Gene name - Toll
Cytological map position - 97D1-2
Function - receptor
Symbol - Tl
Genetic map position - 3-91
Classification - IL-1 type receptor
Cellular location - surface
|Recent literature||Kanoh, H., Kuraishi, T., Tong, L. L., Watanabe, R., Nagata, S. and Kurata, S. (2015). Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract. Biochem Biophys Res Commun 467: 400-406. PubMed ID: 26427875
Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. This study demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, an ex vivo genome-wide RNAi screening was performed in Drosophila cultured cells, and several signaling factors were identified that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that the larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.
|Liu, B., Zheng, Y., Yin, F., Yu, J., Silverman, N. and Pan, D. (2016). Toll receptor-mediated Hippo signaling controls innate immunity in Drosophila. Cell 164: 406-419. PubMed ID: 26824654
The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. This study reports an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. These results elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings.
|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.
Toll is a transmembrane receptor and a member of the 12 gene dorsal group responsible for dorsoventral polarity in the fly. The ligand for Toll is Spätzle, and immediate targets include Pelle, Tube, Dorsal and Cactus. These proteins are held in a state of readiness while in the unfertilized egg. They are primed to carry out the transition from egg to zygote after fertilization occurs. Spätzle is available only in the ventral portion of the egg in the extracellular perivitelline space; when subjected to proteolysis, Spätzle becomes an active ligand for Toll.
Toll signals are first picked up by Tube, a protein in contact with the cell membrane. The signal is transduced to Pelle and subsequently to Cactus, which until its destruction holds Dorsal in the cytoplasm. With Cactus's bond destroyed, Dorsal enters the nucleus where it can serve as activator and repressor of genes involved in dorso-ventral polarity.
Toll is required zygotically in the development of a number of tissues, but Spätzle has not been documented as the ligand in these circumstances. Toll's mammalian homolog is Interleukin-1 (IL-1) receptor, involved in the activation of the immune response. Similarly, the Toll-Cactus-Dorsal system in the fly also activates an immune response.
The extracellular region of the Toll protein does not bear any similarity to the extracellular ligand-binding portion of the IL-1 receptor. Instead, it carries leucine rich repeats (LRR) that are found in molecules as diverse as proteoglycans, adhesion molecules, enzymes, tyrosine kinase receptors and G-protein coupled receptors. LRRs in the fly are found in Toll, Chaoptin and Connectin adhesion molecules and the Slit secreted protein. All these have roles in cell differentiation, morphogenetic events and the migration of cells and axons (Ollendorff, 1994).
The localized activation of Toll was first suggested based on the results of injection of wild-type cytoplasm into mutant flies. Wherever the Toll rescuing activity occurs defines the region from which ventral structures arise. Rescuing activity is not localized ventrally, but distributed uniformly in the wild-type embryo. This implies that the Toll gene product is normally present throughout the embryo but its activity is somehow restricted to ventral regions (Anderson, 1985 and Hashimoto, 1991). It is now understood that the ventral stimulation of Toll is caused by Spätzle, activated only on the ventral side of the egg (Morisato, 1994 and Roth, 1994).
Toll is dynamically expressed later in development by the embryonic musculature. Growth cones of RP3 and other motoneurons normally grow past muscle cells expressing Toll on their surface and innervate more distal muscle cells (muscles 6 and 7), which have down-regulated their Toll expression. The RP3 growth cone likely responds positively to Fasciclin III, an Ig-like cell adhesion molecule expressed on the target muscle cells, but still manages to avoid targeting errors in embryos lacking Fas III. Toll protein preferentially accumulates at muscle-muscle contact sites or "clefts," (the apposition between muscle cells). Later, the Toll-positive ventral muscle cells gradually lose Toll. Late-arriving growth cones innervate the clefts just as Toll expression becomes undetectable (Rose, 1997).
Reciprocal genetic manipulations of Toll proteins can produce reciprocal RP3 phenotypes. In Toll null mutants, the RP3 growth cone sometimes innervates the wrong muscle cells, including those that are normally Toll-positive. In contrast, heterochronic misexpression of Toll in the musculature leads to the same growth cone reaching its correct target region but delaying synaptic initiation. It is proposed that Toll acts locally to inhibit synaptogenesis of specific motoneuron growth cones and that both temporal and spatial control of Toll expression is crucial for its role in development (Rose, 1997).
The LRR (leucine-rich repeats) motif is shared by a number of other Drosophila cell surface molecules: Connectin, Chaoptin, 18-wheeler, Kekkon and Toll-like, as well as mammalian neural receptors, such as NLRR-1 and GARP. Structural analyses indicate that the LRR motifs could mediate protein-lipid as well as homophilic and heterophilic protein-protein interactions. The structurally related Connectin protein, when ectopically expressed in some of the ventral muscle cells, can function as a repulsive signal to motoneuron growth cone. All this evidence suggests a pivotal role for LRR proteins in axon guidance (Rose, 1997 and references).
Little is known about the genetic program that generates synaptic specificity. This study shows that a putative transcription factor, Teyrha-Meyhra (Tey), controls target specificity, in part by repressing the expression of a repulsive cue, Toll. This study focused on two neighboring muscles, M12 and M13, which are innervated by distinct motoneurons in Drosophila. It was found that Toll, which encodes a transmembrane protein with leucine-rich repeats, is preferentially expressed in M13. In Toll mutants, motoneurons that normally innervate M12 (MN12s) form smaller synapses on M12 and instead appear to form ectopic nerve endings on M13. Conversely, ectopic expression of Toll in M12 inhibited synapse formation by MN12s. These results suggest that Toll functions in M13 to prevent synapse formation by MN12s. Tey was identified as a negative regulator of Toll expression in M12. In tey mutants, Toll is strongly upregulated in M12. Accordingly, synapse formation on M12 was inhibited. Conversely, ectopic expression of tey in M13 decreased the amount of Toll expression in M13 and changed the pattern of motor innervation to the one seen in Toll mutants. These results suggest that Tey, which contains no known transcription factor motifs, determines target specificity by repressing the expression of Toll. These results reveal a mechanism for generating synaptic specificity that relies on the negative regulation of a repulsive target cue (Inaki, 2010).
A remarkable feature of the nervous system is the precision of its circuitry. A neural circuit develops through a series of neuronal recognition events. First, neurons find their path, turn at mid-way guideposts, and fasciculate or defasciculate before reaching their final target area. Then, neurons select and form synapses with specific target cells in the target region. The final matching of pre- and post-synapses is thought to be mediated by specific cues expressed on the target cells. However, the regulation and function of such cues remain poorly understood (Inaki, 2010).
The process of neuromuscular targeting in Drosophila features highly stereotypic matchings between 37 motoneurons and 30 target muscle cells, providing a unique model system for the study of neuronal target recognition. Several target cues, including Capricious, Netrin-B and Fasciclin 3, have been identified that are expressed in specific target cells and mediate attractive interactions between the synaptic partners. It has recently been shown that target specificity is also regulated by repulsion from non-target cells. Wnt4, a member of the Wnt family of secreted glycoproteins, is expressed in muscle 13 (M13) and prevents synapse formation by motoneurons targeted to a neighboring muscle, M12. In the absence of Wnt4, motoneurons targeted to M12 form ectopic nerve endings on M13, indicating that Wnt4 repulsion on M13 is required for proper targeting of the motoneurons. In addition to Wnt4, Toll and Semaphorin II (Sema-2a - FlyBase) are known to function as negative regulators of synapse formation in this system. However, whether they have a role in target selection remains unknown (Inaki, 2010).
Another unsolved issue is how the expression of such attractive or repulsive target-recognition molecules is regulated. It is amazing that the expression of these molecules is so precisely regulated that they are present at the right time and place. It is likely that the expression of these molecules is determined as part of the differentiation program of the target cells. However, little is known about the molecules and mechanisms involved. Several transcription factors, such as S59, Krüppel and Vestigial, have been identified as being expressed in subsets of muscle cells. They are expressed from the progenitor stage, and their loss-of-function (LOF) and gain-of-function (GOF) alter the specific characteristics of the individual muscles, such as their size, shape, orientation and attachment sites to the epidermis, indicating that they function as determinants of a particular muscle fate. However, whether these transcription factors regulate the expression of target-recognition molecules and thus determine the innervation pattern is unknown (Inaki, 2010).
A comparative microarray analysis has been conducted of two neighboring target muscles, M12 and M13, that are innervated by distinct motoneurons (Inaki, 2007). By comparing the expression profile of the two muscles, attempts were made to understand the molecular mechanisms that make these muscles distinct targets for the motoneurons. From this screening, ~25 potential target-recognition molecules were identified as preferentially expressed in either muscle cell. Among them was Wnt4, mentioned above. This study reports the functional analyses of two additional genes that were identified in the screening: Toll and teyrha-meyrha (tey). Toll encodes a transmembrane protein with extracellular leucine-rich repeats, and has multiple functions in development. Toll is expressed in subsets of muscles, including 6, 7 and 15-17. Previous studies have shown that Toll inhibits synapse formation by RP3, a motoneuron targeted to muscles 6 and 7. This study shows that Toll is preferentially expressed in M13 over M12 and, like Wnt4, inhibits synapse formation by motoneurons targeted to M12. It was also shown that tey, a previously uncharacterized gene, regulates the expression of Toll in specific muscles. tey is expressed specifically in M12, where it negatively regulates Toll expression. In the absence of tey, Toll is ectopically expressed in M12 and innervation of M12 is inhibited. These results suggest that Tey regulates targeting by downregulation of the repulsive cue Toll specifically in M12. Based on these results, a mechanism is proposed for the generation of synaptic specificity that relies on negative regulation of repulsive target cues (Inaki, 2010).
Toll is preferentially expressed in M13 over M12. The size of M12 terminals was decreased in Toll mutants, with concomitant expansion of M13 terminals. This phenotype is very similar to that of Wnt4 LOF and is likely to be caused by MN12s forming ectopic synapses with M13, although it remains possible that some of the ectopic nerve endings on M13 are formed by other motoneurons. Furthermore, it was observed that the size of M12 terminals is reduced when Toll is misexpressed on the muscle. The LOF and GOF analyses suggest that Toll functions as a repulsive factor in M13 that is important for target selection by MN12s. Thus, Toll provides another example of a repulsive factor that is involved in target selection. How Toll mediates the repulsive signal to motoneurons is currently unknown. A model is that Toll functions as a ligand that is expressed in muscles and signals through receptor(s) expressed in motoneurons. However, no receptor has been identified for Toll. Toll has been shown to function as a receptor, not a ligand, in other systems, such as in dorsoventral patterning and innate immunity. Another possibility is that Toll might mediate the modification or regulation of other targeting molecules, such as Wnt4 (Inaki, 2010).
M13 expresses at least two repulsive cues, Wnt4 and Toll, that are important for the targeting of M12 and M13. It seems that these two molecules contribute to target specificity in a manner that is redundant with yet other molecules because in both single and double mutants of these genes, the connectivity is only partially disrupted. Previously, other potential repulsive cues that are expressed in M13 were identified, including Beat-IIIc and Glutactin (Inaki, 2007). Ectopic expression of these molecules in M12 inhibits synapse formation by MN12s, as observed when Toll and Wnt4 are misexpressed. Although the precise roles of these molecules remain to be verified by LOF analyses, these results suggest the possibility that a single muscle, M13, expresses a number of repulsive cues that are involved in targeting of motoneurons. This is consistent with previous hypotheses that Drosophila neuromuscular connectivity is determined by highly redundant mechanisms. It will be important to determine how the signals from multiple cues are integrated to generate the precise pattern of synaptic connections. It will also be interesting to examine whether other muscles similarly express repulsive cues to prevent inappropriate innervation. The phenotypes of Wnt4 Toll double mutants were of similar severity to those of the single mutants. This might be due to the presence of other targeting molecules, as described above. Toll and Wnt4 might also function in the same signaling pathway. For example, Toll may be involved in the regulation of Wnt4 activity through influencing its secretion, localization or protein modification. Toll and Wnt4 might also act as repellents for distinct MN12s (Inaki, 2010).
This study has shown that a novel nuclear protein, Tey, regulates the expression of Toll and is important for the determination of target specificity. tey regulates the position, orientation and attachment sites of M12. Thus, Tey seems to act as a determinant of several important properties of M12, regulating both the differentiation of the muscle itself and the specificity of nerve innervation. Expression of tey is remarkably specific, being limited within the somatic musculature to a single muscle, M12. Other, known muscle-determinant genes were expressed in broader subsets of muscles (Inaki, 2010).
Tey negatively regulates the expression of Toll in M12. In tey mutants, Toll expression is strongly upregulated in M12. This indicates that tey is required in this muscle to specifically suppress Toll expression. Consistent with this, ectopic expression of tey in M13 partially suppressed Toll expression. Toll is normally expressed in most of the other ventral muscles, including muscles 6, 7, 13-17, but not in M12, suggesting that some positive transcriptional regulator(s) higher up in the hierarchy activate Toll expression in this group of muscles and that negative regulation by Tey is required to suppress Toll expression only in M12. The regulation of Toll by Tey should be at the transcriptional level because the expression of the exogenously introduced Toll enhancer-trap lacZ reporter is affected in tey mutants or when tey is misexpressed. It remains to be determined whether Tey binds directly to the regulatory region of the Toll gene or regulates Toll transcription in an indirect manner (e.g. by regulating other transcription factors). Tey contains no known transcription factor motifs. The expression of another M13-enriched gene, Wnt4, was not affected in tey mutants or when tey was misexpressed. Unlike Toll, Wnt4 is expressed in only two ventral muscles: 13 and 30. Thus, expression of Wnt4 might be regulated in a different manner to Toll, possibly by positive transcription factors that are specifically expressed in these muscles. It will be interesting to determine how the expression of target-recognition molecules is precisely regulated by the combinatorial action of positive and negative transcription factors (Inaki, 2010).
In tey mutants or when tey is misexpressed, neuromuscular connectivity was also altered in a manner consistent with the misregulation of Toll expression. The inappropriate presence of Toll repulsion in tey LOF mutants suppressed synapse formation on M12. Conversely, suppression of Toll expression in M13 in tey GOF mutants led to changes in the innervations of M12 and M13, similar to those observed in Toll mutants. Furthermore, the effects of tey GOF were dramatically reversed when Toll was co-expressed with tey, suggesting that Toll is the major target of tey in causing the GOF phenotypes. These results suggest that Tey regulates neuromuscular connectivity by specifically repressing Toll expression in M12. As noted above, Toll is normally expressed in a number of ventral muscles, but not in M12. Furthermore, Toll is expressed in M12 in the absence of Tey suppression in tey mutants. This suggests that the default state is for Toll to be expressed in all ventral muscles, possibly by the action of positive transcription factor(s) expressed in these muscles. Tey might therefore generate target specificity by suppressing the expression of Toll in one among a group of muscle cells expressing the repulsive cue. The data thus suggest a mechanism of transcriptional control of target specificity, namely, the negative regulation of repulsive cues (Inaki, 2010).
Bases in 5' UTR - 574
Bases in 3' UTR - 1257
A 5.3 kb poly(A)+ ovarian transcript of Toll was purified by hybrid selection with cloned DNA. The sequence of cDNAs suggests that the Toll protein is an integral membrane protein with a cytoplasmic domain and a large extracytoplasmic domain. The putative extracytoplasmic domain contains two blocks (for a total of 15 repeats) of a 24 amino acid, leucine-rich sequence found in both human and yeast membrane proteins. The transmembrane domain is between residues 804 and 828. There are 17 potential glycosylation sites and 17 cysteine residues in 3 clusters (Hashimoto, 1988).
The Toll protein has sequences held in common with the human membrane receptor platelet glycoprotein 1b (Gp1b). These sequences in Toll form disulphide linked extracellular domains that are important for the binding of ligands in the perivitelline space of the embryo. Expression of Toll protein induced in a non-adhesive cell line promotes cellular adhesion, a property held in common with the related Drosophila glycoprotein Chaoptin. Toll protein in such aggregates accumulates at sites of cell-cell interaction, a characteristic displayed by other cellular adhesion molecules (Keith, 1990).
Unusual properties are found for a synthetic LRR peptide derived from the sequence of the Drosophila membrane receptor Toll. In neutral solution the peptide forms a gel revealed by electron microscopy to consist of extended filaments approximately 8 nm in thickness. As the gel forms, the circular dichroism spectrum of the peptide solution changes from one characteristic of random coil to one associated with beta-sheet structures. Molecular modelling suggests that the peptide forms an amphipathic structure with a predominantly apolar and charged surface. Based on these results, models for the gross structure of the peptides filaments and a possible molecular mechanism for cellular adhesion are proposed. The finding that Toll-LRR forms intramolecular ß-sheet structures supports the view that LRRs can participate in protein-protein interactions and homotypic cellular adhesion. It could be that LRRs expressed on the cell surface are initially of disordered structure and that interactions with similarly disordered LRRs on an adjacent cell causes the formation of an extended and stable intermolecular ß structure. Such a mechansim could provide a molecular basis for cellular adhesion mediated by LRRs (Gay, 1991).
date revised: 3 July 97
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