REGULATION (part 2/2)

Dorsal involvement in the immune response

The mammalian transcription factor NF-kappaB regulates a number of genes involved in immune and acute phase responses, by interacting with a nucleotide sequence element, the kappaB-motif, containing a consensus sequence GGGRNNYYCC. Similar motifs participate in the immune response of insects as well. KappaB-like motifs have a regulatory role in the synthesis of cecropins, a set of anti-bacterial peptides, triggered by the presence of bacterial cell wall components in insect blood. The upstream region of the Cecropin gene CecA1 contains elements responsible for inducible and tissue-specific expression (consensus sequence GGGRAYYYYY). A trimer of kappaB-like motis confer high high levels of inducible expression from a reporter gene, after transfection in a Drosophila blood cell line. Stimulation with bacterial lipopolysaccharide induces a nuclear factor that specifically binds to the kappaB-like motif. These data suggest a functional and evolutionary relationship between these insect immune response factors and the mammalian NF-kappaB (Engström, 1993).

The induction of the humoral immune response involves Dif and Dorsal, Rel-containing regulatory proteins related to mammalian NF-kappa B. These regulatory proteins function as sequence-specific transcription factors that induce the expression of immunity genes, including cecropin and diptericin. In mammals, NF-kappa B has been implicated in both lymphocyte differentiation and the acute-phase response. Thus insect and mammalian immunity involve related transcription factors (Ip, 1994).

The nuclear localization of DL during the immune response is controlled by the Toll signaling pathway. In mutants such as Toll or cactus, which exhibit melanotic tumor phenotypes, DL is constitutively nuclear. Together, these results point to a potential link between the Toll signaling pathway and melanotic tumor induction. Although DL has been shown previously to bind to kappa B-related motifs within the promoter of the antibacterial peptide coding gene diptericin, injury-induced expression of diptericin can occur in the absence of DL. Furthermore, the melanotic tumor phenotype of Toll and cactus is not dl dependent. These data underline the complexity of the Drosophila immune response. Like other rel proteins, dl can control the level of its own transcription (Lemaitre, 1995).

Bacterial challenge of larvae or adults of Drosophila induces the rapid transcription of several genes encoding antibacterial peptides with a large spectrum of activity. One of these peptides, the 82-residue anti-gram negative diptericin, is encoded by a single intronless gene and the control of expression of this gene is being characterized. Previous studies using both transgenic experiments and footprint analysis have highlighted the role in the induction of this gene of a 30 nucleotide region which contains three partially overlapping motifs with sequence homology to mammalian NF-kappa B and NF-IL6 response elements and to the GAAANN sequence present in the interferon consensus response elements of some mammalian interferon-induced genes. The latter sequence binds in immune responsive tissues (fat body, blood cells) of Drosophila an approximately 45 kDa polypeptide which cross-reacts with a polyserum directed against mammalian interferon Regulatory Factor-I. Using a transfection assay of Drosophila tumorous blood cells, it is shown that the GAAANN sequence positively regulates the activity of the diptericin promoter. It is propose that this motif cooperatively interacts with the other response elements in the regulation of the diptericin gene expression (Georgel, 1995).

Dorsal is expressed in the fat body of larvae and adults of Drosophila. Its expression is noticeably enhanced upon bacterial (or lipopolysaccharide) challenge. This challenge also induces within 15-30 min a nuclear translocation of the Dorsal protein. The promoters of genes encoding inducible antibacterial peptides in Drosophila contain kappa B-related nucleotide sequences and the Dorsal protein can bind to such motifs and activate them in a Drosophila cell line. However in the absence of Dorsal protein, the genes encoding antibacterial peptides retain their inducibility, suggesting a multifactorial control (Reichhart, 1993).

The receptor Toll, intracellular components of the dorsoventral signaling pathway, Tube, Pelle, and Cactus, and the extracellular Toll ligand, Spätzle, but not the NF-kappaB related transcription factor Dorsal, control expression of the antifungal polypeptide gene drosomycin in adults. Mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. In contrast, drosomycin gene induction is not affected in mutants deficient in gastrulation defective, snake and easter, all upstream of spätze in the dorsoventral pathway. The involvement of Cactus in the drosomycin induction pathway is unexpected, since, in contrast with cat, pll, tub, and Tl, the spz mutant shows no striking zygotic phenotype. The partner of Cact in the drosomycin induction pathway has not yet been identified, but it is probably a member of the Rel family, possibly Dif (Lemaitre, 1996).

There are two distinct regulatory pathways controlling the expression of antimicrobial genes, the dorsoventral pathway and the immune deficiency (imd) gene. In contrast to the results with drosomycin, antibacterial genes, cecropin A1, diptericin, drosocin, attacin, and defensin do not give strong constitutive expression in dorsoventral pathway mutants. However, the level of constitutive expression of anti-bacterial genes in dorsoventral pathway mutants is higher than the basal level, and induction of Cecropin A genes is 4-fold lower in dorsoventral pathway mutants. The transcription of cact, dorsal, dif, pll, tub, Tl and spz genes, but not tub, are clearly up-regulated in response to immune challenge. Even though the same components of the dorsoventral pathway that are involved in antifungal response are also involved in antibacterial response, there is an additional requirement for the as yet uncloned imd gene product (Lemaitre, 1996).

The diptericin and cecropin Al genes, which have been investigated in detail, contain two, respectively one sequence element homologous to the binding site of the mammalian nuclear factor kappaB and other c-rel homologs. A comparative analysis of the transactivating capacities of Dorsal and Dif on reporter genes fused to either the diptericin or the cecropin kappaB-related motifs suggests: (1) the kappaB motifs of the diptericin and cecropin genes are not functionally equivalent; (2) the Dorsal and Dif proteins have distinct DNA-binding characteristics; (3) Dorsal and Dif can heterodimerize in vitro, and (4) mutants containing no copies of dorsal and a single copy of Dif retain their full capacity to express the diptericin and cecropin genes in response to challenge (Gross, 1996).

The dorsoventral regulatory gene pathway, coded for by spatzle, Tolland cactus, controls the expression of several antimicrobial genes during the Drosophila immune response. This regulatory cascade shows striking similarities with the cytokine-induced activation cascade of NF-kappaB during the inflammatory response in mammals. The regulation of the IkappaB homolog Cactus has been studied in the fat body during the immune response. The cactus gene is up-regulated in response to immune challenge. Three hours after a bacterial challenge, cactus gene expression is markedly up-regulated in adults. A faint signal for Cact transcripts is present in unchallenged fat body and adult carcass and a remarkably rapid and strong up-regulation following bacterial challenge is observed. In both larvae and adults, peak values are observed after 2 or 3 h, after which the signals of Cact transcripts level off. These kinetics of induction/up-regulation, frequently referred to as acute phase kinetics, are similar to those of the cecropin A gene in these experiments. In contrast, the drosomycin and the diptericin genes reach their highest level of expression only 6-16 h postchallenge. Two Cact transcripts are observed during development; they are approximately 2.2 kb (referred to as maternal/zygotic) and 2.6 kb (zygotic) and encode proteins of 71 and 69 kDa, respectively, which differ in their C-terminal parts flanking the PEST sequence. Both transcripts are detectable in unchallenged tissues and are clearly up-regulated after bacterial challenge, the 2.2-kb transcript being predominant (Nicolas, 1998).

The immune response enhancer contains several sequence motifs homologous to insect and/or mammalian binding sites for Rel proteins: three sites are present upstream of a first intron P-transposon insertion site and two others are overlapping and located in intron 1. Intron 2 contains four sites, and intron 3 contains one site. These sites all contain the canonical three G residues in the 5' sequence, but differ in their 3' sequences; taken individually, some of these motifs are similar to counterparts in the various promoters of immune inducible genes encoding antimicrobial peptides (Nicolas, 1998).

Interestingly, the expression of the cactus gene is controlled by the spatzle/Toll/cactus gene pathway, indicating that the cactus gene is autoregulated. The two Cactus isoforms are expressed in the cytoplasm of fat body cells and they are rapidly degraded and resynthesized after immune challenge. This degradation is also dependent on the Toll signaling pathway. Altogether, these results underline the striking similarities between the regulation of IkappaB and cactus during the immune response (Nicolas, 1998).

In Drosophila, a septic wound induces the rapid appearance in the hemolymph of a battery of antibacterial peptides that includes the Cecropins, Drosocin, insect Defensin, Metchnikowin, Attacin and one major antifungal peptide, Drosomycin. These peptides are synthesized mostly in the fat body, a functional equivalent of the liver, and secreted into the hemolymph. This reaction constitutes a systemic antimicrobial response. Since experimental wounds are restricted to a single point of entry and since all of the disseminated fat body is responding to the attack, it is thought that a signal is transmitted to the fat body through the hemolymph from the entry site of microorganisms, where non-self recognition presumably occurs. This study asked whether antimicrobial peptides are also expressed in barrier epithelia in Drosophila, independent of a systemic response. This question was specifically addressed regarding the expression of the antifungal peptide Drosomycin in both larvae and adults. Using a drosomycin-green fluorescent protein (GFP) reporter gene, it was shown that in addition to the fat body, a variety of epithelial tissues that are in direct contact with the external environment, including those of the respiratory, digestive and reproductive tracts, can all express the antifungal peptide, suggesting a local response to infections affecting these barrier tissues. As is the case for vertebrate epithelia, insect epithelia appear to be more than passive physical barriers and are likely to constitute an active component of innate immunity. In contrast to the systemic antifungal response, this local immune response is independent of the Toll pathway (Ferrandon, 1998).

NF-kappaB/Rel family proteins regulate genes that are critical for many cellular processes including apoptosis, inflammation, immune response, and development. NF-kappaB/Rel proteins function as homodimers or heterodimers that recognize specific DNA sequences within target promoters. The activities of different Drosophila Rel-related proteins were examined in modulating Drosophila immunity genes by expressing the Rel proteins in stably transfected cell lines. How different combinations of these transcriptional regulators control the activity of various immunity genes were also compared. The results show that Rel proteins are directly involved in regulating the Drosophila antimicrobial response. drosomycin and defensin expression is best induced by the Relish/Dif and the Relish/Dorsal heterodimers, respectively, whereas the attacin activity can be efficiently up-regulated by the Relish homodimer and heterodimers. These results illustrate how the formation of Rel protein dimers differentially regulate target gene expression (Han, 1999).

These results demonstrate that all five immunity genes tested can be regulated by Rel-related proteins. Moreover, heterodimer formation can lead to an increased potential of gene regulatory activity by these factors. At least three pathways have been proposed to be involved in the transcriptional activation of Drosophila anti-microbial gene expression. drosomycin is largely regulated by the Toll/Cactus pathway, whereas attacin and cecropin are regulated by a pathway involving imd and 18wheeler. The third pathway involving imd is employed to regulate diptericin and drosocin. Further evidence indicates that the Drosophila immune system may involve additional regulatory molecules. One example is that attacin and cecropin respond to the p38 mitogen-activated protein kinase signaling pathway. It has also been shown that Dif and Dorsal can be activated independently, implying the presence of multiple signaling components that may lead to the activation of individual Rel proteins. The present results reveal another level of complexity in the regulatory mechanism. Despite some overlapping activations by other combinations, drosomycin expression is primarily activated by Dif/Relish heterodimer; defensin expression is primarily activated by Dorsal/Relish heterodimer. The attacin expression is primarily activated by Relish homodimer, and Dif/Relish and Dorsal/Relish heterodimers can perform the function during LPS stimulation of attacin. Therefore, although the numbers of Rel proteins are limited, they can mediate a broad range of cellular processes by using different combinations of these transcription factors (Han, 1999 and references).

Since these results show that different heterodimers have different preferences with regard to target gene regulation, it is possibile that the cell may regulate Relish/Dif and Relish/Dorsal through different pathways in order to achieve specific needs. This may explain reported observations showing that in different mutant flies the activation of Dif and Dorsal can be independently regulated and that different micro-organisms induce different subsets of antimicrobial peptides. Based on these results, it is also expected that Relish has a broader effect than either Dif or Dorsal on Drosophila immunity gene expression, since Relish is a common subunit of the heterodimers. In summary, it has been established that the differential regulation of Rel proteins can lead to preferential expression of specific target genes (Han, 1999).

An antioxidant system required for host protection against gut infection in Drosophila

A fundamental question that applies to all organisms is how barrier epithelia efficiently manage continuous contact with microorganisms. This study shows that in Drosophila an extracellular Immune-regulated catalase (IRC; CG8913) mediates a key host defense system that is needed during host-microbe interaction in the gastrointestinal tract. Strikingly, adult flies with severely reduced IRC expression show high mortality rates even after simple ingestion of microbe-contaminated foods. However, despite the central role that the NF-kappaB pathway plays in eliciting antimicrobial responses, NF-kappaB pathway mutant flies are totally resistant to such infections. These results imply that homeostasis of redox balance by IRC is one of the most critical factors affecting host survival during continuous host-microbe interaction in the gastrointestinal tract (Ha, 2005; full text of article)

An essential complementary role of NF-kappaB pathway to microbicidal oxidants in Drosophila gut immunity

In the Drosophila gut, reactive oxygen species (ROS)-dependent immunity is critical to host survival. This is in contrast to the NF-kappaB pathway whose physiological function in the microbe-laden epithelia has yet to be convincingly demonstrated despite playing a critical role during systemic infections. A novel in vivo approach was used to reveal the physiological role of gut NF-kappaB/antimicrobial peptide (AMP) system, which has been 'masked' in the presence of the dominant intestinal ROS-dependent immunity. When fed with ROS-resistant microbes, NF-kappaB pathway mutant flies, but not wild-type flies, become highly susceptible to gut infection. This high lethality can be significantly reduced by either re-introducing Relish expression to Relish mutants or by constitutively expressing a single AMP to the NF-kappaB pathway mutants in the intestine. These results imply that the local 'NF-kappaB/AMP' system acts as an essential 'fail-safe' system, complementary to the ROS-dependent gut immunity, during gut infection with ROS-resistant pathogens. This system provides the Drosophila gut immunity the versatility necessary to manage sporadic invasion of virulent pathogens that somehow counteract or evade the ROS-dependent immunity (Ryu, 2006).

The intestinal NF-kappaB activation and subsequent local AMP induction are key elements of gut immunity in Drosophila. Some earlier studies in mammals have also described the in vivo protective role of mammalian AMPs against certain invasive pathogenic infections occurring in the barrier epithelia including the intestine and the skin. In Drosophila gut immunity, it has been shown that ROS-mediated antimicrobial response is essential for host survival during gut infection. In addition to oxidant-dependent immunity, phagocytosis by macrophages also plays an important role in a gut infection model. The present study revealed that in the Drosophila gastrointestinal tract, NF-kappaB/AMP-dependent innate immunity is normally dispensable but provisionally crucial in case the host encounters ROS-resistant microbes. Although the precise mechanism by which ROS-resistant microbes induce epithelial cell damages remains to be investigated, it can be speculated that high numbers of local microbes may produce metabolites toxic to the gut epithelia. Alternatively, it is also possible that excess chronic inflammation due to persistent microbes may cause host gut pathology similar to host immune effector-induced metabolic collapse observed in a Salmonella-infected Drosophila model (Ryu, 2006).

It should be noted that yeast and E. coli are not pathogens for the fly in normal situations and that manipulations to render these microbes ROS resistant may not directly reflect natural infection pathways in the animal. However, since ROS are known to be involved in many of the complex interactions between the invading microorganisms and the host, this approach will likely be a relevant method in understanding the integrative relationship between gut immunity and microbial pathogenesis. Arthropod gut immunity during host-pathogen interactions is particularly interesting because the majority of deadly arthropod-transmitted pathogens/parasites causing illnesses such as malaria, plague, typhus and lyme disease have evolved to use the host's gut as a route of transmission. Within the context of pathogen survival strategies, microbial pathogens must evade or counteract innate immune effectors such as ROS and AMPs in order to disseminate and cause diseases. In a constant competition for survival, the pathogen and the host have developed strategies to overcome the other. Along with the highly efficient microbicidal ROS, the Drosophila gastrointestinal tract has been shown to express at least seven different IMD/NF-kappaB-dependent AMPs, including Drosomycin, each exhibiting a distinct spectrum of in vitro antimicrobial activity. In this context, it is proposed that the different spectra of microbicidal activity encompassed by ROS and AMPs may provide the necessary versatility to the Drosophila gastrointestinal innate immune system to ward off microbial infections. Furthermore, these findings suggest that the diversification of intestinal innate immune effectors into ROS and AMP systems might have been driven by selective pressures exerted on the Drosophila gastrointestinal tract by its constant interactions with a series of different microbial species that employ different immune-evasion strategies (Ryu, 2006).

Dorsal feedback regulating Easter

Proteolytic activation of Spatzle requires the sequential action of four different members of the trypsin family. The first protease in this pathway is encoded by nudel, which is expressed in the somatic follicle cells of the ovary (which secrete the eggshell), whereas the other three proteases are expressed by the germline cells. gastrulation defective (gd) is closely related to the trypsin family, but the protein encoded by gd lacks a number of amino acid residues crucial for protease activity, so its biochemical function is not clear. Downstream of gd is the protease encoded by snake, which acts upstream of Easter, the final protease known in this pathway. The Easter protease is likely to be the direct proteolytic activator of Spatzle. Activated Easter is rapidly converted into a high molecular mass complex, which may contain a protease inhibitor (Misra, 1998).

Easter zymogen activation is controlled by a negative feedback loop from Dorsal, the transcription factor at the end of the signaling pathway. Mutations that block the intracellular signaling pathway leading to the activation of Dorsal do not block the formation of Ea-X. In fact, all these downstream mutants contain 3- to 5-fold more Ea-X than wild-type embryos. Even mutant embryos that lack Spatzle mRNA and protein show increased levels of Ea-X, indicating that Ea-X is not a stable complex of Easter, with its putative substrate, Spatzle. In ventralized cactus mutant embryos, the amount of Ea-X is less than half the amount present in wild-type embryos. Since the amount of Ea-X reflects the amount of activated Easter, these results suggest that a feedback loop regulated by nuclear Dorsal acts back across the plasma membrane of the syncytial embryo to regulate activation of the Easter zymogen. The accumulation of Ea-X in downstream mutants during the early zygotic phase occurs at the same time that signaling through the pathway occurs. While the zymogen (unprocessed) form of Easter is present at fertilization, Ea-X does not appear until 1 hour after fertilization, at approximately the time that the Easter protein is active in the embryo and increased during the next 2 hours of development. As soon as Ea-X is detectable, there is more Ea-X in Toll mutants than there is in wild-type embryos. Thus, well before the final gradient of Dorsal is achieved, at about 2.5 hours after fertilization, the level of Easter processing appears to be modified by this feedback loop (Misra, 1998).

Spatial regulation of microRNA gene expression in the Drosophila embryo: The 8-miR enhancer is regulated by the localized Huckebein repressor, whereas miR-1 is activated by Dorsal and Twist

MicroRNAs (miRNAs) regulate posttranscriptional gene activity by binding to specific sequences in the 3' UTRs of target mRNAs. A number of metazoan miRNAs have been shown to exhibit tissue-specific patterns of expression. This study investigated the possibility that localized expression is mediated by tissue-specific enhancers, comparable to those seen for protein-coding genes. Two miRNA loci in Drosophila melanogaster are investigated, the mir-309–6 polycistron (8-miR) and the mir-1 gene. The 8-miR locus contains a cluster of eight distinct miRNAs that are transcribed in a common precursor RNA. The 8-miR primary transcript displays a dynamic pattern of expression in early embryos, including repression at the anterior and posterior poles. An 800-bp 5' enhancer was identified that recapitulates this complex pattern when attached to a RNA polymerase II core promoter fused to a lacZ-reporter gene. The miR-1 locus is specifically expressed in the mesoderm of gastrulating embryos. Bioinformatics methods were used to identify a mesoderm-specific enhancer located ~5 kb 5' of the miR-1 transcription unit. Evidence is presented that the 8-miR enhancer is regulated by the localized Huckebein repressor, whereas miR-1 is activated by Dorsal and Twist. These results provide evidence that restricted activities of the 8-miR and miR-1 miRNAs are mediated by classical tissue-specific enhancers (Biemar, 2005).

The 8-miR complex is located between two predicted protein-coding genes, CG15125 and CG11018, in the 56E region on the right arm of chromosome 2. To determine the approximate transcription start site of the 8-miR transcription unit, 5' RACE was used. Several independent experiments were carried out, and RACE products corresponding to two different start sites were isolated several times. Consensus sequences for both an initiator and a TATA box are appropriately spaced upstream of the identified start sites. The alignment of this genomic interval with the corresponding regions of the most divergent Drosophilids indicates strong conservation of each of the individual miRNAs within the 8-miR complex (Biemar, 2005).

A digoxigenin-labeled 8-miR antisense RNA probe was hybridized to staged embryos to determine the expression profile of the precursor transcript during development. Expression is initially detected in all of the nuclei of precellular embryos. As expected, staining is restricted to nuclei and not seen in the cytoplasm. The first indication of differential spatial regulation occurs at the midpoint of cellularization, when 8-miR transcripts are lost at the posterior pole. By the completion of cellularization, this loss in staining expands and there is also reduced expression in anterior regions. Staining persists at the anterior tip but is lost from subterminal regions of the anterior pole (Biemar, 2005).

During gastrulation there is both dorsal-ventral and anterior-posterior modulation of the 8-miR-staining pattern. Staining is first lost from the presumptive mesoderm and neurogenic ectoderm in ventral and lateral regions. There are transient stripes of 8-miR expression in the dorsal ectoderm, but they rapidly give way to a single band of staining in central regions. By the onset of the rapid phase of germband elongation, staining is essentially lost except for residual expression at the anterior tip and dorsal ectoderm (Biemar, 2005).

The early loss of staining at the posterior pole suggests that Huckebein (Hkb) might repress 8-miR transcription in the early embryo. To investigate this possibility, colocalization assays were done with snail, which is selectively expressed in the presumptive mesoderm of cellularizing and gastrulating embryos. The posterior border of the snail pattern is established by the localized Hkb repressor. The 8-miR pattern displays a similar posterior border, and there is an expansion of both the snail and 8-miR patterns in hkb-/hkb- mutant embryos (Biemar, 2005).

Further evidence for repression by Hkb was obtained by analyzing torso dominant (torD) mutants. tor encodes a receptor tyrosine kinase that is normally activated only at the poles, where it is required for the localized expression of tailless (tll) and hkb. torD encodes a constitutively activated form of the receptor tyrosine kinase that results in expanded expression of hkb and tll at the poles. This expansion in Hkb causes a severe shift in the posterior border of both the snail and 8-miR expression patterns. The identification of a sequence-specific transcriptional repressor, Hkb, as a likely regulator of 8-miR expression suggests that the dynamic staining pattern is probably controlled at the level of de novo transcription (Biemar, 2005).

Direct support for this possibility was obtained by the identification of an 8-miR enhancer. An ~800-bp genomic DNA fragment extending from the miR-3 region of the 8-miR complex to the predicted start site of CG11018 was attached to a lacZ-reporter gene containing the minimal eve promoter sequence. The resulting fusion gene recapitulates most aspects of the endogenous 8-miR expression pattern. In particular, lacZ transcripts are initially detected throughout precellular embryos but sequentially lost from the posterior pole and anterior regions during cellularization. At the onset of gastrulation, expression is diminished in ventral regions, and the staining detected in the dorsal ectoderm exhibits segmental modulation. Thus, the 5' 8-miR enhancer contains repression elements that mediate silencing by Hkb (and possibly Tll) at the termini in response to Tor signaling (Biemar, 2005).

The preceding analysis provides evidence that cell-specific enhancers regulate miRNA gene expression, as seen for protein coding genes. Further support was obtained by analyzing a second miRNA that displays localized expression in the early Drosophila embryo, miR-1. The mir-1 gene is highly conserved in different animal groups and displays localized expression in a variety of mesodermal lineages, including cardiac mesoderm in vertebrates. The Drosophila mir-1 gene is first expressed in the presumptive mesoderm during the final phases of cellularization. Expression persists in differentiating mesodermal tissues during gastrulation, germband elongation, and segmentation. Mutant embryos that contain the constitutively activated Toll10B receptor display ubiquitous expression of miR-1, concomitant with the transformation of all of the tissues into mesoderm (Biemar, 2005).

Whole-genome tiling arrays were used to obtain an estimate of the miR-1 transcription unit. These high-density oligonucleotide arrays contain 25-nt oligomers spaced on average every 36 bp and cover the entire nonrepetitive Drosophila genome, from one end of each chromosome to the other. Total RNA was extracted from three different mutant strains. Embryos derived from pipe-/pipe- females lack Toll-signaling activity and thereby lack a Dorsal nuclear gradient. As a result, genes normally activated by high, intermediate, and low levels of the gradient are silent, and there is a loss of mesoderm and neurogenic ectoderm. Instead, genes that are repressed by the Dorsal gradient, and normally restricted to the dorsal ectoderm, are now expressed throughout the embryo, causing the transformation of mesoderm and neurogenic ectoderm into dorsal ectoderm. Previous microarray assays have shown that genes expressed in the dorsal ectoderm are overexpressed in mutant embryos derived from pipe-/pipe- embryos. As expected, such mutants display little or no expression of the miR-1 transcription unit. Similarly, embryos derived from Tollrm9/Tollrm10 mutants contain weak Toll signaling and low levels of nuclear Dorsal everywhere. These low levels are insufficient for the activation of mesoderm genes, but are sufficient for the activation of neurogenic genes and the repression of dorsal ectoderm genes. Again, these mutants fail to express miR-1. Toll10B embryos contain strong, ubiquitous Toll signaling and high levels of Dorsal, which activate mesoderm genes throughout the embryo. These embryos display strong expression of the miR-1 transcription unit. The tiling array suggests that the gene is ~2.9 kb in length. The mature, processed miRNA is located roughly in the center of the inferred transcription unit (Biemar, 2005).

The early expression of the miR-1 primary transcript in the mesoderm raises the possibility that the gene might be regulated by the Dorsal gradient. Approximately one-half of all Dorsal-target enhancers also contain binding sites for the basic helix-loop-helix Twist activator. A 50-kb interval encompassing the miR-1 locus was surveyed for clusters of Dorsal and Twist binding sites. The best cluster was identified ~5 kb upstream of the miR-1 start site. There are a total of three Dorsal- and four Twist-binding sites contained over an interval of ~1.1 kb in this distal 5' region (Biemar, 2005).

A genomic DNA fragment encompassing these sites was attached to a lacZ-reporter gene and expressed in transgenic embryos. The reporter gene exhibits localized expression in the ventral mesoderm, beginning at the onset of gastrulation. Expression persists during germband elongation. These observations suggest that miR-1 is directly activated by Dorsal and Twist. However, lacZ transcripts expressed from the miR-1::lacZ transgene are detected somewhat later than the endogenous miR-1 primary transcript, which first appears before the completion of cellularization. It is conceivable that the miR-1 locus contains a second enhancer that directs earlier expression (Biemar, 2005).

The preceding analysis provides evidence that dynamic patterns of miRNA gene expression are controlled by tissue-specific enhancers, and not by the differential processing of miRNA precursor RNAs. Both the 8-miR and miR-1 enhancers produce authentic patterns of lacZ-reporter gene expression when attached to the core promoter region of the eve gene. The 8-miR enhancer appears to be regulated by the Hkb repressor, whereas miR-1 is activated by Dorsal and Twist (Biemar, 2005).

The miR-1 enhancer is somewhat unusual among 'type 1' Dorsal target enhancers, in that it contains a large number of Snail repressor sites. Type 1 enhancers are activated by high levels of the Dorsal gradient in the ventral mesoderm. Previous studies have identified six such enhancers. They all contain multiple low-affinity Dorsal binding sites, but essentially lack Snail repressor sites. The general absence of Snail sites permits activation of type 1 genes in the ventral mesoderm where there are high levels of the repressor. An exception is the type 1 intronic enhancer that regulates Heartless (Htl), one of the two FGF receptor genes in the Drosophila genome (Biemar, 2005).

The htl intronic enhancer is ~800 bp in length and contains two low-affinity Dorsal binding sites and two optimal Twist sites. Each Twist site overlaps a Snail repressor site, but the enhancer nonetheless activates lacZ-reporter gene expression in the presumptive mesoderm before the completion of cellularization. The htl enhancer fails to mediate expression in the neurogenic ectoderm because it lacks the arrangement of optimal Dorsal and Twist sites required for activation by intermediate levels of the Dorsal gradient (type 2 enhancers) (Biemar, 2005).

The miR-1 enhancer contains three weak Dorsal sites, four optimal Twist sites (CACATGT; Kate Senger, unpublished results cited in Biemar, 2005), and five Snail repressor sites (three of the sites overlap the optimal Twist sites and two occur at separate sites). Perhaps the relative increase in the number of Snail repressor sites in the miR-1 enhancer (vs. the htl enhancer) causes late onset of miR-1::lacZ transgene expression. The Snail repressor is transiently expressed in the ventral mesoderm during cellularization but disappears after invagination. It is during the time when Snail levels subside that the miR-1 enhancer first becomes active (Biemar, 2005).

Previous studies have emphasized the importance of the Snail repressor in defining spatially localized patterns of gene expression. Dorsal target genes activated by intermediate (type 2) and low (type 3) levels of the gradient contain Snail repressor sites that keep the genes off in the ventral mesoderm and restricted to the neurogenic ectoderm. The present identification of the distal miR-1 enhancer raises the possibility that Snail also influences the timing of gene expression (Biemar, 2005).

The similarities in miR-1 and Htl regulation raise the possibility that the miR-1 miRNA attenuates the activity of one or more components of the FGF-signaling pathway. FGF is essential for the migration of the invaginated mesoderm along the inner surface of the neurogenic ectoderm. It is also important for the activation of cardiac genes in the dorsal-most mesoderm that forms the heart. miR-1 might attenuate one or more target mRNAs engaged in mesoderm migration and/or heart induction. The mammalian miR-1 miRNA has been shown to attenuate Hnd2 expression, which is essential for the differentiation of ventricular cardiomyocytes (Zhao, 2005). Despite the conservation of the miR-1 miRNA sequence, and a potential role in suppressing heart formation in both flies and mice, it would appear that distinct mechanisms of regulation are used in the two systems: Dorsal and Twist activate miR-1 in flies, whereas distinct regulatory factors, SRF and MyoD, activate miR-1 in the mouse embryo. It is possible however, that later phases of miR-1 expression depend on nautilus (nau), the Drosophila homolog of MyoD (Biemar, 2005).

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).

Calcineurin isoforms are involved in Drosophila Toll immune signaling

Because excessive or inadequate responses can be detrimental, immune responses to infection require appropriate regulation. Networks of signaling pathways establish versatility of immune responses. Drosophila melanogaster is a powerful model organism for dissecting conserved innate immune responses to infection. For example, the Toll pathway, which promotes activation of NF-kappaB transcription factors Dorsal/Dorsal-related immune factor (Dif), was first identified in Drosophila. Together with the IMD pathway, acting upstream of NF-kappaB transcription factor calcineurin A1, acts on Relish during infection. However, it is not known whether there is a role for calcineurin in Dorsal/Dif immune signaling. This article demonstrates involvement of specific calcineurin isoforms, protein phosphatase at 14D (Pp2B-14D)/calcineurin A at 14F (CanA-14F), in Toll-mediated immune signaling. These isoforms do not affect IMD signaling. In cell culture, pharmacological inhibition of calcineurin or RNA interference against homologous calcineurin isoforms Pp2B-14D/CanA-14F, but not against isoform calcineurin A1, decreased Toll-dependent Dorsal/Dif activity. A Pp2B-14D gain-of-function transgene promoted Dorsal nuclear translocation and Dorsal/Dif activity. In vivo, Pp2B-14D/CanA-14F RNA interference attenuated the Dorsal/Dif-dependent response to infection without affecting the Relish-dependent response. Altogether, these data identify a novel input, calcineurin, in Toll immune signaling and demonstrate involvement of specific calcineurin isoforms in Drosophila NF-kappaB signaling (Li, 2014).

The Toll-Dorsal pathway is required for resistance to viral oral infection in Drosophila

Pathogen entry route can have a strong impact on the result of microbial infections in different hosts, including insects. Drosophila melanogaster has been a successful model system to study the immune response to systemic viral infection. This study investigated the role of the Toll pathway in resistance to oral viral infection in D. melanogaster. Several Toll pathway components, including Spatzle, Toll, Pelle and the NF-κB-like transcription factor Dorsal, are required to resist oral infection with Drosophila C virus. Furthermore, in the fat body Dorsal is translocated from the cytoplasm to the nucleus and a Toll pathway target gene reporter is upregulated in response to Drosophila C Virus infection. This pathway also mediates resistance to several other RNA viruses (Cricket paralysis virus, Flock House virus, and Nora virus). Compared with control, viral titres are highly increased in Toll pathway mutants. The role of the Toll pathway in resistance to viruses in D. melanogaster is restricted to oral infection since no phenotype was associated with systemic infection. It was also shown that Wolbachia and other Drosophila-associated microbiota do not interact with the Toll pathway-mediated resistance to oral infection. This study therefore identified the Toll pathway as a new general inducible pathway that mediates strong resistance to viruses with a route-specific role. These results contribute to a better understanding of viral oral infection resistance in insects, which is particularly relevant in the context of transmission of arboviruses by insect vectors (Ferreira, 2014).

Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75NTR receptors and cell death via p75NTR and Sortilin. Drosophila NTs (DNTs; see NT1) bind Toll receptors (see Toll-6 & Toll-7) instead to promote neuronal survival, but whether they can also regulate cell death is unknown. This study show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-κB and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts (Foldi, 2017).

Balancing cell death and cell survival enables structural plasticity and homeostasis, regeneration, and repair and fails in cancer and neurodegeneration. In the nervous system, cell number plasticity is linked to neural circuit formation, adjusting neuronal number to functional requirements. In mammals, the neurotrophin (NT) protein family [NGF, brain-derived neurotrophic factor (BDNF), NT3, and NT4] regulates neuronal number through two mechanisms. First, full-length pro-NTs, comprised of a disordered prodomain and a cystine-knot (CK) domain, induce cell death; in contrast, mature NTs formed of CK dimers promote cell survival. Second, pro-NTs bind p75NTR and Sortilin receptors, inducing apoptosis via JNK signaling, whereas mature NTs bind p75NTR, promoting cell survival via NF-κB and TrkA, B, and C, promoting cell survival via PI3K/AKT and MAPK/ERK. As the NTs also regulate connectivity and synaptic transmission, they couple the regulation of cell number to neural circuitry and function, enabling structural brain plasticity. There is abundant evidence that cell number plasticity occurs in Drosophila melanogaster central nervous system (CNS) development, with neurotrophic factors including NTs and mesencephalic astrocyte-derived neurotrophic factor (MANF), but fruit flies lack p75NTR and Trk receptors, raising the question of how this is achieved in the fly. Finding this out is important, as it could lead to novel mechanisms of structural plasticity for both flies and humans (Foldi, 2017).

The Drosophila NTs (DNTs) Spätzle (Spz), DNT1, and DNT2 share with mammalian NTs the characteristic structure of a prodomain and a conserved CK of 13-15 kD, which forms a disulfide-linked dimer. Spz resembles NGF biochemically and structurally, and the binding of its Toll-1 receptor resembles that of NGF to p75NTR. DNT1 (also known as spz2) was discovered by homology to BDNF, and DNT2 (also known as spz5) as a paralogue of spz and DNT1. DNT1 and 2 promote neuronal survival, and DNT1 and 2, Spz, and Spz3 are required for connectivity and synaptogenesis. Spz, DNT1, and DNT2 are ligands for Toll-1, -7, and -6, respectively, which function as NT receptors and promote neuronal survival, circuit connectivity, and structural synaptic plasticity. Tolls belong to the Toll receptor superfamily, which underlies innate immunity. There are nine Toll paralogues in flies, of which only Toll-1, -5, -7, and -9 are involved in immunity. Tolls are also involved in morphogenesis, cell competition, and epidermal repair. Whether DNTs and Tolls can balance cell number plasticity is unknown (Foldi, 2017).

Like the p75NTR receptor, Toll-1 activates NF-κB (a potent neuronal prosurvival factor with evolutionarily conserved functions also in structural and synaptic plasticity) signaling downstream. Toll-1 signaling involves the downstream adaptor MyD88, which forms a complex with Tube and Pelle. Activation of Toll-1 triggers the degradation of the NF-κB inhibitor Cactus, enabling the nuclear translocation of the NF-κB homologues Dorsal and Dorsal-related immunity factor (Dif), which function as transcription factors. Other Tolls have also been suggested to activate NF-κB. However, only Toll-1 has been shown to bind MyD88, raising the question of how the other Tolls signal in flies (Foldi, 2017).

Whether Tolls regulate cell death is also obscure. Toll-1 activates JNK, causing apoptosis, but its expression can also be activated by JNK to induce nonapoptotic cell death. Toll-2, -3, -8, and -9 can induce apoptosis via NF-κB and dSarm independently of MyD88 and JNK. However, in the CNS, dSarm induces axonal degeneration, but there is no evidence that it can promote apoptosis in flies. In other animals, Sarm orthologues are inhibitors of Toll signaling and MyD88, but there is no evidence that dSarm is an inhibitor of MyD88 in Drosophila. Thus, whether or how Tolls may regulate apoptosis in flies is unclear (Foldi, 2017).

In the mammalian brain, Toll-like receptors (TLRs) are expressed in neurons, where they regulate neurogenesis, apoptosis, and neurite growth and collapse in the absence of any insult. However, their neuronal functions have been little explored, and their endogenous ligands in neurons remain unknown (Foldi, 2017).

Because Toll-1 and p75NTR share common downstream signaling pathways and p75NTR can activate NF-κB to promote cell survival and JNK to promote cell death, this study asked whether the DNTs and their Toll receptors could have dual roles controlling cell survival and death in the Drosophila CNS (Foldi, 2017).

In the first regulatory tier, each DNT has unique features conducive to distinctive functions. Spz, DNT1, and DNT2 share with the mammalian NTs the unequivocal structure of the CK domain unique to this protein family. However, DNT1, DNT2, and Spz have distinct prodomain features and are processed differently, leading to distinct cellular outcomes. Spz is only secreted full length and cleaved by serine proteases. DNT1 and 2 are cleaved intracellularly by conserved furins. In cell culture, DNT1 was predominantly secreted with a truncated prodomain (pro-DNT1), whereas DNT2 was secreted mature. In vivo, both pro- and mature DNTs were produced from neurons. Interestingly, DNT1 also has an isoform lacking the CK domain, and Spz has multiple isoforms with truncated prodomains. Thus, in vivo, whether DNT1 and 2 are secreted full length or cleaved and whether Spz is activated will depend on the proteases that each cell type may express. Pro-DNT1 activates apoptotic JNK signaling, whereas mature DNT1 and 2 activate the prosurvival NF-κB (Dorsal and Dif) and ERK signaling pathways. Mature Spz does not activate ERK. This first tier is evolutionarily conserved, as mammalian pro-NTs can promote cell death, whereas furin-cleaved mature NTs promote cell survival. NF-κB, JNK, and ERK are downstream targets shared with the mammalian NTs, downstream of p75NTR (NF-κB and JNK) and Trks (ERK), to regulate neuronal survival and death. Thus, whether a cell lives or dies will depend on the available proteases, the ligand type, and the ligand cleavage product it receives (Foldi, 2017).

In a second regulatory tier, this study showed that the specific Toll family receptor activated by a DNT matters. Toll-6 and -7 could maintain neuronal survival, whereas Toll-1 had a predominant proapoptotic effect. Because there are nine Tolls in Drosophila, some Tolls could have prosurvival functions, whereas others could have proapoptotic functions. Different Tolls also lead to different cellular outcomes in immunity and development. Thus, the life or death of a neuron will depend on the Toll or combination of Tolls it expresses. Binding of Spz to Toll-1 is most likely unique, but DNT1 and 2 bind Toll-6 and -7 promiscuously, and, additionally, DNT1 and 2 with Toll-6 and -7 activate NF-κB and ERK, whereas pro-DNT1 activates JNK. This suggests that ligand prodomains might alter the affinity for Toll receptors and/or facilitate the formation of heterodimers between different Tolls and/or with other coreceptors to induce cell death. A 'DNT-Toll code' may regulate neuronal numbers (Foldi, 2017).

In a third tier, available downstream adaptors determine the outcome between cell survival and death. Toll-6 and -7 activate cell survival by binding MyD88 and activating NF-κB and ERK (whether ERK activation depends on MyD88 is not known), and Toll-6 can activate cell death via Wek, dSarm, and JNK signaling. Toll-6 was shown to bind MyD88 and Wek, which binds dSarm, and dSarm binds MyD88 and promotes apoptosis by inhibiting MyD88 and activating JNK. Wek also binds MyD88 and Toll-1. So, evidence suggests that Wek recruits MyD88 and dSarm downstream of Tolls. Because Toll-6 binds both MyD88 and Wek and Wek binds both MyD88 and dSarm, Wek functions like a hinge downstream of Toll-6 to facilitate signaling via MyD88 or dSarm, resulting in alternative outcomes. Remarkably, adaptor expression profiles change over time, switching the response to Toll-6 from cell survival to cell death. In the embryo, when both MyD88 and dSarm are abundant, there is virtually no Wek, and Toll-6 can only bind MyD88 to promote cell survival. As Wek levels rise, Toll-6 signaling can also induce cell death. If the Wek-Sarm-JNK route prevails, Toll-6 induces apoptosis; if the Wek-MyD88-NF-κB route prevails, Toll-6 signaling induces cell survival (Foldi, 2017).

Thus, the cellular outcome downstream of DNTs and Tolls is context and time dependent. Whether a cell survives or dies downstream of DNTs and Tolls will depend on which proteases are expressed nearby, which ligand it receives and in which form, which Toll or combination of Tolls it expresses, and which adaptors are available for signaling (Foldi, 2017).

How adaptor profiles come about or change is not understood. A neuronal type may be born with a specific adaptor gene expression profile, or Toll receptor activation may influence their expression. In fact, MyD88 reinforces its own signaling pathway, as Toll-6 and -7 up-regulate Dorsal, Dif, and Cactus protein levels and TLR activation increases Sarm levels. This study showed that apoptosis caused by MyD88 excess depends on JNK signaling. Because JNK functions downstream of Wek and dSarm, this suggests that MyD88, presumably via NF-κB, can activate the expression of JNK, wek, or dsarm. By positively regulating wek expression, MyD88 and dSarm could establish positive feedback loops reinforcing their alternative pathways. Because dSarm inhibits MyD88, mutual regulation between them could drive negative feedback. Positive and negative feedback loops underlie pattern formation and structural homeostasis and could regulate neuronal number in the CNS as well. Whether cell-autonomous or -nonautonomous mechanisms result in the diversification of adaptor profiles, either in time or cell type, remains to be investigated (Foldi, 2017).

Either way, over time the Toll adaptors segregate to distinct neural circuits, where they exert further functions in the CNS. Toll-1, -6, and -8 regulate synaptogenesis and structural synaptic plasticity. Sarm regulates neurite degeneration, and in the worm, it functions at the synapse to determine neuronal identity. The reporters used in this study revealed a potential segregation of MyD88 to the motor circuit and dSarm to the sensory circuit, but this is unlikely to reflect the endogenous complexity of Toll-signaling circuitry, as dsarmMIMIC- has a GFP insertion into one of eight potential isoforms, and dsarm also functions in the motor system (McLaughlin, 2016). Importantly, cell death in the normal CNS occurs mostly in late embryogenesis and in pupae, coinciding with neural circuit formation and remodeling, when neuronal number is actively regulated. Thus, the link by DNTs and Tolls from cell number to circuitry offers a complex matrix of possible ways to regulate structural plasticity in the CNS (Foldi, 2017).

This study has uncovered remarkable similarities between Drosophila Toll-6 and mammalian TLR signaling involving MyD88 and Sarm. All TLRs except TLR3 signal via MyD88 and activate NF-κB . Neuronal apoptosis downstream of TLRs is independent of NF-κB and instead depends on TRIF and Sarm1. Sarm1 is a negative regulator of TLR signaling, an inhibitor of MyD88 and TRIF. sarm1 is expressed in neurons, where it activates JNK and promotes apoptosis. However, the endogenous ligands for TLRs in the normal undamaged brains are not known. Preliminary analysis has revealed the intriguing possibility that NTs either can bind TLRs or induce interactions between Trks, p75NTR, and TLRs. It is compelling to find out whether TLRs regulate structural plasticity in the mammalian brain in concert with NTs (Foldi, 2017).

To conclude, DNTs with Tolls constitute a novel molecular system for structural plasticity in the Drosophila CNS. This could be a general mechanism to be found also in the mammalian brain and in other contexts as well, such as epithelial cell competition and regeneration, and altered in cancer and neurodegeneration (Foldi, 2017).

Return: see Dorsal: Regulation part 1/2

dorsal continued: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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