18 wheeler : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - 18 wheeler
Synonyms - Toll like receptor (tlr)
Cytological map position - 56F8--56F9
Function - Transmembrane receptor
Symbol - 18w
FlyBase ID: FBgn0004364
Genetic map position - 2-
Classification - Leucine rich repeat protein, IL1 like cytoplasmic domain
Cellular location - surface transmembrane protein
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).
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, 18wΔ21, 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, 18wΔ7–35, and in 18wΔ21, 18wΔ7–35 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).
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).
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).
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).
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).
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).
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)
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).
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 18w7-35 homozygous or 18w7-35/Df(2R) hemizygous larvae. Indeed, the 18w7-35-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 18w7-35/Df(2R) hemizygous larvae, in which the concentration of the truncated protein is half that of 18w7-35 homozygous larvae. The 18w7-35 allele affects viability since 18w7-35/Df(2R) flies are recovered in higher numbers than 18w7-35 homozygous flies. This could designate 18w7-35 as a neomorphic mutation indirectly affecting the larval immune response. Alternatively, other unrelated mutations on the 18w7-35 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).
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).
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).
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.
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
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
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
date revised: 30 December 2014
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