tolkin/tolloid-related-1 Biological Overview | Regulation | Developmental Biology | References

Gene name - tolkin

Synonyms - tolloid-related-1 (tlr)

Cytological map position - 96A

Function - protease

Keywords - dorsal-ventral patterning

Symbol - tok

FlyBase ID:FBgn0004885

Genetic map position - 3-[85]

Classification - metalloprotease

Cellular location - secreted

NCBI link: Entrez Gene
tok orthologs: Biolitmine
Recent literature
Zaytseva, O., Mitchell, N. C., Muckle, D., Delandre, C., Nie, Z., Werner, J. K., Lis, J. T., Eyras, E., Hannan, R. D., Levens, D. L., Marshall, O. J. and Quinn, L. M. (2023). Psi promotes Drosophila wing growth via direct transcriptional activation of cell cycle targets and repression of growth inhibitors. Development 150(2). PubMed ID: 36692218
The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Through the first genome-wide binding and expression profiles obtained for a FUBP family protein, this study demonstrates that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFβ signalling). It was further demonstrated that tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.

tolkin (termed here tolloid-related 1) is a member of the BMP family of proteins, like the closely linked tolloid gene. BMP-1, the vertebrate homolog, forms a protein complex with other members of the family. Of interest to Drosophila biologists is mammalian BMP-4, the mammalian homolog of Decapentaplegic. Unlike DPP, both TLD and TLR-1 have metalloprotease domains and two EGF domains. Neither TLD nor TLR-1 are related to mammalian TGF beta, the DPP homolog. By analogy to the vertebrate system, TLD is implicated as a constituent in a multiprotein complex involving DPP.

What distinguishes TLD from TLR-1? Their early expression overlaps, but TLR-1 has several additional expression domains, including expression in anterior and posterior midgut invaginations, in proneural clusters of the peripheral nervous system, in optic lobe precursor cells, in visceral and somatic mesoderm, and in cells that will eventually form the corpus allatum of the ring gland. Thus tlr-1 has a much broader expression during embryogenesis.

TLR-1 and TLD function independently of one another. One does not substitute for the other. Deletion of tlr-1 is lethal, and not amenable to rescue by expression of TLR-1 cDNA driven by the tld promoter (Nguyen, 1994). Therefore, sorting out the particular roles of these two proteins awaits future work.

It has been assumed that Tolloid, through its protease domain, activates DPP, but this may not be true. Mammalian BMP-1 acts on collagen substrates (Kessler, 1996).

Tolloid-related processes Sog in order to help specify the posterior crossvein in the Drosophila wing

Tolloid (Tld) and Tolloid related (Tlr) belong to a family of developmentally important proteases that includes Bone Morphogenetic Protein 1 (Bmp1). Tld is required early in Drosophila development for proper patterning of dorsal embryonic structures, whereas Tlr is required later during larval and pupal stages of development. The major function of Tld is to augment the activity of Decapentaplegic (Dpp) and Screw (Scw), two members of the Bmp subgroup of the TgfbetaŸ superfamily, by cleaving the Bmp inhibitor Short gastrulation (Sog). Evidence is presented that Tlr also contributes to Sog processing. Tlr cleaves Sog in vitro in a Bmp-dependent manner at the same three major sites as does Tld. However, Tlr shows different site selection preferences and cleaves Sog with slower kinetics. To test whether these differences are important in vivo, the role of Tlr and Tld during development of the posterior crossvein (PCV) in the pupal wing was investigated. tlr mutants lack the PCV as a result of too little Bmp signaling. This is probably caused by excess Sog activity, since the phenotype can be suppressed by lowering Sog levels. However, Tld cannot substitute for Tlr in the PCV; in fact, misexpressed Tld can cause loss of the PCV. Reducing levels of Sog can also cause loss of the PCV, indicating that Sog has not only an inhibitory but also a positive effect on signaling in the PCV. It is proposed that the specific catalytic properties of Tlr and Tld have evolved to achieve the proper balance between the inhibitory and positive activities of Sog in the PCV and early embryo, respectively. It is further suggested that, as in the embryo, the positive effect of Sog upon Bmp signaling probably stems from its role in a ligand transport process (Serpe, 2005).

The major distinction between the two Drosophila proteases in terms of their Sog processing function is the time and tissue in which they act. Tld activity is primarily confined to the early embryo, while Tlr is required during pupal wing development. To some extent, the functional differences between them can be attributed simply to differences in expression pattern. In the pupal wing Tlr is far more abundantly expressed than Tld, and this alone might account for the lack of redundancy. In the early embryo, however, the situation is more complex. Both enzymes are expressed with similar profiles, but Tlr does not seem to be capable of providing sufficient Sog processing activity, even when several extra copies are provided as transgenes (Serpe, 2005).

It has been speculated that this difference in activity might reflect differences in activation of the proteases at the level of pro-peptide removal. Like all the members of the Bmp1 family, Tld and Tlr are secreted as pro-enzymes; the processing of the pro-peptide is necessary for the activation of proteolytic activity, since the N-terminal end of the astacin motif is buried inside the catalytic domain forming an internal salt bridge. Mutations at the processing site render the enzymes inactive, whereas removal of the pro-peptides produce activated forms of Tld and Tlr. Tlr has a much longer pro-peptide that could either aid or inhibit activation in a tissue-specific manner. However, the inability of Tlr to rescue Tld mutants does not appear to result from an inefficient activation step. Tld activation, both in the embryo and in S2 cells, is very inefficient with most of the protein found in the pro-enzyme state. By contrast, pro-peptide removal from Tlr is very efficient in S2 cells, and the same is true when Tlr is ectopically expressed in the embryo (Serpe, 2005).

Instead, it seems likely that the difference in kinetics of Sog processing by Tlr is the reason behind the inability of Tlr to rescue tld mutants. Tlr is much less efficient at cleaving Sog in vitro than Tld. Given the rapid developmental time of early embryogenesis, where patterning by Bmps during cellularization occurs within approximately a 30 minute time window, the slower kinetics of Sog processing by Tlr may not support proper patterning. Indeed, computational work has shown that a three to fourfold reduction in kinetic properties of Tld will completely disrupt the patterning process (Serpe, 2005).

Although the slow processing kinetics of Tlr towards Sog may prevent it from functioning effectively in early embryonic patterning, this property may be exactly what is required to achieve proper formation of the PCV. Unlike patterning in the early embryo, formation of the PCV, as assessed by profile of pMad accumulation, occurs over at least a 7 hour time frame. The slower processing rate of Tlr towards Sog may be required to achieve the appropriate balance of Sog destruction and diffusion that is necessary for proper patterning to occur. Consistent with this view, overexpression of various UAS-tld lines under the control of the A9-Gal4 driver in a tlr mutant background does not rescue PCV formation. In fact, in many cases overexpression of an activated Tld, or co-expression of wild type tld and tlr together produce loss of the crossvein tissue in a wild-type background. It is envisioned that, under these conditions, the increased level of enzymatic activity results in over-digestion of Sog, a situation that would phenocopy sog hypomorphs. Consistent with this view, hypomorphic sog allelic combinations also result in the loss of the PCV. In addition, large sog null clones can also result in loss of the PCV (Serpe, 2005).

In a Xenopus assay, it was found that Tlr is only slightly less efficient than Tld at reverting secondary axis induction caused by Sog. Although it is not known how well each enzyme is activated in this animal, it should be noted that the developmental time period over which the patterning process functions in Xenopus is long compared with early Drosophila development. The longer time frame may enable the less efficient protease to produce a similar biological response. Protease domain swap experiments suggest that the reduced processing rate does not involve evolution of intrinsic differences in the catalytic abilities of the protease domain itself, but rather changes occur in the way that the Sog substrate initially interacts with the enzyme. In summary, it is proposed that during evolution there was selection for particular biophysical properties of these two enzymes to properly match the developmental time frame over which the patterning mechanisms operate. It cannot be exclude however, that other differences besides kinetic activity might also play a role in providing functional specificity. For example, it is possible that variation in cleavage site selection might also contribute to the different biological activities of Tlr and Tld. It is worth noting in this regard that different fragments of Sog have been shown to have both positive and negative effects when overexpressed in the wing. However the in vivo roles of endogenous Sog fragments have not been defined (Serpe, 2005).

The results suggest that a proper balance of Sog and protease activity is necessary to pattern the PCV. Interestingly, the same situation holds true in the early embryo. In this case, Sog plays both positive and negative roles in patterning the dorsal domain. It is required in the dorsolateral regions to block Bmp signaling, but it also acts as an agonist to achieve peak levels of Bmp signals at the dorsal midline. Two types of models have been proposed to account for these dual activities. In one model, the different cleavage fragments of Sog are thought to provide either agonist or antagonist function, but the details of the mechanism are unclear. In the other model, both functions are proposed to come about as a result of Sog providing a transport mechanism that spatially redistributes Bmp ligands from the lateral region to the dorsal most cells. This transport mechanism also requires the activity of Tsg, a small cysteine-rich secreted protein which has been shown to form a tripartite complex with Sog and Dpp. The prevailing view is that as Sog diffuses into the dorsal domain it forms a high affinity complex with Tsg and Dpp. This complex is unable to bind to receptors and is responsible for the antagonistic activity of Sog. At the same time, the complex protects Dpp from degradation and receptor binding allowingit to diffuse and accumulate dorsally where it is released by Tld processing. The ability of Sog to redistribute the Bmp ligands accounts for the agonist function of Sog. Computational analyses have provided additional support for this model (Serpe, 2005).

It is proposed that the same type of mechanism may be responsible for patterning the PCV. Recent analysis has provided evidence that the longitudinal veins act as the source of Dpp for PCV specification. Dpp is thought to diffuse from these veins into a PCV competent zone. The exact mechanism by which the competent zone is specified is not clear, but low levels of Sog expression are required. tlr is expressed within the PCV competent zone during the initial stages of crossvein development, suggesting the Tlr:Sog ratio will be higher in this region. Furthermore, because processing of the Sog/Dpp complex by Tld-like enzymes is dependent on the Dpp concentration, the complex will be most efficiently processed in the center of the competent zone (Serpe, 2005).

According to this model, there is limited processing of Sog and therefore limited release of Dpp from its inhibitor in tlr mutants. Conversely, Sog also supplies a positive function for PCV formation, probably by providing a transport mechanism for Dpp, accounting for the partial loss of the PCV in hypomorphic sog mutants and complete loss of the PCV in large sog-null clones. The partial reversion of the tlr mutant phenotype by introduction of hypomorphic sog alleles is also consistent with the view that it is the balance between these two factors that is crucial for proper patterning. Interestingly, this is the way in which Sog was originally identified as an inhibitor of Dpp signaling in the embryo: weak sog alleles were isolated as partial suppressors of tld mutations. One difference is that, in the case of this partial reversion, lowering Sog levels is able to revert a null mutation instead of a hypomorphic condition, as was the case in the embryo. There are at least two possibilities that can explain this suppression effect. First, although these animals may be null for tlr, there could be some low level tld expression in the pupal wing. If so, then these wings would not be devoid of all Sog-processing activity and therefore lowering Sog levels might enable the limited amount of Tld to provide the proper production-destruction balance. Alternatively, neither Sog nor Tlr may be absolutely required for PCV formation. Instead, their functions may be simply to ensure that the patterning occurs reproducibly. Thus, in the absence of both Sog and Tlr, partial PCV formation may occur as a result of some Bmp ligand accumulating in the correct position. However, under these circumstances, the patterning mechanism would be unreliable and would produce different results on case-by-case basis. To prevent this from occurring, it is posited that evolution has selected for supplementary regulatory controls involving Sog and Tlr to ensure that the PCV always forms completely and reliably at the correct position (Serpe, 2005).

Two additional observations make the comparison between formation of the PCV and establishing the high point in embryonic Bmp signaling even more compelling. (1) In the embryo, Tsg is required to enable Sog to bind to Dpp and Scw to achieve peak levels of Bmp signaling. Although Tsg and Scw are not transcribed in the pupal wing, a Tsg-related gene, encoded by the crossveinless (cv) locus, is expressed in the pupal wing. Since cv mutants exhibit a crossveinless phenotype, it seems likely that Cv functionally substitutes for Tsg during PCV formation. (2) Gbb, another Bmp-like ligand, may functionally replace Scw, since gbb hypomorphic mutations lack the PCV and associated Bmp signaling (Serpe, 2005).

A major distinction between embryonic amnioserosa development and PCV formation is that PCV specification also requires the activity of Cv2, a protein that contains cysteine-rich repeats similar to those found in Sog, while amnioserosa specification does not, despite the expression of Cv2 in those cells. Vertebrate homologs of Cv2 can bind Bmps, and act variously as agonists or antagonists of Bmp signaling in different assays. It is not clear by what mechanism Cv2 promotes Bmp signaling during PCV formation. It is also not clear why Cv2 is not required in the early embryo, even though it is expressed in dorsal blastoderm cells (Serpe, 2005).

Finally, Tlr plays additional roles during development, besides processing Sog for specification of the PCV. In contrast to cv and cv2 null mutations, which result in homozygous viable and fertile flies, most tlr mutant animals die during larval stages when there is no known requirement for Sog. In addition, although reducing Sog levels does suppress the PCV defect observed in the tlr mutant escaper flies, it does not increase the frequency of eclosing animals. Therefore Tlr may be required for processing of some other essential component(s) during Drosophila development (Serpe, 2005).


Tolloid-related-1/Tolkin, is located 700 bp 5' to tolloid (Finelli, 1994).

Genomic DNA length - 5.4 kb

cDNA clone length - 5376

Bases in 5' UTR - 328

Exons - five

Bases in 3' UTR - 654


Amino Acids - 1464

Structural Domains

tolloid-related-1 (tlr-1) maps immediately proximal to tld. Sequence analysis indicates that tlr-1 has a large N-terminal extension relative to tld, but otherwise shows the same general organization of sequence motifs found in tld and other BMP-1 family members. These include a region of similarity to astacin, a crayfish metalloprotease, five copies of a repeat first found in complement proteins C1r and C1s, and two copies of an epidermal growth factor-like sequence. tld and tlr-1 arose by gene duplication and each has evolved independently to acquire distinct tissue specific roles in Drosophila development. Both TLD and TLR-1 must undergo proteolysis for activation. A tetra-basic sequence in TLD immediately preceding the first residue on the protease domain is the likely producer of a processing site (Nguyen, 1994).

Tolloid-related-1/Tolkin has an overall structure that is like Tolloid, with an N-terminal metalloprotease domain, five complement subcomponents C1r/C1s, Uegf, and Bmp1 (CUB) repeats and two epidermal growth factor (EGF) repeats (Finelli, 1994).


Protein Interactions

Proper axon pathfinding requires that growth cones execute appropriate turns and branching at particular choice points en route to their synaptic targets. The Drosophila metalloprotease tolloid-related (tlr) is required for proper fasciculation/defasciculation of motor axons in the CNS and for normal guidance of many motor axons enroute to their muscle targets. Tlr belongs to a family of developmentally important proteases that process various extracellular matrix components, as well as several TGF-ß inhibitory proteins and pro-peptides. Tlr is a circulating enzyme that processes the pro-domains of three Drosophila TGF-ß-type ligands, and, in the case of the Activin-like protein Dawdle (Daw), this processing enhances the signaling activity of the ligand in vitro and in vivo. Null mutants of daw, as well as mutations in its receptor babo and its downstream mediator Smad2, all exhibit axon guidance defects that are similar to but less severe than tlr. It is suggestd that by activating Daw and perhaps other TGF-ß ligands, Tlr provides a permissive signal for axon guidance (Serpe, 2006).

Mutants in the metalloprotease tlr cause lethality during larval and pupal stages of development; however, the cause of the lethality has not been determined. Since a small percentage of larvae (about 15%) die as soon as they hatch, the need for Tlr may start during embryogenesis. Beginning at stage 13, Tlr protein expression is found in the muscles, a subset of cells in the central nervous system that include many glia and the corpus allatum portion of the ring gland. The distinct pattern of expression of tlr in the CNS and muscles, together with the observation that rare tlr mutant escapers exhibit impaired movement, prompted an examination of nervous system development in tlr mutants. To look for global defects, all CNS axons in mutant embryos of the strong allelic combination tlrex[2-41]/tlrE1 were stained with the monoclonal antibody mAB BP102. This analysis did not reveal any gross abnormalities in formation of longitudinal or commissural tracts. Next the Fas2 monoclonal antibody mAb 1D4 which, at stages 16 and 17, highlights motor axon tracts in the periphery and six longitudinal bundles within the CNS, was used. In tlrex[2-41]/tlrE1 mutants, the Fas2-positive longitudinal bundles are wavy and irregular and the outer bundle is discontinuous or missing. This phenotype is of variable penetrance because embryos that had mild defects were found as well as embryos with severe, interaxonal adhesion defects (Serpe, 2006).

In abdominal segments A2-A7, motor axons exit the CNS within the intersegmental nerve (ISN) and segmental nerve (SN) roots; these then split into five pathways that innervate 30 muscle fibers. The ISN develops fairly early and reaches the terminus region near muscle 1 at stage 16 of embryogenesis. In tlrex[2-41]/tlrE1 mutants, ISN growth appeared delayed: 85% of ISNs (140 hemisegments examined) reached their final destination by late stage 16, whereas 15% of ISNs were still at the secondary branch point, around muscle 2. By stage 17, the ISN reached its terminal position in most hemisegments of the tlrex[2-41]/tlrE1, but the terminal arbors were thin or bifurcated (Serpe, 2006).

The SNa has a bifurcated morphology. The posterior branch of SNa innervates muscles 5 and 8, and the anterior branch innervates muscles 21-24. To reach muscle 24, the anterior branch makes a characteristic turn at stage 16. In tlrex[2-41]/tlrE1 mutant animals, SNa did not turn, but instead stalled or produced random branches at this point (Serpe, 2006).

The SNb branch innervates the ventral muscles 7, 6, 13 and 12, and contains the axons of RP1, 3, 4 and 5. The development of the SNb involves two key sets of contacts, the first at muscle 28 and 14, where SNb axons leave the common pathway and enter the ventral muscle field, and the second near muscle 30, where stage 16 SNb growth cones shift their trajectory to extend along a more interior muscle layer. At early stage 17, the SNb forms a linear synaptic branch at the muscle 6/7 cleft, a 'blobby' synapse at the proximal edge of muscle 13 (referred as the 13/30 synapse), and a linear synapse at 12/13. In tlrex[2-41]/tlrE1 mutant animals, the appearance of the SNb proximal synapse (6/7) was normal; however, the SNb bundles stalled at the 13/30 'blob' with an occasional thin bundle exiting and extending towards the 12/13 cleft. The thinned SNb appeared to reach the target muscles at random locations and produced very short synapses. The overall appearance was that of stalled growth cones with frail axons perhaps trying to achieve some sort of innervation at the 12/13 synapse. Such unsuccessful attempts to compensate for the lack of proper 12/13 innervation were observed in 46% of the tlrex[2-41]/tlrex[2- 41] hemisegments, in 48% of the tlrex[2-41]/tlrE1 hemisegments, and in 56% of the tldP1/tlrE1 hemisegments. Since tlrP1 is a deletion comprising both tld and tlr genes, and tlrE1 contains a stop codon within the tlr ORF, the slightly lower penetrance of defects in the case of tlrex[2-41]/tlrex[2-41] animals could be due to some residual tlr muscle expression. Such minimal expression might be the result of using an alternative exon, upstream of the breakpoints of the tlrex[2-41] deficiency, that is computationally predicted by Flybase, although it was not recovered in any of the extant tlr cDNAs (Serpe, 2006).

The fact that Tlr was able to rescue mutant animals when supplied from either the muscle or the nerve suggests that its precise spatial expression pattern may not be important for function. Since Tlr is a secreted protein, it is possible that it could gain access to its substrate from the hemolymph. In fact, in addition to expression in muscle and glia, tlr is also heavily expressed in the corpus allatum of the ring gland, a known secretory tissue. To determine if Tlr could rescue mutant animals when expressed exclusively in secretory or circulating cells, a series of ring gland or hemocyte drivers (Cg, hml, phantom) were used and full rescue of tlrex[2-41]/tlrE1 lethality and axon guidance defects was found in all cases. Furthermore, hemolymph samples from wild-type animals, but not from tlr mutants, contained the processed activated Tlr protein. Moreover, significant levels of HA-tagged Tlr were detected in hemolymph samples collected from animals in which a UAS-tlr-HA transgene was overexpressed in various tissues, including glial cells and muscle. These results support the hypothesis that Tlr is secreted and circulates in the hemolymph and need not be supplied locally by either the muscle or glial cells in order to promote proper axon guidance (Serpe, 2006).



tolloid expression can be detected during nuclear division cycle 10-11 at least 40 minutes earlier than tlr-1. In situ hybridization experiments show that tlr-1 expression partially overlaps tld expression in early embryos, but shows unique transcriptional patterns in late stage embryos that are not seen with tld. The dorsal pattern of tlr-1 fades and is replaced by staining in anterior and posterior midgut invaginations. The anterior cells form part of the pharynx, while the posterior cells form part of the visceral mesoderm. There is also expression in patches of cells that may be proneural clusters of the peripheral nervous system. There is transient expression in optic lobe precursor cells and expression in visceral and somatic mesoderm. During dorsal closure, cells that eventually form the corpus allatum of the ring gland show TRL staining. In larval stages, both genes are expressed in identical patterns in imaginal discs and in the optic lobes of the brain, but TLR-1 is more abundant than TLD. Cells forward of the morphogenetic furrow show high levels of tlr-1(Nguyen, 1994).

tolloid-related-1/tolkin expression pattern overlaps that of tolloid and dpp in early embryos and diverges in later stages. In larval tissues, both tolloid and tlr-1 are expressed uniformly in the imaginal disks. In the brain, both tolloid and tlr-1 are expressed in the outer proliferation center, whereas tlr-1 has another stripe of expression near the outer proliferation center (Finelli, 1994).

Effects of mutation or deletion

Deletions that eliminate tlr-1 expression are lethal during larval and pupal stages of development. A small proportion of homozygous mutant flies eclose (hatch) and show wing veination defects. Transgenic animals in which a tlr-1 cDNA is driven by the tld promoter fail to rescue tld mutations, and extra copies of tld fail to rescue tlr-1 mutations, implying that these genes have evolved functionally distinct features (Nguyen, 1994).

Analysis of lethal mutations in tlr-1 indicate it is vital during larval and pupal stages. Analysis of its mutant phenotypes and expression patterns suggests that its functions may be mostly independent of tolloid and dpp (Finelli, 1994).

At the next stop sign turn right: the metalloprotease Tolloid-related 1 controls defasciculation of motor axons in Drosophila

Navigation of motoneuronal growth cones toward the somatic musculature in Drosophila serves as a model system to unravel the molecular mechanisms of axon guidance and target selection. In a large-scale mutagenesis screen, piranha, a motor axon guidance mutant that shows strong defects in the neuromuscular connectivity pattern, was identified. In piranha mutant embryos, permanent defasciculation errors occur at specific choice points in all motor pathways. Positional cloning of piranha revealed point mutations in tolloid-related 1 (tlr1), an evolutionarily conserved gene encoding a secreted metalloprotease. Ectopic expression of Tlr1 in several tissues of piranha mutants, including hemocytes, completely restores the wild-type innervation pattern, indicating that Tlr1 functions cell non-autonomously. Loss-of-function mutants of related metalloproteases do not have motor axon guidance defects and that the respective proteins cannot functionally replace Tlr1. tlr1, however, interacts with sidestep, a transmembrane receptor of the immunoglobulin superfamily that functions as a muscle-derived attractant for motor axons. Double mutant larvae of tlr1 and sidestep show an additive phenotype and lack almost all neuromuscular junctions on ventral muscles, suggesting that Tlr1 functions together with Sidestep in the defasciculation process (Meyer, 2006; full text of article).

Metalloproteases have been implicated in a variety of cellular processes, including cell migration, angiogenesis and metastasis. Neuronal growth cones migrate through an environment that is rich in different extracellular surfaces and may thus exploit similar molecules and mechanisms as migrating cells. Despite the wealth of data on cell migration, only a few reports have been published implicating metalloproteases in axon outgrowth and guidance. With regard to motor axons, only ADM-1 (unc-71), a member of the ADAM family in Caenorhabditis elegans, has been shown to regulate pathfinding. Tlr1 belongs to the astacin family of metalloproteases and is highly related to Drosophila Tolloid. Despite this high degree of conservation, these two proteins have mutually exclusive functions. While Tld cannot functionally replace Tlr1, it is still possible that other metalloproteases with redundant functions assist Tlr1 in defasciculation control, because not all guidance decisions are affected in tlr1 mutants. Loss- and gain-of-function analysis of related metalloproteases, however, did not support this possibility. In addition, no other metalloprotease has so far been recovered from mutant screens. These observations support the idea that Tlr1 may be a key regulatory member of the metalloprotease family in Drosophila that controls motor axon guidance (Meyer, 2006).

As a secreted metalloprotease, Tlr1 is predicted to function extracellularly, either in the extracellular matrix or in the interstitial fluid. Consistent with this prediction, overexpression of Tlr1 in hemocytes or cells of the fat body could fully rescue the tlr1 mutant phenotype. Neither hemocytes nor fat-storing cells have so far been implicated in axon guidance. Hence, it is unlikely that Tlr1 remained associated with the extracellular matrix of these cells, but probably got released into the hemolymph. The circulating hemolymph then distributed it to where it was required. As endogenous Tlr1 is expressed in developing muscles during the period of axonal pathfinding, it is possibly secreted from there into the hemolymph to either proteolytically activate a repellent on motor nerves to induce defasciculation or to activate an attractant on muscles. The axon guidance receptor Sidestep is expressed on muscles and functions as an attractant for motor axons. Based on similarities in their loss-of-function phenotypes, Tlr1 could be required to activate the attractive function of Side. The phenotype of tlr1,side double mutants, however, was clearly stronger compared with each single mutant, indicating that they regulate the same biological process but that they function either independently or that the two functions converge further downstream. Tlr1 could therefore regulate the activity of an alternative pathway. Interestingly, it has been suggested that Tlr1 regulates motor axon guidance in part by activating latent TGF-β ligands. Although the exact molecular function of Tlr1 is currently not known, the data presented in this study clearly demonstrate that the evolutionarily conserved metalloprotease Tolloid-related 1 is necessary for motor axon guidance in Drosophila (Meyer, 2006).


Finelli, A. L. (1995). The tolkin gene is a tolloid/BMP-1 homologue that is essential for Drosophila development. Genetics 141: 271-281. Medline abstract: 8536976

Kessler, E., et al. (1996). Bone morphogenetic protein-1: the type I procollagen C-proteinase. Science 271: 360-362

Meyer, F. and Aberle, H. (2006). At the next stop sign turn right: the metalloprotease Tolloid-related 1 controls defasciculation of motor axons in Drosophila. Development 133(20): 4035-44. Medline abstract: 16971470

Nguyen, T., et al. (1994). Characterization of tolloid-related-1: a BMP-1-like product that is required during larval and pupal stages of Drosophila development. Dev Biol 166: 569-586. Medline abstract: 7813777

Serpe, M., et al. (2005). Matching catalytic activity to developmental function: Tolloid-related processes Sog in order to help specify the posterior crossvein in the Drosophila wing. Development 132: 2645-2656. 15872004

Serpe, M. and O'Connor, M. B. (2006). The metalloprotease Tolloid-related and its TGF-β-like substrate Dawdle regulate Drosophila motoneuron axon guidance. Development 133: 4969-4979. Medline abstract: 17119021

date revised: 22 May 2023 
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