Gene name - spätzle
Cytological map position - 97E2-4
Function - ligand for Toll
Symbol - spz
Genetic map position - 3-92
Classification - novel
Cellular location - perivitelline fluid
|Recent literature||Yamamoto-Hino, M., Muraoka, M., Kondo, S., Ueda, R., Okano, H. and Goto, S. (2015). Dynamic regulation of innate immune responses in Drosophila by Senju-mediated glycosylation. Proc Natl Acad Sci U S A. PubMed ID: 25901322
The innate immune system is the first line of defense encountered by invading pathogens. Delayed and/or inadequate innate immune responses can result in failure to combat pathogens, whereas excessive and/or inappropriate responses cause runaway inflammation. Therefore, immune responses are tightly regulated from initiation to resolution and are repressed during the steady state. It is well known that glycans presented on pathogens play important roles in pathogen recognition and the interactions between host molecules and microbes; however, the function of glycans of host organisms in innate immune responses is less well known. This study shows that innate immune quiescence and strength of the immune response are controlled by host glycosylation involving a novel UDP-galactose transporter called Senju (CG14040). In senju mutants, reduced expression of galactose-containing glycans resulted in hyperactivation of the Toll signaling pathway in the absence of immune challenges. Genetic epistasis and biochemical analyses revealed that Senju regulates the Toll signaling pathway at a step that converts Toll ligand Spatzle to its active form. Interestingly, Toll activation in immune-challenged wild type (WT) flies reduced the expression of galactose-containing glycans. Suppression of the degalactosylation by senju overexpression resulted in reduced induction of Toll-dependent expression of an antimicrobial peptide, Drosomycin, and increased susceptibility to infection with Gram-positive bacteria. These data suggest that Senju-mediated galactosylation suppresses undesirable Toll signaling activation during the steady state; however, Toll activation in response to infection leads to degalactosylation, which raises the immune response to an adequate level and contributes to the prompt elimination of pathogens.
Wu, C., Chen, C., Dai, J., Zhang, F., Chen, Y., Li, W., Pastor-Pareja, J.C. and Xue, L. (2015). Toll pathway modulates TNF-induced JNK-dependent cell death in Drosophila. Open Biol 5. PubMed ID: 26202785
Signalling networks that control the life or death of a cell are of central interest in modern biology. While the defined roles of the c-Jun N-terminal kinase (JNK) pathway in regulating cell death have been well-established, additional factors that modulate JNK-mediated cell death have yet to be fully elucidated. To identify novel regulators of JNK-dependent cell death, this study performed a dominant-modifier screen in Drosophila and found that the Toll pathway participates in JNK-mediated cell death. Loss of Toll signalling suppresses ectopically and physiologically activated JNK signalling-induced cell death. Epistasis analysis suggests that the Toll pathway acts as a downstream modulator for JNK-dependent cell death. In addition, gain of JNK signalling results in Toll pathway activation, revealed by stimulated transcription of Drosomycin (Drs) and increased cytoplasm-to-nucleus translocation of Dorsal. Furthermore, the Spätzle (Spz) family ligands for the Toll receptor are transcriptionally upregulated by activated JNK signalling in a non-cell-autonomous manner, providing a molecular mechanism for JNK-induced Toll pathway activation. Finally, gain of Toll signalling exacerbates JNK-mediated cell death and promotes cell death independent of caspases. Thus, this study identifies another important function for the evolutionarily conserved Toll pathway, in addition to its well-studied roles in embryonic dorso-ventral patterning and innate immunity.
|Capilla, A., Karachentsev, D., Patterson, R. A., Hermann, A., Juarez, M. T. and McGinnis, W. (2017). Toll pathway is required for wound-induced expression of barrier repair genes in the Drosophila epidermis. Proc Natl Acad Sci U S A. PubMed ID: 28289197
The epidermis serves as a protective barrier in animals. After epidermal injury, barrier repair requires activation of many wound response genes in epidermal cells surrounding wound sites. Two such genes in Drosophila encode the enzymes dopa decarboxylase (Ddc) and tyrosine hydroxylase (ple). This paper explores the involvement of the Toll/NF-kappaB pathway in the localized activation of wound repair genes around epidermal breaks. Robust activation of wound-induced transcription from ple and Ddc requires Toll pathway components ranging from the extracellular ligand Spatzle to the Dif transcription factor. Epistasis experiments indicate a requirement for Spatzle ligand downstream of hydrogen peroxide and protease function, both of which are known activators of wound-induced transcription. The localized activation of Toll a few cell diameters from wound edges is reminiscent of local activation of Toll in early embryonic ventral hypoderm, consistent with the hypothesis that the dorsal-ventral patterning function of Toll arose from the evolutionary cooption of a morphogen-responsive function in wound repair. Furthermore, the combinatorial activity of Toll and other signaling pathways in activating epidermal barrier repair genes can help explain why developmental activation of the Toll, ERK, or JNK pathways alone fail to activate wound repair loci.
|Alpar, L., Bergantinos, C. and Johnston, L. A. (2018). Spatially restricted regulation of Spatzle/Toll signaling during cell competition. Dev Cell. PubMed ID: 30146479
Cell competition employs comparisons of fitness to selectively eliminate cells sensed as less healthy. In Drosophila, apoptotic elimination of the weaker "loser" cells from growing wing discs is induced by a signaling module consisting of the Toll ligand Spatzle (Spz), several Toll-related receptors, and NF-kappaB factors. How this module is activated and restricted to competing disc cells is unknown. This study used Myc-induced cell competition to demonstrate that loser cell elimination requires local wing disc synthesis of Spz. Spz processing enzyme (SPE) and modular serine protease (ModSP) are identified as activators of Spz-regulated competitive signaling, and "winner" cells are shown to trigger elimination of nearby WT cells by boosting SPE production. Moreover, Spz requires both Toll and Toll-8 to induce apoptosis of wing disc cells. Thus, during cell competition, Spz-mediated signaling is strictly confined to the imaginal disc, allowing errors in tissue fitness to be corrected without compromising organismal physiology.
|Chowdhury, M., Li, C. F., He, Z., Lu, Y., Liu, X. S., Wang, Y. F., Ip, Y. T., Strand, M. R. and Yu, X. Q. (2019). Toll family members bind multiple Spatzle proteins and activate antimicrobial peptide gene expression in Drosophila. J Biol Chem. PubMed ID: 31088910
The Toll signaling pathway in Drosophila melanogaster regulates several immune-related functions, including the expression of antimicrobial peptide (AMP) genes. The canonical Toll receptor (Toll-1) is activated by the cytokine Spatzle (Spz-1), but Drosophila encodes eight other Toll genes and five other Spz genes whose interactions with one another and associated functions are less well understood. In vitro assays were conducted in the Drosophila S2 cell line with the Toll/interleukin-1 receptor (TIR) homology domains of each Toll family member to determine if they can activate a known target of Toll-1, the promoter of the antifungal peptide gene drosomycin. All TIR family members activated the drosomycin promoter, with Toll-1 and Toll-7 TIRs producing the highest activation. The Toll-1 and Toll-7 ectodomains bind Spz-1, -2, and -5 and also vesicular stomatitis virus (VSV) virions; Spz-1, -2, -5, and VSV all activated the promoters of drosomycin and several other AMP genes in S2 cells expressing full-length Toll-1 or Toll-7. In vivo experiments indicated that Toll-1 and Toll-7 mutants could be systemically infected with two bacterial species (Enterococcus faecalis and Pseudomonas aeruginosa), the opportunistic fungal pathogen Candida albicans and VSV with different survival in adult females and males compared with wild-type fly survival. These results suggest that all Toll family members can activate several AMP genes. These results further indicate that Toll-1 and Toll-7 bind multiple Spz proteins and also VSV, but differentially affect adult survival after systemic infection, potentially because of sex-specific differences in Toll-1 and Toll-7 expression.
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).
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).
Retrograde growth factors regulating synaptic plasticity at the neuromuscular junction (NMJ)
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 Toll10b 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. DNT2e03444 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, DNT2e0344 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, DNT2e0344 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, DNT2e0344 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).
Bases in 5' UTR - 864
Bases in 3' UTR - 298
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
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).
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).
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