dorsal-related immunity factor

DEVELOPMENTAL BIOLOGY

Embryonic

Because DIF mRNA is expressed at very low levels in early embryos, it is unlikely that the gene plays a key role in DV patterning (Ip, 1993).

Larval

Dif is expressed only in fat bodies. There is a rapid accumulation of staining in nuclei when larvae are injected with E. coli prior to dissection. Nuclear staining is detected within 30 minutes after infection. This staining does not require bacterial infection and is observed when larvae are simply damaged by manual puncture (Ip, 1993).

Adult

Although Dif maps close to dorsal, it does not appear to participate in DV patterning, but instead mediates an immune response in Drosophila larvae. Dif is normally localized in the cytoplasm of the larval fat body, but quickly accumulates in the nucleus upon bacterial infection or injury. Once in the nucleus, Dif binds to kappa B-like sequence motifs present in promoter regions of immunity genes. This suggests that mammalian and insect immunity share a common evolutionary origin (Ip, 1993).

Regulation of Toll signaling and inflammation by β-arrestin and the SUMO protease Ulp1

The Toll signaling pathway has a highly conserved function in innate immunity and is regulated by multiple factors that fine tune its activity. One such factor is β-arrestin Kurtz (Krz), which has been implicated in the inhibition of developmental Toll signaling in the Drosophila melanogaster embryo. Another level of controlling Toll activity and immune system homeostasis is by protein sumoylation. This study has uncovered a link between these two modes of regulation and shows that Krz affects sumoylation via a conserved protein interaction with a SUMO protease, Ulp1. Loss of function of krz or Ulp1 in Drosophila larvae results in a similar inflammatory phenotype, which is manifested as increased lamellocyte production; melanotic mass formation; nuclear accumulation of Toll pathway transcriptional effectors, Dorsal and Dif; and expression of immunity genes, such as Drosomycin. Moreover, mutations in krz and Ulp1 show dosage-sensitive synergistic genetic interactions, suggesting that these two proteins are involved in the same pathway. Using Dorsal sumoylation as a readout, it was found that altering Krz levels can affect the efficiency of SUMO deconjugation mediated by Ulp1. These results demonstrate that β-arrestin controls Toll signaling and systemic inflammation at the level of sumoylation (Anjum, 2013).

Effects of Mutation or Deletion

The induction of immunity genes in Drosophila has been proposed to be dependent on Dorsal, Dif, and Relish, the NF-kappaB-related factors. Genetic evidence is provided that Dif is required for the induction of only a subset of antimicrobial peptide genes. The results show that the presence of Dif without Dorsal is sufficient to mediate the induction of drosomycin and defensin. Dif is a downstream component of the Toll signaling pathway in the activation of drosomycin expression. These results reveal that individual members of the NF-kappaB family in Drosophila have distinct roles in immunity and development (Meng, 1999).

A genetic experiment tested whether Dif acts downstream of Toll in regulating drosomycin gene expression. J4 (a null mutation of both Dif and dorsal) and dorsal loss-of-function mutants were crossed with the Toll^10b gain-of-function mutant. The flies that contained different combinations of marker chromosomes were collected and analyzed for the expression of drosomycin. In wild-type flies, drosomycin is expressed at a basal level, and the expression is much elevated in the Toll^10b flies. This Toll^10b activated expression of drosomycin is clearly suppressed by the homozygous J4 chromosome. Because the dorsal mutation itself cannot suppress the Toll^10b effect, the results demonstrate that Dif is an essential component of the Toll signaling pathway in the induction of drosomycin. The possibility that dorsal can replace the function of Dif in Toll signaling has not been ruled out because of the double deletion in the J4 chromosome. Nevertheless, there is no indication that Dorsal performs essential function downstream of Toll during the immune response (Meng, 1999).

Expression of the gene encoding the antifungal peptide Drosomycin in Drosophila adults is controlled by the Toll signaling pathway. The Rel proteins Dorsal and DIF (Dorsal-related immunity factor) are possible candidates for the transactivating protein in the Toll pathway that directly regulates the drosomycin gene. An examination was carried out of the requirement of Dorsal and DIF for drosomycin expression in larval fat body cells, the predominant immune-responsive tissue. The yeast site-specific flp/FRT recombination system was used to generate cell clones homozygous for a deficiency uncovering both the dorsal and the dif genes. In the absence of both genes, the immune-inducibility of drosomycin is lost but can be rescued by overexpression of either dorsal or dif under the control of a heat-shock promoter. This result suggests a functional redundancy between both Rel proteins in the control of drosomycin gene expression in the larvae of Drosophila. Interestingly, the gene encoding the antibacterial peptide Diptericin remains fully inducible in the absence of the dorsal and dif genes. Fat body cell clones homozygous for various mutations were used to show that a linear activation cascade Spaetzle->Toll->Cactus->Dorsal/DIF leads to the induction of the drosomycin gene in larval fat body cells (Manfruelli, 1999).

In contrast to the drosomycin gene, the genes encoding the antibacterial peptides Diptericin, Cecropin and Attacin are not constitutively expressed in Toll^D gain-of-function mutant larvae. The diptericin gene is also fully inducible in larvae deficient for the spz and Tl genes. These data indicate that diptericin induction in larvae is not dependent on the Tl pathway. Diptericin induction, however, is clearly dependent on immune deficiency (imd), a recessive mutation that impairs the inducibility of the genes encoding antibacterial peptides in both larvae and adults, while only marginally affecting the inducibility of the antifungal peptide gene drosomycin. The imd gene, which has not yet been cloned, therefore encodes a component required for the antibacterial response. The expression patterns observed for cecropin and attacinare somewhat different from those of diptericin, since the full induction of these two genes is affected in both spz and imd mutant larvae, indicating that they are regulated both by the Tl pathway and the imd gene product (Manfruelli, 1999).

The larval polyploid fat body cells differentiate from embryonic mesodermal cells whereas the adult fat body cells are derived from larval histoblasts -- presumably from adepithelial cells, associated with the imaginal discs. It was of interest therefore to compare the regulation of antimicrobial genes during an immune response in these relatively different cell types. The results point to an overall similar mode of regulation in larval and adult fat body cells. In essence, the Tl pathway controls drosomycin gene expression whereas the genes encoding the antibacterial peptides require the product of the imd gene (diptericin) or a combination of the imd and Tl pathways (cecropin and attacin). These results with respect to the larval immune response are in keeping with a correlation between the impairment of antifungal gene induction and reduced resistance to fungal infection and, conversely, between the impairment of antibacterial gene induction and reduced resistance to bacterial infection. Northern blot analysis, furthermore, indicates that the inducibility of the drosomycin gene in Tl pathway mutants is less dramatically affected in larvae than in adults. This suggests that another regulatory cascade might partially substitute for the Tl pathway in controlling drosomycin in larval fat body. Drosophila contains several Tl-like receptors, including 18-Wheeler, which is reportedly involved in the control of attacin and, to a lesser extent, cecropin induction in larvae. However, 18-wheeler mutations do not seem to affect drosomycin expression (E. Eldon, personal communication to Manfruelli, 1999). The possible contribution of these receptors to the humoral immune response, and namely to the regulation of the drosomycin gene, awaits further investigation (Manfruelli, 1999).

In larvae, as in adults, the inducibility of the drosomycin gene is slightly reduced in imd mutants. This result, in conjunction with studies on metchnikowin gene expression, leads to the proposition that each antimicrobial peptide gene is regulated by the relative dosage of inputs from several signaling cascades, which are each triggered by distinct stimuli. Current programs of mutagenesis will contribute to the identification of new components of these cascades and help understand the cross-talk between distinct pathways (Manfruelli, 1999).

Depletion of Serpin-27A (Spn27A) from the hemolymph following immune challenge is best explained by assuming that it binds to a cognate protease and that the resulting complex is either removed from circulation or degraded. Two intracellular signaling pathways control the expression of challenge-induced genes in the fat body, the predominant immuno-responsive tissue of Drosophila. DNA microarray data indicate that the infection-induced expression of several putative prophenoloxidase-activating enzyme (PPAE) genes in Drosophila is under the control of the Toll pathway. Moreover, Spn27A transcription is also regulated in an immune-dependent manner. It was of interest to examine whether the melanization observed in the Spn27A^ex32 mutants could be correlated to the well-defined Toll-mediated host response in Drosophila. In Dif or spaetzle (spz) mutants, in which the Toll pathway is blocked, the serpin is not removed from the circulating hemolymph. Interestingly, the serpin is removed in kenny (key) mutants that block the Imd pathway, which is not involved in the control of PPAE gene expression (De Gregorio, 2002a). A testable prediction that can be derived from these results is that in a Toll pathway mutant background PO enzymatic activity following bacterial challenge should be at very low levels. It as indeed observed that Dif- and spz-infected flies have a PO activity comparable to the basal level of non-challenged wild-type flies in contrast to key-infected flies, which show normal PO levels. The data furthermore imply that the protease (or the factor that triggers its activation), which removes Spn27A, may not be present as a zymogen activated by infection, but may need de novo protein synthesis dependent on Toll signal transduction. This was confirmed by infecting wild-type flies with bacteria in the presence of an inhibitor of translation (cycloheximide). Protein synthesis following bacterial infection was examined by mass spectrometry; none of the induced antimicrobial peptides were synthesized in the experimental conditions indicating that protein synthesis was efficiently blocked. Importantly, it was noted that in these flies during the same infection procedures the serpin was not removed from circulation. Taken together, these results on the requirement of Toll signaling pathway and protein synthesis for serpin removal show that the protease(s) removing Spn27A from circulation is synthesized de novo in response to Toll signaling. Alternatively, a component that activates it is dependent on Toll signaling-driven de novo protein synthesis (Ligoxygakis, 2002).

The lesswright (lwr) or semushi gene encodes an enzyme, Ubc9, that conjugates a small ubiquitin-related modifier (SUMO). Since the conjugation of SUMO occurs in many different proteins, a variety of cellular processes probably require lwr function. This study demonstrates that lwr function regulates the production of blood cells (hemocytes) in Drosophila larvae. lwr mutant larvae develop many melanotic tumors in the hemolymph at the third instar stage. The formation of melanotic tumors is due to a large number of circulating hemocytes, which is approximately 10 times higher than those of wild type. This overproduction of hemocytes is attributed to the loss of lwr function primarily in hemocytes and the lymph glands, a hematopoietic organ in Drosophila larvae. High incidences of Dorsal (Dl) protein in the nucleus were observed in lwr mutant hemocytes, and the dl and Dorsal-related immunity factor (Dif) mutations were found to be suppressors of the lwr mutation. Therefore, the lwr mutation leads to the activation of these Rel-related proteins, key transcription factors in hematopoiesis. Also demonstrate was the fact that dl and Dif play different roles in hematopoiesis. dl primarily stimulates plasmatocyte production, but Dif controls both plasmatocyte and lamellocyte production (Huang, 2005). Similar results have been reported by Chiu (2005). Loss-of-function mutations in dUbc9 cause strong mitotic defects in larval hematopoietic tissues, an increase in the number of hematopoietic precursors in the lymph gland and of mature blood cells in circulation, and an increase in the proportion of cyclin-B-positive cells. In the larval fat body, dUbc9 negatively regulates the expression of the antifungal peptide gene drosomycin, which is constitutively expressed in dUbc9 mutants in the absence of immune challenge. dUbc9-mediated drosomycin expression requires Dorsal and Dif. Although both studies are clear in concluding that Ubc9/Lesswright appears to function upstream of Cactus and Dorsal/Dif, the specific target is not yet known. Wild-type Ubc9/Lesswright hold the Toll pathway in check, and could target Cactus, Dorsal/Dif or another upstream component in the pathway (Huang, 2005; Chiu, 2005).

The Toll/NF-kappaB signaling pathway is required for epidermal wound repair in Drosophila

The Toll/NF-kappaB pathway, first identified in studies of dorsal-ventral polarity in the early Drosophila embryo, is well known for its role in the innate immune response. This study revealed that the Toll/NF-kappaB pathway is essential for wound closure in late Drosophila embryos. Toll mutants and Dif dorsal (NF-kappaB) double mutants are unable to repair epidermal gaps. Dorsal is activated on wounding, and Dif and Dorsal are required for the sustained down-regulation of E-cadherin, an obligatory component of the adherens junctions (AJs), at the wound edge. This remodeling of the AJs promotes the assembly of an actin-myosin cable at the wound margin; contraction of the actin cable, in turn, closes the wound. In the absence of Toll or Dif and dorsal, both E-cadherin down-regulation and actin-cable formation fail, thus resulting in open epidermal gaps. Given the conservation of the Toll/NF-kappaB pathway in mammals and the epithelial expression of many components of the pathway, this function in wound healing is likely to be conserved in vertebrates (Carvalho, 2014).

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date revised: 25 March 2015

 

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