizing Mmp1, the actin cytoskeleton generally regulates apical secretion in the embryonic tracheal system; and in cultured neurons, it has been observed that MMP-containing vesicles traffic along microfilaments. Mmp1 mutants that lack a hemopexin domain, or dominant-negative mutants that interfere specifically with the Mmp1 hemopexin domain, are still able to elongate their tubes, indicating that the hemopexin domain is not required for secretion, localization, or activation of Mmp1 in these discrete puncta (Glasheen, 2010).

The interstitial remodeling of tracheal cuticle stands in contrast to other developmental mechanisms of ECM deposition and remodeling that occur during axial growth of rigid or load-bearing structures. During vertebrate long-bone growth, elongation takes place at the growth plates near the ends of the bones, concentrating ECM remodeling and deposition distally. In plants, axial elongation takes place at the meristem regions, which are located distally like growth plates, again confining ECM deposition to distal regions. These cases of spatially restricted remodeling occur in contexts where maintaining the integrity of a rigid ECM presents a structural hurdle to simultaneous remodeling. Interestingly, MMPs appear to be required for both these kinds of growth. Mouse MMP13 mutants cannot remodel the cartilage at the growth plate to make bone, and MMP9 is also required for normal long-bone growth. In Arabidopsis, the MMP mutant At2-mmp1 cannot extend shoots, and in the Loblolly pine, MMP expression is correlated with embryonic root (radicle) protrusion, which is hindered by an MMP inhibitor. In Drosophila larval tracheae, the cuticle is not fully sclerotized and so retains some plasticity, which might be expected as the cuticle does not provide rigidity along the axis but instead protects circumferentially from crushing forces. This different set of structural requirements probably explains why in tracheae deposition of matrix is not confined to distal regions or even to one region per segment, but rather is dispersed across the length of the tissue. The requirement for tube rigidity in the circumferential axis, but not along the body axis, also addresses why tracheal elongation can occur continually, modifying an existing cuticle. In contrast, tube circumferential expansion cannot occur continually but is limited to the molt, when the cuticle is completely replaced. The importance of MMPs in axial growth is underscored by the common requirement for MMPs during all these cases, despite the different structural contexts for matrix remodeling in bone elongation, plant growth, and tracheal elongation (Glasheen, 2010).

In Mmp1 mutants, where the cuticle remains unelongated, it is striking that elongation of the underlying cells and their apical membranes is also impaired. Although there is some excess of apical membrane in Mmp1 mutants, the excess is much less than expected had the cells underlying the cuticle expanded their membranes to the wild-type extent. These results suggest that either Mmp1 activity or cuticle elongation is required for cells and apical membranes to elongate normally (Glasheen, 2010).

How are Mmp1 activity and/or cuticle remodeling coordinated with membrane expansion? Although it is possible that matrix remodeling and membrane expansion represent distinct activities of Mmp1, the simplicity of having a direct causal relationship between matrix remodeling and membrane expansion is appealing. One example of a combined model is that extracellular matrix elongation, mediated by Mmp1, places cells under tension, and cells respond by elongating apical membrane. Another model is that Mmp1 proteolytic activity, which remodels cuticle, may simultaneously generate an inductive signal (or inactivate a negative regulator) that causes the underlying cells to elongate, thus coordinating the elongation of both the rigid cuticle and the underlying cells. Production of such a signal would be analogous to mammalian MMPs cleaving laminin-5 or collagen IV to unmask cryptic signaling sites that promote cell migration; this model would also be consistent with recent findings that release of a collagen IV domain by MT2-MMP is required for branching morphogenesis of the submandibular gland in mice. Consistent with Mmp1 activity generating a cuticle-derived signal, misexpression of Mmp1 in embryonic tracheae causes tube elongation only when cuticle is present (Glasheen, 2010).

These results show that extracellular matrix remodeling is a critical aspect of tube elongation in larval tracheae. Mmp1 mutants cannot remodel cuticle and cannot properly elongate their tubes or degrade unnecessary matrix material to be discarded at molts. These remodeling events are regulated separately from the initial deposition and patterning of cuticle, as Mmp1 mutants are able to secrete normally patterned cuticles. Additionally, Mmp1 appears to regulate cell elongation, perhaps directly by processing a signaling molecule, or indirectly by regulating the ECM. Hence, a matrix metalloproteinase appears to act as a critical coregulator of matrix and cellular growth. Finally, it is significant that tracheal remodeling but not initial tracheal morphogenesis requires a matrix metalloproteinase. This analysis of this remodeling phenotype reinforces the notion that matrix metalloproteinases are specialists for remodeling existing tissues, rather than forming tissues, likely because of the need to alter existing ECM that limits plasticity (Glasheen, 2010).

Requirement of matrix metalloproteinase-1 for intestinal homeostasis in the adult Drosophila midgut

Stem cells are tightly regulated by both intrinsic and extrinsic signals as well as the extracellular matrix (ECM) for tissue homeostasis and regenerative capacity. Matrix metalloproteinases (MMPs), proteolytic enzymes, modulate the turnover of numerous substrates, including cytokine precursors, growth factors, and ECM molecules. However, the roles of MMPs in the regulation of adult stem cells are poorly understood. The present study utilized the Drosophila midgut, which is an excellent model system for studying stem cell biology, to show that Mmp1 is involved in the regulation of intestinal stem cells (ISCs). The results showed that Mmp1 is expressed in the adult midgut and that its expression increases with age and with exposure to oxidative stress. Mmp1 knockdown or tissue inhibitor of metalloproteinase (Timp)-overexpressing flies and flies heterozygous for a viable, hypomorphic Mmp1 allele increased ISC proliferation in the gut, as shown by staining with an anti-phospho-histone H3 antibody and BrdU incorporation assays. Reduced Mmp1 levels induced intestinal hyperplasia, and the Mmp1 depletion-induced ISC proliferation was rescued by the suppression of the EGFR signaling pathway, suggesting that Mmp1 regulates ISC proliferation through the EGFR signaling pathway. Furthermore, adult gut-specific knockdown and whole-animal heterozygotes of Mmp1 increased additively sensitivity to paraquat-induced oxidative stress and shortened lifespan. These data suggest that Drosophila Mmp1 is involved in the regulation of ISC proliferation for maintenance of gut homeostasis (Lee, 2012).

In mammals, previous work has demonstrated a role for MMPs in tumorigenesis. However, the protective role of MMPs against tumorigenesis has also recently become apparent. Thus, the role of MMP in tumorigenesis is controversial. In Drosophila, only two MMP homologs (Mmp1 and Mmp2) have been identified in the genome. It has been suggested that Mmp1, a secreted protein, regulates regenerative proliferation and growth during Drosophila development. This study demonstrates that the guts of Mmp1 knockdown flies and heterozygous mutants have an increased number of proliferating ISCs, which indicates that endogenous Mmp1 can restrict ISC proliferation. Consistent with the current data for Mmp1 knockdown in the gut, this study demonstrated that the ectopic expression of Drosophila Timp, a potent inhibitor of Mmp1 and Mmp2, increased ISC proliferation. Therefore, the findings suggest that MMP has an anti-tumorigenic, protective role. Nevertheless, it was also observed that the overexpression of Mmp1 weakly increased ISC proliferation, suggesting that the expression of Mmp1 must be optimized to maintain homeostasis in the Drosophila intestine (Lee, 2012).

The data from Mmp1 knockdown in ECs and the Flip-out lineage tracing experiment suggest the possibility that ISCs can respond to Mmp1 reduction. Mmp1's regulation of ISC proliferation is at least associated with the EGFR signaling pathway, a key signaling pathway to regulate ISC proliferation, via the suppression of Mmp1 mutant phenotypes by expression of the EGFR^RNAi construct in ISCs/EBs (Lee, 2012).

In mammals, MMPs have been found to modulate growth factor signaling. In Drosophila, it has been reported that the knockdown of Mmp2 in the larval trachea activates ERK. EGFR ligands such as Vein and Spitz are known to be produced by the circular muscle underlying the gut or ECs to stimulate ISC proliferation. Thus, it is possible that Mmp1 directly modulate the level of EGF ligands. However, it was observed that the EGF ligands production was not significantly increased in the Mmp1 insufficient guts, indicating that the increases and activation of ERK by Mmp1 insufficiency may not be associated with the level of EGF ligands. This finding suggests that there may be a possibility that Mmp1 modulates the penetration of EGF ligands to reach ISCs through the modification of the ECM (Lee, 2012).

Both fly MMPs are known to degrade and modulate the basement membrane during development and regeneration. Drosophila ISCs extensively contact the basement membrane and receive signals for the maintenance of ISC 'stemness' from visceral muscle. In the current experimental conditions, however, no significant alteration was found in the expression pattern of vkg-GFP, which expresses a GFP-fusion of collagen IV, a major compartment of the basement membrane, between wild-type and Mmp1 insufficient guts. Thus, it is necessary to investigate the possibility of other ECM components as Mmp1 substrates to control ISC proliferation (Lee, 2012).

It is also possible that Mmp1 may indirectly affect the ISC proliferation in the midgut via the nonautonomous feedback by differentiated cells and EBs. ISC proliferation has been reported to increase upon EC viability or survival affected by several types of intestinal damage. Therefore, Mmp1 insufficiency-related phenotypes could be due to EC damage or death. To verify this possibility, the mRNA level of Upd3, an EC damage-induced cytokine was measured. However, the level of Upd3 was mildly increased in Mmp1 insufficient guts to a relatively lower level (only 2- to 3-fold compared with control) than other reported stresses such as JNK activation in ECs or knockdown of the Hippo tumor suppressor pathway in ISCs/EBs. In addition,no significant increases were observed in apoptotic signals in the guts expressing Mmp1^RNAi or Timp and Mmp1 heterozygous mutant flies using the TUNEL assay. To assure the possibility that Mmp1 depletion may not be associated with EC death, the rescue experiment using the baculovirus protein p35, an apoptosis inhibitor, remains to be completed (Lee, 2012).

Since it was reported that increased ISC proliferation can be due to the EC loss into the lumen, there may be a possibility that Mmp1-related ISC proliferation is associated with this cell loss. However, no sloughing cells were detected in the lumen of the guts expressing Mmp1^RNAi or Timp under Myo1A-Ga80^ts driver and Mmp1 heterozygous mutant flies analyzed through cross-section (Lee, 2012).

In addition, it has been shown that disrupting epithelial integrity increases ISC proliferation via the activation of EGFR and JAK/STAT signaling pathways. Dl-positive polyploid cells were observed in the guts expressing Mmp1^RNAi or Timp under the Myo1A-Ga80^ts driver. The Dl-positive polyploid cells have been reported to be mis-differentiated cells blocked in their ability to terminally differentiate into functional ECs. Furthermore, it was recently reported that EBs can regulate ISC proliferation. Therefore, the possiblity cannot excluded that Mmp1 insufficiency-induced ISC proliferation may be due to the disruption of epithelial integrity and associated with EBs (Lee, 2012).

This study also found that Mmp1 expression in the midgut is increased by oxidative stress or with the age of the fly. In addition, gut-specific knockdown and Mmp1 mutant flies exhibited an additive rather than synergetic sensitivity to oxidative stress-induced ISC proliferation and a shorter lifespan. Other studies have reported on the permissive versus inductive nature of EGFR/ERK and JNK signaling in ISC proliferation. Mmp1-related ISC proliferation may be associated with the modulation of the permissive nature of EGFR signaling. An increased Mmp1 level in aged and oxidative stressed guts may be also required for restriction of ISC proliferation. The level of ISC proliferation has been reported to affect organismal lifespan. MMP expression levels are known to increase upon exposure to several stresses, including inflammation, aging, and mechanical damage. Mmp1 expression has been reported to be regulated by the stress-responsive JNK pathway during larval-tissue regeneration, and this pathway is established to play a protective role in EC stress-tolerance and to regulate Drosophila lifespan. In addition, it has been reported that gut-specific regulation of insulin signaling and free radicals control increased stem cell proliferation, midgut dysplasia, and lifespan. Therefore, the possibility that Mmp1 acts upon extracellular factors in these pathways might be one area to explore (Lee, 2012).

These data demonstrate that Drosophila Mmp1 plays a role in the regulation of ISC proliferation for the maintenance of gut homeostasis (Lee, 2012).

The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth

Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, this study identified grindelwald (grnd; CG10176), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. This study shows that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of RasV12/scrib-/- tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth (Andersen, 2015).

A genome-wide screen was carried to identify molecules that are required for neoplastic growth. The condition used for this screen was the disc-specific knockdown of avalanche, also known as syntaxin 7), a gene encoding a syntaxin that functions in the early step of endocytosis2. avl-RNAi results in ectopic Wingless (Wg) expression, neoplastic disc overgrowth, and a 2-day delay in larva-to-pupa transition. A collection of 10,100 transgenic RNA interference (RNAi) lines were screened for their ability to rescue the pupariation delay, and 121 candidate genes were identified. Interestingly, only eight candidate genes also rescued ectopic Wg expression and neoplastic overgrowth. These included five lines targeting core components of the JNK pathway (Bendless, Tab2, Tak1, Hemipterous and Basket. Using a puckered enhancer trap (puc-lacZ) as a readout for JNK activity, it was confirmed that JNK signalling is highly upregulated in avl-RNAi discs. One of the remaining lines targets CG10176, a gene encoding a transmembrane protein. Reducing expression of CG10176 by using two different RNAi lines was as efficient as tak1 silencing to restore normal Wg pattern and suppresses JNK signalling and neoplastic growth in the avl-RNAi background. Sequence analysis of GC10176 identified a cysteine-rich domain (CRD) in the extracellular part with homology to vertebrate TNFRs harbouring a glycosphingolipid-binding motif (GBM) characteristic of many TNFRs including Fas. CG10176 was named grindelwald (grnd) , after a village at the foot of Eiger, a Swiss mountain that lent its name to the unique Drosophila TNF, Egr. Immunostaining and subcellular fractionation of disc extracts confirmed that Grnd localizes to the membrane. Moreover, co-immunoprecipitation experiments showed that both Grnd full-length and Grnd-intra, a form lacking its extracellular domain, directly associate with Traf2, the most upstream component of the JNK pathway. This interaction is disrupted by a single amino acid substitution within a conserved Traf6-binding motif (human TRAF6 is the closest homologue to Traf2. Overexpression of Grnd-intra, but not full-length Grnd, is sufficient to induce JNK signalling, ectopic Wg expression and apoptosis, and Grnd-intra-induced apoptosis is efficiently suppressed in a hep (JNKK) mutant background, confirming that Grnd acts upstream of the JNK signalling cascade (Andersen, 2015).

The Drosophila TNF Egr activates JNK signalling and triggers cell death or proliferation, depending on the cellular context. Therefore tests were performed to see whether Grnd is required for the small-eye phenotype generated by Egr-induced apoptosis in the retinal epithelium (via Egr overexpression). Inhibition of JNK signalling by reducing tak1 or traf2 expression, or by overexpressing puckered, blocks Egr-induced apoptosis and rescues the small-eye phenotype. In contrast to a previous report, RNAi silencing of wengen (wgn) , a gene encoding a presumptive receptor for Egr, does not rescue the small-eye phenotype. Furthermore, the small-eye phenotype is not modified in a wgn-null mutant background, confirming that Wgn is not required for Egr-induced apoptosis in the eye. By contrast, reducing grnd levels partially rescues the Egr-induced small-eye phenotype, producing a 'hanging-eye' phenotype that is not further rescued in a wgn-knockout mutant background. A similar phenotype was previously reported as a result of non-autonomous cell death induced by a diffusible form of Egr. This suggests that Grnd prevents Egr from diffusing outside of its expression domain. Co-immunoprecipitation experiments show that both full-length Grnd and Grnd-extra, a truncated form of Grnd lacking the cytoplasmic domain, associate with Egr through its TNF-homology domain. Although Grnd-extra can bind Egr, it cannot activate JNK signalling. Therefore, it was reasoned that Grnd-extra expression might prevent both cell-autonomous and non-autonomous apoptosis by trapping Egr and preventing its diffusion and binding to endogenous Grnd. Indeed, GMR-Gal4-mediated expression of grnd-extra fully rescues the Egr small-eye phenotype. To confirm that the removal of Grnd induces Egr-mediated non-autonomous cell death, wing disc clones were generated expressing egr alone, egr + tak1 RNAi, or egr + grnd RNAi. As expected, reducing tak1 levels in egr-expressing clones prevents their elimination by apoptosis. Similarly, reducing grnd levels prevents autonomous cell death, but also induces non-autonomous apoptosis. This suggests that Egr, like its mammalian counterpart TNF-α, can be processed into a diffusible form in vivo whose interaction with Grnd limits the potential to act at a distance. Flies carrying homozygous (grnd^Minos/Minos) or transheterozygous (grnd^Minos/Df) combinations of a transposon inserted in the grnd locus express no detectable levels of Grnd protein and are equally resistant to Egr-induced cell death. In addition, grnd^Minos/Minos mutant flies are viable and display no obvious phenotype, suggesting that Grnd, like Egr, participates in a stress response to limit organismal damage. Collectively, these data demonstrate that Grnd is a new Drosophila TNF receptor that mediates most, if not all, Egr-induced apoptosis (Andersen, 2015).

TNFs probably represent a danger signal produced in response to tissue damage to rid the organism of premalignant tissue or to facilitate wound healing. Disc clones mutant for the polarity gene scribbled (scrib) induce an Egr-dependent response resulting in the elimination of scrib mutant cells by JNK-mediated apoptosis. To test the requirement for Grnd in this process, scrib-RNAi and scrib-RNAi + grnd-RNAi clones obtained 72 h after heat shock induction were compared. As expected, scrib-RNAi cells undergo apoptosis and detach from the epithelium. By contrast, scrib-RNAi clones with reduced grnd expression survive, indicating that Grnd is required for Egr-dependent elimination of scrib-RNAi cells. Similar results were obtained by generating scrib mutant clones in the eye disc (Andersen, 2015).

In both mammals and flies, TNFs are double-edged swords that also have the capacity to promote tumorigenesis in specific cellular contexts. Indeed, scrib minus eye disc cells expressing an activated form of Ras (Ras^V12) exhibit a dramatic tumour-like overgrowth and metastatic behaviour, a process that critically relies on Egr. Ras^V12/scrib^-/- metastatic cells show a strong accumulation of Grnd and Mmp1, and invade the ventral nerve cord. Primary tumour cells reach peripheral tissues such as the fat body and the gut, where they form micro-metastases expressing high levels of Grnd. Reducing grnd levels in Ras^V12/scrib^-/- clones is sufficient to restore normal levels of Mmp1 and abolish invasiveness in a way similar to that observed in an egr mutant background. Therefore, Grnd is required for the Egr-induced metastatic behaviour of Ras^V12/scrib^-/- tumorous cells. Similarly, reducing grnd, but not wgn levels, strongly suppresses Mmp1 expression in Ras^V12/dlg-RNAi cells and limits tumour invasion, indicating that Wgn does not have a major role in the progression of these tumours (Andersen, 2015).

Perturbation of cell polarity is an early hallmark of tumour progression in epithelial cells. In contrast to small patches of polarity-deficient cells, for example, scrib mutant clones, organ compartments or animals fully composed of polarity-deficient cells become refractory to Egr-induced cell death and develop epithelial tumours. The formation of these tumours requires JNK/MAPK signalling, but not Egr, suggesting Egr-independent coupling between loss of polarity and JNK/MAPK-dependent tumour growth. In line with these observations, it was noticed that, in contrast to Grnd, Egr is not required to drive neoplastic growth in avl-RNAi conditions. This suggests that, in addition to its role in promoting Egr-dependent functions, Grnd couples loss of polarity with JNK-dependent growth independently of Egr. Disc immunostainings revealed that Grnd co-localizes with the apical determinant Crb in the marginal zone, apical to the adherens junction protein E-cadherin (E-cad) and the atypical protein kinase C (aPKC). In avl-RNAi discs, Grnd and Crb accumulate in a wider apical domain. Apical accumulation of Crb is proposed to be partly responsible for the neoplastic growth induced by avl knockdown, since overexpression of Crb or a membrane-bound cytoplasmic tail of Crb (Crb-intra) mimics the avl-RNAi phenotype. Therefore whether Grnd might couple the activity of the Crb complex with JNK-mediated neoplastic growth was examined. Indeed, reducing grnd levels, but not wgn, in ectopic crb-intra discs suppresses neoplastic growth as efficiently as inhibiting the activity of the JNK pathway. Notably, Yki activation is not rescued in these conditions, illustrating the ability of Crb-intra to promote growth independently of Grnd by inhibiting Hippo signalling through its FERM-binding motif (FBM). Indeed, neoplastic growth and polarity defects induced by a form of Crb-intra lacking its FBM (CrbΔFBM-intra) are both rescued by Grnd silencing. As expected, the size of ectopic crbΔFBM-intra;grnd-RNAi discs is reduced compared to the size of ectopic crb-intra; grnd-RNAi discs (Andersen, 2015).

Crb, Stardust (Sdt; PALS1 in humans), and Pals1-associated tight junction protein (Patj) make up the core Crb complex, which recruits the adaptor protein Veli (MALS1-3 in humans). In agreement with previous yeast two-hybrid data, this study found that Grnd binds directly and specifically to the PDZ domain of Veli through a membrane-proximal stretch of 28 amino acids in its intracellular domain. Grnd localization is unaffected in crb and veli RNAi mutant clones. However, reducing veli expression rescues the patterning defects and disc morphology of ectopic crb-intra mutant cells, suggesting that Grnd couples Crb activity with JNK signalling through its interaction with Veli. Interestingly, aPKC-dependent activation of JNK signalling also depends on Grnd. aPKC is capable of directly binding and phosphorylating Crb, which is important for Crb function. This suggests that aPKC, either directly or through Crb phosphorylation, activates Grnd-dependent JNK signalling in response to perturbation of apico-basal polarity (Andersen, 2015).

These data are consistent with a model whereby Grnd integrates signals from Egr, the unique fly TNF, and apical polarity determinants to induce JNK-dependent neoplastic growth or apoptosis in a context-dependent manner. Recent work reveals a correlation between mammalian Crb3 expression and tumorigenic potential in mouse kidney epithelial cells. The conserved nature of the Grnd receptor suggests that specific TNFRs might carry out similar functions in vertebrates, in which the link between apical cell polarity and tumour progression remains elusive (Andersen, 2015).

Interplay among Drosophila transcription factors Ets21c, Fos and Ftz-F1 drives JNK-mediated tumor malignancy

This study defines TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (Ras^V12) and loss of the tumor suppressor Scribble (scrib¹). Malignant transformation of the ras^V12scrib¹ tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to ras^V12scrib¹ tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in ras^V12scrib¹ tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with Ras(V12) in inducing malignant clones that, like ras^V12scrib¹ tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While ras^V12ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, this study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes. (Kulshammer, 2015).

Genome-wide transcriptome profiling in the Drosophila epithelial tumor model has generated a comprehensive view of gene expression changes induced by defined oncogenic lesions that cause tumors of an increasing degree of malignancy. These data allowed discovery of how a network of collaborating transcription factors confers malignancy to Ras^V12scrib¹ tumors (Kulshammer, 2015).

This study revealed that the response of transformed Ras^V12scrib¹ epithelial cells is more complex in comparison to those with activated Ras^V12 alone with respect to both the scope and the magnitude of expression of deregulated genes. Aberrant expression of more than half of the genes in Ras^V12scrib¹ tumors requires JNK activity, highlighting the significance of JNK signaling in malignancy. Importantly, the tumor-associated, JNK-dependent transcripts cluster with biological functions and processes that tightly match the phenotypes of previously described tumor stages. Furthermore, the Ras^V12scrib¹ transcriptome showed significant overlap (27% upregulated and 15% downregulated genes) with microarray data derived from mosaic EAD in which tumors were induced by overexpressing the BTB-zinc finger TF Abrupt (Ab) in scrib¹ mutant clones as well as with a transcriptome of scrib¹ mutant wing discs. It is proposed that 429 misregulated transcripts (e.g. cher, dilp8, ets21c, ftz-f1, mmp1, upd), shared among all the three data sets irrespective of epithelial type (EAD versus wing disc) or cooperating lesion (Ras^V12 or Ab), represent a 'polarity response transcriptional signature' that characterizes the response of epithelia to tumorigenic polarity loss. Genome-wide profiling and comparative transcriptome analyses thus provide a foundation to identify novel candidates that drive and/or contribute to tumor development and malignancy while unraveling their connection to loss of polarity and JNK signaling (Kulshammer, 2015).

In agreement with a notion of combinatorial control of gene expression by an interplay among multiple TFs, this study identified overrepresentation of cis-acting DNA elements for STAT, GATA, bHLH, ETS, BTB, bZIP factors and NRs in genes deregulated in Ras^V12scrib¹ mosaic EAD, implying that transcriptome anomalies result from a cross-talk among TFs of different families. Many of the aberrantly expressed genes contained binding motifs for AP-1, Ets21c and Ftz-F1, indicating that these three TFs may regulate a common set of targets and thus cooperatively promote tumorigenesis. This is consistent with the occurrence of composite AP-1-NRRE (nuclear receptor response elements), ETS-NRRE and ETS-AP-1 DNA elements in the regulatory regions of numerous human cancer-related genes, such as genes for cytokines, MMPs (e.g., stromelysin, collagenase) and MMP inhibitors (e.g., TIMP) (Kulshammer, 2015).

Interestingly, Drosophila ets21c and ftz-f1 gene loci themselves contain AP-1 motifs and qualify as polarity response transcriptional signature transcripts. Indeed, this study has detected JNK- and Fos-dependent upregulation of ets21c and ftz-f1 mRNAs in Ras^V12scrib¹ tumors. While JNK-mediated control of ftz-f1 transcription has not been reported previously, upregulation of ets21c in the current tumor model is consistent with JNK requirement for infection-induced expression of ets21c mRNA in Drosophila S2 cells and in vivo. Based on these data, it is proposed that Ftz-F1 and Ets21c are JNK-Fos-inducible TFs that together with AP-1 underlie combinatorial transcriptional regulation and orchestrate responses to cooperating oncogenes. Such an interplay between AP-1 and Ets21c is further supported by a recent discovery of physical interactions between Drosophila Ets21c and the AP-1 components Jun and Fos (Rhee, 2014). Whether regulatory interactions among AP-1, Ets21c and Ftz-F1 require their direct physical contact and/or the presence of composite DNA binding motifs of a particular arrangement to control the tumor-specific transcriptional program remains to be determined (Kulshammer, 2015).

Importantly, some of the corresponding DNA elements, namely AP-1 and STAT binding sites, have recently been found to be enriched in regions of chromatin that become increasingly accessible in Ras^V12scrib¹ mosaic EAD relative to control. This demonstrates that comparative transcriptomics and open chromatin profiling using ATAC-seq and FAIRE-seq are suitable complementary approaches for mining the key regulatory TFs responsible for controlling complex in vivo processes, such as tumorigenesis (Kulshammer, 2015).

The prototypical form of AP-1 is a dimer comprising Jun and Fos proteins. In mammals, the Jun proteins occur as homo- or heterodimers, whereas the Fos proteins must interact with Jun in order to bind the AP-1 sites. In contrast to its mammalian orthologs, the Drosophila Fos protein has been shown to form a homodimer capable of binding to and activating transcription from an AP-1 element, at least in vitro (Kulshammer, 2015).

The role of individual AP-1 proteins in neoplastic transformation and their involvement in pathogenesis of human tumors remain somewhat elusive. While c-Jun, c-Fos and FosB efficiently transform mammalian cells in vitro, only c-Fos overexpression causes osteosarcoma formation, whereas c-Jun is required for development of chemically induced skin and liver tumors in mice. In contrast, JunB acts as a context-dependent tumor suppressor. Thus, cellular and genetic context as well as AP-1 dimer composition play essential roles in dictating the final outcome of AP-1 activity in tumors (Kulshammer, 2015).

This study shows that, similar to blocking JNK with its dominant-negative form, Bsk, removal of Fos inhibits ets21c and ftz-f1 upregulation, suppresses invasiveness, improves epithelial organization and differentiation within Ras^V12scrib¹ tumors and allows larvae to pupate. Strikingly, depletion of Jun had no such tumor-suppressing effects. It is therefore concluded that in the malignant Ras^V12scrib¹ tumors, Fos acts independently of Jun, either as a homodimer or in complex with another, yet unknown partner. A Jun-independent role for Fos is further supported by additional genetic evidence. Fos, but not Jun, is involved in patterning of the Drosophila endoderm Ÿand is required for expression of specific targets, e.g., misshapen (msn) and dopa decarboxylase (ddc), during wound healing. Future studies should establish whether the JNK-responsive genes containing AP-1 motifs, identified in this study, are indeed regulated by Fos without its 'canonical' partner (Kulshammer, 2015).

The current data identify Fos as a key mediator of JNK-induced MMP1 expression and differentiation defects in Ras^V12scrib¹ tumors. Only Fos inhibition caused clear suppression of MMP1 levels and restoration of neurogenesis within clonal EAD tissue, thus mimicking effects of JNK inhibition. Improved differentiation and reduced invasiveness are, however, not sufficient for survival of animals to adulthood, because interfering with Fos function in Ras^V12scrib¹ clones always resulted in pupal lethality (Kulshammer, 2015).

The systems approach of this paper, followed by genetic experiments, identified Ets21c and Ftz-F1 as being essential for Ras^V12scrib¹-driven tumorigenesis. It was further shown that mutual cooperation of both of these TFs with Fos is required to unleash the full malignancy of Ras^V12scrib¹ tumors (Kulshammer, 2015).

TFs of the ETS-domain family are key regulators of development and homeostasis in all metazoans, whereas their aberrant activity has been linked with cancer. ets21c encodes the single ortholog of human Friend leukemia insertion1 (FLI1) and ETS-related gene (ERG) that are commonly overexpressed or translocated in various tumor types. While FLI1 is considered pivotal to development of Ewing's sarcoma, ERG has been linked to leukemia and prostate cancer. As for Ftz-F1 orthologs, the human liver receptor homolog-1 (LRH-1) has been associated with colonic, gastric, breast and pancreatic cancer, whereas steroidogenic factor 1 (SF-1) has been implicated in prostate and testicular cancers and in adrenocortical carcinoma. However, the molecular mechanisms underlying oncogenic activities of either the ERG/FLI1 or the SF-1/LRH-1 proteins are not well understood (Kulshammer, 2015).

This study shows that removal of Ftz-F1 markedly suppressed invasiveness of Ras^V12scrib¹ tumors, restoring the ability of tumor-bearing larvae to pupate. Additionally, and in contrast to Fos, Ftz-F1 inhibition also partly reduced tumor growth in the third-instar EAD and allowed emergence of adults with enlarged, rough eyes composed predominantly of non-clonal tissue. The reduced clonal growth coincided with downregulation of the well-established Yki target, expanded, implicating Ftz-F1 as a potential novel growth regulator acting on the Hpo/Yki pathway. It is further speculated that reduced viability of Ras^V12scrib¹ftz-f1RNAi clones and induction of non-autonomous compensatory proliferation by apoptotic cells during the pupal stage could explain the enlargement of the adult eyes. The precise mechanism underlying compromised growth and invasiveness of Ras^V12scrib¹ftz-f1RNAi tumors and improved survival of the host remains to be identified (Kulshammer, 2015).

In contrast, effects of Ets21cLONG knockdown in Ras^V12scrib¹ tumors appeared moderate relative to the clear improvement conferred by either Fos or Ftz-F1 elimination. ets21c^LONG RNAi neither reduced tumor mass nor suppressed invasiveness, and pupation was rescued only partly. However, unlike ftz-f1RNAi, ets21c^LONG RNAi significantly reduced expression of dilp8 mRNA. Based on abundance of Ets21c binding motifs in the regulatory regions of tumor-associated genes and the normalized expression of >20% of those genes upon removal of Ets21c, it is further suggested that Ets21c acts in Ras^V12scrib¹ tumors to fine-tune the tumor gene-expression signature (Kulshammer, 2015).

Dilp8 is known to be secreted by damaged, wounded or tumor-like tissues to delay the larval-to-pupal transition. This study has corroborated the role of JNK in stimulating dilp8 expression in Ras^V12scrib¹ tumor tissue, and has further implicated Ets21c and Fos as novel regulators of dilp8 downstream of JNK. However, the data also show that elevated dilp8 transcription per se is not sufficient to delay metamorphosis. Unlike the permanent larvae bearing Ras^V12scrib¹ tumors, those with Ras^V12scrib¹ftz-f1RNAi tumors pupated despite the excessive dilp8 mRNA. Likewise, pupation was not blocked by high dilp8 levels in larvae bearing EAD clones overexpressing Abrupt. As Dilp8 secretion appears critical for its function, it is proposed that loss of Ftz-F1 might interfere with Dilp8 translation, post-translational processing or secretion (Kulshammer, 2015).

Consistent with the individual TFs having unique as well as overlapping functions in specifying properties of Ras^V12scrib¹ tumors, knocking down pairwise combinations of the TFs had synergistic effects on tumor suppression compared with removal of single TF. This evidence supports the view that malignancy is driven by a network of cooperating TFs, and elimination of several tumor hallmarks dictated by this network is key to animal survival. An interplay between AP-1, ETS-domain TFs and NRs is vital for development. For example, the ETS-factor Pointed has been shown to cooperate with Jun to promote R7 photoreceptor formation in the Drosophila adult eye. In mosquitoes, synergistic activity of another ETS-factor, E74B, with the ecdysone receptor (EcR/USP) promotes vitellogenesis. It is thus proposed that tumors become malignant by hijacking the developmental mechanism of combinatorial control of gene activity by distinct TFs (Kulshammer, 2015).

Despite the minor impact of ets21c^LONG knockdown on suppressing Ras^V12scrib¹ tumors, Ets21c^LONG is the only one of the tested TFs that was capable of substituting for loss of scrib in inducing malignant clonal overgrowth when overexpressed with oncogenic Ras^V12 in EAD. While invasiveness of such Ras^V12ets21c^LONG tumors required JNK activity, JNK signaling appeared dispensable for tumor growth. Importantly, the overgrowth of Ras^V12ets21c^LONG tumors was primarily independent of a prolonged larval stage, because dramatic tumor mass expansion was detected already on day 6 AEL. How cooperativity between Ets21c^LONG and Ras^V12 ensures sufficient JNK activity and the nature of the downstream effectors driving tumor overgrowth remain to be determined. In contrast, co-expression of either Ftz-F1 or Fos with Ras^V12 resulted in a non-invasive, Ras^V12-like hyperplastic phenotype (Kulshammer, 2015).

Why does Ets21c^LONG exert its oncogenic potential while Fos and Ftz-F1 do not? Simple overexpression of a TF may not be sufficient, because many TFs require activation by a post-translational modification (e.g., phosphorylation), interaction with a partner protein and/or binding of a specific ligand. Full activation of Fos in response to a range of stimuli is achieved through hyperphosphorylation by mitogen-activated protein kinases (MAPKs), including ERK and JNK. Indeed, overexpression of a FosN-Ala mutated form that cannot be phosphorylated by JNK was sufficient to phenocopy fos deficiency, indicating that Fos must be phosphorylated by JNK in order to exert its oncogenic function. Consistent with the current data, overexpression of FosN-Ala partly restored polarity of lgl mutant EAD cells. It is therefore conclude that the tumorigenic effect of Fos requires a certain level of JNK activation, which is lacking in EAD co-expressing Fos with Ras^V12. Nevertheless, the absence of an unknown Fos-interacting partner cannot be excluded (Kulshammer, 2015).

Interestingly, MAPK-mediated phosphorylation also greatly enhances the ability of SF-1 and ETS proteins to activate transcription. Two potential MAPK sites can be identified in the hinge region of Ftz-F1, although their functional significance is unknown. Whether Ets21c or Ftz-F1 requires phosphorylation and how this would impact their activity in the tumor context remains to be determined. Genetic experiments demonstrate that at least the overgrowth of Ras^V12ets21c^LONG tumors does not require Ets21c phosphorylation by JNK (Kulshammer, 2015).

In addition, previous crystallography studies revealed the presence of phosphoinositides in the ligand binding pocket of LHR-1 and SF-1 and showed their requirement for the NR transcriptional activity. Although developmental functions of Drosophila Ftz-F1 seem to be ligand independent, it is still possible that Ftz-F1 activity in the tumor context is regulated by a specific ligand. An effect of Ftz-F1 SUMOylation cannot be ruled out (Kulshammer, 2015).

In summary, this work demonstrates that malignant transformation mediated by Ras^V12 and scrib loss depends on MAPK signaling and at least three TFs of different families, Fos, Ftz-F1 and Ets21c. While their coordinated action ensures precise transcriptional control during development, their aberrant transcriptional (Ets21c, Ftz-F1) and/or post-translational (Fos, Ftz-F1, Ets21c) regulation downstream of the cooperating oncogenes contributes to a full transformation state. The data implicate Fos as a primary nuclear effector of ectopic JNK activity downstream of disturbed polarity that controls ets21c and ftz-f1 expression. Through combinatorial interactions on overlapping sets of target genes and acting on unique promoters, Fos, Ftz-F1 and Ets21c dictate aberrant behavior of Ras^V12scrib¹ tumors. Although originally described in Drosophila, detrimental effects of cooperation between loss of Scrib and oncogenic Ras has recently been demonstrated in mammalian tumor models of prostate and lung cancer. This study and further functional characterization of complex TF interactions in the accessible Drosophila model are therefore apt to provide important insight into processes that govern cancer development and progression in mammals (Kulshammer, 2015).

JNK- and Fos-regulated Mmp1 expression cooperate with Ras to induce invasive tumor in Drosophila

Loss of the epithelial polarity gene scribble in clones of Drosophila imaginal disc cells can cooperate with Ras signaling to induce malignant tumors. Such mutant tissue overproliferates, resists apoptosis, leaves its place of origin and invades other organs, ultimately causing lethality. This study shows that increased Jun N-terminal kinase (JNK) signaling resulting from the loss of scribble promotes the movement of transformed cells to secondary sites. This effect requires Fos-dependent transcriptional activation of a matrix metalloprotease gene mmp1 downstream of JNK. Expression of the Mmp inhibitor Timp or Mmp RNAi knockdown suppresses cell invasiveness. The proinvasive function of the JNK pathway is revealed in a tumor context when active Ras signaling prevents the apoptotic response to JNK activity as it occurs in nontransformed cells. Based on these results, a model is presented that explains the oncogenic cooperation between JNK and Ras, and describes how aberrant regulation of cell survival, proliferation and mobilization cooperate to incite malignant tumor formation (Uhlirova, 2006).

At this point, it is not clear what causes the activation of JNK in scribble-deficient cells, and further experiments are required to elucidate this question. One credible speculation would be the involvement of cell competition in which faster growing wild-type cells eliminate slower growing scrib^−/− neighbors from the eye imaginal disc epithelium via inducing JNK-dependent apoptosis. The loss of epithelial polarity would further sensitize the scrib^−/− cells to cell competition. As predicted by this hypothesis, clonal growth is restored when apoptosis of scrib^−/− clones is blocked by expression of the caspase inhibitor p35 or DIAP. Similarly, it is conceivable that cell competition caused by the difference in proliferation rate coupled with abnormal cell-cell interactions would underlie the observation of occasional areas of JNK activity (evidenced by mmp1 gene induction in imaginal discs harboring clones of over proliferating Ras^V12 cells. The interaction between clones of transformed cells and their normal cell environment is probably of key relevance in malignant tumor progression and the model system used in this study offers a great opportunity to explore such relationships (Uhlirova, 2006).

In scribble-deficient cells, active JNK signaling stimulates both invasiveness and cell death. Interestingly, either response requires the AP-1 transcription factor Fos. RNAi-mediated knockdown of Fos function inhibits both the invasiveness of ras^V12, scrib^−/− tumors as well as apoptosis of scrib-deficient cells. Fos-mediated control of apoptosis might be achieved by transcriptional activation of the hid gene (Uhlirova, 2006).

Because of the apoptotic function of JNK signaling, its stimulatory effect on cell mobilization becomes evident, and can be functionally dissected, only when the proapoptotic effect of the pathway is repressed. This occurs under conditions of increased Ras signaling, or experimentally when apoptosis is inhibited by the expression of p35. A model of the signaling interactions between Ras gain-of-function and scribble loss-of-function mutations is presented that would explain the cooperative induction of tumor invasiveness and malignancy (Uhlirova, 2006).

A plausible transcriptional target of JNK-Fos signaling with relevance to cell invasiveness in the tumor model in this study is the mmp1 gene. This study shows that JNK and Fos are required for the induction of Mmp1 in malignant tumor tissue. It has to be stressed that this JNK-induced Mmp1 expression is essential for the establishment of the invasive phenotype, as shown by using Timp expression or Mmp1 RNAi, but that Mmp1 activation alone is most likely not sufficient for this effect. It was found that overexpression of Mmp1 in a raf gain-of-function, but scrib wild-type background does not give rise to an invasive phenotype. It appears that JNK activation causes multiple cellular changes that are important for cell mobilization. In addition to the loss of basement membrane integrity that correlates with elevated Mmp1 expression, changes in the actin cytoskeleton are observed as a consequence of JNK activation in ras^V12, scrib^−/−-transformed cells. This cytoskeletal rearrangement phenotype is not due to Mmp1 induction, since blocking of Mmp activity by Timp or RNAi does not influence it, a conclusion that is also supported by the observation that in the embryo JNK induces actin reorganization without turning on the mmp1 gene. Interestingly, the only other mmp gene in Drosophila, mmp2, has recently been implicated in Src-induced and JNK-mediated mobilization of wing imaginal disc cells (Vidal, 2006). This analysis shows clearly that mmp2 is not transcriptionally induced by JNK. However, the RNAi experiments indicate that both Mmp1 and Mmp2 act in conjunction to mediate cell invasiveness. This might occur by a threshold effect where the combined activity of Mmp1 and Mmp2 has to exceed a certain level required for cell invasion. Alternatively, the two Drosophila Mmps might have nonoverlapping substrate specificities, which are both essential for the tumor cell to escape from its tissue of origin. Strikingly, under conditions of Timp expression and Mmp downregulation by RNAi growth of the tumor tissue was not reduced, and even seemed to be more pronounced compared to ras^V12, scrib^−/−. This accumulation of clonal tissue could be explained simply by the fact that proliferation and growth of ras^V12, scrib^−/−, timp and ras^V12, scrib^−/−, mmp1^RNAi, mmp2^RNAi clones is similar to ras^V12, scrib^−/−; however, their reduced ability to degrade ECM, spread and penetrate new organs forces them to expand in volume (Uhlirova, 2006).

This study illustrates how the derailing of several signaling systems is required to create a malignant cancer phenotype in which apoptosis is suppressed, epithelial integrity breaks down, tissue barriers are breached and cells proliferate and mobilize. Using the eye imaginal disc model, it is possible to deconstruct these complex signaling mechanisms and their interplay from the molecular to the tissue level. Future studies that can take full advantage of the experimental power of Drosophila genetics may improve understanding and ultimately the management of pathological cell movements (Uhlirova, 2006).

Src42A modulates tumor invasion and cell death via Ben/dUev1a-mediated JNK activation in Drosophila

Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, this study identified Src42A, the ortholog of mammalian Src, as a key modulator of both Ras^V12/lgl ^-/-triggered tumor invasion and loss of cell polarity gene-induced cell migration. The genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, this study showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with Ras^V12 to promote tumor overgrowth and invasion. In addition, it was found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. These data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a (Ma, 2013).

Notch and Mef2 synergize to promote proliferation and metastasis through JNK signal activation in Drosophila

Genetic analyses in Drosophila revealed a synergy between Notch and the pleiotropic transcription factor Mef2 (myocyte enhancer factor 2), which profoundly influences proliferation and metastasis. This study shows that these hyperproliferative and invasive Drosophila phenotypes are attributed to upregulation of eiger, a member of the tumour necrosis factor superfamily of ligands, and the consequent activation of Jun N-terminal kinase signalling, which in turn triggers the expression of the invasive marker MMP1. Expression studies in human breast tumour samples demonstrate correlation between Notch and Mef2 paralogues and support the notion that Notch-MEF2 synergy may be significant for modulating human mammary oncogenesis (Pallavi, 2012).

A genetic modifier screen was undertaken in Drosophila and a number of genetic modifiers of Notch signals were identified that affect proliferation. Further examination of one of these modifiers, Mef2, established that its synergy with Notch signals directly triggers expression of the Drosophila JNK pathway ligand eiger, consequently activating JNK signalling that profoundly influences proliferation and metastatic behaviour. It might perhaps be worth noting that metastatic behaviour in Drosophila may not be completely equivalent to mammalian metastasis, notwithstanding the fact that they share molecular signatures, for example, MMP activation (Pallavi, 2012).

Cancer is characterized by the deregulation of the balance between differentiation, proliferation and apoptosis; thus, it is not surprising that the Notch signalling pathway, which plays a central role in all these developmental events, is increasingly implicated in oncogenic events. The rationale of this study is based on the fact that synergy between Notch and other genes is key in understanding how Notch signals contribute to oncogenesis. It remains a remarkable fact that while activating mutations in the Notch receptor have been associated with >50% of T-cell lymphoblastic leukemias (T-ALLs), a search for mutations in other cancers, despite a few suggestive reports, remains essentially unfruitful. Yet, correlative studies have linked Notch activity with a broad spectrum of human cancers and work in mice suggests that while Notch activation promotes proliferation, it is the synergy between Notch and other factors that eventually leads to cancer. Similar synergies have been identified before but the extraordinary complexity of the gene circuitry that modulates the Notch pathway suggests that more such relationships will be uncovered as exemplified by the discovery of Mef2 as a Notch synergistic partner affecting proliferation (Pallavi, 2012).

The transcription factor Mef2 plays an essential role in myogenic differentiation, but several studies have also shown a broad pleiotropic role of Mef2. Mef2 can integrate signals from several signalling cascades through chromatin remodelling factors and other transcriptional regulators to control differentiation events. This study extends the functionality of Mef2 by uncovering the profound effect it can have on proliferation and metastatic cell migration in synergy with Notch signals (Pallavi, 2012).

This is not the first study to link Mef2 with Notch. They have been linked before in the context of myogenesis both in Drosophila and in vertebrates. A ChIP-on-chip analysis of Mef2 target regions identified several Notch pathway components as potential Mef2 targets during Drosophila myogenesis. In human myoblasts, Mef2C was suggested to bind directly to the intracellular domain of Notch via the ankyrin repeat region, suppressing Mef2C-induced myogenic differentiation. Mef2 has also been reported to interact with the Notch coactivator MAML1 and suppress differentiation (Pallavi, 2012).

While upregulation of Mef2 alone does not show overt proliferation effects, these analyses demonstrate that in vivo it can activate MMP1. Even though Mef2 was ectopically expressed in the whole wing pouch, MMP1 expression was confined around the D/V boundary, where endogenous Notch signals are active. This effect of Mef2 overexpression depends on Notch signals, a notion corroborated by the fact that inhibiting Notch activity by RNAi reverses the effects of Mef2 on MMP1 (Pallavi, 2012).

The polarity gene scribble cooperates with Ras signalling to upregulate the JNK pathway, promoting invasiveness and hyperplasticity. However, the synergy seen in this study appears to be scribble independent. The fact that both the scribbled/Ras and the Notch/Mef2 metastatic pathways converge at the level of JNK signal activation suggests that JNK is a crucial regulator of oncogenic behaviour, which is controlled by inputs from multiple signals. Even though there is little evidence that twist activates JNK signalling, it is a crucial regulator of epithelial-to-mesenchymal transition and metastasis and has also been independently linked to both Mef2 and Notch in myogenesis. However, it is noted that the Notch-Mef2 synergy seems to be independent of twist, as Twist cannot replace Mef2 in the synergistic relationship (Pallavi, 2012).

Numerous reports link JNK signalling to normal developmental events requiring cell movement and to metastatic phenomena both in Drosophila and in vertebrates. JNK signals seem to be crucial for controlling gene activities involved in epithelial integrity and the observations from Drosophila suggest that the N^act and Mef2 synergy may be important in JNK-linked carcinogenesis. A role for Notch in controlling JNK signals has been reported previously. While studies carried out using breast cancer samples can only be correlative now, the observations suggest that metastatic breast tumours harbour higher levels of Notch and Mef2 paralogue pairs, consistent with observations in Drosophila (Pallavi, 2012).

Although the majority of studies on Mef2 are focused on muscle development/differentiation, some intriguing links between Mef2C and leukaemias are noteworthy. MEF2C and Sox4 synergize to cause myeloid leukaemia in mice. Analysis of T-ALL patient samples revealed increased levels of Mef2C; however, Mef2C alone could not cause cellular transformation of NIH3T3 cells, but it could do so in the presence of RAS or myc. Given the role of Notch in T-ALL it will be important to examine how activated Notch mutations, often the causative oncogenic mutation, correlate with Mef2 family members. The functional differences between the different Mef2 homologues in humans are not well understood and the specific role each may play in the Notch synergy remains to be elucidated (Pallavi, 2012).

This analysis clearly indicates that, in Drosophila, the underlying molecular mechanism of the Notch/Mef2 synergy relies on the direct upregulation of expression of the prototypical TNF ligand egr through the binding of Mef2 and Su(H), the effector of Notch signals, to regulatory sequences on the egr promoter. In Drosophila, egr is the only JNK ligand while in humans, the superfamily is large and includes the cytokines TNFα (TNF), TRAIL and RANKL which have been associated with tumour progression in numerous human cancers including breast. RANKL plays a key role in bone metastasis of breast cancer, and is the target of a therapeutically effective monoclonal antibody. In breast cancer cells, TNFα, which can signal through several pathways, including JNK and NF-κB, affects proliferation and promotes invasion and metastasis (Pallavi, 2012).

In human breast cancer, clinical relapse after initial treatment is almost always accompanied by metastatic spread and it is almost invariably lethal. ER- tumours tend to respond well to first-line chemotherapy, but a significant subset of these tumours recur. Recurrent ER- tumours are typically resistant to chemotherapy and radiation, and are highly lethal. The current data suggest that ER- tumours that recur but not ER- tumours that do not recur show significant positive correlation between NOTCH1 and all four MEF2 paralogues. Further, the data show that even within the recurrent subset, NOTCH1 expression predicts poor survival but MEF2 expression does not. While these observations do not establish causality, they are consistent with the hypothesis that NOTCH1/MEF2 coexpression identifies a set of breast cancers that are more likely to relapse, and that MEF2 genes act as NOTCH cofactors rather than independently of NOTCH (Pallavi, 2012).

In conclusion, this study in Drosophila uncovers a new functional role for Mef2, which in synergy with Notch affects proliferation and metastasis. Mechanistically, this synergy relies on the direct upregulation of the JNK pathway ligand eiger. The correlation analysis and tumour staining of human cancer samples suggests that the observations in Drosophila may well be valid in humans, defining Notch-Mef2 synergy as a critical oncogenic parameter, one that may be associated with metastatic behaviour, emphasizing the value of model systems in gaining insight into human pathobiology (Pallavi, 2012).

Distinct functions for the catalytic and hemopexin domains of a Drosophila matrix metalloproteinase

Human matrix metalloproteinases (MMPs) are believed to contribute to tumor progression. Therapies based on inhibiting the catalytic domain of MMPs have been unsuccessful, but these studies raise the question of whether other MMP domains might be appropriate targets. The genetic dissection of domain function has been stymied in mouse because there are 24 related and partially redundant MMP genes in the mouse genome. This study presents a genetic dissection of the functions of the hemopexin and catalytic domains of a canonical MMP in Drosophila melanogaster, an organism with only 2 MMPs that function nonredundantly. The phenotypes of Mmp1 null alleles were compared with alleles that have specific hemopexin domain lesions, and phenotypes of dominant-negative mutants were also examined. Although the catalytic domain appears to be required for all MMP functions including extracellular matrix remodeling of the tracheal system, the hemopexin domain is required specifically for tissue invasion events later in metamorphosis but not for tracheal remodeling. Thus, it was found that this MMP hemopexin domain has an apparent specialization for tissue invasion events, a finding with potential implications for inhibitor therapies (Glasheen, 2009).

It seems likely that the hemopexin domain is a protein-protein interaction domain, as previously thought, but that it only binds to a subset of Mmp1's binding partners and substrates; others bind directly to the catalytic domain. These hemopexin binding partners could be substrates, localization determinants, or have other functions. It is reasonable to assume that Mmp1 substrates in tracheal elongation and in disc eversion are different, because the tracheae are lined with a chitinous apical extracellular matrix that must be remodeled during elongation, whereas imaginal disc eversion requires remodeling of a basement membrane containing collagen IV and laminin. Importantly, in addition to its role in developmental tissue invasion, Mmp1 is required in Drosophila for the local invasion of both transplanted and clonally induced tumors. These tissue invasion events require the hemopexin domain, because it has been shown that tumors induced in Q273* homozygous mutants have reduced invasive capacity; thus the hemopexin domain mediates both developmental and pathological tissue invasion (Glasheen, 2009).

Dendrite-specific remodeling of Drosophila sensory neurons requires matrix metalloproteases, ubiquitin-proteasome, and ecdysone signaling

During neuronal maturation, dendrites develop from immature neurites into mature arbors. In response to changes in the environment, dendrites from certain mature neurons can undergo large-scale morphologic remodeling. A group of Drosophila peripheral sensory neurons, the class IV dendritic arborization (C4da) neurons completely degrade and regrow their elaborate dendrites. Larval dendrites of C4da neurons are first severed from the soma and subsequently degraded during metamorphosis. This process is controlled by both intracellular and extracellular mechanisms: The ecdysone pathway and ubiquitin-proteasome system (UPS) are cell-intrinsic signals that initiate dendrite breakage, and extracellular matrix metalloproteases are required to degrade the severed dendrites. Surprisingly, C4da neurons retain their axonal projections during concurrent dendrite degradation, despite activated ecdysone and UPS pathways. These results demonstrate that, in response to environmental changes, certain neurons have cell-intrinsic abilities to completely lose their dendrites but keep their axons and subsequently regrow their dendritic arbors (Kuo, 2005).

To visualize abdominal C4da neurons during Drosophila metamorphosis, a pickpocket (ppk)-EGFP reporter line was used. Filleted white pupae (WP), at the onset of metamorphosis, were stained with an anti-EGFP antibody to reveal three C4da neurons, vdaB (V), v'ada (V'), and ddaC (D), in each hemisegment. Because the soma and dendritic projections of these neurons remained very close to the body surface during pupariation, live imaging was used to follow these neurons throughout metamorphosis (Kuo, 2005).

Initially at the WP stage, the C4da neurons exhibited intact, complex class IV dendritic branches that covered much of the pupal surface. Shortly after the white pupal stage, 2 h after puparium formation (APF), fine terminal dendritic branches began to disappear. By 10 h APF, most major dendritic branches were severed from the soma. This severing of dendrites has also been observed in a recent study of da neuron remodeling. During the next 8 h, which coincided with head eversion during metamorphosis, these severed and blebbing dendrites are degraded. By 20 h APF, the process of larval dendrite removal is complete, leaving C4da neurons with their axonal projections but devoid of larval dendrites. Axons from all three C4da neurons project into the VNC. By this time, V' and D neurons begin to extend fine dendritic projections. The V neurons, which do not show new dendritic projections, disappear between 30 and 35 h APF, leaving V' as the surviving neuron in the ventral hemisegment (Kuo, 2005).

Compared with the rapid sequence of larval dendritic pruning, the process of pupal dendrite regrowth is slow. By 70 h APF, both V' and D neurons begin to take on the shape of their respective adult neurons. By 95 h APF, shortly before adult eclosion, the dendritic patterns of abdominal V' neurons closely resemble larval C4da neurons before pupariation. In contrast, the D neurons take on a more elongated dendritic field, perhaps reflecting a functional divergence between these two neurons in the adult fly. These results show that C4da neurons can completely degrade their elaborate larval dendrites during early metamorphosis, survive these changes, and subsequently regrow their dendritic arbors (Kuo, 2005).

During Drosophila metamorphosis, most larval organs are replaced by adult structures. To understand the cellular environment during C4da dendrite degradation, the expression of Armadillo, an adhesive junction protein that outlines the epithelial monolayer during early metamorphosis, was examined. High-level Armadillo staining at the WP stage is completely abolished by 13 h APF but subsequently returns at 20 h APF when the pupal epithelium is reformed. Thus, the pruning of C4da neuron dendrites occurs concurrently with epithelial remodeling during metamorphosis. To determine whether the degradation of larval dendrites is a result of local tissue remodeling or neuron-intrinsic signaling, focus was placed on enzymes that are important for tissue remodeling (Kuo, 2005).

Drosophila matrix metalloproteases (metalloproteinases) Mmp1 and Mmp2 regulate tissue remodeling during metamorphosis (Page-McCaw, 2003). The weaker alleles of both genes, Mmp1^Q273 and Mmp2^W307, survive past head eversion to midpupariation, making it possible to visualize dendritic pruning of ppk-EGFP-expressing C4da neurons. Remarkably, there were abundant C4da neuron larval dendrites in both Mmp1 and Mmp2 mutants after head eversion. Whereas in WT pupae at 20 h APF all larval dendrites from C4da neurons were cleared from the extracellular space, in both Mmp1 and Mmp2 mutants, larval dendrites that are severed from the soma remain. These larval dendrites persist to much later stages at 50 and 35 h APF, just before the lethal phases of Mmp1^Q273 and Mmp2^W307 mutants, respectively. The ineffective removal of larval dendrites in Mmp mutants is not caused by a generalized delay in metamorphosis because Mmp mutant pupae had completed head eversion and epidermal remodeling, thus indicating a specific defect in dendrite degradation. Because Mmp1;Mmp2 double mutants do not survive to pupariation, it was not possible to look at dendritic pruning in the double mutant background (Kuo, 2005).

To determine whether Mmps functions on the cell surface of dendrites to regulate degradation, an Mmp inhibitor was expressed in C4da neurons to see whether the survival of larval dendrites can be prolonged. Using the ppk promoter to express transcriptional activator Gal4 (ppk-Gal4), the UAS-Gal4 system was used to express the Drosophila tissue inhibitor of metalloproteases (TIMP) in C4da neurons. Fly TIMP is closely related to mammalian TIMP-3, which associates with extracellular membrane surfaces to modulate Mmp activities. In control animals expressing GFP at 20 h APF, identical pruning of larval dendrites as in ppk-EGFP flies was seen. In contrast, when TIMP is overexpressed in C4da neurons, larval dendrites remain in the extracellular space at 20 h APF (Kuo, 2005).

The fact that TIMP inhibition can successfully delay the degradation of larval dendrites confirms Mmp's involvement in this process. But these enzymes could be synthesized by either the C4da neurons or by the surrounding cells. To identify the source of this Mmp activity, MARCM studies were performed to generate C4da clones that in both Mmps. Mmp1^Q112Mmp2^W307 double mutant C4da clones not only show dendritic branching patterns similar to WT clones during early pupariation, but live time-lapse imaging revealed complete larval dendrite removal after head eversion at 20 h APF, just like WT controls. These results show that cell-intrinsic Mmps are not required for dendritic pruning and that extracellular Mmp activity is sufficient for degrading severed larval dendrites during metamorphosis. A possible source of this extracellular activity could be phagocytic blood cells, because they have been shown to engulf dendritic debris during metamorphosis (Kuo, 2005).

Whereas removal of severed dendrites requires extrinsic metalloproteases, C4da neurons in Mmp mutants still retain their ability to sever larval dendrites from the soma during metamorphosis. To look for cell-intrinsic pathways in cleaving larval dendrites, the role of ecdysone, a steroid hormone that regulates much of Drosophila metamorphosis, was examined. Binding of ecdysone to its nuclear receptor heterodimers, consisting of Ultraspiracle (Usp) and one of three EcR isoforms (EcR-A, EcR-B1, and EcR-B2), mediates a transcriptional hierarchy that regulates tissue responses during metamorphosis. To determine whether EcR signaling plays a role in initiating dendritic pruning, EcR expression patterns were examined in the ppk-EGFP transgenic line that specifically labels C4da neurons (Kuo, 2005).

Staining with the EcR-C antibody, which recognizes the common regions of EcR family members, and staining with EcR-A and EcR-B1 specific antibodies during different stages of late larval through early pupal development, revealed that all three C4da neurons exhibit similar staining patterns. In third-instar larvae, when the ecdysone level is low before the onset of pupariation, EcR expression in C4da neurons is relatively low when compared with surrounding cells that already exhibit a high level of nuclear EcR. At the WP stage, with a transient rise in ecdysone level, EcR in C4da neurons becomes concentrated in the nucleus. Over the next 7 h, EcR gradually redistributes throughout the soma of C4da neurons, which corresponds to a rapid drop-off in ecdysone levels in the pupae after initiation of metamorphosis. Strong nuclear EcR localization in C4da neurons returns at 20 h APF, correlating with the onset of midpupal ecdysone release. Antibodies specific to either EcR-A or B1 show that whereas EcR-A expression is diffuse and weak throughout metamorphosis, EcR-B1 expression in C4da neurons corresponds to the dynamic nuclear localization patterns seen with the EcR-C antibody (Kuo, 2005).

To examine the functional significance of EcR expression, attempts were made to disrupt ecdysone signaling specifically in C4da neurons. EcR mutants either do not survive to the pupal stage or die shortly after the onset of metamorphosis; therefore, it is not possible to look at dendritic remodeling in those mutants. The cytological location of EcR genes also precludes MARCM studies; therefore, use was made of a set of dominant-negative (DN) EcR proteins to inhibit EcR activity. When ecdysone signaling is inhibited by EcR-DN proteins, C4da neurons lose their ability to initiate larval dendrite pruning at 20 h APF. To determine whether the defects are specific to the ecdysone signaling pathway, attempts were made to rescue the EcR-DN phenotype. Coexpression of both EcR-DN and WT EcR-B1 proteins in C4da neurons resulted in complete rescue of dendritic pruning defects in all three neurons. This rescue is complete with two copies of ppk-Gal4 in C4da neurons, showing that the rescue is not caused by reduced expression of DN protein in the coexpression experiments (Kuo, 2005).

Because dimerization of EcR-B1 with its obligatory hormone receptor partner Usp is essential for transcriptional regulation, the involvement of Usp in dendrite remodeling was examined. Usp mutants do not survive to metamorphosis; however, it was possible to generate Usp MARCM clones for analysis. At 20 h APF, Usp mutant C4da clones fail to prune their larval dendrites, and this genetic mutation shows an identical phenotype to the EcR-DN experiments. Given the severity and full penetrance of this phenotype, together with the timing of EcR-B1 nuclear localization, it is concluded that the ecdysone signaling pathway plays an important cell-intrinsic role in initiating dendritic pruning in C4da neurons during metamorphosis (Kuo, 2005).

What might be the cellular machineries that carry out dendrite pruning in C4da neurons? One attractive model is a caspase-mediated local digestion and degradation of dendrites. However, overexpression of p35, an effective inhibitor of fly caspases, in C4da neurons did not prevent or delay larval dendrite degradation during metamorphosis. Another protein degradation pathway, the ubiquitin protease system (UPS), has been shown to regulate both axon and dendrite pruning of mushroom body neurons during fly metamorphosis. To test the involvement of UPS in C4da neuron remodeling, use was made of ppk-Gal4 to overexpress UBP2, a yeast ubiquitin protease, in the C4da neurons. By reversing the process of substrate ubiquination, UBP2 is an effective UPS inhibitor in the fly. Some of the C4da neurons expressing UBP2 aberrantly retained their larval dendritic arbors. Note that this pruning defect is very different from that seen in Mmp mutants. Whereas Mmp mutants accumulated severed larval dendrites in the extracellular space, UBP2 inhibition prevented efficient severing of dendrites from the soma (Kuo, 2005).

To further examine the involvement of the UPS machinery in dendritic pruning, the MARCM system was used to generate C4da clones that were either deficient in ubiquitin activation enzyme 1 (Uba1) or had mutation in the 19S particle of the proteasome (Mov34). Time-lapse imaging of Uba1 and Mov34 mutant C4da clones at WP stage and 20 h APF showed that, unlike WT clones, both mutant clones failed to efficiently sever their larval dendrites during metamorphosis. These results confirmed the requirement for an activated UPS in the severing of larval dendrites from C4da neurons during metamorphosis (Kuo, 2005).

To compare the defects in larval dendritic pruning caused by different mutations, the number of large (primary and secondary) dendritic branches that remain attached to C4da neuron soma was counted. In WT pupae at the start of metamorphosis, C4da neurons extended close to 20 large dendritic branches, none of which was retained after head eversion at 20 h APF. Mutations that disrupt ecdysone signaling, such as EcR-DN expression or Usp-deficient clones, result in the retention of 85%-90% of large dendritic branches after head eversion. Mutations in the UPS pathway, such as Uba1 and Mov34, resulted in the retention of 45%-49% of large dendritic branches at 20 h APF. Mmp1 or Mmp2 mutants retained only 3%-8% of large dendritic branches after head eversion, and Mmp1;Mmp2 mutant clones did not retain larval dendrites at 20 h APF. These data suggest that dendrite remodeling in C4da neurons starts with signals from ecdysone and UPS that result in the cleavage of larval dendrites from the soma, which then allows for the degradation of severed dendrites by Mmp activity in the extracellular matrix (Kuo, 2005).

It is possible that UPS is an upstream regulator of EcR and can lead to EcR misexpression in UPS mutants; however, normal EcR expression patterns are observed in both Uba1 and Mov34 C4da MARCM clones. It is also conceivable that EcR signaling is upstream of the UPS cascade, but this idea is difficult to demonstrate experimentally. It was reasoned that in this case, inhibition of EcR signaling should result in lower levels of protein ubiquination. However, staining in EcR-DN-expressing C4da neurons showed no significant differences in the level of ubiquitin/polyubiquitin between undegraded larval dendrites and WT dendrites before degradation. This finding does not rule out an EcR function upstream of UPS during dendritic remodeling, because EcR signaling may regulate critical factors in the UPS cascade after protein ubiquination at the level of ubiquitin ligases. The identities of such ligases are currently unknown (Kuo, 2005).

To test whether dendritic pruning in C4da neurons involves concurrent axonal remodeling, axon tracks of C4da neurons were examined in the Drosophila VNC during early metamorphosis. Direct live imaging of the ppk-EGFP transgenic line at the WP stage showed axon tracks from three C4da neurons. Axon tracing of EGFP-expressing C4da neurons at 6 h APF showed continuous axon tracks between all three C4da neurons and the VNC. At 10 h APF, the VNC appeared more compact, presumably as a result of the various remodeling events that occur in the nervous system during metamorphosis. At 20 h APF, axon tracks of EGFP-expressing C4da neurons can still be clearly identified at the VNC and are continuous with the soma, despite the complete removal of dendritic arbors of these same neurons (Kuo, 2005).

Drosophila peripheral sensory neurons generally have simple axon projections into the VNC that terminate locally. To visualize C4da neuron axon terminals during metamorphosis, the UAS>CD2>CD8-GFP system was used, together with ppk-Gal4, to generate single clones of surviving V' and D neurons. The V' C4da neuron was found to project its axon ipsilaterally upon entering the VNC to the segment immediately anterior during the WP stage. At 20 h APF, after complete pruning of larval dendrites, the V' C4da neuron keeps this axonal projection intact in the VNC. The D C4da neuron axon, in addition to having an ipsilateral branch that projects to the anterior segment, sends a commissural branch that crosses the midline at the segment where the axon enters the VNC. Likewise, at 20 h APF, the D C4da neuron keeps both axonal terminal branches intact. These data show that C4da neurons do not significantly modify their larval axons during concurrent dendrite degradation, despite the activated ecdysone and UPS pathways, which are known to facilitate axon remodeling and degradation (Kuo, 2005).

What might account for the dendrite-specific remodeling in C4da neurons, as opposed to the previously reported concurrent remodeling of both axons and dendrites? It is possible that local environments may play a role. A recent study in Manduca found central versus peripheral hormonal differences affecting axon versus dendrite remodeling. However, it remains to be tested whether the ecdysone levels are different in the fly epidermis and the VNC during metamorphosis. Anatomically, C4da neurons have distinct axon-dendrite polarity in that the cell bodies send out multiple primary dendritic arbors to the surrounding environment while each extends a single axon toward the VNC. This morphology is in contrast to most insect neurons, such as femoral depressor motoneurons and mushroom body gamma-neurons, which extend a single branch from the soma that later gives rise to both dendrites and axons. As such, C4da neurons may have developed separate mechanisms at the soma to remodel just the dendrites. Just what these mechanisms might include is currently unknown (Kuo, 2005).

This study has provided evidence that certain mature neurons have the ability to selectively degrade their dendritic projections in vivo and regrow new ones. Although fly metamorphosis is a specialized developmental process, dendrite-specific remodeling may provide a paradigm for neurons to retain part of their connections in the neuronal circuitry while responding to environmental changes such as tissue degeneration near their dendrites. Certain conditions in mammalian systems, such as trauma and injury, can induce localized degeneration and remodeling and may mimic the active tissue remodeling during metamorphosis. In the human CNS, for example, significant reorganization of granule cell projections in the dentate gyrus after human temporal lobe epilepsy has been observed. Thus, it would be of great interest to examine whether dendritic-specific remodeling of C4da neurons in Drosophila represents an evolutionarily conserved mechanism for neurons to respond to drastic changes in their environment, and to determine whether mammalian neurons have similar capacities to remodel their dendrites (Kuo, 2005).

Drosophila matrix metalloproteinases are required for tissue remodeling, but not embryonic development

The matrix metalloproteinase (MMP) family is heavily implicated in many diseases, including cancer. The developmental functions of these genes are not clear, however, because the >20 mammalian MMPs can be functionally redundant. Drosophila has only two MMPs, which are expressed in embryos in distinct patterns. Mutations were created in both genes: Mmp1 mutants have defects in larval tracheal growth and pupal head eversion, and Mmp2 mutants have defects in larval tissue histolysis and epithelial fusion during metamorphosis; neither is required for embryonic development. Double mutants also complete embryogenesis, and these represent the first time that all MMPs have been disrupted in any organism. Thus, MMPs are not required for Drosophila embryonic development, but, rather, for tissue remodeling (Page-McCaw, 2003).

This paper reports the identification, expression, and phenotypic analysis of the two Drosophila MMPs, as well as the phenotype of the double MMP mutant. Flies have only two MMPs, compared with more than 20 in mammals, and both are inhibited by the single fly Tissue inhibitor of metalloproteases (Timp). Mmp1 RNA is expressed in a few tissues during embryogenesis, and Mmp1 protein is detected in embryos and in larval tracheal cells. Mmp2 RNA is expressed in many tissues during embryogenesis, including a subset of the nervous system, and is expressed widely in omaginal discs. Mutant analysis of alleles generated for this study reveals that Mmp1 is required for growth of the larval tracheal system and is involved in release of cuticle at molting and head eversion during pupation. Mmp2 is required for fusion of imaginal tissue and histolysis of larval tissue during metamorphosis. The two MMPs may be partially redundant for the morphogenetic event of pupal head eversion. Neither MMP is required for embryonic development. Interestingly, the double Mmp2 Mmp1 mutant is able to complete embryonic and much of larval development, dying with a phenotype similar to that of Mmp1 mutants, indicating that MMPs are not essential for embryonic development in flies (Page-McCaw, 2003).

Mmp1 tracheal defects may be caused by an inability of cells to detach from the tracheal cuticle, a multilayered extracellular matrix that lines the apical (lumenal) surface of the entire tracheal system. In wild-type animals, the tracheal trunks must expand 7-fold in diameter and 14-fold in length during larval growth, all without cell division. Increases in tracheal diameter occur during rapid dilation events at the two larval molts, when cells loosen their attachments to the old cuticle, dilate their lumens, and secrete new cuticle; molting is known to require extensive proteolysis of the old cuticle. The irregular diameter of Mmp1 dorsal trunks suggests that tracheal cells have difficulty detaching from the cuticle during the dilations. A failure of detachment may also explain why some Mmp1 animals remain stuck to old cuticles after molting. In contrast, increases in tracheal length occur gradually throughout larval development, and the tracheal cuticle must elongate concomitantly. Mmp1 trunks cannot properly elongate beginning in first instar larvae, before the first molt, as evidenced by the stretching of the tracheal system. This stretching probably causes the broken trunks and internalized spiracles. Tracheal tube elongation may also require that cells loosen their cuticle attachments in order to spread, and, if so, then the failure to elongate could also be caused by a failure of detachment. Alternatively, tube elongation may be constrained in mutants by an inability of the cuticle itself to elongate, perhaps because cuticle elongation requires localized proteolysis. As obstructions that block oxygen diffusion accumulate in the tracheae, the animals appear hypoxic, with increased wandering behavior and eventual death (Page-McCaw, 2003).

During metamorphosis larval organs and tissues are destroyed and replaced with those of the adult; one example is the larval midgut, which is histolyzed and replaced by the developing adult midgut in a process that requires significant apoptosis of the larval cells. Mmp2 is required for complete histolysis of larval midgut structures. In the vertebrate parallel process, MMPs are known to be important for frog metamorphosis. In both cases, larval tissue histolysis is under hormonal regulation, with Drosophila metamorphosis controlled by ecdysone and Xenopus metamorphosis controlled by thyroid hormone. In Xenopus, different MMP family members play different roles in histolysis of the larval intestine: stromelysin-3 is involved in triggering apoptosis, presumably by interrupting contact between the cells and their basement membranes, and collagenase-3, collagenase-4, and gelatinase A are expected to be involved in clearing away ECM after cell death. It will be interesting to see whether Mmp2 mutant larval midguts are able to undergo apoptosis, but not matrix degradation, or whether apoptosis itself is blocked (Page-McCaw, 2003).

Mmp2 also appears to be required for fusion of the epithelial tissue of the notum. Normally, the wing discs fuse along the midline of the notum early in morphogenesis, before head eversion, timing that is consistent with the first appearance of the black thoracic spots in mutants. The discs spread medially from their lateral locations, with the cells at the advancing edge (termed S cells) crawling over preexisting larval epithelium; the larval cells are then replaced by imaginal cells in the disc. The S cells from the two discs meet at the midline and then are lost. It is possible that the black spots in mutants could represent larval cells that persist after they should be removed, similar to the persistence of the larval midgut; S cells that fail to be removed from the tissue; or a defect in spreading of the imaginal tissue over the larval tissue. The role of MMPs in epithelial fusion has been examined in the developing mouse palate, where epithelial cells at the advancing edge of the mesenchymal tissue disappear after they meet at the midline. MMPs are expressed at the fusion site, and addition of an MMP inhibitor in vitro causes a failure of epithelial fusion. Moreover, in TGF-β3 mutants, MMP-2 and MMP-13 fail to be expressed at the fusion site, the epithelial cells persist, and the palates do not fuse, resulting in mice with cleft palates (Blavier, 2001). Weaker Mmp2 mutants develop cleft notums, and it is interesting to note that mutation of the Drosophila TGF-β gene dpp also causes a cleft along the midline of the notum The roles of MMPs in these mouse and fly processes appear to be homologous (Page-McCaw, 2003).

It is useful to consider the similarities and differences between MMPs in flies and mammals. It appears that the two fly MMPs diverged when insects and mammals shared a common ancestor, so they may represent an ancient divergence in the MMP family. As with mammals, fly Timp inhibits fly MMPs, and Llano (2000) found that Mmp1 is also inhibited by mammalian TIMP-2 and -4. In addition to MMPs and Timp, the fly genome contains a single gene (CG5392) homologous to mammalian RECK, a recently identified MMP inhibitor (Oh, 2001). Thus, it appears that flies have many of the same players as mammals in the MMP pathway. As with mammalian MMPs, fly MMPs may remain associated with the cell surface after secretion. Mmp2 is predicated to contain a GPI anchor sequence, suggesting that it might be membrane associated (Llano, 2002). An Mmp1 cDNA fragment also contained a predicted GPI anchor sequence (Page-McCaw, 2003).

In mammals, MMPs can compensate for the loss of one family member with the upregulation of others, as has been demonstrated in heart tissue and mouse involuting uterus. It seemed likely that the mild phenotypes observed in many MMP mutant mice may be a result of similar MMP compensation. The question of MMP function was addressed more definitively in Drosophila because there are only two MMPs. To rule out the formal possibility that a third MMP gene might lie in a repeat-rich, heterochromatic portion of the genome that is not clonable in BACs, BLAST searches were carried of all the sequence traces generated in the whole genome shotgun, which should include any nonrepetitive sequence from the heterochromatin, and no significant homology to MMPs other than that accounted for by Mmp1 and Mmp2 was found. The result of this comprehensive genome search argues strongly against the existence of a third Drosophila MMP gene. Since Mmp2 Mmp1 double mutant animals, including those derived from germline clones, hatch and grow to third instar larvae, it is clear that Drosophila embryos do not require any MMPs to complete embryogenesis and develop to third instar (Page-McCaw, 2003).

Structural and enzymatic characterization of Drosophila Dm2-MMP, a membrane-bound matrix metalloproteinase with tissue-specific expression

A cDNA has been isolated and characterized encoding Dm2-MMP, the second matrix metalloproteinase (MMP) identified in the Drosophila melanogaster genome. The cloned cDNA codes for a polypeptide of 758 residues that displays a domain organization similar to that of other MMPs, including signal peptide, propeptide, catalytic, and hemopexin domains. However, the structure of Dm2-MMP is unique because of the presence of an insertion of 214 amino acids between the catalytic and hemopexin domains that is not present in any of the previously described MMPs. Dm2-MMP also contains a C-terminal extension predicted to form a cleavable glycosylphosphatidylinositol anchor site. Western blot and immunofluorescence analysis of S2 cells transfected with the isolated cDNA confirmed that Dm2-MMP is localized at the cell surface. Production of the catalytic domain of Dm2-MMP in Escherichia coli and analysis of its enzymatic activity revealed that this proteinase cleaves several synthetic peptides used for analysis of vertebrate MMPs. This proteolytic activity was abolished by MMP inhibitors such as BB-94, confirming that the isolated cDNA codes for an enzymatically active metalloproteinase. Reverse transcription-PCR analysis showed that Dm2-MMP is expressed at low levels in all of the developmental stages of Drosophila as well as in adult flies. However, detailed in situ hybridization at the larval stage revealed a strong tissue-specific expression in discrete regions of the brain and eye imaginal discs. According to these results, it is proposed that Dm2-MMP plays both general proteolytic functions during Drosophila development and in adult tissues and specific roles in eye development and neural tissues through the degradation and remodeling of the extracellular matrix (Llano, 2002. Full text of article).

Dm1-MMP, a matrix metalloproteinase from Drosophila with a potential role in extracellular matrix remodeling during neural development

A cDNA encoding Dm1-MMP, the first matrix metalloproteinase (MMP) identified in Drosophila melanogaster, has been cloned and characterized. The isolated cDNA encodes a protein of 541 residues that has a domain organization identical to that of most vertebrate MMPs including a signal sequence, a prodomain with the activation locus, a catalytic domain with a zinc-binding site, and a COOH-terminal hemopexin domain. Northern blot analysis of Dm1-MMP expression in embryonic and larval adult tissues revealed a strong expression level in the developing embryo at 10-22 h, declining thereafter and being undetectable in adults. Western blot analysis confirmed the presence of pro- and active forms of Dm1-MMP in vivo during larval development. In situ hybridization experiments demonstrated that Dm1-MMP is expressed in a segmented pattern in cell clusters at the midline during embryonic stage 12-13, when neurons of the central nervous system start to arise. Recombinant Dm1-MMP produced in Escherichia coli exhibits a potent proteolytic activity against synthetic peptides used for analysis of vertebrate MMPs. This activity is inhibited by tissue inhibitors of metalloproteinases and by synthetic MMP inhibitors such as BB-94. Furthermore, Dm1-MMP is able to degrade the extracellular matrix and basement membrane proteins fibronectin and type IV collagen. On the basis of these data, together with the predominant expression of Dm1-MMP in embryonic neural cells, it is proposed that this enzyme may be involved in the extracellular matrix remodeling taking place during the development of the central nervous system in Drosophila (Llano, 2000. Full text of article).

Search PubMed for articles about Drosophila Mmp2

Ahmed, Z., Dent, R. G., Leadbeater, W. E., Smith, C., Berry, M. and Logan, A. (2005). Matrix metalloproteases: degradation of the inhibitory environment of the transected optic nerve and the scar by regenerating axons. Mol. Cell. Neurosci. 28: 64-78. PubMed ID: 15607942

Andersen, D. S., Colombani, J., Palmerini, V., Chakrabandhu, K., Boone, E., Rothlisberger, M., Toggweiler, J., Basler, K., Mapelli, M., Hueber, A. O. and Leopold, P. (2015). The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth. Nature. PubMed ID: 25874673

Basile, J. R., Holmbeck, K., Bugge, T. H. and Gutkind, J. S. (2007). MT1-MMP controls tumor-induced angiogenesis through the release of semaphorin 4D. J. Biol. Chem. 282: 6899-6905. PubMed ID: 17204469

Blavier, L., Lazaryev, A., Groffen, J., Heisterkamp, N., DeClerck, Y. A. and Kaartinen, V. (2001). TGF-beta3-induced palatogenesis requires matrix metalloproteinases. Mol. Biol. Cell 12: 1457-1466. PubMed ID: 11359935

Demestre, M., Wells, G. M., Miller, K. M., Smith, K. J., Hughes, R. A., Gearing, A. J. and Gregson, N. A. (2004). Characterisation of matrix metalloproteinases and the effects of a broad-spectrum inhibitor (BB-1101) in peripheral nerve regeneration. Neuroscience 124: 767-779. PubMed ID: 15026117

Endo, K., Takino, T., Miyamori, H., Kinsen, H., Yoshizaki, T., Furukawa, M. and Sato, H. (2003). Cleavage of syndecan-1 by membrane type matrix metalloproteinase-1 stimulates cell migration. J. Biol. Chem. 278: 40764-40770. PubMed ID: 12904296

Fambrough, D., Pan, D., Rubin, G. M. and Goodman, C. S. (1996). The cell surface metalloprotease/disintegrin Kuzbanian is required for axonal extension in Drosophila. Proc. Natl. Acad. Sci. 93: 13233-13238. PubMed ID: 8917574

Fox, A. N. and Zinn, K. (2005). The heparan sulfate proteoglycan syndecan is an in vivo ligand for the Drosophila LAR receptor tyrosine phosphatase. Curr. Biol. 15: 1701-1711. PubMed ID: 16213816

Galko, M. J. and Tessier-Lavigne, M. (2000). Function of an axonal chemoattractant modulated by metalloprotease activity. Science 289: 1365-1367. PubMed ID: 10958786

Glasheen, B. M., Kabra, A. T. and Page-McCaw, A. (2009). Distinct functions for the catalytic and hemopexin domains of a Drosophila matrix metalloproteinase. Proc Natl Acad Sci 106(8): 2659-2664. PubMed ID: 19196956

Glasheen, B. M., et al. (2010). A matrix metalloproteinase mediates airway remodeling in Drosophila. Dev. Biol. 344(2): 772-83. PubMed ID: 20513443

Guha, A., Lin, L. and Kornberg, T. B. (2009). Regulation of Drosophila matrix metalloprotease Mmp2 is essential for wing imaginal disc:trachea association and air sac tubulogenesis. Dev. Biol. 335(2): 317-26. PubMed ID: 19751719

Hattori, M., Osterfield, M. and Flanagan, J. G. (2000). Regulated cleavage of a contact-mediated axon repellent. Science 289: 1360-1365. PubMed ID: 10958785

Heine, W., Conant, K., Griffin, J. W. and Hoke, A. (2004). Transplanted neural stem cells promote axonal regeneration through chronically denervated peripheral nerves. Exp. Neurol. 189: 231-240. PubMed ID: 15380475

Hehr, C. L., Hocking, J. C. and McFarlane, S. (2005). Matrix metalloproteinases are required for retinal ganglion cell axon guidance at select decision points. Development 132: 3371-3379. PubMed ID: 15975939

Jia, Q., Liu, S., Wen, D., Cheng, Y., Bendena, W. G., Wang, J. and Li, S. (2017). Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation. J Biol Chem 292(52): 21504-21516. PubMed ID: 29118190

Kulshammer, E., Mundorf, J., Kilinc, M., Frommolt, P., Wagle, P. and Uhlirova, M. (2015). Interplay among Drosophila transcription factors Ets21c, Fos and Ftz-F1 drives JNK-mediated tumor malignancy. Dis Model Mech 8: 1279-1293. PubMed ID: 26398940

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date revised: 22 December 2023

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