BIOLOGICAL OVERVIEW

Two signals initiate the events that will engender dorso-ventral patterning in the egg and in the embryo (Schumpbach, 1994). The first prepares the scene for the second. It is a signal from the oocyte to follicle cells (maternal cells surrounding the egg) to enter a dorsal differentiation program, and it requires the ligand Gurken and the receptor Torpedo. This first signal negatively delimits the ventral zone within the follicle cell epithelium, restricting the formation of the ligand of the second signal to a spatially restricted area in the ventral part of the egg. Having its boundaries prepared, the second signal awaits activation upon fertilization. It is this second signal that involves the ligand Spätzle and the receptor Toll.

Spätzle is one of 11 dorsal group genes identified by their completely dorsalized phenotype, lacking ventral, lateral and dorsolateral pattern elements. The dorsal group genes activate the Dorsal transcription factor, expressed in the ventral part of the embryo and responsible for ventralization of the embryo.

The area around the egg is filled with a perivitelline fluid capable of restoring dorsal-ventral polarity to mutant easter, snake and Spätzle embryos. This has been demonstrated by injecting perivitelline fluid into the perivitelline space of mutant eggs (Stein, 1992).

One component of the perivitelline fluid is zymogen serine protease, a member of the trypsin family coded for by the easter gene. Easter is one of three proteins in the perivitelline fluid required for Dorsal protein activation. Spätzle is the only dorsal group gene upstream of the Toll receptor that is required for Easter to exert its effect on the dorsal-ventral pattern (Chasen, 1992). Easter is responsible for activating Spätzle, so that Spätzle can then interact with Toll. The Easter protein activates Spätzle by proteolysis, producing a ligand that can bind the Toll receptor. Toll in turn activates Dorsal in a process involving a change in location of Dorsal protein from the cytoplasm to the nucleus.

The cloning of the spätzle gene necessitated employment of a reversion to the wild type of a dominant mutant of spätzle. Why a dominate mutant? Whatever the cause of a dominant mutation, inactivation of such a mutation might involve destruction of the gene itself. The dominant mutant allele was mutated by a viral insertion element (P) integrated in the midst of the spätzle gene, thereby inactivating it. Use of a P insertion element allows for the recovery of the chromosome element in which it inserts (by fishing for the P element).

A genomic library was constructed from the P revertant type, and a P positive clone was identified that hybridized with the same genome region to which spätzle maps (Marisato, 1994). This is proof that the P vector contains DNA homologous to spätzle. In a different lab, spätzle activity was purified using an assay for ventralization by perivitelline fluid (Schneider, 1994).

Constitutive activation of Toll-mediated antifungal defense in serpin-deficient Drosophila

The antifungal defense of Drosophila is controlled by the spätzle/Toll/cactus gene cassette. A loss-of-function mutation in the gene encoding a blood serine protease inhibitor, Spn43Ac, has been shown to lead to constitutive expression of the antifungal peptide Drosomycin, and this effect is mediated by the spätzle and Toll gene products. Spätzle is cleaved by proteolytic enzymes to its active ligand form shortly after immune challenge; cleaved Spätzle is constitutively present in Spn43Ac-deficient flies. Hence, Spn43Ac negatively regulates the Toll signaling pathway, and Toll does not function as a pattern recognition receptor in the Drosophila host defense (Levashina, 1999).

Flies carrying ethylmethane sulfonate-induced mutations in the necrotic (nec) locus were used. The locus, which maps at position 43A, generates three transcripts encoding putative serine protease inhibitors of the serpin family. The nec mutants exhibit brown spots along the body and the leg joints, corresponding to necrotic areas in the epidermis. This mutant phenotype is rescued by a single transgenic copy of one of the serpin genes, Spn43Ac. Because the absence of a functional Spn43Ac serpin may affect proteolytic cascades involved in the host defense of Drosophila, the level of expression of the antimicrobial peptide genes were examined in nec mutants. All genes were induced 6 hours after challenge in wild-type (WT) flies; however, in nec mutants the gene encoding drosomycin is strongly expressed in the absence of immune challenge. The expression is further enhanced by immune challenge. The gene encoding the peptide metchnikowin, which has both antibacterial and antifungal activities, also exhibits constitutive expression in nec mutants, although the response is less marked than for drosomycin. In contrast, no constitutive expression is observed by genes encoding diptericin and cecropin A1, whose expression is either independent of the Toll signaling pathway or requires a signal from an additional pathway, depending on the immune deficiency (imd) gene (Levashina, 1999).

Overexpression of the Spn43Ac gene in nec flies abolishes the constitutive expression of drosomycin, whereas overexpression of a different serpin gene from the same cluster, Spn43Aa, has no effect on this phenotype. In a Tl or spz loss-of-function background, the nec-mediated constitutive expression of drosomycin is abolished, indicating that Spn43Ac acts upstream of spz and Tl. However, when the nec mutation is combined with gastrulation defective (gd) or snake (snk) loss-of-function mutations, constitutive expression of drosomycin is still observed, confirming that these proteases are not necessary for the Toll-controlled antifungal response. Furthermore, the constitutive expression of drosomycin is not affected when the nec mutation is in an imd mutant background, suggesting that the imd-mediated expression of the antibacterial peptide genes is independent of the proteolytic cascade controlled by Spn43Ac (Levashina, 1999).

The expression of the Tl gene and that of the downstream genes in the signaling cascade is up-regulated by immune challenge. The transcription of the Spn43Ac gene is up-regulated by immune challenge. This up-regulation is not observed in a Tl loss-of-function background. Conversely, Tl gain-of-function mutants exhibit a constitutive expression of Spn43Ac. In imd mutants, the up-regulation of Spn43Ac by immune challenge is similar to that in wild-type flies. Thus, Spn43Ac is an immune-responsive gene, and its expression is under the positive control of the Toll pathway. This could represent a negative feedback mechanism to shut down the activation of Toll by inhibiting the upstream proteolytic cascade (Levashina, 1999).

To function as a negative regulator of the Toll pathway upstream of Spätzle and Toll, Spn43Ac should be present in the hemolymph of adult flies. Indeed, immunoblotting with an antiserum directed against recombinant Spn43Ac reveals a band of ~60 kD in the blood of WT flies. This band is absent from the hemolymph of flies deficient for the Spn43Ac gene. The size of the mature Spn43Ac protein predicted from the cDNA sequence is smaller (52kD) than the size of the immunoreactive protein, possibly reflecting posttranslational modifications (because serpins are generally glycoproteins). After immune challenge, a band of ~50 kD is observed, which may correspond to the Spn43Ac serpin that has undergone cleavage by activated protease or proteases (Levashina, 1999).

During dorsoventral patterning of the embryo, the 382-residue Spätzle protein is cleaved to a 106-residue COOH-terminal active ligand form. Experiments on the putative proteolytic cleavage of Spätzle in the host defense have not been reported so far, and protein extracts from naive and immune-challenged flies were examined by protein immunoblotting, using two polyclonal antisera directed against recombinant COOH-terminal Spätzle. These antisera recognize the full-length Spätzle protein and a smaller COOH-terminal fragment of 16- to 18-kD. In experiments with unchallenged flies, a band corresponding to a protein of 40 to 45 kD is detected in denatured extracts. It is also present in extracts of hemolymph. One hour after immune challenge, the 40- to 45-kD band has disappeared, whereas an immune-reactive doublet of ~16- to 18-kD is apparent, which is assumed to correspond to the processed form of Spätzle protein. The Spätzle protein has glycosylation sites, which may account for slightly larger molecular sizes than predicted from the cDNA sequences. The 16- to 18-kD doublet is detected in unchallenged nec flies, together with the 40- to 45-kD protein corresponding to uncleaved Spätzle. This result is in agreement with the working hypothesis that in nec mutants the absence of the functional serpin leads to the constitutive cleavage of Spätzle. Finally, the strong signal of the 40- to 45-kD form of Spätzle together with that of the 16- to 18-kD form in nec mutants confirms at the protein level that the expression of the spz gene is regulated by a positive-feedback loop (Levashina, 1999).

These data indicate that in the absence of a functional product of the Spn43Ac serpin gene in the blood of adult flies, the Spätzle protein is spontaneously cleaved, leading to constitutive activation of the Toll signaling pathway. This phenotype can be rescued, either by a functional Spn43Ac transgene or by a spz- or Tl-deficient background. It is not known whether the protease, which cleaves Spätzle, is a direct target of the serpin (Levashina, 1999).

Conceptually, the activation of Spätzle by blood protease zymogens is similar to the coagulation cascade in the horseshoe crab, which can be activated by binding of LPS to an upstream multidomain recognition protein. Several serpins, which fall into the same class as Spn43Ac, can specifically inhibit the proteases of the coagulation cascade (Levashina, 1999 and references therein).

These results, and the parallels with the horseshoe crab coagulation cascade, imply that non-self recognition is an upstream event. Toll does not qualify as a bona fide pattern recognition receptor in Drosophila, in contrast to what has been proposed for Toll-like receptors in mammals. The actual pattern recognition receptor, which initiates the cascade leading to the cleavage of Spätzle and activation of Toll, remains to be identified. Genetic aberrations and deficiencies of mammalian serpin genes have been correlated with clinical syndromes, such as pulmonary emphysema, angioedema, and coagulopathies, as a result of inappropriate inhibition of their respective target proteases. The demonstration that a serpin functions in the regulation of the Drosophila immune response highlights the similarities between innate immunity in insects and mammals and reinforces the idea of a common ancestry of this system (Levashina, 1999 and references therein).

Neuron-type specific functions of DNT1, DNT2 and Spz at the Drosophila neuromuscular junction

Retrograde growth factors regulating synaptic plasticity at the neuromuscular junction (NMJ) in Drosophila have long been predicted but their discovery has been scarce. In vertebrates, such retrograde factors produced by the muscle include GDNF and the neurotrophins (NT: NGF, BDNF, NT3 and NT4). The NT family of proteins in Drosophila is formed of DNT1, DNT2 and Spätzle (Spz), with sequence, structural and functional conservation relative to mammalian NTs. This study investigated the functions of Drosophila NTs (DNTs) at the larval NMJ. All three DNTs are expressed in larval body wall muscles, targets for motor-neurons. Over-expression of DNTs in neurons, or the activated form of the Spz receptor, Toll, in neurons only, rescued the semi-lethality of spz and DNT1;DNT2 double mutants, indicating retrograde functions in neurons. In spz mutants, DNT1;DNT2 double mutants, and upon over-expression of the DNTs, NMJ size and bouton number increased. Boutons were morphologically abnormal. Mutations in spz and DNT1,DNT2 resulted in decreased number of active zones per bouton and decreased active zone density per terminal. Alterations in DNT function induced ghost boutons and synaptic debris. Frequency and amplitude of spontaneous events were reduced in spz² mutants suggesting defective neurotransmission. These data indicate that DNTs are produced in muscle and are required in neurons for synaptogenesis. Most likely, alterations in DNT function and synapse formation induce NMJ plasticity leading to homeostatic adjustments that increase terminal size restoring overall synaptic transmission (Sutcliffe, 2013).

These data show that the Drosophila NTs DNT1, DNT2 and Spz are produced in muscles, required at the larval NMJ synapse with neuron type specificity, that alterations in their function affect synaptic structure and, at least in the case of Spz, also physiology. The spz² allele is a mutation in the pro-domain that interferes with the secretion of Spz in cell culture. These data show that the semi-lethality of the spz² allele can be rescued with the over-expression of activated Toll^10b in neurons implying that the spz² mutation causes a reduction in normal spz function. The mechanism by which the spz² allele affects function is not currently known, and this is a question that should be solved in the future. The DNT1⁴¹ and DNT1⁵⁵ mutant alleles are null and produce no protein. DNT2^e03444 is a Piggy-Bac insertion allele that is hypomorphic. It has not been possible to generate a DNT2 null allele, thus it is conceivable that a null might have revealed more dramatic phenotypes. It was shown that DNT1, DNT2 and spz are expressed in the larval body wall muscle, and the temperature sensitive semi-lethality of the mutants was used to address the question of whether the DNTs might be functional in neurons. The data showed that over-expression of cleaved DNT1CK’+, DNT2CK and spzCK restricted to neurons can rescue the semi-lethality of the mutants. No link was hypothesized between the larval muscle expression and adult survival, and it does not necessarily exist. The data show importantly that DNT1, DNT2 and Spz can function in neurons and that these neuronal functions are essential for viability (Sutcliffe, 2013).

Reduced Spz, DNT1 and DNT2 function increased terminal size and bouton number, caused abnormal post-synaptic bouton morphology, reduced number of active zones per bouton, reduced active zone density per NMJ terminal and increased shedding of synaptic material. The deficit in active zones in DNT1 DNT2 double mutants could be rescued by the over-expression of either DNT1CK3’+ or DNT2CK in neurons, demonstrating that this phenotype was the direct result of loss of DNT1 and DNT2 function. The experiments did not reveal rescue of the spz² phenotype, which could be due to technical reasons or to the nature of spz² allele (Sutcliffe, 2013).

To ease the analysis, an automatic method was developed to quantify anti-Brp (nc82) staining at the NMJ that was named DeadEasy Synapse. Data was acquired both using conventional manual counting and DeadEasy Synapse. Both methods revealed the same results, thus validating DeadEasy Synapse, which will be of great use to the Drosophila NMJ community. The plug-in works with ImageJ and will be made publicly available through the lab webpage (Sutcliffe, 2013).

Aberrant Spz function in the spz² allele resulted in reduced frequency and amplitude of spontaneous mEJPs. However, evoked EJPs were normal for all mutants examined (Sutcliffe, 2013).

Altogether, the data strongly suggest that DNT1, DNT2 and Spz are required for synaptogenesis. They also suggest that the increase in NMJ terminal size and bouton number in the mutants corresponds to a homeostatic structural adjustment that compensates for the reduced number of active zones, thus restoring the overall number of functional release sites and synaptic transmission. Remarkably, Spz functions at the muscle 4 NMJ, whereas the combined functions of DNT1 and DNT2 are required for the muscle 6,7 NMJ (Sutcliffe, 2013).

It has long been known that homeostatic compensatory mechanisms adjust the NMJ terminal to muscle size as the larva grows, to maintain synaptic efficacy within an appropriate physiological range. Increased synaptic growth is accompanied by a decrease in transmitter release per bouton resulting in normal muscle excitation. Conversely, mutants with fewer boutons have normal physiology, as each bouton has more active zones maintaining a constant overall number per NMJ. An analogous scenario is seen in mammals: in NT4 knockout mice, a reduction in post-synaptic AChR density induces a compensatory increase in NMJ terminal area. In Drosophila, it has long been anticipated that retrograde factors produced in the muscle regulate pre-synaptic neurotransmitter release, perhaps by regulating the number of presynaptic active zones in each bouton or some aspect of the presynaptic release mechanism, but their discovery has been scarce. One retrograde growth factor at the Drosophila NMJ is Gbb (Sutcliffe, 2013).

The data are consistent with the DNTs functioning as retrograde growth factors. Firstly, they are expressed at the muscle, and muscle over-expression of spz and DNT2 can rescue the semi-lethality of spz² mutants. Secondly, activated Toll rescues the semi-lethality of spz² mutants when expressed in neurons but not in muscle. Thirdly, DNT2 transcripts are localised at the boutons and over-expression of DNT2CK in neurons rescues the semi-lethality of DNT1⁴¹, DNT2^e0344 double mutants. However, further evidence that over-expressed cleaved spzCK, DNT1CK3’+ and DNT2CK using the GAL4 system can be secreted and taken up normally by receiving cells would be desirable. There is robust evidence that spzCK, DNT1CK3’+ and DNT2CK are functional cell-autonomously in the cells in which they are over-expressed using the GAL4/UAS system. However, inappropriate secretion upon over-expression of the cleaved forms could explain why over-expression from muscle is not as effective as over-expression from neurons at rescuing the semi-lethality of the mutants (Sutcliffe, 2013).

Autocrine and anterograde functions of the DNTs in neurons and/or muscle are also likely. Toll is expressed in muscle where it functions as an inhibitor of synaptogenesis. Targeting by motoraxons coincides with a downregulation of Toll at the target muscle, and over-expression of Toll in muscle reduces bouton number at the muscle 6,7 NMJ. The DNTs may also have bidirectional functions. DNT2 transcripts are localised post-synaptically, reminiscent of the post-synaptic expression of BDNF and NT4. Their transcripts are translated in response to neuronal activity and the combination of anterograde and retrograde functions results in synaptic potentiation. DNTs, perhaps particularly DNT2, may also have bidirectional functions at the synapse. The full characterisation of retrograde and/or anterograde functions of the DNTs must await the discovery of the receptors functioning at the NMJ, and the production of good antibodies that enable visualisation of the endogenous ligands (Sutcliffe, 2013).

The data indicate that the DNTs are required for synaptogenesis. DNT loss of function mutants display increased bouton number and increased terminal size but reduced number of active zones per bouton and normal EJPs. Most likely, the increase in terminal size and bouton number is a homeostatic compensation for the deficient formation and function of active zones. In DNT1⁴¹, DNT2^e0344 mutants, muscles are smaller compared to wild-type and active zone density is reduced, but the NMJ is larger. This suggests that since in DNT mutants boutons have fewer synapses, the NMJ expands making more boutons, thus compensating for the synaptic deficits and maintaining overall normal function. Over-expression of DNTs in neurons increases muscle size, ghost boutons and shed synaptic debris, phenotypes that are consistent with a function of the DNTs in promoting axonal and muscle growth and/or in synaptic transmission. On the other hand, the observation that loss and gain of DNT function results in increased production of synaptic debris and ghost boutons, could also reflect defective clearance of synaptic material (e.g. by muscle or glia) upon interference with DNT function (Sutcliffe, 2013).

In spz² mutants in physiological calcium levels no changes in synaptic transmission were observed; lowering the extracellular calcium concentration reveals reduced frequency and amplitude of mEJPs, correlating with a reduction in the number of active zones per bouton. Despite the aberrant frequency and amplitude of spontaneous mEJPs in spz² mutants in low calcium, evoked EJPs were normal under all conditions for all mutants examined and loss of DNTs did not affect synaptic transmission (Sutcliffe, 2013).

These phenotypes are reminiscent of those found upon manipulation of another invertebrate neurotrophin superfamily member in mollusks, Aplysia neurotrophin (ApNT). Alterations in ApNT levels influence synaptic structure and the formation of synaptic varicosities. Expression of a dominant negative form of the receptor Ap-Trk-DN in cultured neurons had no effect in synaptic transmission following a single pulse of 5-HT, and effects were only seen in Long Term Facilitation after 5 consecutive pulses. Similarly, expression of ApNT or bathing cells in ApNT also led to more pronounced increases in evoked potential in Long Term Facilitation. Conceivably, high frequency stimulation might reveal enhanced effects in synaptic transmission upon manipulation of DNT function, and this is something worth testing in the future (Sutcliffe, 2013).

To conclude, in the case of Drosophila, it is most likely that the increase in NMJ terminal size and bouton number in the DNT mutants corresponds to a homeostatic structural adjustment that compensates for the reduced number of active zones, restoring the overall number of functional release sites and subsequent synaptic transmission (Sutcliffe, 2013).

The DNTs are the first growth factors to be identified to have neuron-type specificity at the Drosophila NMJ: the spz² mutation affected the muscle 4 NMJ, and DNT1⁴¹, DNT2^e0344 double mutants affected the muscle 6,7 NMJ. This observed neuron-type specificity is reminiscent of the neuronal modality of mammalian NT and Trk receptor function in the central and peripheral nervous systems. However, this study analysed the muscles 4 and 6,7 NMJs, not all muscles in the larva, thus the possibility cannot be ruled out that the DNTs may have more redundant and less specific functions in other muscles. How the observed specificity comes about is not yet understood, since the current evidence indicates that the three ligands are expressed throughout the muscles. In future work, antibodies to the DNTs may provide higher resolution and reveal distinct distribution patterns. For now, the distribution of DNT2 transcripts in synaptic boutons is a significant difference. In any case, the data also show that there is some functional redundancy between Spz, DNT1 and DNT2. DNTs may be promiscuous ligands that in some circumstances (e.g. upon over-expression) can bind multiple receptors, and thus neurons may be able to respond to the excess of any of the DNTs. This is reminiscent of the redundancy between vertebrate NT ligands, and the fact that different NTs can bind the same Trk receptor. In the normal larva, the distinct NMJ-specific effects may reflect the distribution of the DNT receptors in distinct neuronal types, and testing this hypothesis will await the identification of the receptors for DNT1 and DNT2. In any case, the NMJ specificity precisely reflects the neuron type specificity for the DNTs that also takes place during motoraxon targeting in the embryo: DNT1 is required for targeting of ISNb/d and Spz for targeting of SNa motoraxons. Intriguingly, since specific DNTs appear to be required for both motor-axon targeting and synapse formation at the NMJ, this would suggest that such neuron-type specific functions serve to shape motor-neuron-muscle connectivity required for the organisation of locomotor behaviour. Future progress will tackle the identification of the DNT receptors, the basis of this neuron-type specificity and the relevance for behaviour (Sutcliffe, 2013).

Spz/Toll-6 signal guides organotropic metastasis in Drosophila

Targeted cell migration plays important roles in developmental biology and disease processes including metastasis. Drosophila tumors exhibit traits characteristic of human cancers, providing a powerful model to study developmental and cancer biology. This study finds that cells derived from Drosophila eye disc tumors also display organ specific metastasis to invade receptive organs but not wing disc. Toll receptors are known to affect innate immunity and tumor inflammatory microenvironment by modulating the NF-kappaB pathway. RNAi screen and genetic analyses show that Toll-6 is required for migration and invasion of the tumor cells. Further, receptive organs express Toll-ligands, Spz family molecules, and ectopic Spz expression renders wing disc receptive to metastasis. Finally, Toll-6 promotes metastasis by activating JNK signaling, a key regulator of cell migration. Hence, this study reports Toll-6 and Spatzle as a new pair of guidance molecules mediating organ-specific metastasis behavior and highlight a novel signaling mechanism for Toll family receptors (Mishra-Gorur, 2019).

Targeted cell migration and invasion play important roles in a variety of biological and disease processes. This phenomenon is exemplified by organotropic metastasis in cancer progression. Tumor metastasis is the leading cause of death for cancer patients. A fundamental feature of metastasis is the ability of distinctive tumor types to colonize different organ sites, which depends on the inherent properties of the tumor cells and their interaction with the host tissue. However, the mechanism of organotropic metastasis is not well delineated. Deciphering the underlying molecular mechanism(s) of interactions between tumor cells and secondary host tissue is essential for understanding metastasis (Mishra-Gorur, 2019).

Almost a century ago, two theories were stated to explain organotropic tumor metastasis. The 'anatomical mechanical theory' has bee proposed to account for cancer metastasis. It was theorized that the patterns of blood flow from the primary tumor can predict the first metastasized organs. On the other hand, the 'seed and soil' theory hypothesized that tumor cells migrate to tissues that support their growth. In other words, it was suggested that the site of metastasis depended on the affinity of the tumor for the microenvironment. A modern version of the seed and soil theory focuses on a 'homing mechanism', which suggests that tumor cells are drawn to specific organ sites because of complex signaling crosstalk between the tumor cells and the cells of the organ (Fidler, 2002). Consistent with this idea, recent studies in mammalian systems indicate that chemokines and their receptors are involved in organ-specific tumor metastasis (Mishra-Gorur, 2019).

The strength of forward genetic screens and mosaic analysis in Drosophila has empowered investigators to utilize this model organism for identifying genes of cancer relevance, defining cancer signaling pathways and deciphering cancer biology. Previously a genetic screen was performed and a Drosophila model was developed for tumor metastasis (Pagliarini, 2003). Somatic cells expressing oncogenic Ras protein (RasV12) and simultaneously carrying a loss-of-function mutation in any of the cell polarity genes, including scribbled (scrib), lethal giant larvae (lgl) or discs large (dlg), develop into malignant tumors (known as RasV12/cell-polarity-defect tumors). This fly tumor model displays the main hallmarks of human metastatic cancers, including uncontrolled growth, basement membrane (BM) degradation, loss of E-cadherin, migration, invasion, and secondary-tumor formation at distant organs. Further studies have revealed that JNK (c-Jun N-terminal kinase) signaling is activated in the tumor cells and is required for tumor-cell migration and invasion. It was also learned that JNK signaling can be activated non-autonomously or under stress conditions and can propagate to collaborate with RasV12-expressing cells to induce tumorigenesis (Mishra-Gorur, 2019).

This study consisted of a pathological and chronological examination of tumor progression and invasion in the fly tumor model. Tumor cells were found to metastasize to distal organs in a tissue-specific pattern. To identify genes responsible for organotropic metastasis, fly tumor cell lines were successfully established from the imaginal disc of a single larva, and a genome-wide RNA interference (RNAi) screen was performed. Toll-6, a Toll-receptor family member, was identified as a crucial gene for tumor cell migration. It was also shown that Toll-6 is required in tumor cells for organ-specific metastasis in vivo by inducing JNK signaling activation. Finally, the expression of the Spötzle (Spz) ligands in targeted organs serves as a cue for the guided migration and invasion. The Spz/Toll-6 system provides a novel molecular mechanism for organotropic metastasis (Mishra-Gorur, 2019).

Metastasis is the leading cause of mortality in cancer patients. Befittingly, both the words 'cancer' (Latin: 'crab') and 'metastasis' (Greek: 'displacement') refer to cell movement: the crab-like invasion of cancer into healthy tissue and the migration of cancer cells to secondary sites. Since Paget's initial observation more than 100 years ago, pathologists have recognized that the movement of cancer cells is not random and that different types of cancer have different destinations or organ-specific metastasis. For example, colon carcinomas usually metastasize to liver and lung but rarely to bone, skin or brain and almost never to kidneys, intestine or muscle. In contrast, other tumors, such as breast carcinomas, frequently form metastases in most of these organs, while prostate cancer metastasis occurs most prominently in bone. A similar phenomenon occurs in the spread of malignant tumors in Drosophila. The RasV12/cell-polarity-defect tumors exhibit organ-specific metastasis. Tumor cells originating from the eye-antennal imaginal disc in the larvae metastasize to almost all organs (mouth hook, VNC, SG, leg discs, haltere disc, gut, FB) except the wing disc (Mishra-Gorur, 2019).

In mouse cancer models and human patients, it is known that tumor cells are disseminated through vascular networks such as the blood and lymphatic vessels. The tracheal system in Drosophila is a tubular network for supplying oxygen, functioning as the equivalent of human lungs and blood vessels. Interestingly, in flies, it was also observed that tumor cells from primary tumors can metastasize to the trachea, suggesting that the trachea might function as an essential media to facilitate tumor metastasis. Consistent with this hypothesis, a recent study by Ross Cagan and Benjamin Levine showed that trachea-derived tumor cells can migrate significant distances (Levine, 2016). Additionally, RasV12/cell-polarity-defect tumors have been shown to express tracheal markers and migrate along the trachea (Grifoni, 2015). It will be interesting to further study the role of the trachea in organotropic metastasis, and possibly investigate whether a similar conserved mechanism exists in mammalian tumor cell migration (Mishra-Gorur, 2019).

Targeted cell migration plays a key role in normal development. Studies of neural development have identified multiple receptors and their ligands that regulate guided neuronal migration (e.g. Robo/Slit, Trk receptors/neurotrophins). For organotropic metastasis, the 'seed and soil' theory states that metastases develop only when the 'seed' (tumor cells) and the 'soil' (target organs) are compatible. It is only recently that studies have begun to reveal some of the identities of the 'seed and soil' molecules that regulate the spread of tumor cells. Experiments with breast cancer cell lines showed that inhibition of the chemokine receptor CXCR4 by a neutralizing antibody abrogates their metastasis to the lung, which expresses the corresponding chemokine ligand, CXCL12. However, the CXCR4/CXCL12 interaction is unlikely involved in the metastasis of breast cancers to the liver The diverse types of cancers and their complex metastatic patterns indicate that other 'seed and soil' molecules must be involved in organ-specific metastasis (Mishra-Gorur, 2019).

This study reports that Toll-6, a Toll-family neurotrophin receptor, plays an essential role in tumor cell metastasis in Drosophila. Toll-6 is expressed in the RasV12/cell-polarity-defect tumors, and downregulation of Toll-6 in the tumor cells blocks their migration and invasion. Interestingly, this study also showed that Toll-6-expressing tumor cells migrate towards and invade the organs that express Spz or Spz-related molecules. Furthermore, the Toll-6-expressing tumor cells do not metastasize to the wing disc, an organ with no detectable Spz or Spz-related gene expression in this system, strongly arguing that Spz or a Spz-related molecule could be serving as a cue for guiding Toll-6-expressing tumor cells. Interestingly, a recent study shows that the wing disc produces extremely low levels of Spz in a tightly regulated spatially restricted pattern with no resultant signaling events. Indeed, artificial overexpression of SpzACT in the wing disc converts it to a tissue receptive for the migration and invasion by Toll-6-expressing tumor cells. Together, these data indicate that Spz/Toll-6 serve as the 'seed and soil' molecules for organotropic metastasis in the fly. In support of this conclusion, clinical studies have revealed a correlation between increased expression levels of Toll-like receptors (TLRs) and malignancy of multiple cancers. TLRs have also been shown to affect malignancy by altering the tumor inflammatory microenvironment. Given that the Toll-family receptors are evolutionarily conserved, consisting of nine members in Drosophila and ten TLRs in mammals, this study raises the possibility that mammalian TLRs could play a similar role in mediating organotropic metastasis and cell migration (Mishra-Gorur, 2019).

In Drosophila, Toll regulates dorsoventral patterning in embryos and anti-fungal defense in adults. Toll has also been found to have an inhibitory role in the formation of neuromuscular junctions and, recently, 18 wheeler, a Toll-like receptor protein, was found to play a role in border cell migration. Studies in both flies and mammals show that Toll-family receptors mediate NF-κB signaling activation. A study using biochemical inhibitors suggests that Toll molecules could affect random migration of neutrophils by activating ERKs. This study shows that Toll-6 activates JNK signaling, which is in accordance with the recent study by Foldi (2017) that shows the role of Toll-6 and JNK in cell death. However, JNK signaling is also a key regulator for cell migration during development, and previous work has shown that JNK signaling is activated in RasV12/cell-polarity-defect tumors and is essential for metastasis (Igaki, 2006). This study reports that, first, Toll-6 knockdown in RasV12/cell-polarity-defect tumors completely blocks metastasis by effectively reducing JNK signaling; second, the role of JNK activation by Toll-6 is highlighted in the in vitro assay, as Toll-6 knockdown reduced migration of the tumor cells even when discs were not placed, suggesting that JNK activation by Toll-6 might be important for general cell migratory behavior. Finally, ectopic expression of Toll-6ACT in the wing disc results in JNK activation. These data indicate that Toll-6 regulates metastasis by activating JNK signaling. Recently, Grindelwald (Grnd) has been identified as a novel tumor necrosis factor receptor (TNFR) mediating RasV12/scrib^-/--induced tumor growth and invasion (Andersen, 2015). The current data suggest that Toll-6 could be providing a second signal for the activation of JNK. Indeed, epistasis data support the model that Toll-6 genetically interacts with the JNK pathway. It is possible that inputs from both Toll-6 and Grnd signals could result in or are required for a high level of JNK activation (Mishra-Gorur, 2019).

In addition to Spz, there are five Spz-related genes in the Drosophila genome, which could serve as ligands for Toll-6. Toll-6 has been recently reported to function as a neurotrophin receptor in regulating motor neuron targeting and survival, and it also physically binds to Spz5. Consistently this study found that co-expression of Toll-6 and Spz5 synergistically promote collective cell invasion in the developing wing, phenocopying Toll-6 and SpzACT co-expression. Interestingly, although both SpzACT and Spz5 can activate Toll-6-mediated JNK signaling, it was found that, unlike Spz5, expression of SpzACT alone is not sufficient to activate JNK. Given the current results, it is inferred that Spz and Spz5 might display some redundancy. Indeed, a previous study by has demonstrated the redundancy of Spz2 (DNT1) and Spz5 (DNT2) in binding to Toll-6. Furthermore, a redundancy between Spz, Spz2 and Spz5 has also been demonstrated, suggesting that Spz proteins may function as promiscuous ligands in some circumstances (e.g., upon overexpression) and can bind multiple Toll receptors. Under overexpression conditions, Spz is thought to be capable of activating Toll-6 (albeit at lower levels than is Spz5). Therefore, this study finds that different Spz proteins might act redundantly to induce Toll-6-mediated JNK activation and cell migration. In conclusion, these genetic and biochemical data show that Toll-6 and Spz compose a new pair of guidance molecules for directing cell migration and that their interaction mediates organotropic metastasis by activating JNK signaling (Mishra-Gorur, 2019).

Dynamics of Spaetzle morphogen shuttling in the Drosophila embryo shapes gastrulation patterning

Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible extracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, was followed in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dorsal into the nuclei at the final division cycle plays an instructive role, as the residence time of Dorsal in each nucleus is translated to the amount of zygotic transcript that will be produced, thereby guiding graded accumulation of specific zygotic transcripts that drive patterned gastrulation. It is concluded that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos (Rahimi, 2019).

The early Drosophila embryo provides extreme challenges for the generation and maintenance of extracellular morphogen gradients. Most notably, the peri-vitelline fluid surrounding the embryo facilitates rapid diffusion of molecules. In addition, the alteration in the surface of the plasma membrane at every nuclear division provides an active mixing force. Thus, analysis of the early morphogen gradients operating in this environment, including ventral Spz/Toll activation and the subsequent BMP gradient patterning the dorsal aspect, should consider this highly dynamic environment. In the case of the Toll pathway, the active Spz ligand is generated by proteolytic processing within the extra-embryonic peri-vitelline fluid in a broad ventral region, defined by the activation domain of the Easter (Ea) protease. The generation of a sharp Spz activation gradient within this broad ventral domain of processing takes place by diffusion-based shuttling. Previous work demonstrated that the pro-domain of Spz plays an instructive role in delivering the active, cleaved ligand towards the ventral midline. While a variety of experiments and computational analyses indicated the utilization of a 'self-organized shuttling' mechanism in this context, it was imperative to visualize the actual dynamics of the process (Rahimi, 2019).

It was possible to infer the dynamics of the extracellular Spz gradient by following the kinetics of Dl-GFP nuclear accumulation in individual live embryos during the final syncytial nuclear division cycles and the early phase of NC 14. Nuclear levels of Dl are not a direct readout of the extracellular gradient, since accumulation of Dl in the nuclei is re-initiated at the onset of every nuclear cycle. Nevertheless, it is possible to infer key features of the extracellular Spz gradient from this dynamic behavior. Using this approach hallmarks of ligand shuttling were identified, most notably the lateral overshoot and the presence of two lateral peaks which converge to a central ventral peak. This convergence takes place within a timeframe of minutes, and repeats at every nuclear cycle. Since new protein molecules of the extracellular components are continually translated, the ongoing activity of the shuttling process is vital. Therefore, shuttling is important not only for generating the gradient, but also for maintaining it, in the face of rapid diffusion and mixing within the peri-vitelline fluid. Importantly, by ~10-15 minutes into NC 14, when the robust induction of transcription of the cardinal zygotic Dl-target genes twt and sna ensues, the nuclear gradient of Dl is sharp and a single ventral peak is resolved (Rahimi, 2019).

Having described the dynamics of Dl-nuclear entry and gradient formation, an experimental approach was used in order to examine regulatory processes affecting Toll signaling. The Wnt family ligand WntD provides an essential buffering system to variations in Toll signaling between embryos. wntD is an early zygotic gene that is expressed initially at the posterior-ventral region of the embryo, and its expression levels depend on the magnitude of Toll signaling. Although WntD is produced locally, the rapid secretion and diffusion of the protein in the peri-vitelline space generates a uniform attenuation of Toll signaling throughout the embryo surface. The activity of WntD leads in different embryos to convergence of the variable global Toll activation gradient to a similar pattern, which is dictated by the fixed final signaling level that shuts off wntD expression. This paradigm, termed 'distal pinning', is achieved in this case by an induction-contraction mechanism (Rahimi, 2019).

Secreted WntD is recruited to the plasma membrane by binding to its receptor Fz4. Epistasis assays have indicated that WntD exerts its inhibitory effect on Toll signaling by associating with the extracellular domain of Toll, thereby reducing the number of Toll receptors that are available for binding Spz. Bearing the cardinal features of shuttling in mind, this mode of inhibition implies that the effect of WntD would be global and non-autonomous, and will actually change the shape of the gradient, making it sharper. The observed dynamics of Dl-GPF in wntD mutant embryos indeed confirms this prediction. The shuttling process is driven by competition between the inhibitory Spz pro-domain and the Toll receptor for binding free, active Spz. Binding to the pro-domain is favored in the lateral part of the embryo, where its concentration is higher, while in more ventral regions binding to Toll takes over. Since WntD impinges on the extracellular properties of the Toll receptor, the active ligand is deposited in more ventral regions, where the concentration of the pro-domain is lower. Thus, WntD does not simply reduce the overall profile of Toll activation, but actually re-directs the ligand from the lateral regions to the ventral domain. Previous work has shown that accumulation of excess ligand in the peak by shuttling is an effective mechanism to buffer noise. Since activation in this region is already maximal, the excess ligand will not alter the resulting cell fates (Rahimi, 2019).

The rapid timing of processes in the early embryo and the short duration of interphases between nuclear divisions raises the question of whether it is actually possible to produce sufficient levels of WntD that will drive the morphogen profile to the desired equilibrium. When monitoring wntD transcription directly utilizing the MS2 system, most, if not all embryos were shown to express wntD indicating that Toll signaling overshoots in most embryos. Furthermore, within single embryos the number of nuclei expressing wntD was reduced between NCs 12 and 13, and completely terminated by NC 14, implying that WntD impinges on the Toll gradient and its own expression by this time. The intronless arrangement of the wntD gene and the rapid secretion of the protein, which does not require post-translational modifications (Herr et al., 2012), may facilitate the process (Rahimi, 2019).

The ventral cohort of zygotic target genes including twi and sna is induced by the Toll activation gradient, and the threshold for their induction corresponds to ~50% of maximal Dl-nuclear localization. Within the ventral domain, nuclei exhibit a similar level of sna transcription. These genes are triggered at NC 14 after the Dl gradient is stabilized and a distinct activation peak generated (Rahimi, 2019).

Are there zygotic target genes that respond to the dynamics of Dl nuclear targeting, before it stabilizes? This appears to be the case for T48, which encodes a transmembrane protein that facilitates recruitment of RhoGEF2 and ultimately Rho and actomyosin, to mediate apical constriction of invaginating mesodermal cells. Graded distribution of myosin II was shown to be critical for proper invagination of these cells to form the ventral furrow (Rahimi, 2019).

This study provides evidence that the graded distribution of T48 mRNA results from the dynamics of Dl-nuclear re-entry at NC 14. The ventral-most cells reach the threshold of T48 induction earlier than more lateral cells, and hence will express the gene longer. Integration of the length of expression along the D-V axis then leads to a gradient of cytoplasmic T48 mRNA accumulation. This example represents a unique case, where graded morphogen activation instructs the generation of a gradient of target-gene expression. The strict dependence on the timing of transcription initiation provides another mechanism to generate differences between adjacent nuclei along the D-V axis (Rahimi, 2019).

In conclusion, this work has utilized live imaging of Toll pathway activation, to identify and characterize the hallmarks of ligand shuttling. This process is rapid and takes place continuously throughout the final nuclear division cycles, to generate and maintain a sharpactivation gradient in the diffusible environment of the peri-vitelline fluid. WntD impinges on Spz shuttling, and is responsible not only for buffering variability between embryos, but also for generating a sharp activation peak. This peak is utilized to induce a graded expression of a zygotic target gene that is essential for executing processes that drive gastrulation. Thus, diffusion-based ligand shuttling, coupled with a dynamic readout, establishes a refined pattern within the environment of early embryos (Rahimi, 2019).

GENE STRUCTURE

Toll, the receptor for Spätzle and the spätzle gene are tightly linked, both found within 0.4cm of one another (Morisato, 1994).

Bases in 5' UTR - 864

Bases in 3' UTR - 298

PROTEIN STRUCTURE

Two transcripts are found, one of 2.1 kb and a second of 2.3 kb. The 2.3 kb transcript codes for an extra 73 amino acids inserted into the N-terminal region of the protein (Morisato, 1994).

Amino Acids - 253

Structural Domains

The spätzle gene encodes a novel secreted protein that appears to require activation by a proteolytic processing reaction, controlled by genes acting upstream of spätzle in the genetic pathway. A deletion mutant that retains only the C-terminal 106 amino acids of spz can activate Toll in the absence of the genes normally required for SPZ activity. This domain includes 7 of the 9 cysteine residues of SPZ. The activity of the C-terminal domain is inhibited in the full-length precursor. Because the deletion that retains the C-terminal 168 amino acids rescues the spz mutant phenotype with normal polarity, and requires easter for activity, it is inferred that this truncated protein retains the inhibitory activity (Morisato, 1994).

Biochemical interactions underlying the generation of the ventralising signal during Drosophila embryogenesis were investigated by the expression of recombinant Easter and Spatzle proteins. An active form of Easter protease cleaves the Spatzle protein, generating a carboxyterminal polypeptide fragment which, when microinjected into the perivitelline space of a spatzle deficient embryo, directs production of ventrolateral pattern elements. This Spatzle carboxyterminal fragment is a disulfide-linked dimer. Modelling suggests that the core disulfide bonds and dimer arrangement of this fragment are highly similar to vertebrate nerve growth factor. Each polypeptide contains three intrachain disulfide bridges in a 1-3, 2-6 and 3-7 arrangement. Cysteine number five is assigned to a single interchain disulfide bridge resulting in the 24-kD dimer. The disulfide involved in dimerization contributes significantly but is not absolutely required for the activity of this protein. It is concluded that the 24-kD Spatzle carboxyterminal dimer must adopt a parallel (head-to-head) structure that is similar to NGF. Thus Spatzle may be considered a member of a new family of neurotrophin-like signaling molecules in invertebrate development (DeLotto, 1998).

EVOLUTIONARY HOMOLOGS

The ligand for the Toll receptor is thought to be Spätzle, a secreted protein that is activated by proteolytic cleavage. trunk, a gene required for activity of the Torso receptor, encodes a protein that resembles SPZ in several respects. In particular, the sequence suggests that TRK is a secreted protein and that it contains an internal site for proteolytic cleavage. Furthermore, the carboxy-terminal domain of TRK has a similar arrangement of cysteines to that of SPZ. It has been proposed that trk encodes an extracellular ligand involved in specifying terminal body pattern and suggested by analogy with SPZ that a cleaved form of TRK constitutes the ligand for the Torso receptor (Casanova, 1995).

The Spatzle/Toll signaling pathway controls ventral axis formation in Drosophila by generating a gradient of nuclear Dorsal protein. Dorsal controls the downstream regulators dpp and sog, whose patterning functions are conserved between insects and vertebrates. Although there is no experimental evidence that upstream events are conserved as well, the following question was posed: can a vertebrate embryo respond to maternal components of the fly Dorsal pathway? A dorsalizing activity is demonstrated for the heterologous Easter, Spatzle and Toll proteins in UV-ventralized Xenopus embryos; dorsalization is inhibited by a co-injected dominant Cactus variant. Thus the epistatic relationships between upstream and downstream components of the Drosophila dorsoventral (d/v) pathway are maintained in the frog, as is evident from the inhibtion of Spz and Easter activity by the dominant Cactus mutation. It is concluded that the Dorsal signaling pathway is a component of the conserved d/v patterning system in bilateria (Armstrong, 1998).

date revised: 2 January 2023

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