InteractiveFly: GeneBrief

Tollo: Biological Overview | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Specific glycan expression is an essential characteristic of developing tissues. Molecular characterization of a mutation in Tollo that abolishes neural-specific glycosylation in the Drosophila embryo demonstrates that cellular interactions influence glycan expression. The HRP epitope is an N-linked oligosaccharide expressed on a subset of neuronal glycoproteins. In wild-type embryos, Tollo is expressed by ectodermal cells that surround differentiating neurons and precedes HRP-epitope appearance. Re-introduction of Tollo into null embryos rescues neural-specific glycan expression. Thus, loss of an ectodermal cell surface protein alters glycosylation in juxtaposed differentiating neurons. Neural differentiation and axon extension, to the extent that they are revealed by standard monoclonal antibody markers, are unaffected by loss of Tollo function. The ability to induce specific glycan expression complements the previously identified developmental and innate immune functions of Toll-like receptors (Seppo, 2003).

Eukaryotic cells are enveloped within a complex coating of carbohydrate. Composed of the pendant oligosaccharide moieties of glycoproteins, glycolipids and proteoglycans, the glycocalyx constitutes the interface at which cells interact with each other and with their environment. Glycans mediate cellular recognition and adhesion, facilitate protein maturation, regulate protein activity through allostery and bioavailability, influence receptor ligation, and modulate transmembrane signaling. Specific oligosaccharide structural elements participate in each of these processes, requiring spatial and temporal regulation of glycan synthesis in developing and mature organisms. As a consequence of regulated expression, oligosaccharide structures are among the most discriminating markers for cellular differentiation in complex tissues. Despite extensive descriptions of specific oligosaccharide distributions, the cellular and molecular mechanisms by which cells achieve expression of their characteristic portfolio of surface glycans are largely unknown (Seppo, 2003 and references therein).

Antibodies raised against the plant glycoprotein, Horseradish Peroxidase (HRP), crossreact with an N-linked oligosaccharide epitope that is distributed throughout the Drosophila melanogaster nervous system and is also expressed in a small, well characterized subset of non-neural tissues (Jan, 1982; Snow, 1987). Two mutations abolish expression of the HRP epitope. In the first, designated nac, the epitope is absent in the larval, pupal and adult nervous system (Katz, 1988). The molecular nature of the nac mutation is unknown, but affected adults exhibit sensory afferent defasciculation and behavioral phenotypes (Whitlock, 1993; Phillis, 1993). The second mutation that abolishes HRP-epitope expression is carried on the TM3 balancer chromosome, an extensively rearranged form of the third chromosome (Snow, 1987). TM3 homozygotes do not express the HRP epitope in the embryonic nervous system but do produce the glycan in the expected non-neural tissues. Therefore, it is likely that the structural genes necessary for synthesis of the HRP epitope are intact and that the TM3 mutation alters a gene that regulates tissue-specific glycosylation (Seppo, 2003).

The TM3 locus that abolishes HRP-epitope expression has been characterized. The affected gene, which has been named 'tollo' encodes a member of the family of cell surface receptors with homology to the Toll protein (Toll-like receptors, TLRs). Genome sequence characterization re-identified the tollo locus, resulting in its designation as 'toll-8' (Tauszig, 2000). The founding member of the TLR family (Toll) was originally identified as a component of the signaling pathway that induces dorsal-ventral polarity in the Drosophila embryo. Subsequently, TLRs have also been shown to participate in innate immune responses in Drosophila and other organisms by transducing pattern recognition signals. The induction of tissue-specific glycosylation can now be added to the list of TLR functions (Seppo, 2003).

Nearly a half-century of molecular biochemistry has documented tissue-specific, cell-specific, stage-specific, and disease-specific oligosaccharide presentation. Despite this wealth of information, few descriptions exist of molecular mechanisms that control the specificity of glycan expression. Glycan synthesis has been modulated by manipulating various transmembrane signaling pathways, indicating that receptor-mediated events at the cell surface can influence oligosaccharide profiles. Although the surface receptors that transmit these signals have not been identified, the results of this study demonstrate that the Tollo/Toll-8 transmembrane protein influences cell-specific glycan expression in the Drosophila embryo (Seppo, 2003 and references therein).

The localization of Tollo expression to non-neural ectodermal cells and the rescue of neural-specific glycosylation by transgenic tollo both demonstrate that non-homologous cells modulate glycan expression in adjacent tissues. The close proximity of Tollo-expressing ectodermal cells to differentiating neurons is consistent with a molecular mechanism in which neural glycosylation is influenced by the activity of a neuronal surface receptor that directly binds ectodermal Tollo. Alternatively, the molecular activity of tollo may reside entirely within the ectodermal cell, exerting an indirect influence on neural glycosylation by propagating or attenuating instructive signals subsequently interpreted by local neurons. At present, the data cannot unambiguously distinguish whether the direct or indirect mechanism applies. However, the results of HRP-epitope rescue and Tollo misexpression studies indicate requirements that both models must satisfy (Seppo, 2003).

Neuronal synthesis of the HRP-glycan is rescued in Brd¹⁵/Brd¹⁵ embryos when Tollo is expressed in its wild-type ectodermal pattern (tollo-Gal4/UAS-tollo). However, ELAV-Gal4/UAS-tollo and rho-Gal4/UAS-tollo embryos fail to rescue the HRP epitope, despite driving misexpression in neurons and glia that would present Tollo to neuronal surfaces at developmental stages coincident with the normal Tollo expression pattern. Therefore, if tollo acts directly to alter neural glycosylation, ectodermal presentation of Tollo must be unique in comparison with expression in other cell types that also share contact with neurons; either the Tollo protein requires an ectodermal-specific post-translational modification for activity or an ectodermal co-factor is necessary for appropriate presentation to neurons. If tollo indirectly affects neural glycosylation by generating or influencing paracrine signals sensed by differentiating neurons, then the cellular context in which Tollo is expressed determines induction of the HRP epitope; either the relevant paracrine influence is specifically of ectodermal origin or a required tollo intracellular signaling pathway is absent from neurons and glia (Seppo, 2003).

The indirect mechanism is consistent with the function of other TLRs and is supported by two additional observations. (1) The discontinuous distribution of tollo mRNA in the neurogenic ectoderm of the ventral nerve cord indicates that tollo expression is limited to ectodermal cells contacting only a subset of the total differentiating neuron pool. Therefore, global CNS expression of the HRP epitope requires a signal unrestricted by the need for cell-cell contact. (2) HRP-epitope expression is rescued in PNS sensory neurons located near salivary glands that ectopically express Tollo, consistent with the generation of a locally active signal. Expression of the HRP epitope is not rescued in the CNS nor in more remote parts of the PNS by hsp70-Gal4/UAS-tollo, implying that temporal and physical barriers can limit Tollo activity (Seppo, 2003).

The unexpected, ectopic expression of the HRP epitope in the secretory epithelium of the salivary gland indicates that some developing tissues are only one signal away from assuming an altered glycosylation phenotype. At least within the salivary gland, this result also indicates that Tollo can act directly or can generate an autocrine signal that autonomously modulates glycosylation. For neural tissue, though, elaboration of the HRP epitope is a non-autonomous neuronal behavior that requires ectodermal Tollo expression. By analogy to Toll, soluble protein ligands (like Spätzle) are prime candidates for the Tollo activator, but the full diversity of TLR ligands has yet to be characterized in any organism (Seppo, 2003).

In plants and in the Drosophila adult, HRP-epitope structure has been demonstrated to contain an extensively trimmed high-mannose core carrying an alpha3-linked Fuc residue on the internal GlcNAc of the chitobiose. To generate the described Drosophila HRP epitopes, high-mannose oligosaccharides must first be trimmed to a Man₃GlcNAc₂ or Man₂GlcNAc₂ core. The core structure is then di-fucosylated (alpha3 and alpha6), requiring the activity of two distinct fucosyltransferases. Addition of Fuc alpha3 to the core requires previous and transient addition of GlcNAc to a terminal Man, yielding a di-fucosylated Man_2/3GlcNAc₂ oligosaccharide. Therefore, trimming mannosidases, two fucosyltransferases, an N-acetylglucosaminyltransferase and a hexosaminidase constitute the minimal set of processing activities required to generate an HRP epitope. Of these activities, addition of the alpha3 Fuc imparts antibody recognition to the oligosaccharide (Seppo, 2003).

A Drosophila fucosyltransferase that adds Fuc in alpha3 linkage to core GlcNAc has been characterized (Fabini, 2001). Designated 'FucTA', the enzyme exhibits in vitro acceptor specificity appropriate for synthesis of the HRP epitope and the gene maps to 71B2, 87 kb distal to tollo. Although this lies within the Brd¹⁵ deletion, combining tollo-Gal4 with UAS-tollo results in rescue of HRP epitope expression in Brd¹⁵ homozygotes. Therefore, glycan expression is rescued by Tollo/Toll-8 in a FucTA null background. The relevance of FucTA activity to HRP-epitope expression in the embryonic nervous system remains to be determined, but Drosophila requires alpha3 fucosyltransferase activity and the resulting capacity to synthesize the HRP epitope in multiple contexts. Mutants that lack the HRP epitope in larval and adult stages express the oligosaccharide embryonically and epitope expression in embryonic non-neural tissue is maintained in mutants that lack the embryonic neural oligosaccharide (Snow, 1987; Katz, 1988). Thus, multiple pathways, under independent control and active in different tissues and developmental stages, lead to synthesis of the HRP epitope (Seppo, 2003).

These results suggest superficially that loss of the HRP epitope is of relatively little consequence. However, the component of the HRP epitope structure that imparts antibody recognition may be distinct from the functional domain of the oligosaccharide. Therefore, mutations in genes such as tollo, which affect specific carbohydrate expression, may not immediately reveal oligosaccharide function. The nac mutant, which lacks larval, pupal and adult expression of the HRP epitope, exhibits grossly normal nervous system morphology (Katz, 1988; Phillis, 1993). Highly penetrant axon defasciculation errors are present in the nac adult but only become apparent when afferent projections arising from discrete subsets of dye-labeled sensory neurons in the wing margin are visualized at their entry point into the central nervous system (Whitlock, 1993). Until techniques of similar resolution are applied to embryos that lack the HRP epitope, the functional significance of loss of this tissue-specific glycan cannot be fully evaluated (Seppo, 2003).

In Drosophila, the HRP epitope is present on several neural proteins, many of which are also expressed in non-neural tissue where they lack the glycan (Desai, 1994; Snow, 1987; Sun, 1995; Wang, 1994). Thus, cells determine whether or not to construct the HRP epitope on a particular glycoprotein based on the tissue in which the protein is expressed, rather than on a signal intrinsic to the polypeptide. While tollo/toll-8 demonstrates that such tissue-specific glycan expression can be achieved through the activity of a Toll-like receptor, the correlation between Drosophila TLR expression and specific glycosylation patterns cannot be comprehensively assessed before glycan characterization in the Drosophila embryo is greatly expanded. Nonetheless, distributions of other TLRs exhibit spatial and temporal overlap with Tollo expression, raising the possibility that TLRs sculpt embryonic glycosylation patterns through combinatorial activation of glycosylation pathways in interacting domains of developing tissues (Seppo, 2003).

TLRs mediate pattern recognition (frequently glycan-based) as part of the innate, non-adaptive immune response in Drosophila and vertebrates. However, only a subset of Drosophila TLRs induce defensive responses. TLR family members appear divided into clans that function in innate immunity or that fulfill developmental needs (Tauszig, 2000). The capacity to control glycosylation could unite the TLR family in support of a common cause, to produce appropriate spatial and temporal patterns of cell-specific glycosylation. Expressed by immune cell types that participate in tissue surveillance, TLRs are positioned to locally influence cellular glycosylation in response to pathogen, thereby coupling innate detection of non-self patterns with expression of protective glycans on host cells. In addition, further analysis of the distribution and function of TLRs may indicate that the constitutive maintenance of diverse tissue glycan profiles is generally an active process in which glycan expression is continually renewed or responsively modified by TLR-mediated signaling. In mature tissues and in the embryo, the expression of glycans must be orchestrated to coincide with the appearance of relevant carbohydrate binding proteins that mediate cell adhesion and recognition. Therefore, broader mechanisms that impart specificity to cell-cell interactions are likely to be revealed with further characterization of the pathway by which tollo/toll-8 controls oligosaccharide expression (Seppo, 2003).

DEVELOPMENTAL BIOLOGY

Embryonic

Expression of tollo mRNA is first detected in the cellular blastoderm, initially as prominent bands at both ends of the embryo. Very rapidly, tollo mRNA appears in dorsoventral bands repeated along the entire length of the embryo. As germband retraction begins, late in stage 12, the bands of tollo mRNA span the entire width of the germband, placing Tollo expression within ectodermal domains from which ventral nerve cord precursor cells differentiate and delaminate. By the time germband retraction is complete (stage 13), tollo mRNA expression disappears from the ventral ectoderm that underlies the delaminated, discrete nerve cord. Expression of the HRP epitope in the ventral nerve cord is first detected reproducibly at stage 14, shortly after tollo mRNA decreases. Thus, Tollo expression in ventral ectoderm coincides with a period of maximal contact with differentiating neurons and disappears once neurons segregate from the ectoderm to form a consolidated ventral nerve cord (Seppo, 2003).

In the lateral ectoderm, tollo mRNA is found in distinct, segmentally repeated domains at stage 13. Together, these repeated domains form continuous anteroposterior stripes of ectodermal expression. Within each domain, expression is not uniform. Cells at the segment boundaries express higher levels of tollo mRNA, forming ectodermal pockets that are partly lined with Tollo-expressing cells. By early stage 15, tollo mRNA is greatly reduced in the lateral ectoderm and expressing domains are attenuated to a few cells immediately adjacent to segment boundaries. Expression of the HRP epitope in the peripheral nervous system is first detected reproducibly at stage 15, shortly after tollo mRNA expression has decreased in the lateral ectoderm (Seppo, 2003).

Consistent with the determination that the TM3 chromosome has not lost the entire tollo gene, hybridization signal was detected in TM3/TM3 embryo. Thus, RNA that contains tollo sequence is produced in TM3 embryos despite being undetectable in blotted poly-A⁺ mRNA preparations. Hybridization to Brd¹⁵/Brd¹⁵ embryos was not detected at any stage, indicating that the anti-sense probe is specific for tollo and does not cross-hybridize to other embryonically expressed Toll-like receptors (Seppo, 2003).

The position of Tollo-expressing domains along the dorsoventral axis of the lateral ectoderm closely approximates the site of proneural cluster formation. Therefore, tollo mRNA expression was localized relative to the position of differentiating neurons in the peripheral nervous system. Within the lateral domains of Tollo expression found in each segment, maturing neurons occupy patches that display reduced or undetectable tollo mRNA. At stage 14, all but the earliest neurons to differentiate (which have actively begun to migrate away from their birthplace towards their final embryonic positions) are found in close association with ectodermal cells that express tollo mRNA. Thus, in the peripheral nervous system, as in the ventral nerve cord, Tollo expression coincides temporally with periods of neural differentiation that are characterized by maximal contact between the ectoderm and neural precursor cells (Seppo, 2003).

To determine whether tollo is sufficient to rescue expression of the HRP epitope in the Brd¹⁵ homozygote, a transformation construct was generated (pUASTtollo) that placed tollo-coding sequence under the control of UAS elements. A second transformation construct was prepared (ptolloGal4) that placed Gal4 expression under control of 2.5 kb of Drosophila genomic DNA found immediately upstream of the tollo initiation codon. Tollo-Gal4 transformant lines were crossed to a UAS-lacZ reporter line and embryo collections were stained for ß-galactosidase activity. Both in the germband extended embryo at stage 12 and in the lateral ectoderm of the stage 13 embryo, lacZ activity matched the distribution of tollo mRNA detected by in situ hybridization. Thus, the 2.5 kb of genomic DNA incorporated into the ptolloGal4 transformation vector contains control sequences sufficient to recapitulate normal Tollo expression (Seppo, 2003).

UAS-tollo and tollo-Gal4 transformant lines were separately prepared in the Brd¹⁵/TM3 background. HRP-epitope expression is absent from embryos collected from lines bearing either construct alone. However, when UAS-tollo and tollo-Gal4 lines are crossed to each other, HRP-epitope expression is rescued in embryos that lack (Brd¹⁵/TM3 and TM3/TM3 genotypes) and in embryos that possess (Brd¹⁵/Brd¹⁵) the head involution defect associated with the Brd¹⁵ deletion. Thus, the head involution defect is independent of HRP-epitope expression. Tollo-Gal4/UAS-tollo rescues HRP-epitope expression in the ventral nerve cord and in the peripheral nervous system (Seppo, 2003).

Other Gal4 driver lines were screened for their ability to rescue the HRP epitope in UAS-tollo transformants. Neither a pan-neural driver (ELAV-Gal4) nor a mesectodermal/midline glial driver (rhomboid-Gal4) rescued oligosaccharide expression when crossed to UAS-tollo, despite their ability to drive expression in cells that make extensive contact with neuronal surfaces. Therefore, simple juxtaposition of Tollo protein and a neuron is insufficient; induction of the neuronal HRP epitope requires Tollo expression in appropriate non-neural ectodermal cells (Seppo, 2003).

Heat-shock driven expression of Tollo in all cells (hsp70-Gal4/UAS-tollo) generates early embryonic lethality that precludes assessment of HRP-epitope rescue. However, in the course of these experiments, hsp70-Gal4/UAS-tollo embryos not subjected to heat shock were also collected and stained with anti-HRP antibody. Unexpectedly, unshocked embryos older than stage 15 express the HRP epitope in the salivary gland and in sensory neurons most proximal to the gland. Other neuronal populations were not stained, whether in the CNS or in more posterior segments of the PNS. Thus, leaky Gal4 expression in the salivary gland (verified by UAS-lacZ reporter) is sufficient to induce the HRP epitope in a tissue that does not normally express the glycan and is able to rescue the epitope in nearby sensory neurons (Seppo, 2003).

Antiserum against horseradish peroxidase (anti-HRP Ab) labels the surfaces of neurons in both Drosophila and grasshopper. The anti-HRP Ab immunoprecipitates at least 17 different membrane glycoproteins from the Drosophila embryo CNS (and a similar array from grasshopper). Anti-HRP Ab recognizes a neural-specific carbohydrate moiety expressed by most if not all of these proteins. Although the anti-HRP Ab stains all axon pathways, 2 of the anti-HRP glycoproteins, Fasciclin I and II, are expressed on specific subsets of axon pathways in the grasshopper embryo (Snow, 1987).

In embryos, the fasciclins are localized to axonal subsets, while the RPTPs appear to be expressed on most or all CNS axons. To identify other neuronal cell surface glycoproteins in the Drosophila embryo, a biochemical approach has been taken. This is based on the observation that antisera against horseradish peroxidase (HRP) recognize a carbohydrate epitope that is selectively expressed in the insect nervous system. A large number of neuronal glycoproteins (denoted 'HRP proteins') apparently bear the HRP carbohydrate epitope. Polyclonal anti-HRP antibodies have been used to purify these proteins from Drosophila embryos, and protein sequences have been obtained from seven HRP protein bands. These data define three major HRP proteins as Neurotactin, Fasciclin I, and an RPTP, Ptp69d. Fasciclin II, Neuroglian, Ptp10D, and Ptp99A are also HRP proteins (Desai, 1994).

Antibodies recognizing horse radish peroxidase (HRP) stain neurons in Drosophila and other insects. Western blots were used to analyze and characterize some of the anti-HRP reactive components from Drosophila melanogaster. Anti-HRP reactive components can be reproducibly detected during all developmental stages, although the pattern changes at different developmental times. In adults, there are at least 10 reproducibly stained components. Two of the bands, with molecular sizes of 42 and 80 kDa are likely to be the major contributors to neuronal anti-HRP staining in Drosophila. These components are enriched in adult fly heads. In contrast, many of the other anti-HRP reactive components in adults are enriched in abdomen and are present exclusively or at much higher levels in male flies. Two of the male specific components with molecular sizes of 62 and 48 kDa have been purified and characterized. Partial N-terminal amino acid sequencing revealed that the 62 kDa protein is identical to a part of D. melanogaster carboxylesterase, while the 48 kDa protein does not match any known sequences. Esterase-6 has previously been shown to be enriched in male accessory gland and consistent with this, anti-HRP antibodies also have been shown to stain this gland (Wang, 1994).

Characterizing the gene product(s) recognized by anti-HRP antibodies is of interest because it may be important for nervous system function and/or development. An anti-HRP-reactive Mr 42K glycoprotein has been identified and purified from adult Drosophila heads that is likely to be the major contributor to neuronal specific anti-HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between Mr 38K and 42K that vary as a function of developmental age. These components have been collectively named Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti-HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti-HRP staining in adult nervous system (Sun, 1995).

A positional Toll receptor code directs convergent extension in Drosophila

Elongation of the head-to-tail body axis by convergent extension is a conserved developmental process throughout metazoans. In Drosophila, patterns of transcription factor expression provide spatial cues that induce systematically oriented cell movements and promote tissue elongation. However, the mechanisms by which patterned transcriptional inputs control cell polarity and behaviour have long been elusive. This study demonstrates that three Toll family receptors, Toll-2 (18 wheeler), Toll-6 and Toll-8, are expressed in overlapping transverse stripes along the anterior-posterior axis and act in combination to direct planar polarity and polarized cell rearrangements during convergent extension. Simultaneous disruption of all three receptors strongly reduces actomyosin-driven junctional remodelling and axis elongation, and an ectopic stripe of Toll receptor expression is sufficient to induce planar polarized actomyosin contractility. These results demonstrate that tissue-level patterns of Toll receptor expression provide spatial signals that link positional information from the anterior-posterior patterning system to the essential cell behaviours that drive convergent extension (Pare, 2014).

Together, these results demonstrate that the spatial signals that establish planar polarity and direct polarized cell behaviour during convergent extension in Drosophila are encoded at the cell surface by three Toll family receptors expressed in overlapping stripes along the AP axis of the embryo. Simultaneous disruption of Toll-2, Toll-6 and Toll-8 significantly impairs planar polarity, cell intercalation, and convergent extension, and removing one or two receptors disrupts planar polarity in distinct subsets of cells, indicating that these proteins serve non-redundant and highly localized functions. These findings support a model in which planar polarity is induced by interactions between neighbouring cells with different levels of Toll receptor activity. Therefore, Drosophila Toll receptors provide the basis of a spatial code that translates patterned Eve and Runt transcriptional activity into planar polarized actomyosin contractility, linking positional information provided by the embryonic AP patterning system to the essential cell behaviours that drive convergent extension. The Toll receptor code is incomplete in certain regions, such as the parasegmental boundaries, suggesting the existence of additional polarity cues at these interfaces. Toll-2,6,8 mutants are similar to runt mutants with respect to all measures of cell rearrangement and planar polarity, but are not as severe as eve mutants. Thus, although Toll-2,6,8 mutants recapitulate much of the eve mutant phenotype, Eve likely has additional targets important for planar polarity (Pare, 2014).

Toll family receptors have a highly conserved structure in vertebrates and invertebrates, including extracellular LRR motifs that are often present in proteins involved in cell adhesion and cell-cell recognition. Although individual receptors are not orthologous between flies and humans, mammalian Toll-like receptors are required for epithelial regeneration and wound healing, processes that involve dynamic and spatially regulated changes in cell adhesion. In the innate immune system, pathogen detection by Toll family receptors activates transcriptional pathways mediated by NF-κB and MAP kinase signalling. However, the spatial information provided by patterned Toll receptor expression in Drosophila, as well as the rapid timescale of cell rearrangements during convergent extension, suggest a more direct connection between Toll receptor signalling and the cellular contractile machinery. Consistent with this possibility, activation of mammalian Toll-like receptors in dendritic cells induces a rapid remodelling of the actin cytoskeleton and mammalian Toll-like receptors can inhibit neurite outgrowth and trigger rapid growth cone collapse in neurons, reminiscent of Toll receptor functions in the Drosophila nervous system. Elucidating the mechanisms that link Toll family receptors to dynamic changes in cell polarity and behaviour may provide insight into conserved and relatively unexplored aspects of Toll receptor signalling (Pare, 2014).

An ancient defense system eliminates unfit cells from developing tissues during cell competition

Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. This study reports that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. Toll-related receptors (TRRs) and the cytokine Spatzle (Spz) lead to NFκB-dependent apoptosis. Null mutations in Toll-3, Toll-8, or Toll-9 suppress elimination of loser cells, increasing loser clone size and cell number per clone, but do not alter control clones. Diverse 'loser' cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. These findings demonstrate a functional repurposing of components of TRRs and NFkappaB signaling modules in the surveillance of cell fitness during development (Meyer, 2014).

Altogether, these results demonstrate that the conceptual resemblance between cell competition and innate immunity is matched with genetic and mechanistic similarities. Thus, cells within developing tissues that are recognized as mutant or compromised are competitively eliminated via a TRR- and NFκB-dependent signaling mechanism. Although similar core signaling components are activated in both processes, cell competition culminates in local expression of proapoptotic genes rather than systemic induction of antimicrobial genes. Because cell competition is initiated by the emergence of cells of different fitness than their neighbors in a tissue, it is surmised that the initiating signal is common to many competitive contexts. The genetic data leads to a proposal of a model for how this signal is detected and transduced. The results point to a role for Spz in signal detection, as it is a secreted protein that is required for the killing activity of competitive conditioned medium (cCM), is a known ligand for the Toll receptor, and is produced by several tissues in the larva. Thus, it is speculated that Spz functions as a ligand for one or more TRR in cell competition. Because Spz must be activated through a series of proteolytic steps, the relevant proteases may respond directly to the initiating signal in cell competition. It is proposed that the genetic identity or context of the competing populations influences activation of different TRR signaling modules and that the precise configuration of TRRs on loser cells dictates which of the three Drosophila NFκB proteins is activated. How signaling to the NFκBs is restricted to the loser cells is not known, but higher expression of Toll-2, Toll-8, and Toll-9 in loser cells could bias signal transduction. PGRP-LC, a receptor known to bind only bacterial products, also plays a role in Myc-induced competition. As commensal gut microflora is known to influence larval growth, this raises the possibility that it also contributes to the competitive phenotype (Meyer, 2014).

Throughout evolution, signaling modules have adapted to fulfill different functions even within the same species. This study has provided evidence for adaptation of TRR-NFκB signaling modules in an organismal surveillance system that measures internal tissue fitness rather than external stimuli. It is noteworthy that the killing of WT cells by supercompetitor cells is a potentially pathological form of cell competition that could propel expansion of premalignant tumor cells. If so, activated TRR-NFκB signaling modules in nonimmune tissues could be diagnostic markers, and their competitive functions could serve as therapeutic targets for cancer prevention (Meyer, 2014).

EFFECTS OF MUTATION

The nature of anti-HRP antigens was investigated in Drosophila and found to include a complex set of developmentally regulated proteins. Their common epitope appears to be a carbohydrate that shares features with the sugar moiety of pineapple stem bromelain, a plant glycoprotein whose carbohydrate structure has been determined. A mutation was identified that eliminates staining by the antibody in imaginal and adult neural tissue. Tissue specific glycoconjugates, although widespread in the animal kingdom, are little understood. This mutation provides a unique opportunity to address the consequences of altering a neural specific carbohydrate moiety in an otherwise intact and behaving animal. The mutation maps to 84F. A second mutation, contained on the third chromosome balancer, TM3, eliminates anti-HRP staining in embryos. These mutations appear to be separate genes (Katz, 1988).

Embryos homozygous for the TM3 balancer chromosome fail to express the HRP epitope in the ventral nerve cord and peripheral nervous system, although expression is maintained in three non-neural tissues. TM3 rearrangement breakpoints provide likely candidates for regions of the third chromosome that affect HRP-epitope expression. To assess the relevance of these breakpoints and to ensure that a gene located between TM3 breakpoints was not missed, overlapping deletion stocks that together cover approximately 80% of the third chromosome were screened for their ability to complement loss of the HRP epitope in a TM3 background. The smallest non-complementing deletion (breakpoints 71A1/2-71C1/2) is carried in a Bearded stock designated Brd¹⁵. The combination Brd¹⁵/TM3 or Brd¹⁵/TM3-lacZ produces viable and fertile adults (Seppo, 2003).

Of the relevant genotypes, only Brd¹⁵ homozygotes display gross morphologic aberrations. Brd¹⁵/Brd¹⁵ embryos develop normally until stage 14 when defects in the formation of anterior terminal structures become apparent. In particular, retraction of the clypeolabrum is stalled in Brd¹⁵ homozygotes, causing the supraesophageal ganglia (embryonic brain lobes) to appear exteriorized. The head involution defect provides an unambiguous, reliable diagnostic for the Brd¹⁵ homozygous genotype. Examination of neural tissue integrity by monoclonal antibody staining demonstrates that longitudinal and commissural bundles are present in the central nervous system (mAb BP102), appropriate cell numbers and approximate cellular relations are preserved in the peripheral nervous system (mAb 22C10), and efferent motor pathways develop normally (mAb 1D4) in Brd¹⁵ homozygotes. Thus, neural differentiation and axon extension, to the extent that they are revealed by these mAb markers, are unaffected by Brd¹⁵ (Seppo, 2003).

The proximal breakpoint of the Brd¹⁵ deletion (71C1/2) overlaps a TM3 rearrangement breakpoint at 71C. Therefore, P1 phage clones that map to the 71C1/2 interval were obtained and probed with ³²P-end-labeled mRNA prepared from embryo collections from OreR or Brd¹⁵/TM3 stocks. The P1 phage clone designated DS06206 contains a 2.4 kb EcoRI fragment and a 1.1 kb BamHI/XbaI fragment that are both transcribed in OreR but not detected in Brd¹⁵/TM3 embryos. Subsequent sequence analysis placed the 1.1 kb fragment within the 2.4 kb fragment. Probe prepared from the 2.4 kb EcoRI fragment was used to probe Northern blots of poly-A⁺ RNA isolated from OreR or Brd¹⁵/TM3 embryos. A 6.5-7.0 kb band was identified in the OreR preparation that was not detected in Brd¹⁵/TM3 poly-A⁺ RNA. Genomic Southern analysis demonstrates multiple restriction fragment length polymorphisms in the Brd¹⁵/TM3 genotype. Probe prepared from the 2.4 kb EcoRI fragment hybridizes to a 1.5 kb HindIII fragment in OreR that is shifted to approximately 6 kb in Brd¹⁵/TM3. In turn, probe prepared from the 1.5 kb HindIII fragment identifies the same polymorphism as well as 876 bp HindIII/Xba1 and 280 bp PstI fragments that also differentiate the two genotypes. The sequenced 2.4 kb EcoRI genomic fragment yielded an open reading frame of 1250 bp which was extended to 4038 bp in length by further genomic sequencing (Seppo, 2003).

A total of 6735 nucleotides were sequenced, extending from 17 bp upstream of the ORF to 2677 bp beyond the first in-frame stop codon, and found to be co-linear with genomic sequence in GenBank Accession Number AE003531 and with Drosophila cDNA sequence LD33590. The sequence predicts that the HindIII, XbaI and PstI polymorphisms observed in Brd¹⁵/TM3 lie in the 3' UTR of the gene. To more precisely define the polymorphism, 3'-RACE was performed on poly-A⁺ RNA isolated from OreR and Brd¹⁵/TM3 embryos. The fragment amplified from Brd¹⁵/TM3 embryos yielded 969 nucleotides of sequence of which the first 237 matched previously sequenced genomic DNA. However, TM3 sequence diverged from wild-type at a position corresponding to nucleotide 5635, 1.6 kb downstream from the first in-frame stop codon of tollo (nucleotide 4039) and within the 3' UTR predicted by mRNA size. Sequence obtained for the first 732 bases of divergence matches a Drosophila transposable element designated '412', GenBank Accession Number X04132. It was not determined whether the divergent sequence reflects the insertion of an intact transposable element or identifies the site of the TM3 rearrangement breakpoint previously mapped to 71C (Seppo, 2003).

Toll-8/Tollo negatively regulates antimicrobial response in the Drosophila respiratory epithelium

Barrier epithelia that are persistently exposed to microbes have evolved potent immune tools to eliminate such pathogens. If mechanisms that control Drosophila systemic responses are well-characterized, the epithelial immune responses remain poorly understood. This study consisted of a genetic dissection of the cascades activated during the immune response of the Drosophila airway epithelium i.e. trachea. Evidence is presented that bacteria induced-antimicrobial peptide (AMP) production in the trachea is controlled by two signalling cascades. AMP gene transcription is activated by the inducible IMD pathway that acts non-cell autonomously in trachea. This IMD-dependent AMP activation is antagonized by a constitutively active signalling module involving the receptor Toll-8/Tollo, the ligand Spätzle2/DNT1 (Neurotrophin 1) and Ect-4, the Drosophila ortholog of the human Sterile alpha and HEAT/ARMadillo motif (SARM). The data show that, in addition to Toll-1 whose function is essential during the systemic immune response, Drosophila relies on another Toll family member to control the immune response in the respiratory epithelium (Akhouayri, 2011).

Epithelial responses are local responses to prevent the epithelium from unnecessary immune reactions. Since the recognition steps in Drosophila respiratory epithelia involve the transmembrane receptor PGRP-LC and occur within the extracellular space, it is expected that molecular mechanisms must be at work to prevent constitutive or excessive immune response in this tissue, particularly essential for animal growth and viability. This report presents data demonstrating that the transmembrane receptor Tollo is part of a signalling network, whose function is to specifically down-regulate AMP production in the trachea. Tollo antagonizes IMD pathway activation in the respiratory epithelium, and DNT1/Spz2 and Ect4/SARM are putative Tollo ligand and transducer, respectively, in this process. These data demonstrate that, in addition to the family founder Toll-1, another member of the Leucine-Rich-Repeats family of Toll proteins, is regulating the Drosophila innate immune response. Although it has been abundantly documented that every single mammalian TLR has an immune function, the putative implication of Toll family members, other than Toll-1 itself, in the Drosophila immune response has been a subject of controversy. Data showing that Drosophila Toll-9 over-expression was sufficient to induce AMPs expression in vivo has prompted the idea that Toll-9 could maintain significant levels of anti-microbial molecules, thus providing basal protection against microbes. However, a recent analysis of a complete Toll-9 loss-of-function allele has shown that this receptor is neither implicated in basal anti-microbial response nor required to mount an immune response to bacterial infection (Narbonne-Reveau, 2011. The present data are also fully consistent with a recent report showing that Toll-6, Toll-7 and Toll-8 are not implicated in systemic AMP production in flies, and demonstrate that a Toll family member, Tollo, is a negative regulator of local airway epithelial immune response upon bacterial infection. In contrast to Toll-1, whose activation is inducible in the fat body, Tollo pathway activation seems to be constitutive in the trachea. Despite these differences, both receptors use a member of the Spz family as ligand. Interestingly, sequence similarities, intron's size and conservation of key structural residues, indicate that Spz2/DNT1 is phylogenetically the closest family member to the Toll ligand Spz. Furthermore, both Spz and Spz2/DNT1 have been shown to have neurotrophic functions in flies. It would be of great interest to test whether Tollo also mediates Spz2 function in the nervous system (Akhouayri, 2011).

Both during embryonic development and immune response, Spz is activated by proteolytic cleavage. This step depends upon the Easter protease that is implicated in D/V axis specification and on SPE for Toll pathway activation by microbes. Since Spz orthologs are also produced as longer precursors, they are likely to be activated by proteolysis. The fact that Tollo and Spz2 loss-of-function phenotypes correspond to excessive AMP production, suggests that in wild-type conditions, the Tollo pathway is constitutively activated by an active form of the Spz2 ligand. This situation is reminiscent to that observed in the embryonic ventral follicle cells, in which a Pipe-mediated signal induces a constitutive activation of the Easter cascade leading to Spz cleavage, Toll activation and, in turn, ventral fate acquisition. It should be noted that Easter and one Pipe isoform are very strongly expressed in the trachea cells, and are candidate proteins in mediating Tollo activity in the respiratory epithelia (Akhouayri, 2011).

The fact that Ect4, but not dMyd88 mutant, loss-of-function mutant phenocopies Tollo mutant suggest that Ect4 could be the TIR domain adaptor transducing Tollo signal in the tracheal cells. Alternatively, Ect4/SARM could mediate Tollo function by interfering with IMD pathway signalling. In mammals, SARM is under the transcriptional control of TLR and negatively regulates TLR3 signalling by directly interfering with the association between the RHIM domain-containing proteins TRIF and RIP (Carty, 2006). Since PGRP-LC contains a RHIM domain as TRIF, and IMD is the Drosophila counterpart of RIP, one can envisage that Drosophila SARM could act by interfering with the PGRP-LC/IMD association required for IMD pathway signalling. Similarly to its function as a negative regulator in fly immunity, SARM is the only TIR domain-containing adaptor that acts as a suppressor of TLR signalling (Akhouayri, 2011).

One obvious question relates to the mode of action of Tollo on IMD pathway downregulation. Two mechanisms have been recently described that result in the down-regulation of the IMD pathway. The first one regulates PGRP-LC membrane localization, and is dependent on the PIRK protein (Lhocine, 2008). Upon infection, the intracellular PIRK protein is up-regulated and, in turn, represses PGRP-LC plasma membrane localization leading to the shutdown of the IMD signalling (Lhocine, 2008). In infected pirk mutants, IMD-dependent AMPs are overproduced in both the gut and the fat body. In the conditions used in this study, however, inactivation of PIRK specifically in the trachea did not influence Drosomycin activation in trachea. To verify whether Tollo is acting via a mechanism similar to PIRK, PGRP-LC membrane localization was examined using a UAS-PGRP-LC::GFP construct. PGRP-LC membrane localization was identical in wild-type and Tollo mutant tracheal cells. The second mechanism that modulates IMD activation, acts directly on the promoters of IMD target genes. Caudal transcription factor has been shown to sit on some of the IMD target promoters preventing their activation by Relish. The putative implication of Caudal in Tollo signalling was tested by using Drs-GFP reporter transgenes containing either wild-type Caudal Responsive Elements (CDREs) or mutated versions unresponsive to Caudal activity. Upon infection, Drs-GFP with mutated CDREs was activated in fat body but not in gut or trachea. In conclusion, Caudal acts as a transcriptional activator, rather than a repressor, for the Drs-GFP reporter in trachea. These results indicate that Tollo does not regulate the IMD pathway via PGRP-LC membrane localization or through promoter targeting of Caudal. One challenging task for the future will be to identify the mechanism used by Tollo to counter-balance tracheal PGRP-LC activation. It has been reported that the loss of Tollo function in ectodermal cells during embryogenesis alters glycosylation in nearby differentiating neurons. Since the pattern of oligosaccharides expressed in a cell can influence its interactions with others and with pathogens, Tollo could function by modifying glycosylation pattern in response to microbes. It could be envisaged that Tollo mediates PGRP-LC glycosylation, and thereby reduces its ability to respond to bacterial elicitors. Further work will be required to address the above hypothesis, whereby Tollo activity and glycosylation modification could be linked in order to regulate the IMD pathway activation in trachea (Akhouayri, 2011).

REFERENCES

Search PubMed for articles about Drosophila Tollo

Akhouayri, I., Turc, C., Royet, J. and Charroux, B. (2011). Toll-8/Tollo negatively regulates antimicrobial response in the Drosophila respiratory epithelium. PLoS Pathog. 7(10): e1002319. PubMed Citation: 22022271

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Lhocine, N., et al. (2008). PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling. Cell Host Microbe 4: 147-158. PubMed Citation: 18692774

Meyer, S. N., Amoyel, M., Bergantinos, C., de la Cova, C., Schertel, C., Basler, K. and Johnston, L. A. (2014). An ancient defense system eliminates unfit cells from developing tissues during cell competition. Science 346: [Epub ahead of print]. PubMed ID: 25477468

Narbonne-Reveau, K., Charroux, B. and Royet, J. (2011). Lack of an antibacterial response defect in Drosophila Toll-9 mutant. PLoS One 6: e17470. PubMed Citation: 21386906

Pare, A. C., Vichas, A., Fincher, C. T., Mirman, Z., Farrell, D. L., Mainieri, A. and Zallen, J. A. (2014). A positional Toll receptor code directs convergent extension in Drosophila. Nature 515(7528):523-7. PubMed ID: 25363762

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date revised: 25 September 2023

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