Stage 10 egg chambers show a strong DL protein staining in nurse cells. Dorsal is transported into the oocyte by a cytoskeletal based mechanim later when nurse cell contents are distributed to the oocyte (Rushlow, 1989). After fertilization there is a graded nuclear localization of DL protein. The ventral region of the embryo shows complete nuclear transport, the lateral portion shows intermediate transport and the dorsal portion shows no transport. In the absence of Cactus function, the DL protein is found in nuclei on the dorsal as well as the ventral side (Roth, 1989). In mutants of the dorsal group of genes, including easter, spätzle and Toll, no nuclear localization is observed (Steward, 1989).

Dorsal-ventral polarity of the Drosophila embryo is established by a nuclear gradient of Dorsal protein, generated by successive gurken-Egfr and spätzle-Toll signaling. Overexpression of extracellular Spätzle dramatically reshapes the Dorsal gradient: the normal single peak is broadened and then refined to two distinct peaks of nuclear Dorsal, to produce two ventral furrows. This partial axis duplication, which mimics the ventralized phenotype caused by reduced gurken-Egfr signaling, arises from events in the perivitelline fluid of the embryo and occurs at the level of Spätzle processing or Toll activation. The production of two Dorsal peaks is addressed by a model that invokes the action of a diffusible inhibitor, which is proposed to normally regulate the slope of the Dorsal gradient (Morisato, 2001).

The Dorsal gradient in the wild-type embryo possesses a characteristic shape. The domain of peak nuclear Dorsal in the embryo can vary over a wide range, depending on the level of Spätzle production, but never exceeds the limits presaged by the expression of pipe in the ovary. In contrast, the slope of the Dorsal gradient, as measured by the extent of sog expression, is relatively constant under these conditions (Morisato, 2001).

The shape of the Dorsal gradient is dramatically changed in embryos laid by females carrying mutations in the gurken-Egfr signaling pathway. Not only do these embryos expand Twist expression, as a consequence of a reduction in the dorsalizing signal that establishes egg chamber asymmetry, but they exhibit two distinct peaks within the Twist domain that give rise to two ventral furrows. In the experiments described here, this partial axis duplication is not evident during oogenesis, because pipe RNA was found to be expressed in a single broad domain in follicle cells. The production of two Dorsal peaks could be mimicked by injecting high levels of spz RNA into the pre-cellular embryo cytoplasm, suggesting that pattern refinement occurs during embryogenesis. It is suggested that while the size of the ventral domain is expanded in grk and Egfr ovarian egg chambers, the partial axis duplication observed in mutant embryos is caused by reactions occurring later in the embryo (Morisato, 2001).

It may have been easier to imagine how the selection of one or two gradient peaks would involve signaling within the follicular epithelium, because spatial information could then be stably maintained and transmitted by cells. The elaboration of the two dorsal appendages in the Drosophila eggshell results from a series of such intercellular signaling events. Activation of Egfr by Gurken stimulates transcriptional induction of Argos, a secreted Egfr inhibitor, which then downregulates Egfr activity in the initial central domain, leaving two lateral domains of signaling (Morisato, 2001).

In fact, the findings described in this paper argue that events involving the diffusion of an extracellular morphogen not only regulate the gradient slope, but perhaps unexpectedly, determine the position and number of maxima within the axis in response to the broad cues generated during oogenesis. Reaction-diffusion models have been applied to analyze the respective contributions of the gurken-Egfr and spätzle-Toll pathways in generating embryonic pattern. The current studies provide experimental support for this theoretical work, and present opportunities for understanding the underlying mechanisms (Morisato, 2001).

Formation and maintenance of the Dorsal gradient appear dynamic. The shape of the Dorsal gradient in the wild-type embryo does not change markedly after nuclear translocation is first detected. In embryos laid by grk females or embryos expressing high levels of Spätzle, however, the shape of the Dorsal gradient is subtly modified. In particular, the minimum lying between the two Dorsal peaks becomes deeper in older embryos. This observation suggests that signaling takes place over a period of time, and explains how an initial asymmetry, in the form of the broad stripe of pipe, might be gradually refined into a gradient of positional information (Morisato, 2001).

Evidence is presented for the following model, which accounts for many of the observations described above. The initial shape of the gradient (at t0) is established by the proteolytic activation of Spätzle in a relatively broad domain, reflecting the ventral region of the egg chamber that expresses pipe RNA. It is proposed that the Spätzle processing reaction generates an inhibitor that negatively regulates the production of the ventral signal, possibly at the level of Easter protease activity or the interaction between processed Spätzle and Toll. Whereas processed C-terminal Spätzle is believed to bind to Toll quickly and show limited movement after cleavage, it is postulated that the hypothetical inhibitor undergoes broader diffusion. In the wild-type embryo, inhibitor action is responsible for establishing the region of high nuclear Dorsal, corresponding to the Twist domain, to be narrower than the ventral region of the egg chamber expressing pipe RNA. The final shape of the Dorsal gradient (at t1) is generated over time by the opposing effects of processed Spätzle and the inhibitor (Morisato, 2001).

In embryos produced by grk females, it is inferred that Spätzle processing is occurring at wild-type levels, but the reaction is distributed over a broader domain. The ventral region becomes sufficiently expanded such that the difference between the diffusion rates of processed Spätzle and the inhibitor can reshape the ventral domain itself. In particular, rapid diffusion of the inhibitor results in a lower concentration at each border, compared with the center of the domain. This change in the ratio of processed Spätzle to inhibitor eventually produces a peak at each border of the expanded domain. By this reasoning, an expanded ventral domain never generates more than two peaks because there are never more than two borders (Morisato, 2001).

Embryos synthesizing high levels of precursor Spätzle increase the amount of processed Spätzle, thereby expanding the domain of high nuclear Dorsal. In contrast to embryos produced by grk females, where a wild-type level of processed Spätzle is distributed over a broader area, an increased level of processed Spätzle appears to generate a broader domain in these injected embryos. Pattern refinement is observed only at the highest levels of Spätzle production, perhaps because only in this situation can the minimum domain size be created (Morisato, 2001).

The complexity of the patterning process is underscored by the observation that partial axis duplication can be induced by both an increase and decrease in spz dosage, depending on the extent of pipe expression dictated by gurken-Egfr signaling. A deeper understanding of this dynamic behavior will probably require the application of mathematical approaches (Morisato, 2001).

In order to explain the production of two Dorsal peaks, the inhibitor must be generated in a spatially asymmetric manner. In the model outlined here, inhibitor production has been linked to the proteolytic processing of Spätzle to satisfy this condition. The results described above raise the possibility that N-terminal processed Spätzle is acting as an inhibitor to shape the Dorsal gradient, although the model does not exclude action of other negative regulators. For example, pattern refinement may involve maternal Dpp signaling, acting parallel or downstream of Toll, which has been shown to reduce the magnitude of Dorsal nuclear translocation. At the mechanistic level, the N-Spätzle inhibitor could be recruiting molecule X, found in a stable complex with Easter and suggested to be a serpin, or it could be acting on one of the proteases that act upstream of Easter. Alternatively, the inhibitor could be negatively regulating the binding of processed Spätzle to Toll (Morisato, 2001).

The strongest genetic support for diffusion playing a role in the formation of the Dorsal gradient comes from mosaic analysis. Ventral pipe- clones not only result in the absence of Twist expression, but also produce a corresponding loss of sog expression in lateral regions of the embryo. These results rule out the presence of a pre-existing gradient in the follicular epithelium. The requirement for a ventral source of the Dorsal gradient suggests that the slope is generated by the diffusion of a component in the embryonic signaling pathway, although a sequential induction mechanism within the follicular epithelium is not formally excluded (Morisato, 2001).

An output dependent on the ratio of the respective diffusion rates of activator and inhibitor, rather than diffusion of the activator alone, may allow the embryo to generate a more stable gradient shape in response to the broad spatial signals defined during oogenesis. Moreover, such a mechanism may help the embryo cope with changes in perivitelline fluid viscosity, caused by fluctuations in temperature and humidity after egg deposition, that would otherwise result in developmental defects. Coupling diffusion of an activator and inhibitor may represent a general strategy for regulating extracellular signaling in other patterning reactions (Morisato, 2001).

Modeling of the Dorsal gradient across species reveals interaction between embryo morphology and Toll signaling pathway during evolution

Morphogenetic gradients are essential to allocate cell fates in embryos of varying sizes within and across closely related species. The maternal NF-kappaB/Dorsal (Dl) gradient has acquired different shapes in Drosophila species, which result in unequally scaled germ layers along the dorso-ventral axis and the repositioning of the neuroectodermal borders. This study combined experimentation and mathematical modeling to investigate which factors might have contributed to the fast evolutionary changes of this gradient. To this end, a previously developed model was developed that employs differential equations of the main biochemical interactions of the Toll (Tl) signaling pathway, which regulates Dl nuclear transport. The original model simulations fit well the D. melanogaster wild type, but not mutant conditions. To broaden the applicability of this model and probe evolutionary changes in gradient distributions, a set of 19 independent parameters was adjusted to reproduce three quantified experimental conditions (i.e. Dl levels lowered, nuclear size and density increased or decreased). Next, the most relevant parameters were sought that reproduce the species-specific Dl gradients. Adjusting parameters relative to morphological traits (i.e. embryo diameter, nuclear size and density) alone is not sufficient to reproduce the species Dl gradients. Since components of the Tl pathway simulated by the model are fast-evolving, it was next asked which parameters related to Tl would most effectively reproduce these gradients, and a particular subset was identified. A sensitivity analysis reveals the existence of nonlinear interactions between the two fast-evolving traits tested above, namely the embryonic morphological changes and Tl pathway components. The modeling further suggests that distinct Dl gradient shapes observed in closely related melanogaster sub-group lineages may be caused by similar sequence modifications in Tl pathway components, which are in agreement with their phylogenetic relationships (Ambrosi, 2014; PubMed).

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

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

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

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

The Friend of GATA transcriptional co-regulator, U-Shaped, is a downstream antagonist of Dorsal-driven prohemocyte differentiation in Drosophila
Recent studies suggest that mammalian hematopoietic stem and progenitor cells (HSPCs) respond directly to infection and inflammatory signaling. These signaling pathways also regulate HSPCs during steady-state conditions (absence of infection), and dysregulation may lead to cancer or age-related loss of progenitor repopulation capacity. Toll-like receptors (TLRs) are a major class of pathogen recognition receptors, and are expressed on the surface of immune effector cells and HSPCs. TLR/NF-κB activation promotes HSPCs differentiation; however, the mechanisms by which this signaling pathway alters the intrinsic transcriptional landscape are not well understood. Although Drosophila prohemocytes are the functional equivalent of mammalian HSPCs, a prohemocyte-specific function for Toll signaling has not been reported. Using Drosophila transgenics, this study identified prohemocyte-specific roles for Toll pathway members, Dorsal and Cactus. It was shown that Dorsal is required to limit the size of the progenitor pool. Additionally, activation of Toll signaling in prohemocytes drives differentiation in a manner that is analogous to TLR/NF-κB-driven HSPC differentiation. This was accomplished by showing that over-expression of Dorsal, or knockdown of Cactus, promotes differentiation. The study also investigated whether Dorsal and Cactus control prohemocyte differentiation by regulating a key intrinsic prohemocyte factor, U-shaped (Ush), which is known to promote multipotency and block differentiation. It was found that Dorsal represses Ush expression levels to promote differentiation, whereas Cactus maintains Ush levels to block differentiation. Additionally, another Toll antagonist, Lesswright, also maintains the level of Ush to block differentiation and promote proliferative quiescence. Collectively, these results identify a novel role for Ush as a downstream target of Toll signaling (Gao, 2016).

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient
The transcription factor NF-kappaB plays an important role in the immune system, apoptosis and inflammation. Dorsal, a Drosophila homolog of NF-kappaB, patterns the dorsal-ventral axis in the blastoderm embryo. During this stage, Dorsal is sequestered outside the nucleus by the IkappaB homolog Cactus. Toll signaling on the ventral side breaks the Dorsal/Cactus complex, allowing Dorsal to enter the nucleus to regulate target genes. Fluorescent data show that Dorsal accumulates on the ventral side of the syncytial blastoderm. This study used modeling and experimental studies to show that this accumulation is caused by facilitated diffusion, or shuttling, of the Dorsal/Cactus complex. Active Toll receptors are limiting in wild-type embryos, which is a key factor in explaining global Dorsal gradient formation. These results suggest that shuttling is necessary for viability of embryos from mothers with compromised dorsal levels. Therefore, Cactus not only has the primary role of regulating Dorsal nuclear import, but also has a secondary role in shuttling. Given that this mechanism has been found in other, independent, systems, it is suggested that it might be more prevalent than previously thought (Carrell, 2017).

Effects of Mutation or Deletion

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 TollD 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).

Dorsal-ventral specification of the Drosophila embryo is mediated by signaling pathways that have been very well described in genetic terms. However, little is known about the physiology of Drosophila development. By imaging patterns of free cytosolic calcium in Drosophila embryos, it has been found that several calcium gradients are generated along the dorsal-ventral axis. The most pronounced gradient is formed during stage 5, in which calcium levels are high dorsally. Manipulation of the stage 5 calcium gradient affects specification of the amnioserosa, the dorsal-most region of the embryo. This calcium gradient is inhibited in pipe, Toll, and dorsal mutants, but is unaltered in decapentaplegic or punt mutants, suggesting that the stage 5 calcium gradient is formed by a suppression of ventral calcium concentrations. It is concluded that calcium plays a role in specification of the dorsal embryonic region (Creton, 2000).

During early development (0 -2.5 h), calcium concentrations were elevated in the ventral region of the embryo and oscillate along with the cell cycle. Early Drosophila development is characterized by nuclear division without cell division, indicating that these calcium oscillations are associated with the embryo's nuclear cycle. The lowest levels of calcium are observed at the end of stage 4 (2.5 h). These low calcium concentrations represent the 'resting level' of calcium and average 72 nM. Cell formation (stage 5) lasts for about an hour and calcium levels increase during this time. This calcium increase is most pronounced at the dorsal region, thus creating a stage 5 calcium gradient with high calcium dorsally. The calcium concentrations in the dorsal region average 107 nM. The calcium gradient remains visible until the end of stage 6 (3 h 35 min). Calcium levels increase further in late embryonic development. These calcium elevations seem to be associated with gross morphological changes such as germ band extension and stomodeal invagination. Calcium levels reach a maximum of 137 nM during germ band extension (4 h 15 min). At 5.5 h, calcium gradients reverse for a second time to give high calcium concentrations ventrally. Thus, a total of three calcium gradients was observed along the dorsoventral axis during the first 6 h of development (Creton, 2000).

Injected BAPTA-type calcium buffers are known to permanently suppress calcium-dependent development by repeatedly carrying calcium from a source (such as a leaky region of the plasma membrane) to a calcium sink (such as the subsurface ER) into which this calcium is released. Such 'shuttle buffers' thereby suppress the development of high calcium zones. To determine if BAPTA injection affects specification of the amnioserosa, the dorsal-most region of the embryo was used, from which a Drosophila line was generated in which the amnioserosa element of the Kruppel upstream region drives the expression of lacZ. The Kr-lacZconstruct is exclusively expressed in the amnioserosa cells of stage 14 embryos. Injection of dibromo-BAPTA during stage 5 strongly inhibits Kr-lacZ expression. Thus the dorsal calcium zone is needed for the development of dorsal-specific gene expression. Kr-lacZ expression is fully restored or 'rescued' by raising the injectate's calcium to the micromolar level. This rescue further confirms that the observed inhibition is a direct effect of suppressing the dorsal calcium zone (Creton, 2000).

Embryos injected with dibromo-BAPTA at a final concentration of 3 mM show severe developmental abnormalities. The head fold and dorsal folds are mostly reduced or missing. In some cases, a head fold is formed that is symmetric along the dorsal-ventral axis. Cuticles are not formed in most of the embryos. Embryos that form cuticles show a reduced number of denticle belts, which are localized at their normal ventral position and do not seem to be extended or reduced along the dorsoventral axis. A normal mouth, present in treated embryos, includes mouth hooks, H-pieces, and ventral arms. However, the dorsal bridge and dorsal arms are often absent. This latter defect indicates a mild ventralization of the embryo. The lack of cuticle formation in the majority of the embryos may be expected, since calcium is a widely used messenger that probably affects many aspects of development. Nonetheless, the signs of ventralization further support the concept that a high calcium zone is needed for dorsal development, The expression of Kr-lacZ is significantly inhibited in treated morphologically normal stage 14 embryos; i.e., only half (49%) of the stage 14 embryos express Kr-lacZ. This experiment indicates that low concentrations of dibromo-BAPTA can inhibit specification of the amnioserosa without affecting the overall morphology of the stage 14 embryo (Creton, 2000).

Dorsalized pipe mutant embryos show highly elevated levels of calcium during stage 5 in the dorsal as well as the ventral regions. The stage 5 calcium gradient is lost in these embryos. Pipe is a key regulator of dorsoventral specification during oogenesis. Thus, the formation of the stage 5 calcium gradient is far downstream of this event. Calcium patterns were subsequently imaged in the Toll10B mutants. Toll10B mutant embryos are ventralized due to the overactive plasma membrane receptor Toll. These embryos do not show the typical dorsal calcium elevation during stage 5 and the stage 5 calcium gradient is lost. This indicates that the stage 5 calcium gradient is formed downstream of Toll. The dorsalized dl2 embryos show highly elevated levels of calcium during stage 5 in the dorsal as well as the ventral region. The stage 5 calcium gradient is thus lost in these embryos. This shows that the formation of the calcium gradient is downstream of dl. Other experiments show that the calcium pattern is upstream or independent of the Dpp signaling pathway (Creton, 2000).

The analysis of calcium patterns in mutant embryos shows that the formation of the stage 5 calcium gradient is downstream of dl and suggests that the Dl protein plays a role in formation of this calcium gradient by inhibiting ventral calcium elevations. At present it is not clear by which mechanisms Dl inhibits calcium concentrations in the ventral region. Possibly, nuclear Dl affects transcription of genes coding for calcium channels or calcium pumps. This modulation of gene expression may be a direct effect of Dl, or may be mediated by other proteins such as Twist or Snail. It is also not clear which mechanisms are responsible for the observed calcium elevation in the dorsal region during stage 5. It is speculated that this calcium increase may be caused by formation of the cleavage furrows, which activate stretch-sensitive calcium channels. This would cause a general calcium increase during stage 5, which would subsequently be inhibited on the ventral side by the Dl protein (Creton, 2000).

Drosophila Sex-peptide stimulates female innate immune system after mating via the Toll and Imd pathways

Insect immune defense is mainly based on humoral factors like antimicrobial peptides (AMPs) that kill the pathogens directly or is based on cellular processes involving phagocytosis and encapsulation by hemocytes. In Drosophila, the Toll pathway (activated by fungi and gram-positive bacteria) and the Imd pathway (activated by gram-negative bacteria) leads to the synthesis of AMPs. But AMP genes are also regulated without pathogenic challenge, e.g., by aging, circadian rhythms, and mating. This study shows that AMP genes are differentially expressed in mated females. Metchnikowin (Mtk) expression is strongly stimulated in the first 6 hr after mating. Sex-peptide (SP), a male seminal peptide transferred during copulation, is the major agent eliciting transcription of Mtk and of other AMP genes. Both pathways are needed for Mtk induction by SP. Furthermore, SP induces additional AMP genes via the Toll (Drosomycin) and the Imd (Diptericin) pathways. SP affects the Toll pathway at or upstream of the gene spätzle, and the Imd pathway at or upstream of the gene imd. Mating may physically damage females and pathogens may be transferred. Thus, endogenous stimulation of AMP transcription by SP at mating might be considered as a preventive step to encounter putative immunogenic attacks (Peng, 2005).

The Toll and Imd signaling cascades are the major and best-characterized pathways involved in the activation of AMPs after pathogenic challenges. The effect of SP on AMP expression was studied by comparing the expression of Mtk, Drs, and Dipt in wt females or in females mutant in the Toll and Imd pathways, respectively, before and after mating with wt males. RNA was extracted from virgin and mated females and analyzed by quantitative PCR (Peng, 2005).

With the exception of dorsal (dl), all loss-of-function mutants of the Toll and Imd pathways abolish or strongly reduce Mtk expression after mating. Thus, Mtk expression induced by SP is dependent on both pathways. Furthermore, since spz and imd females fail to induce Mtk transcription after mating, SP must act on or upstream of spz and imd. dl and its functional homolog dif have been reported to be involved in AMP gene transcription under pathogenic challenge in the larval stage, but not functional in the adult immune defense. A partial response is observed in dl females, indicating that dl may be partially involved in the innate immune response elicited by SP in adult females (Peng, 2005).

Drs expression, controlled by the Toll pathway, is completely abolished in spz and Tl mutants. Correspondingly, Dipt expression, which is controlled by the Imd pathway, is completely abolished in the Imd pathway loss-of-function mutants imd, Tak1, and rel. It is concluded that SP can activate the Toll and the Imd pathways. The Toll pathway is essential for Drs expression, whereas the Imd pathway is essential for Dipt expression (Peng, 2005).

The SP-induced immune response activates the transcription of all three AMP genes studied. After pathogenic infections, Drs is induced by the Toll pathway and Dipt by the Imd pathway, whereas both pathways induce Mtk expression. The results obtained with the loss-of-function mutants follow this scheme. Whereas loss-of-function mutants of both pathways reduce or abolish Mtk expression after mating, induction of Drs expression is only abolished by loss-of-function mutants of the Toll pathway, whereas induction of Dipt expression is only lost in mutants of the Imd pathway. In sum, the classical pathways are activated to induce the transcription of AMP genes after mating as after microbial or fungal infections (Peng, 2005).

Detection of microorganisms and triggering the appropriate pathway is achieved by pattern recognition receptors (PRRs), immune proteins recognizing general microbial components. Two families of PRRs have been identified in Drosophila: the peptidoglycan recognition proteins (PGRPs) and the gram-negative binding proteins (GNBPs). Some of the 13 PGRPs encoded in the D. melanogaster genome have been implicated in the activation of specific immune responses. However, the signaling cascades between the PRRs and the Toll and the Imd pathways are not well characterized. Since in spz and imd null mutants AMP induction by SP is specifically abolished, the inducing signals must affect the signaling cascades at or upstream of those genes. At this stage, it cannot be determined whether SP enters the pathways at the PRR level or at an intermediate level between the PRRs and spz or imd, respectively. Furthermore, the induction of AMPs may occur systemically (e.g., in the fat body) or locally in the reproductive tract. Microarray analysis of AMP expression after mating of wt females with either wt or SP0 males, respectively, suggests that AMPs are mainly induced in the abdomen, but it does not discriminate between a systematic response in the abdomen and a specific response in the genital tract (Peng, 2005).

Drosophila females undergo dramatic physiological changes after mating, predominantly induced by SP. Mating may also physically damage females and may expose the female to pathogens transferred by the male as shown for the milkweed leaf beetle. Thus, the activation of the innate immune system to encounter putative immunogenic attacks during this sensitive phase of the life history of females makes biological sense. The signal is plausibly coupled to copulation in the form of SP transferred in the seminal fluid. Such a mechanism might allow the female to respond preventively to potential threats. In sum, these findings may describe the result of an optimal economical balance between spending costly energy for the innate immune response and preventive measures to fight a putative pathogenic attack (Peng, 2005).

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).

dorsal continued: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | References

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