18 wheeler

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).

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)

Effects of mutation or deletion

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).

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

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.

date revised: 25 August 2007 

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