InteractiveFly: GeneBrief

18 wheeler : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

18 wheeler, so named because its segmental expression pattern resembles that of the segment polarity genes, and as well, the tied down, seemingly segmented tarpaulin over the rear half of an 18 wheeled semi (a large tractor-trailor truck), was characterized in two laboratories at the same time. In the Bellen lab (Eldon, 1994), 18w was characterized in a search for full length cDNAs of the couch potato gene (Bellen, 1992). In the Beachy lab, 18w was characterized because of the segmentally reiterated transverse stripes of beta-galactosidase expression in a P-element insertion line (Chiang, 1994).

18 wheeler is expressed early in head segments and in an alternating segment polarity pattern corresponding to the domains of wingless and engrailed expression. A comparison of the late expression patterns of 18w and Toll, coding for another leucine rich repeat protein, indicates that the genes are coexpressed in many tissues: the tracheal placodes, some midline cells, the developing salivary glands, the epidermis at the intersegmental furrows, the pharynx, the cells that form the dorsal vessel, the Malpighian tubules, and the hindgut. Although the expression patterns show differences, the genes are frequently coexpressed in regions undergoing extensive cell movements. Both proteins are approximately the same size and share similar motifs throughout most of the protein, including Interleukin 1 receptor homology in the cytoplasmic domain. Both proteins behave as heterophilic cell adhesion molecules when tested for adhesion in Schneider 2 cells, suggesting that they must be able to interact with one or more proteins that are constitutively expressed by these cultured cells. In spite of these similarities, there are no obvious genetic interactions between various mutant alleles of 18w and Toll (Eldon, 1994)

18-Wheeler (18W), is a critical component of the humoral immune response. 18-wheeler is expressed in the larval fat body, the primary organ of antimicrobial peptide synthesis. 18W is also detected in the lymph gland, garland cells and salivary glands. Transcript levels of 18w increase after bacterial infection. In the absence of the 18W receptor, larvae are more susceptible to bacterial infection. Nuclear translocation of the Rel protein Dorsal-like immunity factor (Dif) is inhibited, though nuclear translocation of another Rel protein, Dorsal, is unaffected. Induction of several antibacterial genes is reduced following infection, relative to wild-type: attacin is reduced by 95%, cecropin by 65% and diptericin by 12% (Williams, 1997).

18 wheeler regulates apical constriction of salivary gland cells via the Rho-GTPase-signaling pathway

Rho GTPase and its upstream activator, guanine nucleotide exchange factor 2 (RhoGEF2), have emerged as key regulators of actin rearrangements during epithelial folding and invagination (Nikolaidou, 2004). A Rho-GTPase-signaling pathway is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation. This study shows that Drosophila 18 wheeler (18W), a Toll-like receptor protein, is a novel component of the Rho-signaling pathway involved in epithelial morphogenesis. 18w mutant embryos have salivary gland invagination defects similar to embryos that lack components of the Rho pathway, and ubiquitous expression of 18W results in an upregulation of Rho signaling. Transheterozygous genetic interactions and double mutant analysis suggest that 18W affects the Rho-GTPase-signaling pathway not through Fog and RhoGEF2, but rather by inhibiting Rho GTPase activating proteins (RhoGAPs). RhoGAP5A and RhoGAP88C/Crossveinless-c (CV-C) are required for proper salivary gland morphogenesis, implicating them as potential targets of 18W (Kolesnikov, 2007).

Based on microarray experiments, 18w is downregulated in Scr mutants, implicating it in salivary gland development. To follow 18w expression in more detail, RNA in situ hybridizations were performed. The results show that in wild-type embryos 18w RNA is not maternally contributed but is expressed in salivary gland cells prior to and throughout their invagination. Within the salivary placode, 18w expression first becomes evident at stage 11 as a small spot in the dorsal posterior region. During early stage 12, following the beginning of invagination, 18w expression spreads throughout the placode. During the remainder of stage 12, 18w transcripts can be detected in both gland cells that have yet to invaginate as well as those that have already internalized, albeit at a reduced intensity. At stage 13, 18w transcripts cease to be expressed in salivary gland cells but are evident in salivary duct cells. In addition to salivary glands and ducts, 18w RNA is also detected in other tissues undergoing morphogenesis, including the tracheal placodes and the hindgut. As anticipated from microarray experiments, 18w transcripts are absent exclusively from parasegment two in Scr mutants while expression in the rest of the embryo remains unaltered. Overall, performing microarray experiments with Scr mutant embryos has proven to be an effective method for identifying salivary gland genes (Kolesnikov, 2007).

While 18w is expressed in several tissues undergoing morphogenesis, embryonic defects have yet to be identified in 18w mutant embryos. The striking 18w expression within the invaginating salivary gland prompted a more carefully investigation of the role of 18w in embryonic salivary gland development. In examining a null allele of 18w, 18w^Δ21, it was discovered that initiation of invagination in the dorsal posterior region of the placode appears normal. However, during the next phase of invagination, in which the remaining cells of the salivary placode normally internalize in a strict sequential order, defects become apparent. In 18w mutants invagination is less synchronized; too many placode cells invaginate simultaneously rather than sequentially, thereby causing a wider lumen in 66% of 18w mutants when compared to wild-type embryos. Thus, it is the timely progression of invagination, not the invagination process itself, that is affected in 18w mutants. Due to the lack of proper coordination in 18w, the internalization of the gland cells appears to be delayed causing the cells that internalize last to remain near the ventral surface instead of reaching their final, more dorsal, position within the embryo. Eventually all of the salivary gland cells do internalize in 18w mutants, but the proximal part of the gland, which remains abnormally close to the ventral surface, becomes caught in the anterior movement of the ectoderm during head involution. As a result, the glands end up much closer to the anterior end of the embryo than in wild-type embryos. Similar defects are seen in mutants homozygous for the loss of function 18w allele, 18w^Δ7–35, and in 18w^Δ21, 18w^Δ7–35 transheterozygous mutant embryos. Based on these results, 18w is an important component in coordinating salivary gland invagination (Kolesnikov, 2007).

Activation of the Rho-signaling pathway results in the phosphorylation of the myosin II regulatory light chain, encoded by the spaghetti squash (sqh) gene. The phosphorylated form of Sqh can interact with actin and cause actomyosin-based contractility at the apices of cells. To verify that 18W is part of the Rho pathway, whether overexpression of 18W results in an upregulation of Rho signaling, evident as an increase in phosphorylation of Sqh, was checked. Immunoblot analysis on embryonic extracts using anti-phospho-Sqh antibody reveals that overexpressing 18W throughout the embryo results in a two-fold increase of P-Sqh when compared to wild type. Similar results are seen when a constitutively active form of Rho is overexpressed ubiquitously in the embryo. Moreover, introducing one copy of a phosphomimetic sqh transgene, sqhE20E21, into an 18w mutant rescues the 18w invagination defects, indicating that 18W acts upstream of Sqh phosphorylation. Thus, both genetic and biochemical evidence indicate that 18W is a novel component of the Rho signaling pathway (Kolesnikov, 2007).

To determine whether 18W negatively regulates the RhoGAP branch of the Rho pathway, it was necessary to identify the RhoGAPs involved in salivary gland development. Of the 20 distinct RhoGAPs encoded by the Drosophila genome, 17 UAS-RhoGAP dsRNA lines were examined. Each of these lines was crossed to flies containing a salivary gland-expressing driver, scabrous-GAL4, and the progeny were screened for salivary gland defects. Only RhoGAP5A dsRNA expression resulted in salivary gland defects. Invagination defects are evident from more anteriorly placed glands, while migration defects resulted in wavy glands. Similar defects are seen upon expressing high levels of constitutively active Rho in the salivary gland with the scabrous-GAL4 driver. Overexpressing low levels of Rho in the gland results in mostly migratory defects, indicating that gland migration is more sensitive to the levels of Rho signaling than is the process of invagination. Invagination defects are seldom accompanied by migration defects, presumably because glands that have not properly internalized do not reach the visceral mesoderm upon which they normally migrate (Kolesnikov, 2007).

Since the GAL4/UAS dsRNA system may not result in a complete loss-of-function phenotype, strong alleles of two GAPs, RhoGAP68F and RhoGAP88C/Cv-c, which will be referred to simply as Cv-c. These have previously been shown to regulate Rho activity but lack salivary defects using the dsRNA interference technique. While neither rhoGAP68F nor cv-c mutations cause invagination defects, the cv-c mutants do display migration defects similar to those seen in embryos expressing RhoGAP5A dsRNA. To determine whether RhoGAP5A and Cv-c act redundantly within the salivary gland to regulate Rho activity, RhoGAP5A dsRNA within the gland was examined in a cv-c mutant. These embryos should have reduced activity of both RhoGAPs. They have gland invagination and migration defects that are more severe and penetrant than embryos that lack just one of them, indicating that these RhoGAPs are, in fact, partially redundant during salivary gland development (Kolesnikov, 2007).

In situ hybridization in wild-type embryos shows that cv-c RNA is not maternally contributed but is expressed in several tissues undergoing morphogenesis. In addition to its expression within the developing trachea and mesoderm, cv-c, similar to 18w, is expressed in the salivary glands prior to and during their invagination. Unlike 18w, however, cv-c expression does not appear to originate at the initial invagination site but rather initiates expression throughout most of the placode. During the onset of invagination at stage 12, cv-c expression intensifies and continues to be expressed within cells that have internalized until the conclusion of invagination at stage 13 (Kolesnikov, 2007).

To examine whether 18W regulates Rho activity by inhibiting Cv-c, several overexpression and genetic interaction experiments were performed. As might be expected if 18W negatively regulates Cv-c, overexpressing 18w within the salivary gland results in migratory defects similar to those seen in cv-c mutant embryos. In addition to migratory defects, however, some embryos overexpressing 18w also exhibit invagination defects, suggesting that Cv-c may not be the only RhoGAP negatively regulated by 18W. Moreover, lowering the dose of cv-c enhances the defects caused by 18w overexpression and suppresses the 18w mutant invagination defects, further supporting the role of 18W as a negative regulator of RhoGAP signaling. Therefore, genetic interaction experiments indicate that 18W regulates Rho signaling in the salivary gland by inhibiting at least one known RhoGAP (Kolesnikov, 2007).

Previous studies have shown that the Fog ligand activates RhoGEF2 through an as yet unidentified receptor, leading to the apical constriction of cells that form the ventral furrow and posterior midgut. Similar to salivary gland cells in 18w mutants, cells of the ventral furrow and posterior midgut in fog mutants do eventually invaginate but in an uncoordinated and delayed fashion. Since 18w and fog mutants have similar invagination defects, and 18W is a receptor protein that activates Rho signaling, whether 18W might be the FOG receptor was examined. This seems unlikely, however, because FOG overexpression within the salivary gland rescues the 18w mutant salivary gland defects. Since fog mutations do not completely eliminate apical constriction during ventral furrow and posterior midgut formation but RhoGEF2 mutations do, it has been argued that additional pathways must regulate apical constrictions via RhoGEF2. However, since 18w RhoGEF2 double mutants have more severe defects than either of the single mutants, 18W is not one of the additional upstream activators of RhoGEF2. Although neither present downstream of FOG nor upstream of RhoGEF2, 18W does appear to be positioned upstream of Sqh phosphorylation since the 18w salivary gland mutant phenotype can be rescued by introducing one copy of a phosphomimetic allele of sqh (Kolesnikov, 2007).

One possible way that 18W might activate Rho signaling is by negatively regulating RhoGAPs. Two RhoGAPs, RhoGAP5A and Cv-c, were identified that function partially redundantly during salivary gland morphogenesis. Embryos defective for both RhoGAPs exhibit invagination and migration defects similar to those observed when 18W is overexpressed within the salivary glands. Comparable defects are also seen upon expression of activated Rho, supporting the role of both RhoGAPs and 18W in Rho signaling (Kolesnikov, 2007).

Although overexpression and genetic interaction data demonstrate that 18W does indeed work in opposition to Cv-c activity, whether 18W actually negatively regulates RhoGAPs or if it controls Rho signaling through an alternate and unknown pathway has yet to to be deciphered. Another RhoGAP, RhoGAPp190, is regulated by the Src family of tyrosine kinases in both mammals and Drosophila. Depending on the site of phosphorylation, mammalian RhoGAPp190 can be either activated or inhibited by Src, while the Drosophila RhoGAPp190 appears to be only negatively regulated by the Drosophila Src homolog, Src64B. Genetic interactions and double mutant analysis with 18w and either Src64B or the other Drosophila Src gene, Src42A, however, suggest 18W does not regulate RhoGAPs via Src kinases in Drosophila (Kolesnikov, 2007).

Considering that 18W is a member of the Toll family of receptors, it might signal through the pathway used by Toll itself. Upon activation by its ligand, Spätzle, Toll signals via the cytoplasmic proteins MyD88, Tube, and Pelle to promote the degradation of the Cactus protein. This degradation releases the sequestered transcription factor Dorsal, allowing it to enter the nucleus and activate transcription. Although both 18w and Toll are expressed in the salivary gland, no evidence was found to suggest that 18W signals through the Toll-pathway or that it functions redundantly with Toll. Zygotic tube, pelle, MyD88, or Toll mutant embryos do not have salivary gland defects and MyD88 does not physically interact with any of the Toll-like receptors except for Toll itself. Similarly, there are no obvious genetic interactions between mutant alleles of 18w and Toll based both on lethality and salivary gland abnormalities (Kolesnikov, 2007).

Similar to the Toll family of receptors, many RhoGAPs and RhoGEFs are found in both mammals and flies. The Drosophila genome encodes 21 RhoGAPs and 20 RhoGEFs but only seven Rho-family GTPases. Since a specific RhoGTPase can be regulated by multiple RhoGAPs, there may be some redundancy in the function of the RhoGAPs. This appears to be the case during salivary gland development. Of the 17 RhoGAPs analyzed by RNAi, by available alleles, or by both, two resulted in distinct defects in the salivary glands. Mutant embryos that lack both of these RhoGAPs have more severe and penetrant gland defects than embryos that only lack one, indicating that the two have redundant roles during gland development (Kolesnikov, 2007).

Since 18W is expressed in several tissues undergoing morphogenesis, it will be interesting to establish whether it is important for the development of additional tissues other than the salivary gland. It will also be interesting to determine whether 18W functions in opposition to the particular RhoGAPs that are active within these other tissues. Overall, since very little is known about pathways controlling RhoGAP activity during apical constriction, identifying additional genes that interact with 18W may prove to be important not only in elucidating RhoGAP regulation but also in understanding the process of epithelial invagination (Kolesnikov, 2007).

A Toll-receptor map underlies structural brain plasticity

Experience alters brain structure, but the underlying mechanism remained unknown. Structural plasticity reveals that brain function is encoded in generative changes to cells that compete with destructive processes driving neurodegeneration. At an adult critical period, experience increases fiber number and brain size in Drosophila. This study asked if Toll receptors are involved. Tolls demarcate a map of brain anatomical domains. Focusing on Toll-2, loss of function caused apoptosis, neurite atrophy and impaired behaviour. Toll-2 gain of function and neuronal activity at the critical period increased cell number. Toll-2 induced cycling of adult progenitor cells via a novel pathway, that antagonized MyD88-dependent quiescence, and engaged Weckle and Yorkie downstream. Constant knock-down of multiple Tolls synergistically reduced brain size. Conditional over-expression of Toll-2 and wek at the adult critical period increased brain size. Through their topographic distribution, Toll receptors regulate neuronal number and brain size, modulating structural plasticity in the adult brain (Li, 2020).

Structural brain plasticity and neurodegeneration reveal generative and destructive processes operating in the brain. Plasticity reflects adaptations of the brain to environmental change, involving adult neurogenesis, growth of neurites and synapses, which correlate with learning, experience, physical exercise and anti-depressant treatment; conversely, neuroinflammation, neurodegeneration, loss of neurons, neurites and synapses, correlate with ageing, stress, depression and disease. Structural brain plasticity affects the brain topographically, influencing the specific regions involved in experience-dependent processing. These manifestations suggest that brain function is encoded in physical changes to cells (Li, 2020).

Structural plasticity occurs in the Drosophila brain. Breeding adult flies in constant darkness decreases, and in constant light increases brain volume. Breeding adult flies in isolation vs. crowded conditions, or in single sex vs. mixed groups, also causes brain volume changes. The affected modules include the optic lobe, the mushroom body calyx and central complex. Changes in brain volume are prominent in a critical period spanning from adult eclosion to day 5, and correlate with changes in fiber number. The molecular mechanisms underlying structural brain plasticity are unknown, and discovering them is crucial to understand the normal functionality of the brain as well as its pathological responses to disease (Li, 2020).

Primary candidates to regulate brain plasticity are the neurotrophins. In the mammalian brain, neurotrophins (BDNF, NGF, NT3, NT4) regulate cell proliferation, cell survival, circuit connectivity, synaptic transmission and potentiation. Alterations in neurotrophins underlie brain disease, and anti-depressants increase the levels of the neurotrophin BDNF. NTs have dual functions, as they promote plasticity via p75NTR activating NF-κB, and via Trk receptors activating AKT, ERK and CREB downstream, and they promote neurodegeneration via p75NTR and JNK signalling. Drosophila neurotrophins (DNTs) also regulate neuronal survival and death, connectivity and synaptic structural plasticity. However, there are no canonical tyrosine-kinase-Trk and p75NTR receptors in Drosophila, and instead, DNTs are ligands for the Kekkons, kinase-less members of the Trk family, and Tolls (McIlroy, 2013; Foldi, 2017; Ulian-Benitez, 2017). Drosophila Toll and mammalian Toll-Like-Receptors (TLRs) are best known for their universal function in innate immunity, but also have non-immune functions in development and in the central nervous system (CNS). In neurons, Tolls and TLRs can promote neuronal survival via MyD88 and neuronal death via Sarm, both in flies and mammals. In humans, alterations in TLR function underlie brain diseases from stroke and neurodegeneration to multiple sclerosis and neuroinflammation. Most attention has focused on TLR functions in microglia, their response to damage or infection, and in neuroinflammation. However, TLRs are also in neurons, but functions in neurons and neural progenitor cells are largely unknown. Importantly, TLRs can influence neurogenesis, neuronal survival and death, neurite growth, synaptic transmission and behaviour, including learning and memory. These findings suggest that TLRs could regulate structural brain plasticity, but this remains little explored (Li, 2020 and references therein).

Tolls regulate cell number plasticity in the Drosophila ventral nerve cord (VNC) through a three-tier mechanism (Foldi, 2017). In embryos and larvae, Toll-6 and Toll-7 maintain neuronal survival via MyD88 and NF-κB (McIlroy, 2013; Foldi, 2017). However, in pupae, they can also promote apoptosis via Weckle (Wek), Sarm and JNK (Foldi, 2017). Furthermore, different Tolls lead to different outcomes, for instance, Toll-1 is more pro-apoptotic than Toll-6 (Foldi, 2017). Whether a neuron lives or dies in the CNS depends on the ligand and its cleavage state it receives, the Toll or combination of Tolls it expresses, and the downstream adaptors available for signalling (Foldi, 2017). Thus, cell number control is context dependent. The ability of DNTs and Tolls to regulate cell number by promoting both cell survival and cell death is crucial for the modulation of structural brain plasticity, homeostasis and neurodegeneration (Li, 2020).

This study asked whether Toll receptors influence developmental and structural plasticity in the Drosophila brain (Li, 2020).

At least seven of the nine Toll-receptors are expressed topographically, mapping the distinct modules that form the brain. Toll receptors regulate cell number and brain size in development and structural brain plasticity in the adult, through their ability to promote either cell survival or death, progenitor cell quiescence or proliferation. Evidence indicates that Tolls can underlie the changes that experience brings about in the adult brain, and that structural plasticity and neurodegeneration are two faces of Toll-driven cellular responses in the brain (Li, 2020).

Toll-2 promotes neuronal survival and proliferation, both in development and in the adult brain. Toll-2 is neuroprotective as loss of function caused neurodegeneration: it increased apoptosis and caused neuronal loss, and Toll-2 mutant neurons that survived had dendrite loss, axon atrophy and misrouting. Toll-2 loss of function also impaired climbing and walking, and decreased lifespan, phenotypes characteristic of neurodegeneration. Toll-2 promotes cell survival through the canonical MyD88-NFκB pathway, as previously found for the pro-survival functions of Toll-6 and −7 in development (McIlroy, 2013; Foldi, 2017). Both in flies and mammals, Tolls and TLRs promote cell survival via MyD88 and cell death via Sarm, which activates the pro-apoptotic function of JNK and inhibits MyD88. Distinct Tolls and TLRs can preferentially promote cell survival or cell death, as for instance, Toll-1 is more pro-apoptotic than Toll-6, and in mammals TLR4 promotes neuronal survival and TLR8 neuronal death. This study showed that Toll-2 over-expression did not cause cell loss, meaning that Toll-2 is not pro-apoptotic in the brain. Altogether, the data showed that Toll-2 is neuroprotective in the brain (Li, 2020).

A novel molecular pathway underlies the ability of Toll-2 to regulate cell proliferation in development and structural brain plasticity in the adult. A remarkable finding was that Toll-2 gain of function in the pupal or adult brain not only maintained neurons alive, but also induced cell cycling. This waswas visualized with standard cell proliferation markers PCNA-GFP for S-phase, Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) for G1, G2, G2/M, and Stg and nuclear Yki for G2/M and M phases. Toll-2 increased cell number and brain size. This study showed that there are progenitor cells in the adult brain that are kept quiescent by MyD88, and loss of MyD88 at the adult critical period increased neuronal number and brain size. As MyD88 is the general adaptor for canonical Toll signalling, this implies that Toll signalling maintains progenitor cells quiescent. Cell proliferation was induced when the repression by MyD88 was overcome by Toll-2 over-expression and signalling via the alternative adaptor, Wek. Conditional over-expression of Toll-2 or wek, or knock-down of MyD88, at the adult critical period increased neuronal number and brain size. Furthermore, the effect of Toll-2 was dependent on both Wek and Yki, a well-known inducer of cell proliferation. Over-expression of Toll-2 induced nuclear translocation and shuttling of Yki, correlating with nuclear localization of Stg, target of Yki, in the brain. Furthermore, genetic epistasis analysis showed that the increase in cell number caused by Toll-2 over-expression could be rescued with either yki-RNAi or wek-RNAi knockdown. Thus, Toll-receptor signalling can switch between promoting quiescence via MyD88 to promoting cell proliferation via Wek. Whether Wek might activate cell proliferation directly, or activate Yki or JNK first, was not solved. Wek also induces cell death, by linking Tolls to Sarm, which activates pro-apoptotic JNK signalling (Foldi, 2017). JNK can also induce cell proliferation, and Yki is activated downstream of JNK and Toll in other contexts. Thus, Wek could activate cell proliferation directly and independently of Yki, or it could activate Yki either directly, or via Sarm and/or JNK. Any of these options is possible, as in immunity and in cell competition (which also involves regulation of cell number), Toll-1 can regulate Yki downstream via JNK-dependent and independent pathways. Either way, the current data showed that Toll-2 can prevent or induce progenitor cell proliferation, through alternative MyD88 or Wek signalling pathways downstream (Li, 2020).

Knock-down of multiple Tolls through development severely altered brain structure and reduced brain size. Most likely, Tolls promote either cell survival or cell proliferation or both during brain development, and together they modulate brain formation. How each of them may influence the adult brain, is more difficult to dissect. Through their ability to elicit multiple cellular outcomes, Tolls can have distinct, redundant, synergistic, antagonistic or compensatory functions. For instance, whereas Toll-1 and -6 can have pro-apoptotic functions in the pupal VNC (Foldi, 2017), Toll-2 was not pro-apoptotic in the brain. Altering Toll-2 function alone did not affect Kenyon Cells, but simultaneous persistent knock-down of three Tolls reduced KC number and disorganized KC clusters; and whereas conditional knock-down of Toll-2, and -6 at the adult critical period reduced KC number, conditional knock-down of Toll-6 and -7 increased KC number. Thus, although all Tolls could access the same downstream signalling pathways, each Toll modulates these pathways in their own way. As a consequence, knock-down of one or more Tolls most likely induced complex responses by other Tolls in the same or neighbouring cells, compounding the phenotypes. What enables Tolls to elicit different cellular outcomes is an intriguing question (Li, 2020).

Adult neurogenesis is much debated. Neurogenesis occurs in the adult brain of humans and other animals, but the extent of it is unknown, and solving this is important to understand how the brain works and brain disease. In Drosophila, developmental neural stem cells are eliminated by the end of pupal life. However, neurogenesis has been reported in the adult brain: naturally occurring cell death induces cell proliferation in the adult brain; cell proliferation was reported with mitotic recombination clones; mir-31 mutations induce glial and neuroblast proliferation in the adult brain; injury induces gliogenesis and neurogenesis; the partner of Yki, scalloped, is expressed in the adult brain; immuno-histochemistry and single cell transcriptomics revealed that neuroblast or intermediate neural progenitor (INP) markers eyeless, dichate, grainy-head, dpn, miranda and inscutable are expressed in the adult brain; and interference with the normal regulation of cell survival and cell death - processes that Tolls can influence - results in ectopic and/or persistent neuroblasts in the adult brain. Consistently with these findings, in the adult brain there are MyD88+ Dpn+ Stg-GFP+ Yki-GFP+ FUCCI+ cells in S-phase or G2/M in the optic lobes, and in S, G1 and G2/M in the central brain. MyD88+ progenitor cells are normally quiescent and Toll-2 gain of function can induce their cell cycling and proliferation at the adult critical period (Li, 2020).

Adult progenitor cells may be distinct from developmental neuroblasts. In fact, the fate of quiescent INPs has not been determined, suggesting some could also exist in the adult brain. In other insects, adult progenitor cells differ from developmental neuroblasts, and instead originate from hemocytes. In the mammalian brain, adult progenitors originate from glia. Some of the large Toll-2+ and MyD88+ Dpn+ cells also had the glial marker Repo. Thus, adult progenitor cells may not originate from canonical developmental larval neuroblasts (Li, 2020).

Experience alters brain structure topographically, altering the regions involved in experience-dependent processing. For instance, rearing flies in constant light or constant darkness alters the size of brain modules involved in vision (e.g., optic lobes). Neuronal activity increased cell number in the optic lobes in a Toll-2 dependent manner, and conditional over-expression of Toll-2 or wek in the adult brain, at the critical period, increased both cell number and brain size. The anatomical segregation of the seven Toll receptors enables them to modulate cell number within distinct brain modules. This implies that: (1) in development, Tolls could adjust brain neuronal populations topographically to the motor and sensory circuits, enabling appropriate behaviour. (2) In evolution, Tolls could have driven changes in brain shape, enabling adaptation to distinct environments and behavioural diversification. (3) In adults, Tolls can enable structural brain plasticity, by adjusting brain neuronal populations topographically to experience-dependent inputs, and drive behavioural adaptation throughout the life-course (Li, 2020).

TLRs could operate in analogous ways in the human brain. TLRs are expressed throughout the mammalian brain. Some TLRs have neuro-protective and other TLRs pro-apoptotic functions, for instance TLR-2 and -4 promote cell survival, and TLR-3 and -8 apoptosis, neurite retraction and neurodegeneration. TLRs can regulate neural stem cell proliferation, formation or elimination of neurites and neurons, including during learning and long-term memory (Li, 2020).

To conclude, through their topographic distribution, Tolls modulate cell number and brain size, in development and in structural plasticity in the adult. They do so by engaging different molecular pathways that regulate neuronal survival or death, and progenitor quiescence via MyD88 or proliferation via Wek. It will be compelling to test if the combination of TLR topography and diversity of signalling options downstream, also underlies neurodegeneration and structural plasticity in the human brain (Li, 2020).

Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity

Toll-like receptors are essential for animal development and survival, with conserved roles in innate immunity, tissue patterning, and cell behavior. The mechanisms by which Toll receptors signal to the nucleus are well characterized, but how Toll receptors generate rapid, localized signals at the cell membrane to produce acute changes in cell polarity and behavior is not known. This study shows that Drosophila Toll receptors direct epithelial convergent extension by inducing planar-polarized patterns of Src and PI3-kinase (PI3K) activity. Toll receptors target Src activity to specific sites at the membrane, and Src recruits PI3K to the Toll-2 complex through tyrosine phosphorylation of the Toll-2 cytoplasmic domain. Reducing Src or PI3K activity disrupts planar-polarized myosin assembly, cell intercalation, and convergent extension, whereas constitutive Src activity promotes ectopic PI3K and myosin cortical localization. These results demonstrate that Toll receptors direct cell polarity and behavior by locally mobilizing Src and PI3K activity (Tamada, 2021).

Cell-surface receptors convert extracellular signals into changes in cell behavior that are essential for the formation, remodeling, and repair of multicellular tissues. Toll-like receptors (TLRs) are a conserved family of receptors with roles in dorsal-ventral patterning and cell-pathogen recognition in the innate immune system. More recently, members of the Toll-receptor family have also been shown to regulate cell behavior during the development of epithelial tissues and the nervous system. Toll receptors organize cell movement, direct nervous system wiring, repair wounds, and eliminate less fit cells through cell competition, defining new functions for this receptor family. In the immune system, Toll-like receptor (TLR) signaling activates the expression of immune-response genes by promoting the nuclear translocation of transcriptional regulators in the NF-κB, AP-1, and IRF families. However, how Toll receptors elicit fast-acting and localized responses in cells to generate spatially regulated changes in cell behavior is not understood (Tamada, 2021).

Tyrosine phosphorylation is a rapid and reversible mechanism that transduces extracellular information into molecular changes within cells. Cell-surface receptors such as growth-factor receptors initiate signaling cascades through their intrinsic tyrosine-kinase activity, which is activated by ligand binding and receptor dimerization. In addition, receptors that lack kinase activity can also participate in tyrosine-kinase signaling by recruiting nonreceptor tyrosine kinases. Src-family nonreceptor tyrosine kinases are essential for signaling by growth-factor receptors, T and B cell receptors, integrins, and cadherins, and have diverse roles in cell-cell adhesion, and cytoskeletal organization. A critical event in receptor-initiated tyrosine kinase signaling is often phosphorylation of the receptor itself, which generates binding sites that recruit downstream effector proteins. Although canonical TLR signaling involves a serine/threonine kinase cascade triggered by the recruitment of TLR-associated adaptor proteins, tyrosine kinases in the Src family have also been shown to facilitate the response to TLR signaling in cultured cells. However, the effects of Src-family kinases on Toll-receptor signaling in vivo, and whether tyrosine-kinase pathways mediate the rapid and localized effects of Toll receptors on cell behavior, are not known (Tamada, 2021).

Convergent extension is a conserved morphogenetic process that shapes developing organs and elongates the body axis of multicellular organisms. In Drosophila, three Toll-family receptors expressed in distinct striped patterns provide critical spatial cues that orient cell rearrangements and elongate the embryo along the head-to-tail axis. Toll receptors guide cell movements by directing the planar-polarized localization of proteins involved in actomyosin contractility and cell adhesion. However, the signaling pathways that generate spatially regulated changes in the organization and activity of the force-generating machinery downstream of Toll receptors are unknown. This study shows that a Src-mediated tyrosine-kinase pathway is essential for planar polarity and Toll-receptor signaling during convergent extension in Drosophila. Toll receptors generate a planar-polarized pattern of Src kinase activity, and Src in turn phosphorylates the Toll-2 C-terminal domain, promoting the association of Toll-2 with PI3-kinase (PI3K). Drosophila Src-family kinases, the regulatory and catalytic subunits of the PI3K complex, and tyrosine phosphorylation of the Toll-2 cytoplasmic domain are all necessary for planar polarity, cell intercalation, and convergent extension. These results identify a localized signaling mechanism by which Toll receptors induce rapid changes in cell polarity and behavior during development (Tamada, 2021).

Toll-family receptors are best known for their roles in signaling to the nucleus to activate the expression of immune-response genes. This study shows that Toll receptors also direct cell polarity and behavior during convergent extension by promoting the localized activities of Src and PI3K at the cell membrane. Toll receptors recruit active Src kinases to planar-polarized membrane domains, and localized Src activity generates spatially regulated actomyosin contractility by promoting the phosphorylation of two clusters of tyrosines in the Toll-2 cytoplasmic domain, creating docking sites for PI3K-reg. Src and PI3K are critical effectors of Toll-receptor signaling, as the loss of Src or PI3K recapitulates the effects of Toll-receptor mutants on planar polarity, cell intercalation, and convergent extension. Moreover, disruption of a single cluster of tyrosines in the Toll-2 C-terminal domain, which is phosphorylated in a Src-dependent fashion, inhibits Toll-2/PI3K complex formation and abolishes Toll-2 function during convergent extension. Taken together, these results identify Src and PI3K-reg as critical effectors of Toll-receptor signaling during convergent extension and reveal a noncanonical mechanism by which Toll receptors induce spatially regulated cell behavior by mobilizing two potent signaling proteins to specific subcellular domains (Tamada, 2021).

These results demonstrate that a critical symmetry-breaking event during convergent extension in Drosophila is the formation of localized Toll-2/PI3K complexes that generate tissue-wide patterns of actomyosin contractility. The upstream signals that activate Toll receptors in specific cellular domains are not known but could involve a localized extracellular ligand that increases Toll-2 binding to Src or enhances Src activity. Alternatively, mechanisms that promote Toll-receptor clustering could amplify the activity of associated Src kinases by enhancing their proximity to multiple substrates. Toll-2 and Toll-6 both interact with PI3K-reg and are tyrosine phosphorylated by Src kinases in vitro, suggesting that a Src/PI3K-signaling mechanism could link both Toll-2 and Toll-6 to PI3K. By contrast, Toll-8 is not phosphorylated by Src kinases, although it does associate weakly with PI3K-reg. Toll-8 could signal through a distinct mechanism, or it could communicate with Src and PI3K indirectly through heterodimerization with receptors such as Toll-6, which is expressed in many of the same cells. The defects in embryos lacking Src or PI3K appear to be more severe than the defects in Toll-2,6,8 mutants, suggesting that Src and PI3K may represent a common mechanism that mediates signaling downstream of multiple receptors during convergent extension (Tamada, 2021).

Tyrosine phosphorylation is a widely used signaling mechanism in multicellular organisms, with a particular enrichment of tyrosine phosphorylation at sites of contact between cells and with the extracellular matrix. Identifying the substrates required for the diverse roles of tyrosine-kinase signaling in vivo remains a formidable challenge. This study demonstrates that Src-dependent phosphorylation of a single cluster of tyrosines in the Toll-2 C-terminal domain is sufficient to eliminate Toll-2 function during convergent extension. In addition to Toll-2, Src kinases may also promote the phosphorylation of other proteins involved in Toll-receptor signaling. For example, Src-family kinases can phosphorylate Rho-kinase and recruit Rho-kinase and myosin II to focal adhesions and adherens junctions in fibroblasts and epithelial cells, and can phosphorylate and activate the Abl tyrosine kinase, which regulates planar polarized junctional dynamics during convergent extension. In addition, Src kinases can stabilize cell adhesion, enhance E-cadherin dynamics, influence cytoskeletal organization, and modulate cell polarity. The recruitment of active Src kinases to Toll-receptor complexes in Drosophila demonstrates an essential role for tyrosine-kinase pathways in signaling by Toll receptors and suggests a mechanism by which Toll receptors could trigger a wide range of cellular events that contribute to epithelial remodeling (Tamada, 2021).

PI3K is a conserved lipid kinase with widespread roles in cell growth, proliferation, survival, and polarity. This study demonstrates that PI3K directs planar polarity in the Drosophila embryo by promoting the localized assembly of contractile myosin networks in epithelial cells. These results are consistent with the altered cell topology and deregulated myosin activity observed in epithelial cells lacking the PI3K-antagonizing enzyme PTEN. The mechanisms by which PI3K influences tissue structure are not well understood. PI3K promotes the activity of the Rac GTPase in a number of cell types, but PI3K can also influence epithelial organization by activating PDK1, and PI3K can regulate the localization and activity of RhoA and Rho-kinase in fibroblasts and endothelial cells. PI3K could influence planar polarity by regulating Rho GTPase signaling, which has been linked to Toll-2 and is essential for convergent extension. The effectors that link Toll-2/Src/PI3K signaling to changes in actomyosin organization remain to be identified. These could include classical PI3K effectors that signal to the actin cytoskeleton or other proteins such as RhoGEFs that interact with the phospholipid product of PI3K, phosphatidylinositol (3,4,5)-trisphosphate (PIP3) (Tamada, 2021).

TLRs initiate signaling by recruiting adaptor proteins that assemble large multiprotein signaling complexes. The Toll-receptor signaling complexes involved in morphogenesis have not been defined. This study demonstrated that the C-terminal-most tyrosine cluster of Toll-2 is targeted by Src kinases and recruits Src and PI3K to the Toll-2 complex. This tyrosine cluster bears homology to immunoreceptor tyrosine-based activation motifs (ITAMs and hemITAMs). These motifs are present in several classes of immunoreceptors and their associated adaptor proteins and, when phosphorylated, create binding sites for downstream effector proteins. Toll-2 therefore displays features of multiple receptors in the immune system: an extracellular domain and intracellular TIR domain characteristic of TLRs and a C-terminal extension that couples to effectors through Src-family kinases and PI3K, reminiscent of other immunoreceptors such as C-type lectin-like receptors and the T cell receptor/CD3 complex. Although mammalian TLRs do not display this bipartite structure, tyrosine-rich adaptor proteins that associate with mammalian TLRs, such as BCAP and SCIMP, could create docking sites for the assembly of downstream signaling components. Toll-family receptors have conserved roles in regulating spatially regulated cell behaviors in flies and mammals, including axon growth, synapse formation, epithelial remodeling, and wound healing. An intriguing possibility is that Src and PI3K could represent a core module of a spatially regulated branch of Toll-receptor signaling that promotes the rapid and directional mobilization of cells during tissue development and immune defense (Tamada, 2021).

This study demonstrates a functional connection between Toll receptors and localized Src and PI3K activity during convergent extension in Drosophila. However, several limitations remain. First, phosphospecific antibodies to the C2 tyrosine cluster of Toll-2 could not be generated, and the other phosphospecific Toll-2 antibodies that were generated in this study worked only for western blots, but not for immunostaining. Therefore, it was not possible to analyze the spatiotemporal pattern of Toll-2 tyrosine phosphorylation in the embryo. Second, embryos were analyzed that had only a partial loss of Src42, PI3K-reg, and PI3K-cat function, due to essential roles of these proteins at earlier stages of development. Therefore, these proteins may have additional functions in this tissue that were not detected in this study. Third, focus was placed on protein localization and cell behavior in the adherens junction domain, and the results do not exclude the possibility that defects in basolateral regions of these cells or in other parts of the embryo could contribute to the phenotypes observed (Tamada, 2021).

REGULATION

Transcriptional Regulation

Analysis of the expression of 18w in different mutant backgrounds shows that it is under control of segment polarity and homeotic genes. Initial accumulation of 18w is normal in wingless mutants. However, by full germband extension, the ventrolateral expression of 18w is narrower than in wild type. These changes appear well before cell death is seen in wg mutants. In patched mutants, the domains of wg and of 18w expand to include the expression domains of wingless and engrailed. These results suggest that wg and en positively regulate 18w expression within the ventromedial stripes. The cytological bands to which 18w maps correspond to one of the most prominent binding sites of the Ubx protein on polytene chromosome (Juan Botas, personal communication to Eldon, 1994). Expanded 18w expression is observed in Ubx mutants (Eldon, 1994).

The 18w stripes require pair rule gene function for their establishment and later become dependent upon segment-polarity gene function for their maintenance. The establishment of the first even-numbered stripes of 18w depends on the function of the pair-rule gene fushi tarazu; the appearance of odd-numbered stripes depends on the function of even skipped. In wingless and hedgehog mutants, 18w expression rapidly declines as the germband reaches full extension, with the exception of small regions including a subset of neuroblasts along the midline. In engrailed mutant embryos, although general loss of expression is consistently observed, the loss of expression is not as striking as in wg and hh mutants. In naked and patched mutant embryos, the 18w stripes expand to about twice their normal width with occasional broadening in naked mutants such that the space between stripes is obliterated (Chiang, 1995).

Segmental modulation of 18w expression later in the tracheal system is dependent upon the function of the homeotic genes of the bithorax complex. In Ultrabithorax mutant embryos, a larger, more intense patch of 18w extends to cells surrounding the tracheal pits in T2 and T3, indicative of a role for Ultrabithorax protein in repression of 18w in T2 and T3 tracheal pits and consistent with a homeotic transformation in Ubx mutants of posterior T2 and T3 towards a T1 identity. Expanded 18w similarly extends posteriorly to A6 in flies lacking both Ubx and abd-A functions and to A7 in a triple mutant also deficient in Abd-B, indicating a role for abd-A and Abd-B in the repression of 18w in the posterior abdominal segment. In the triple mutant, loss of intense posterior spiracle staining suggests that Abd-B may also be required in A8 for positive regulation of 18w. It is not known whether BX-C regulation of 18w is direct or indirect (Chiang, 1995).

Targets of Activity

The Drosophila immune response uses many of the same components as the mammalian innate immune response, including signaling pathways that activate transcription factors of the Rel/NK-kappaB family. In response to infection, two Rel proteins, Dorsal immune factor (Dif) and Dorsal, translocate from the cytoplasm to the nuclei of larval fat-body cells. The Toll signaling pathway, which controls dorsal-ventral patterning during Drosophila embryogenesis, regulates the nuclear import of Dorsal in the immune response, but the Toll pathway is not required for nuclear import of Dif. Dif is properly translocated from fat-body cytoplasm to nuclei in response to infection in Toll and pelle mutant larvae. Cytoplasmic retention of both Dorsal and Dif depends on Cactus protein; nuclear import of Dorsal and Dif is accompanied by degradation of Cactus. Therefore the two signaling pathways that target Cactus for degradation must discriminate between Cactus-Dorsal and Cactus-Dif complexes. New genes have been identified that are required for normal induction of transcription of antibacterial peptide diptericin during the immune response. Mutations in three of these genes prevent nuclear import of Dif in response to infection, and define new components of signalling pathways involving Rel. The 18-wheeler gene, which encodes transmembrane protein that is homologous to Toll, is important for the nuclear localization of Dif during the immune response, so two of these genes may encode products that are necessary for 18-wheeler activation or cytoplasmic components that act downstream of 18-wheeler. Mutations in three other genes, constituting a second class of mutations, cause constitutive nuclear localization of Dif; these mutations may block Rel protein activity by a novel mechanism. The gene immune deficiency (imd) belongs to this second class, as both Dif and Dorsal are constitutively nuclear in imd mutants. Cactus does not appear to be found in the nucleus in class II mutants. One hypothesis that fits these observations is that the class II genes are required to allow the formation of a nuclear complex of Dif with other proteins, and that this complex is required both for activation of diptericin transcription and for turnover of the nuclear Rel proteins (Wu, 1998).

Protein Interactions

Expression of 18W protein in non-adhesive Schneider 2 cells promotes rapid and robust aggregation of cells. 18W appears to be a heterophilic adhesion molecule, as non-transfected cells are found in aggregates of transfected cells (Eldon, 1994).

DEVELOPMENTAL BIOLOGY

Embryonic

18 wheeler transcripts accumulate in embryos in a pattern reminiscent of segment polarity genes. Just prior to cellular blastoderm formation, during nuclear elongation, expression is initiated in several domains. The most anterior two primary stripes (S1 and S2) in the presumptive head region are each 5-6 cells wide. These stripes do not encircle the embryo. The third stripe (S3) is 3 cells wide and does encircle the embryo. Prior to cellularization, the central region of the embryo is faintly labelled. As cellularization proceeds, this domain rapidly sudivides in five circling stripes, each two to three cells wide (S4-8). The two most posterior stripes, S9 and S10, each 4-5 cells wide, are more intensely labelled than S4-8 (Eldon, 1994).

At late cellular blastoderm, secondary stripes begin to appear between all but the first two primary stripes. They appear first at the anterior end, are initially only one cell wide, and lead to the transient expression of a total of 18 stripes during early gastrulation (hence the name 18 wheeler). The secondary stripes rapidly widen and darken, and two additional stripes appear in the head region. During gastrulation the cephalic furrow forms immediately anterior to stripe 3 (of 18 stripes). Stripe 10 encircles the pole cells in the presumptive hindgut and anal region. As the ventral furrow invaginates, 18w expression is initially retained in the presumptive mesoderm (Eldon, 1994)

At maximum germ band elongation the pattern becomes still more complex. Lateral expression in each stripe is lost, while ventral and dorsal expression is retained. Along the ventral midline a small cluster of cells begin to express 18w on the the posterior side of each ventral stripe. In addition, transcripts accumulate around each invaginating tracheal pit and at the sites of the salivary gland placodes. Staining is also observed in five regions in the presumptive head. At stage 12, striped expression fades, and the developing tracheal system, the salivary gland anlagen and the anlagen for the anal plate, the posterior spiracles, and the clypeolabrum each express 18w. In addition, a row of cells at the leading edge of the two epidermal sheets that converge toward the dorsal midline are labelled. These cells participate in the formation of the dorsal vessel (heart) and continue to express 18w into the late phases of embryonic development. In stage 15 and 16, 18w is observed in two cells in the head region, the dorsal portion of the pharynx, a portion of the stomach and the hindgut. Transcript is present in each metamere of the central nervous system (Eldon, 1994).

The pattern of 18w expression at the extended germ band stage is characterized by 15 transverse stripes in the gnathal and trunk segments, with an additional four patches of expression corresponding to head segments and one more patch of expression in the presumptive hindgut. The segmentally repeated 18w stripes in the trunk overlap both the wingless and engrailed stripes and thus span the parasegment boundary (Eldon, 1994 and Chiang, 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).

Larval

The 18w gene also is prominently expressed in the imaginal discs, including antennal, leg, wing and haltere discs. In the eye disc, this expression occurs in two stripes at the anterior and posterior margins of the morphogenetic furrow. Expression in the leg disc is found in concentric rings that evaginate during pupation to give rise to the segments of the legs. The outer most rings are less intensely stained and staining increases in intensity towards the center of the discs. Similar staining patterns can also be observed in antennal imaginal discs. Expression in the wing disc is localized to the base of the wing blade and to portions of the notum. 18w expression is apparent in the brain of third instar larvae in a portion of the optic lobe. Transcript is also present in adult females (Eldon, 1994 and Chiang, 1995)

Oogenesis

A combinatorial code for pattern formation in Drosophila oogenesis

Two-dimensional patterning of the follicular epithelium in Drosophila oogenesis is required for the formation of three-dimensional eggshell structures. Analysis of a large number of published gene expression patterns in the follicle cells suggests that they follow a simple combinatorial code based on six spatial building blocks and the operations of union, difference, intersection, and addition. The building blocks are related to the distribution of inductive signals, provided by the highly conserved epidermal growth factor receptor and bone morphogenetic protein signaling pathways. The validity of the code is demonstrated by testing it against a set of patterns obtained in a large-scale transcriptional profiling experiment. Using the proposed code, 36 distinct patterns were distinguished for 81 genes expressed in the follicular epithelium, and their joint dynamics were characterize over four stages of oogenesis. The proposed combinatorial framework allows systematic analysis of the diversity and dynamics of two-dimensional transcriptional patterns and guides future studies of gene regulation (Yakoby, 2008b).

Drosophila eggshell is a highly patterned three-dimensional structure that is derived from the follicular epithelium in the developing egg chamber. The dorsal-anterior structures of the eggshell, including the dorsal appendages and operculum, are formed by the region of the follicular epithelium, which is patterned by the highly conserved epidermal growth factor receptor (EGFR) and bone morphogenetic protein (BMP) signaling pathways. The EGFR pathway is activated by Gurken (GRK), a transforming growth factor α-like ligand secreted by the oocyte. The BMP pathway is activated by Decapentaplegic (DPP), a BMP2/4-type ligand secreted by the follicle cells stretched over the nurse cells (Yakoby, 2008b).

Acting through their uniformly expressed receptors, these ligands establish the dorsoventral and anteroposterior gradients of EGFR and DPP signaling and control the expression of multiple genes in the follicular epithelium. Under their action, the expression of a Zn finger transcription factor, Broad (BR), evolves into a pattern with two patches on either side of the dorsal midline. The BR-expressing cells form the roof (upper part) of the dorsal appendages. Adjacent to the BR-expressing cells are two stripes of cells that express rhomboid (rho), a gene that is directly repressed by BR and encodes ligand-processing protease in the EGFR pathway. These cells form the floor (lower part) of the appendages (Yakoby, 2008b).

The patterns of genes expressed during the stages of egg development that correspond to appendage morphogenesis are very diverse. At the same time, inspection of a large number of published patterns suggests that they can be 'constructed' from a small number of building blocks. For instance, the T-shaped pattern of CG3074 is similar to the domain 'missing' in the early pattern of br, while the two patches in the late pattern of br appear to correspond to the two 'holes' in the expression of 18w. Based on a number of similar observations, it was hypothesized that all of the published patterns could be constructed from just six basic shapes, or primitives, which reflect the anatomy of the egg chamber and the spatial structure of the patterning signals (Yakoby, 2008b).

In computer graphics, representation of geometrical objects in terms of a small number of building blocks is known under the name of constructive solid geometry, which provides a way to describe complex shapes in terms of just a few parameters -- the types of the building blocks, such as cylinders, spheres, and cubes, their sizes, and operations, such as difference, union, and intersection. Thus, information about a large number of structures can be stored in a compact form of statements that contain information about the types of the building blocks and the operations from which these structures were assembled. This study describes a similar approach for two-dimensional patterns and demonstrate how it enables the synthesis, comparison, and analysis of gene expression at the tissue scale (Yakoby, 2008b).

The six building blocks used in the annotation system can be related to the structure of the egg chamber and the spatial distribution of the EGFR and DPP signals. The first primitive, M (for 'midline'), is related to the EGFR signal. It reflects high levels of EGFR activation and has a concave boundary, which can be related to the spatial pattern of GRK secretion from the oocyte. The second primitive, denoted by D (for 'dorsal'), reflects the intermediate levels of EGFR signaling during the early phase of EGFR activation by GRK, and is defined as a region of the follicular epithelium that is bounded by a level set (line of constant value) of the dorsoventral (DV) profile of EGFR activation. The boundary of this shape is convex and can be extracted from the experimentally validated computational model of the GRK gradient. The third primitive, denoted by A (for 'anterior'), is an anterior stripe which is obtained from a level set of the early pattern of DPP signaling in the follicular epithelium. This pattern is uniform along the DV axis, as visualized by the spatial pattern of phosphorylated MAD (P-MAD). Thus, the D, M, and A primitives represent the spatial distribution of the inductive signals at the stage of eggshell patterning when the EGFR and DPP pathways act as independent AP and DV gradients (Yakoby, 2008b).

Each of the next two primitives, denoted by R (for 'roof') and F (for 'floor'), is composed of two identical regions, shaped as the respective expression domains of br and rho, and reflect spatial and temporal integration of the EGFR and DPP pathways in later stages of eggshell patterning. The mechanisms responsible for the emergence of the F and R domains are not fully understood. It has been shown that the R domain is established as a result of sequential action of the feedforward and feedback loops within the EGFR and DPP pathways. The formation of the F domain requires the activating EGFR signal and repressive BR signal, expressed in the R domain. Thus, at the current level of understanding, the R and F domains should be viewed as just two of the shapes that are commonly seen in the two-dimensional expression patterns in the follicular epithelium. The sixth primitive, U (for 'uniform'), is spatially uniform and will be used in combination with other primitives to generate more complex patterns (Yakoby, 2008b).

While a number of patterns, such as those of jar and Dad, can be described with just a single primitive, more complex patterns are constructed combinatorially, using the operations of intersection (∩), difference ( ), and union (∪) For example, the dorsal anterior stripe of argos expression is obtained as an intersection of the A and D primitives (A∩D). The ventral pattern of pip is obtained as a difference of the U and D primitives (U D). The pattern of 18w is constructed from the A, D, and R primitives, joined by the operations of union and difference (A∪D R). For a small number of published patterns, the annotations reflect the experimentally demonstrated regulatory connections. For example, the U D annotation for pip reflects that actual repression of pip by the dorsal gradient of EGFR activation. For a majority of genes, the annotations should be viewed as a way to schematically represent a two-dimensional pattern and as a hypothetical description of regulation (Yakoby, 2008b).

The geometric operations of intersection, difference, and union can be implemented by the Boolean operations performed at the regulatory regions of individual genes. Boolean operations evaluate expression at each point and assign a value of 0 (off) or 1 (on). As an example, consider a regulatory module, hypothesized for argos, that performs a logical AND operation on two inputs: the output of the module is 1 only when both inputs are present. When both of the inputs are spatially distributed, the output is nonzero only in those regions of space where both inputs are present, leading to an output that corresponds to the intersection of the two inputs. Similarly, a spatial difference of the two inputs can be realized by a regulatory module that performs the ANDN (ANDNOT) operation. This is the case for pip, repressed by the DV gradient of GRK signaling and activated by a still unknown uniform signal. Finally, a regulatory module that performs an OR operation is nonzero when at least one of the inputs is nonzero. When the inputs are spatially distributed, the output is their spatial union (Yakoby, 2008b).

Boolean operations on primitives lead to patterns with just two levels of expression (the gene is either expressed or not). In addition to Boolean logic, developmental cis-regulatory modules and systems for posttranscriptional control of gene expression can perform analog operations, leading to multiple nonzero levels of output. Consider a module that adds the two binary inputs, shaped as the primitives. The output is nonzero in the domain shaped as the union of the two primitives, but is characterized by two nonzero levels of expression. This type of annotation is reserved only for those cases where the application of Boolean operations would lead to a loss of the spatial structure of the pattern (such as the A + U expression pattern of mia at stage 11 of oogenesis. For example, the union of the A and U primitives is a U primitive, whereas the sum of these primitives is an anterior band superimposed on top of a spatially uniform background (Yakoby, 2008b).

Signaling pathways guide organogenesis through the spatial and temporal control of gene expression. While the identities of genes controlled by any given signal can be identified using a combination of genetic and transcriptional profiling techniques, systematic analysis of the diversity of induced patterns requires a formal approach for pattern quantification, categorization, and comparison. Multiplex detection of gene expression, which has a potential to convert images of the spatial distribution of transcripts into a vector format preferred by a majority of statistical methods, is currently feasible only for a small number of genes and systems with simple anatomies. This paper presents an alternative approach based on the combinatorial construction of patterns from simple building blocks (Yakoby, 2008b).

In general, the building blocks can be identified as shapes that are overrepresented in a large set of experimentally collected gene expression patterns. This approach can be potentially pursued in systems where mechanisms of pattern formation are yet to be explored. At the same time, in well-studied systems, the building blocks can be linked to identified patterning mechanisms. This study chose six primitives based on the features that are commonly observed in real patterns and related to the structure of the tissue as well as the spatial distribution of the inductive signals. A similar approach will be useful whenever a two-dimensional cellular layer is patterned by a small number of signals, when cells can convert smoothly varying signals into spatial patterns with sharp boundaries, and when the regulatory regions of target genes have the ability to combinatorially process the inductive signals. One system in which this approach could be feasible is the wing imaginal disc, which is patterned by the spatially orthogonal wingless and DPP morphogens (Yakoby, 2008b).

The six primitives are sufficient to describe the experimentally observed patterns during stages 10-12 of oogenesis. A natural question is whether it is possible to accomplish this with a smaller number of primitives. Two of the primitives, R and F, could be potentially constructed from the D, M, and A primitives, which are related to the patterns EGFR and DPP activation during the earlier stages of eggshell patterning. Specifically, recent studies of br regulation suggest that the R domain is formed as a difference of the D, A, and M patterns (Yakoby, 2008a). Furthermore, the formation of the F domain requires repressive action in the adjacent R domain. With the R and F domains related to the other four primitives, the size of the spatial alphabet will be reduced even further (from six to four), but at the expense of increasing the complexity of the expressions used to describe various spatial patterns (Yakoby, 2008b).

Previously, the question of the diversity of the spatial patterns has been addressed only in one-dimensional systems. For example, transcriptional responses to the Dorsal morphogen gradient in the early Drosophila embryo give rise to three types of patterns in the form of the dorsal, lateral, and ventral bands. This work provides an attempt to characterize the diversity and dynamics of two-dimensional patterns. Thirty-six qualitatively different patterns were constructed, and it is proposed that each of them can be constructed using a compact combinatorial code. The sizes of the data sets from the literature and from transcriptional profiling experiments are approximately the same (117 and 96 patterns, respectively. Based on this observation, it is expected that discovered patterns will be readily described using this annotation system (Yakoby, 2008b).

A gene expressed in more than one stage of oogenesis is more likely to appear in different patterns, and it was found that groups of genes sharing the same pattern at one time point are more likely to scatter in the future than to stay together. More detailed understanding of the dynamics of the spatial patterns of the EGFR and DPP pathway activation is crucial for explaining these trends and the two observed scenarios for the emergence of complex patterns. A gene that makes its first appearance as a complex pattern, such as the A∩D pattern of argos at stage 10B, can be a direct target of the EGFR and DPP signal integration. In contrast, a gene such as Cct1, which changes from the A to the R pattern, can be a dedicated target of DPP signaling alone, and changes as a consequence of change in the spatial pattern of DPP signaling. Future tests of such hypotheses require analysis of cis-regulatory modules responsible for gene regulation in the follicular epithelium. While only a few enhancers have been identified at this time, this categorization of patterns should accelerate the identification of enhancers for a large number of genes (Yakoby, 2008b).

Proposed for the spatial patterns of transcripts, these annotations can also describe patterns of protein expression, modification, and subcellular localization. For example, the stage 10A patterns of MAD phosphorylation and Capicua nuclear localization can be accurately described using the A and U D annotations, respectively. The ultimate challenge is to use the information about the patterning of the follicular epithelium to explore how it is transformed into the three-dimensional eggshell. A number of genes in the assembled database encode cytoskeleton and cell adhesion molecules, suggesting that they provide a link between patterning and morphogenesis. It is hypothesized that the highly correlated expression patterns of these genes give rise to the spatial patterns of force generation and mechanical properties of cells that eventually transform the follicular epithelium into a three-dimensional eggshell (Yakoby, 2008b).

EFFECTS OF MUTATION

Mutations in 18w cause death during larval development and early adulthood. Escaping mutant adults often display leg, antenna, and wing deformities, presumably resulting from improper eversion of imaginal discs. Antennae are often abnormally positioned and appear larger than usual (Eldon, 1994).

There is a genetic interaction between an 18 wheeler mutation and an eye-specific allele of hedgehog (Chiang, 1994).

Essential aspects of innate immune responses to microbial infections appear to be conserved between insects and mammals. In particular, in both groups, transmembrane receptors of the Toll superfamily play a crucial role in activating immune defenses. The Drosophila Toll family member 18-Wheeler had been proposed to sense Gram-negative infection and direct selective expression of peptides active against Gram-negative bacteria. The role of 18-Wheeler was reexamined; in adults it is dispensable for immune responses. In larvae, 18wheeler is required for normal fat body development, and in mutant larvae induction of all antimicrobial peptide genes, and not only of those directed against Gram-negative bacteria, is compromised. 18-Wheeler does not qualify as a pattern recognition receptor of Gram-negative bacteria (Ligoxygakis, 2002).

The data presented in this study confirm the idea that a functional 18wheeler gene is required for larvae to mount a wild-type antimicrobial response. Rather than restricting this effect to the induction of attacin, the data show that the inducibility of all antimicrobial peptides is affected. As exemplified by the expression of the unrelated Fbp1 gene in mutant fat body, the data point to a general developmental delay of the fat body as a result of the mutation. A developmental role of 18W has already been postulated. It is an open question whether the effects that were observed in the fat body directly result from the absence of functional copies of the 18W protein in 18w^7-35 homozygous or 18w^7-35/Df(2R) hemizygous larvae. Indeed, the 18w^7-35-encoded N-terminal truncated TIR domain could have a dominant-negative effect on a neighboring pathway by analogy with the effect of overexpression of the MyD88 TIR domain on interleukin-1 receptor-induced NF-kappaB activation. This would account for the less drastically affected development in 18w^7-35/Df(2R) hemizygous larvae, in which the concentration of the truncated protein is half that of 18w^7-35 homozygous larvae. The 18w^7-35 allele affects viability since 18w^7-35/Df(2R) flies are recovered in higher numbers than 18w^7-35 homozygous flies. This could designate 18w^7-35 as a neomorphic mutation indirectly affecting the larval immune response. Alternatively, other unrelated mutations on the 18w^7-35 chromosome, which lie outside of the deficiency used, could contribute to this difference (Ligoxygakis, 2002).

It is concluded that there is no stringent evidence to consider the transmembrane receptor 18Wheeler as a sensor of Gram-negative infection. In fact, three independent studies point to a paramount role of the peptidoglycan recognition protein PGRP-LC in sensing Gram-negative infection. Data on the other receptors of the Toll family indicate that they do not signal to any of the bacterial peptide genes, and that only Toll-5 and Toll-9 are able to signal to the antifungal peptide gene Drosomycin. In the context of the valuable cross-talk between the studies on Drosophila host defense and on mammalian innate immunity, it is relevant to realize that Toll signaling cannot be fully equated with that of the TLRs. The data available in Drosophila indicate that Toll is not activated by direct (or even indirect) interaction with microbial ligands, but rather responds to the cleavage product of the cytokine Spaetzle. Cleavage of Spaetzle, in turn, depends on a proteolytic amplification cascade that is triggered when upstream proteins interact with microbial patterns in the hemolymph. This is in stark contrast to the situation described for the TLR family, whose members appear to directly interact with and discriminate between distinct microbial patterns. Furthermore, the data, taken in conjunction with those from other members of the Toll family, leads one to question whether Tolls are involved at all in the defense against Gram-negative sepsis in Drosophila (Ligoxygakis, 2002).

Expression of 18-wheeler in the follicle cell epithelium affects cell migration and egg morphology in Drosophila

The Drosophila ovary is a model system for examining the genetic control of epithelial morphogenesis. The somatic follicle cells form a polarized epithelium surrounding the 16-cell germ line cyst. The integrity of this epithelium is essential for the successful completion of oogenesis. Reciprocal signaling between germ line and somatic cells establishes embryonic and eggshell polarity. The follicle cells are responsible for shaping the egg and secreting the eggshell. Follicle cells at the boundary between the nurse cells and the oocyte migrate centripetally to cover the anterior end of the oocyte and secrete the operculum. Dorsal anterior main body follicle cells undergo elaborate patterning to produce the dorsal appendages. The expression of the Toll-like receptor, 18-wheeler (18w), was examined in the ovary; it is restricted to subpopulations of follicle cells. Females carrying loss-of-function 18w mutant clones in their ovaries show delayed follicle cell migrations. The eggs laid by such females also show morphological defects in egg shape and dorsal appendage morphology. It is proposed that the 18W protein plays an adhesive or signaling role in regions of the epithelium engaged in cell migration (Kleve, 2006).

All the cells expressing 18w are post-mitotic, and thus 18w expression may reflect an early stage in fate determination. The intriguing pattern of 18w expression in posteriorly migrating cuboidal epithelial cells, centripetally migrating cells, and likely the floor cells secreting the dorsal appendages is consistent with a role in cells that migrate as sheets. Although expression of the 18w enhancer detector was detected in the stalk cells of young egg chambers, it does not appear to be expressed in the polar or terminal cells, nor in the border cell clusters that they become. It is not clear whether stalk cell expression is correlated with a role for 18w in cell migration. Some aberrantly long stalks separating egg chambers were observed in females bearing heat shock-induced loss-of-function clones. However, marked clones were needed to carry out a statistical analysis of this effect. The T155 driver used to induce marked mutant clones is not expressed in stalk cells (Kleve, 2006).

Failure of expression of 18w in main body follicle cells in mid-oogenesis results in delayed cell migrations. These delays are apparently affecting the morphology of the eggs that are produced by females carrying loss-of-function clones. The follicle cells are responsible for the shape of the egg in addition to secreting the chorion. Delayed cell migration may prevent the follicle layer from constraining egg diameter during nurse cell dumping and would produce shorter, rounder eggs. The shorter eggs laid by females expressing a gain-of-function form of 18w could be caused by a failure of follicle cells to allow sufficient egg expansion during nurse cell dumping. These eggs, however, do not have the open anterior cup phenotype associated with many dumpless phenotypes (Kleve, 2006).

A striking percentage of deflated eggs was observed in some collections from females carrying loss-of-function clones. They could not be measured accurately, and were not included in a statistical analysis of egg shape. These eggs frequently took up the purple color of the grape juice egg lay plates and often showed dramatically enlarged opercula. Some eggs in these collections were extremely fragile and prone to rupture during normal handling. All these characteristics are consistent with a failure of the follicle cell epithelium to secrete an intact chorion. The chorion is secreted by the follicle cells during the last day of oogenesis, stages 9-14, so delayed migration could disrupt this carefully choreographed process (Kleve, 2006).

What does delayed cell migration reveal about the normal function of 18w in the ovary? Cell migration depends upon cells recognizing their position and polarity, and reconfiguring their cytoskeletal components to allow changes in shape and adhesive properties. Cell polarity could involve detecting gradients of signaling molecules and transducing that signal to the cytoskeleton. It is also possible that 18w acts as an adhesion molecule promoting migration. Multiple signaling pathways act in the developing egg chamber. If the 18W receptor were acting as a competence factor, then its loss would make cells less responsive to signals, either slowing their migration or reducing their directionality. If 18W were acting as an adhesion molecule, then its loss might reduce cells' ability to remodel their cytoskeleton or to change their cell-cell contacts for normal migration (Kleve, 2006).

In addition to understanding what 18w is doing in the ovary, it would be of interest to know how its expression is regulated. It is possible that early expression in the stalk cells is regulated differently than later expression in the main body cells. Such temporal differences in control of gene expression during oogenesis have been noted for Broad Complex transcripts. The expression of 18w in the centripetally migrating cells suggests that 18w normally responds to the anterior TGF-β signal, Dpp, which is responsible for setting the operculum size. The expression domain of 18w along the anterior dorsal midline is likely to be affected by EGF receptor signaling in addition to Dpp. 18w-expressing cells contribute to dorsal appendage synthesis and subtle appendage defects are observed when that contribution is missing. Ultimately, it is of interest to understand how 18w contributes to the extremely complex signaling that occurs in the dorsal anterior region of the mid- to late-stage egg chamber to execute the elaborate morphogenetic events of eggshell patterning (Kleve, 2006).

This study has presented evidence that a Toll-like receptor, 18-wheeler, is expressed in the follicle cell epithelium and plays a role in the timely migration of main body and centripetally migrating follicle cells. Eggs laid by females carrying 18w mutant follicle cell clones show defects in the eggshell, both structurally and morphologically. Further analysis will be required to examine how 18w interacts with other signaling and adhesion molecules to contribute to normal epithelial migration and to the production of viable eggs (Kleve, 2006).

Genetic variation in Drosophila melanogaster resistance to infection: a comparison across bacteria

Insects use a generalized immune response to combat bacterial infection. Natural populations of D. melanogaster harbor substantial genetic variation for antibacterial immunocompetence and that much of this variation can be mapped to genes that are known to play direct roles in immunity. It was not known, however, whether the phenotypic effects of variation in these genes are general across the range of potentially infectious bacteria. To address this question, the same set of D. melanogaster lines were reinfected with Serratia marcescens, the bacterium used in the previous study, and with three additional bacteria that were isolated from the hemolymph of wild-caught D. melanogaster. Two of the new bacteria, Enterococcus faecalis and Lactococcus lactis, are gram positive. The third, Providencia burhodogranaria, is gram negative like S. marcescens. Drosophila genotypes vary highly significantly in bacterial load sustained after infection with each of the four bacteria, but mean loads are largely uncorrelated across bacteria. Statistical associations were tested between immunity phenotypes and nucleotide polymorphism in 21 candidate immunity genes. Molecular variation was found in some genes, such as Tehao, to contribute to phenotypic variation in the suppression of only a subset of the pathogens. Variation in SR-CII and 18-wheeler, however, has effects that are more general. Although markers in SR-CII and 18-wheeler explain >20% of the phenotypic variation in resistance to L. lactis and E. faecalis, respectively, most of the molecular polymorphisms tested explain less than 10% of the total variance in bacterial load sustained after infection (Lazzaro, 2006).

Polymorphic sites in 18-wheeler and SR-CII are associated with variation in resistance to all of the bacteria tested in this study. These associations may be somewhat unexpected. Despite early reports to the contrary, the direct involvement of 18-wheeler in mounting a systemic induced immune response in adult flies has been called into question. 18-wheeler is, however, required for proper development of the larval fat body and may play a role in inducible larval defenses and hematopoesis. There is no direct evidence that SR-CII is involved in immune defense, even though SR-CI, the closest Drosophila paralog to SR-CII, is known to be involved in phagocytosis of bacteria. SR-CII expression is thought to be maximal early in Drosophila development, and molecular evolutionary analysis reveals SR-CII to be on a distinctly more conservative evolutionary trajectory the other three SR-Cs in Drosophila. Therefore it is suggested that the associations observed between polymorphism in 18-wheeler and SR-CII and variation in resistance to bacterial infection may stem from roles those genes play in physiological processes such as fat body development and cell proliferation, which are essential for organismal immunocompetence but may not be components of the inducible adult immune response per se (Lazzaro, 2006).

REFERENCES

Search PubMed for articles about Drosophila 18 wheeler

Chiang, C. and Beachy, C. A. (1995). Expression of a novel Toll-like gene spans the parasegment boundary and contributes to hedgehog function in the adult eye of Drosophila. Mech. Dev. 47(3): 225--239.

Eldon, E., et al. (1994). The Drosophila 18 wheeler is required for morphogenesis and has striking similarities to Toll. Development 120 (4): 885-899.

Foldi, I., Anthoney, N., Harrison, N., Gangloff, M., Verstak, B., Nallasivan, M. P., AlAhmed, S., Zhu, B., Phizacklea, M., Losada-Perez, M., Moreira, M., Gay, N. J. and Hidalgo, A. (2017). Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila. J Cell Biol 216(5): 1421-1438. PubMed ID: 28373203

Kleve, C. D., Siler, D. A., Syed, S. K. and Eldon, E. D. (2006). Expression of 18-wheeler in the follicle cell epithelium affects cell migration and egg morphology in Drosophila. Dev. Dyn. 235(7): 1953-61. PubMed citation: 16607637

Kolesnikov, T. and Beckendorf, S. K. (2007). 18 wheeler regulates apical constriction of salivary gland cells via the Rho-GTPase-signaling pathway. Dev. Biol. 307(1): 53-61. Medline abstract: 17512518

Lazzaro, B. P., Sackton, T. B. and Clark, A. G. (2006). Genetic variation in Drosophila melanogaster resistance to infection: a comparison across bacteria. Genetics 174(3): 1539-54. PubMed citation: 16888344

Ligoxygakis, P., Bulet, P. and Reichhart, J.-M. (2002). Critical evaluation of the role of the Toll-like receptor 18-Wheeler in the host defense of Drosophila. EMBO Reports 3: 666-673. 12101100

McIlroy, G., Foldi, I., Aurikko, J., Wentzell, J. S., Lim, M. A., Fenton, J. C., Gay, N. J. and Hidalgo, A. (2013). Toll-6 and Toll-7 function as neurotrophin receptors in the Drosophila melanogaster CNS. Nat Neurosci 16(9): 1248-1256. PubMed ID: 23892553

Li, G., Forero, M. G., Wentzell, J. S., Durmus, I., Wolf, R., Anthoney, N. C., Parker, M., Jiang, R., Hasenauer, J., Strausfeld, N. J., Heisenberg, M. and Hidalgo, A. (2020). A Toll-receptor map underlies structural brain plasticity. Elife 9. PubMed ID: 32066523

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

Rock, F. L., et al. (1998). A family of human receptors structurally related to Drosophila Toll. Proc. Natl. Acad. Sci. 95: 588-593

Tamada, M., Shi, J., Bourdot, K. S., Supriyatno, S., Palmquist, K. H., Gutierrez-Ruiz, O. L. and Zallen, J. A. (2021). Toll receptors remodel epithelia by directing planar-polarized Src and PI3K activity. Dev Cell. PubMed ID: 33932332

Ulian-Benitez, S., Bishop, S., Foldi, I., Wentzell, J., Okenwa, C., Forero, M. G., Zhu, B., Moreira, M., Phizacklea, M., McIlroy, G., Li, G., Gay, N. J. and Hidalgo, A. (2017). Kek-6: A truncated-Trk-like receptor for Drosophila neurotrophin 2 regulates structural synaptic plasticity. PLoS Genet 13(8): e1006968. PubMed ID: 28846707

Williams, M. J., et al. (1997). The 18-wheeler mutation reveals complex antibacterial gene regulation in Drosophila host defense. EMBO J. 16(20): 6120-6130

Wu, L. P. and Anderson, K. V. (1998). Regulated nuclear import of Rel proteins in the Drosophila immune response. Nature 392(6671): 93-97.

Yakoby, N., Lembong, J., Schupbach, T. and Shvartsman, S. Y. (2008a). Drosophila eggshell is patterned by sequential action of feedforward and feedback loops. Development 135: 343-351. PubMed Citation: 18077592

Yakoby, N., et al. (2008b). A combinatorial code for pattern formation in Drosophila oogenesis. Dev. Cell 15(5): 725-37. PubMed Citation: 19000837