Toll

DEVELOPMENTAL BIOLOGY

Embryonic

Toll mRNA is maternally provided and is found in the ovary and early embryo. Toll protein does not accumulate in the embryo, however, until the syncytial blastoderm stage. Toll protein is not detectable in ovaries and protein levels substantially increase during the first 2 hours of embryogenesis. Toll protein expression peaks at the syncytial blastoderm stage, consistent with whole-mount staining results. These results are consistent with translational activation of Toll mRNA during early embryogenesis or with Toll protein instability during the first hour of development. No evidence for differential protein stability is found during the first few hours of embryogenesis. Therefore, the favored interpretation is that Toll mRNA is translationally activated during early development (Schisa, 1998).

Toll is a glycoprotein tightly associated with embryonic membranes. During the syncytial stage when dorsal-ventral polarity is established, the maternal Toll protein is associated with the plasma membrane around the entire embryo. During later embryonic stages, the Toll protein is expressed zygotically on many cell surfaces, including mesectoderm, stomodeum, proctodeum, anterior and posterior midgut and splanchnopleura, the prospective visceral mesoderm. Still later in embryogenesis it is found in salivary glands, foregut, hindgut, Malpighian tubules and the epidermis at intersegmental boundaries (Hashimoto, 1991).

Dorsoventral (DV) patterning of the Drosophila embryo is initiated by a broad Dorsal nuclear gradient, which is regulated by a conserved signaling pathway that includes the Toll receptor and Pelle kinase. What are the consequences of expressing a constitutively activated form of the Toll receptor, Toll(10b), in anterior regions of the early embryo? Using the bicoid 3' UTR, localized Toll(10b) products result in the formation of an ectopic, anteroposterior (AP) Dl nuclear gradient along the length of the embryo. The analysis of both authentic Dorsal target genes and defined synthetic promoters suggests that the ectopic gradient is sufficient to generate the full repertory of DV patterning responses along the AP axis of the embryo. For example, mesoderm determinants are activated in the anterior third of the embryo, whereas neurogenic genes are expressed in central regions. These results raise the possibility that Toll signaling components diffuse in the plasma membrane or syncytial cytoplasm of the early embryo (Huang, 1997).

Loss of one or both copies of the Toll gene leads to widespread defects in motoneuron number and muscle patterning. Loss of motoneurons prevents certain muscle fibers from receiving their wild-type innervation. Denervation in the mutants results in collateral sprouting from nearby nerve branches and leads to the appearance of ectopically placed motor endings. The limited expressivity observed suggests that Toll is only one of several genes required for proper motoneuron and muscle specification (Halfon, 1995).

Toll is dynamically expressed later in development by the embryonic musculature. Growth cones of RP3 and other motoneurons normally grow past Toll-positive muscle cells and innervate more distal muscle cells (muscles 6 and 7), which have down-regulated their Toll expression. The RP3 growth cone likely responds positively to Fasciclin III, an Ig-like cell adhesion molecule expressed on the target muscle cells, but still manages to avoid targeting errors in embryos lacking Fas III. Expression of Toll occurs in three stages. At stage 14 (approximately 12 hours into embryogenesis), five ventral (proximal) muscle cells (7, 15, 16, 17 and 28) express Toll at high levels. This is before SNb growth cones exit the CNS. Toll protein preferentially accumulates at muscle-muscle contact sites or "clefts," that is, the apposition between muscle cells 6 and 17, and between 15 and 16. During stage 15, the second phase of Toll expression, the SNb growth cones begin to contact the ventral-most oblique muscle cells 15, 16 and 17. The 6/7 cleft, the normal synaptic site of RP3, loses Toll during this period. During stage 16, the SNb growth cones extend further and innervate their targets. The remaining Toll-positive ventral muscle cells gradually lose Toll until the protein remains weakly detectable only at the 15/16 cleft. Later-arriving growth cones of SNd, which include the growth cone of motoneuron RP5, innervate this cleft just as its Toll expression becomes undetectable. It is concluded that Toll, a muscle cell surface molecule, locally inhibits synaptic initiation of the RP3 motoneuron growth cone in Drosophila (Rose, 1997).

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).

Effects of Mutation or Deletion

Certain dominant Toll alleles encode proteins that behave as partially ligand-independent receptors, causing embryos containing these proteins to become ventralized. In extracts of embryos derived from mothers carrying these dominant alleles, using antibodies to Toll, a polypeptide of approximately 35 kDa in addition to full-length Toll polypeptides is found. The smaller polypeptide is a truncated form of Toll lacking extracellular domain sequences. The truncated Toll protein elicits the most ventral cell fate independently of the wild-type Toll protein and its ligand (Winans, 1995).

Three of five recessive loss-of-function alleles of Toll are caused by point mutations in the region of the cytoplasmic domain of Toll, possessing a similar sequence to the mammalian interleukin-1 receptor. This supports the hypothesis that Toll acts as a signal-transducing receptor. Mutations in the extracellular domain lead to dominant gain-of-function alleles, causing Toll to be active in dorsal, as well as ventral, regions of the embryo. Cysteine-to-tyrosine changes immediately outside the transmembrane domain in three of the dominant alleles appear to cause the protein to be constitutively active. All six of the remaining dominant alleles require the presence of a wild-type transmembrane Toll protein for their ventralizing effect, and all encode truncated proteins that lack the transmembrane and cytoplasmic domains (Schneider, 1991).

The requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway have now been analyzed. spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, the spatial requirements for Toll expression were examined during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. A 6.5-kb enhancer element drives expression solely in mesodermally derived tissues. A 1.4-kb enhancer drives expression in the epidermis, CNS midline, gut, salivary glands, Malpighian tubules, pharynx and esophagus, but not in mesodermal tissues. These two enhancers were used to drive expression of Toll in transgenic flies. The 1.4-kb enhancer express Toll in the epidermis (in a narrow strip of cells that includes the EMA cells) as well as in a cluster of cells in the lateral, mid-bodywall region of each segment. This lateral region contains the cells where the lateral transverse muscle fibers have their insertions. Flies with 1.4-kb enhancer driven Toll expression show complete rescue of the muscle error phenotype. These results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process (Halfon, 1998).

Although loss of single minded, a regulator of the Toll pathway in the central midline, causes positioning and insertion errors in a group of the most ventral muscles, these defects are qualitatively different from those observed in Toll mutants. The errors due to sim mutation are thus not likely due to loss of Toll expression; Toll expression in the midline appears to be uninvolved in muscle patterning. It is known that signaling from the muscle fibers induces the expression of beta1-tubulin in the EMA cells and regulates the maintenance of expression of other attachment site-specific genes such as delilah, groovin, and stripe. The nature of the Toll muscle phenotype (most of which consists of duplicated and deleted muscle fibers) suggests that Toll may be acting early in the development process, during the time of founder specification or early muscle fiber growth. The remaining errors (those in muscle insertion) may be either early or late in origin: they may be secondary to mis-specification of muscle identity (early), or alternatively, might indicate a further requirement for Toll during the insertion process (late) (Halfon, 1998).

A variety of cell recognition pathways affect neuronal target recognition. However, whether such pathways can converge at the level of a single growth cone is not well known. The RP3 motoneuron in Drosophila has previously been shown to respond to the muscle cell surface molecules Toll and Fasciclin 3 (Fas3), which are normally encountered during RP3 pathfinding in a sequential manner. Toll and Fas3, putative 'negative' and 'positive' recognition molecules, respectively, affect RP3 antagonistically. Under normal conditions, Toll and Fas3 together improve the accuracy of RP3 target recognition. When presented with concurrent Toll and Fas3 expression, RP3 responds to both, integrating their effects. This was demonstrated most succinctly by single cell visualization methods. When a balance in relative expression levels between the two antagonistic cues is achieved, the RP3 growth cone exhibits a phenotype virtually identical to that seen when neither Toll nor Fas3 is misexpressed. Thus, growth cones are capable of quantitatively evaluating distinct recognition cues and integrating them to attain a net result, in effect responding to the 'balance of power' between positive and negative influences. It is suggested that the ability to integrate multiple recognition pathways in real-time is one important way in which an individual growth cone interprets and navigates complex molecular environments (Rose, 1999).

Toll and Fas3 protein were simultaneously misexpressed in the entire embryonic musculature during the period of motoneuron-muscle interaction. Testing with Toll and Fas3 antibodies indicates that the two proteins are simultaneously expressed at elevated levels in all muscles. As with other muscle cell surface molecules, both Toll and Fas3 appear to accumulate at muscle-muscle contact sites. Misexpression occurs throughout most of the period of motoneuron axon pathfinding and synaptogenesis, i.e., hours 12-20 of embryogenesis. Despite transgenic expression, there is no difference in the number or overall morphology of muscles. Therefore, motoneuron growth cones leaving the CNS will experience both Toll and Fas3 expression on all muscle surfaces that they encounter. This contrasts the wild-type situation in which growth cones first encounter Toll-expressing proximal muscles (15, 16, 17, and 28) before reaching FAS3-expressing proximal-medial muscles a few hours later. Any motoneuron axon defects that result from co-misexpression of Toll and Fas3 may be interpreted as a likely direct result of motoneuron growth cones encountering Toll and Fas3 proteins outside of their normal context. Nevertheless, the nervous system was found to develop normally in Toll/Fas3 co-misexpressor embryos. In each hemisegment of the peripheral nervous system, all five major nerve branches [intersegmental nerve, segmental nerve a (SNa), SNb, SNc, and SNd] extend outside the CNS into characteristic muscle regions. Previous studies using the Toll and Fas3 misexpressing lines have revealed that subsets of motoneuron growth cones are often misguided when encountering either ectopic Toll or Fas3 expression in the musculature. Toll/Fas3 co-misexpressor embryos remain innervated at a high frequency when examined with immunocytochemistry This apparent return to a wild-type innervation pattern could be attributable to one of at least two separate situations. Either RP3 or MN6/7b, another motoneuron that innervates the 6/7 cleft, could once again be attracted to the cleft when the levels of Toll and Fas3 expression there are equalized. Alternatively, the presence of mAb 1D4 staining at the cleft could indicate that a motoneuron other than RP3 or MN6/7b is innervating the cleft ectopically. The latter would imply that co-misexpression of Toll and Fal3 permits ectopic innervation at the 6/7 cleft more readily than misexpression of either cue alone. The former would suggest that the 6/7 cleft becomes 'normalized' when both Toll and Fas3 are co-misexpressed in the musculature. To distinguish between these possibilities, the RP3 growth cone was visualized directly. In all cases, the RP3 axon extends out of the CNS normally. It first crosses the dorsal midline of the CNS and then exits via the intersegmental nerve, one segment posterior to its origin. This supports the notion that co-misexpression of Toll and Fas3 in the embryonic musculature does not affect RP3 axonal pathfinding within the CNS. Once outside the CNS and when encountering concurrent misexpression of Toll and Fas3, the most common decision of the RP3 growth cone is to navigate through the normal peripheral pathway and to innervate the 6/7 cleft, its wild-type site of innervation. Overall, whereas Toll is capable of preventing RP3 innervation of its normal target site, co-misexpression of Fas3 and Toll leads to a phenotype that is similar to wild type. These observations are interpreted as RP3 growth cone integration of two antagonistic signals during its target recognition. Thus the RP3 growth cone is competent to respond to concurrently misexpressed Toll and Fas3, two structurally and physiologically distinct molecules normally expressed by the targets of the growth cone and surrounding cells. Furthermore, in vitro, the growth cone is capable of evaluating the relative contributions of each molecule and responding appropriately. These results support the general idea that signal integration at individual growth cones is an important mechanism by which neural networks are established (Rose, 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).

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 Tol^l10B mutants. Tol^l10B 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).

The Drosophila Pelle kinase plays a key role in the evolutionarily conserved Toll signaling pathway, but the mechanism responsible for its activation has been unknown. In vivo and in vitro evidence is presented establishing an important role for concentration-dependent autophosphorylation in the signaling process. Pelle phosphorylation can be detected transiently in early embryos, concomitant with activation of signaling. Importantly, Pelle phosphorylation is enhanced in a gain-of-function Toll mutant (Toll^10b), but decreased by loss-of-function Toll alleles. Pelle is phosphorylated in transfected Schneider L2 cells in a concentration-dependent manner such that significant modification is observed only at high Pelle concentrations, which coincide with levels required for phosphorylation and activation of the downstream target, Dorsal. Pelle phosphorylation is also enhanced in L2 cells co-expressing Toll^10b, and is dependent on Pelle kinase activity. In vitro kinase assays reveal that recombinant, autophosphorylated Pelle is far more active than unphosphorylated Pelle. Importantly, unphosphorylated Pelle becomes autophosphorylated, and activated, by incubation at high concentrations (Shen, 2002).

In Drosophila, the dorsoventral axis is set up by the action of the dorsal group of genes and cactus, all of which have been ordered genetically in a linear pathway. krapfen (kra) has been identified as a new member of the dorsal-group genes. kra encodes for the Drosophila homolog of MyD88, an adapter protein operating in the mammalian IL-1 pathway. Epistasis experiments reveal that Myd88/krapfen acts between the receptor Toll and the cytoplasmic factor Tube. There is a direct interaction between Krapfen and Tube presumably mediated by the death domains present in both proteins. Tube in turn interacts with its downstream effector Pelle through death domain association. It is therefore suggested that upon Toll activation, Krapfen associates with Pelle and Tube, in an heterotrimeric complex (Charatsi, 2002).

Originally, the krapfen (kra) mutation kra⁵⁶ was identified in a genetic screen for new maternal genes involved in embryonic pattern formation. The embryos laid by homozygous kra⁵⁶ females fail to gastrulate properly and die as hollow tubes of dorsal cuticle. This phenotype is undistinguishable from those caused by mutations in the dorsal group of genes. However, kra⁵⁶ did not fall into any known complementation group. In order to investigate the role of kra in the dorsal pathway, the presence of the mesoderm, which is formed by the ventral most cells, was tested for. The expression of the mesodermal marker Twist was examined in early blastoderm embryos. In contrast to the wild type situation where upon Toll activation Twist is expressed ventrally, in kra mutant embryos, as in other dorsal group gene mutants, Twist is absent. These results show that Myd88 is a new member of the dorsal group of genes (Charatsi, 2002).

Whether Kra participates in the activation of the Toll receptor or cooperates in the signal transduction downstream of Toll was investigated. The effect of loss of maternal kra was tested in a dominant Toll background, Toll^9Q, which is a ligand-independent gain-of-function allele of Toll. Embryos laid by Toll^9Q heterozygous flies show a strongly ventralized phenotype due to the constitutively active Toll that signals throughout the embryo circumference. Embryos laid by females with the genotype kra¹/kra¹; Toll^9Q/TM3 show a complete dorsalized phenotype. Thus, kra suppresses the constitutive Toll signal, indicating that Kra acts downstream of the Toll receptor. This finding suggests that Kra operates in the cytoplasmic compartment of the Drosophila early embryo. In order to place Kra upstream or downstream of the cytoplasmic protein Tube, the phenotype of kra embryos was analzyed after microinjection of the gain-of-function construct of Tube, pBtor⁴⁰²¹Tube. In this construct, the intracellular kinase domain of a gain-of-function allele of the receptor tyrosine kinase torso is replaced by the tube coding sequence. pBtor⁴⁰²¹ fusions show that Tube operates upstream of Pelle. Whereas uninjected kra embryos develop only dorsal epidermis, kra embryos injected with pBtor⁴⁰²¹Tube RNA can specify ventrolateral fates and restore ventrolateral pattern elements, such as ventral denticle belts and Filzkörper, that are never observed in kra mutant embryos. Through these methods kra can be placed downstream of Toll and upstream of tube (Charatsi, 2002).

The genetic positioning of kra between Toll and tube, however, is not informative as to the physical interactions that take place during signal transduction. In order to investigate the molecular role of kra, a yeast two hybrid assay was performed. In the yeast two hybrid assay, wild type Kra as well as Kra⁵⁶, which is an EMS allele carrying a missense mutation within the TIR domain, were both able to interact strongly with Tube. This indicates that the TIR domain is not required for a Kra-Tube interaction (Charatsi, 2002).

Pelle interacts with Tube through death domain association. In the same yeast two hybrid experiment, no direct interaction was found between Pelle and Kra. This suggests that Tube could mediate the formation of a complex by association with both Pelle and Kra (Charatsi, 2002).

The epistasis experiment placed Kra downstream of the receptor Toll. Both Toll and Kra contain TIR domains, which could potentially mediate their interaction. Additionally, the kra⁵⁶ allele, which shows a dorsalized phenotype presumably caused by a defective TIR domain, strongly suggests that this cytoplasmic domain plays an essential role in signal transduction. An interaction between Kra and Toll, which is not necessarily direct, is supported by immunoprecipitation experiments in which Kra/Myd88 coimmunoprecipitates with Toll. However, in the yeast two hybrid assay, no direct interaction was found between the TIR cytoplasmic domain of Toll and Kra (Charatsi, 2002).

In order to understand how the signal is transduced to Kra through Toll, the possibility that Kra homodimerizes was investigated. In mice, MyD88 is known to form homodimers in vivo through death domain-death domain and TIR-TIR interactions. Kra does not homodimerize in the yeast two hybrid assay nor in immunoprecipitation experiments (Charatsi, 2002).

Microarray chips (Affymetrix) were used to study the gene expression profiles of gastrulating embryos derived from wild-type, dorsal^–/– and Toll^10b flies. Toll^10b codes for a gain-of-function Toll receptor. Many known target genes, such as twist, snail, short gastrulation, tinman and mef2, showed lower expression in the dorsal^–/– sample and increased expression in the Toll^10b sample, as predicted. Among the novel targets, the annotated gene CG8458 was selected for further study because it encodes a member of the Wnt family (WntD), and Wnt proteins have been implicated in controlling cell polarity and cell movement in many organisms (Ganguly, 2005).

Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo

Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). This study used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. It is proposed that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo (Mavrakis, 2008).

To study the organization of the PM and the spatiotemporal dynamics of membrane components in living Drosophila embryos, transgenic animals were generated expressing different PM proteins tagged with Cerulean or Venus fluorescent proteins. The proteins were selected because they have different modes of membrane attachment and potentially different PM distributions. They included: (1) Venus fused to the first 20 amino acids of growth-associated protein 43 (GAP43), which contain a dual palmitoylation signal that tightly anchors the protein to the inner leaflet of the PM, (2) Cerulean fused to the pleckstrin-homology domain of phospholipase C delta 1, PH(PLCδ1), which binds specifically to the phosphoinositide PI(4,5)P2, and (3) Venus fused to full-length Toll receptor, a type I transmembrane protein that is required for dorsal-ventral embryonic polarity (Mavrakis, 2008).

This study provides evidence that the plasma membrane of the fly syncytial blastoderm exhibits a polarized, epithelial-like organization prior to cellularization. Previously, it was thought that the PM of the blastoderm had no specialized organization prior to the formation of cell boundaries at cellularization. The results show that despite the absence of cell boundaries, the PM of the syncytial blastoderm has apical- and basolateral-like domains surrounding individual cortical nuclei and that PM proteins do not exchange between PM regions surrounding adjacent nuclei. This organization is maintained throughout syncytial mitotic division cycles and is dependent on an intact F-actin network (Mavrakis, 2008).

Support for these conclusions came from live imaging and fluorescent highlighting experiments in living embryos. Using a variety of membrane markers, two distinct PM regions were distinguished. One region was above individual nuclei and had apical-like characteristics, including the presence of microvilli and an enrichment in PI(4,5)P₂, a key determinant of apical PM biogenesis, as well as in GAP43, a protein that localizes to raft-like membranes, which typically compose apical PM surfaces in epithelial cells. The second PM region was lateral to nuclei, and was enriched in markers typically associated with basolateral membranes and junctions, including the cell-cell adhesion molecule E-cadherin, the multi-PDZ domain scaffolding protein DPatj. FRAP experiments showed that the molecules could freely diffuse in the PM domains surrounding individual nuclei but did not diffuse outside them, suggesting the presence of a diffusion barrier between the domains during interphase. Moreover, optical pulse-chase experiments showed that these components did not diffuse outside PM domains surrounding mitotic units throughout the time period of syncytial divisions. Thus, during mitosis, the polarized organization and restricted diffusion pattern of proteins in the PM did not change. Finally, the requirement of an intact F-actin network was supported by drug-induced actin depolymerization, which disrupted PM association of DPatj and Peanut and abolished the restricted diffusion pattern in the PM (Mavrakis, 2008).

The finding that the PM of the syncytial blastoderm is organized as a pseudoepithelium prior to cellularization has several important implications for understanding many aspects of embryo development. First, it directly impacts on how dorsal-ventral and terminal patterning are set up prior to cellularization. These are dependent on Toll and Torso membrane receptors. Toll is distributed uniformly along the syncytial PM, but is activated only ventrally. Similarly, Torso is uniformly expressed along the surface membrane of early embryos, but its activation occurs only at the anterior and posterior poles. Given that membrane receptors have the capacity to diffuse across the PM, it has been unclear why the activation zones of these receptors do not spread widely across the PM. The results revealing the compartmentalized character of the PM during interphase and syncytial nuclear divisions now provide a potential answer. Receptors diffuse locally within the PM surrounding a particular nucleus, but they do not diffuse to PM regions associated with other nuclei. Consequently, activation zones of receptors (set up by the localized spatial signal of ligands) do not spread, allowing robust downstream signaling events in particular regions of the embryo. This possibility is supported by the spreading of the Dorsal gradient to more anterior and posterior regions in embryos treated with latA. LatA-induced actin depolymerization abolished the confined diffusion pattern in the PM suggesting that an intact actin network is likely to be important for containing activated Toll diffusion and thus maintaining a robust downstream Dorsal gradient (Mavrakis, 2008).

The molecular basis for the compartmentalized diffusion in the PM of the syncytial embryo appears to be due to the presence of bona fide diffusion barriers in the PM regions directly between adjacent nuclei. The finding that septins and components of junctions are specifically enriched in this PM region raises the possibility that these molecules together with other cytoskeletal components organize a barrier to diffusion in the plane of the PM in a way similar either to the organization of septin rings at the yeast bud neck or of adherens junctions in epithelial cells. Moreover, the loss of PM association of DPatj and Peanut, as well as the abolishment of the restricted diffusion pattern in latA-treated embryos, suggest that an intact F-actin network is required both to localize and/or maintain septins and junctional components to specialized PM regions and to contain diffusion of proteins in PM units around individual syncytial nuclei. An intact F-actin network was recently shown to be required for compartmentalizing furrow canals during cellularization further supporting that F-actin organizes lateral diffusion of proteins in the PM. Future studies will need to genetically dissect the molecular machineries involved in organizing such diffusion barriers (Mavrakis, 2008).

A second implication of the observed PM dynamics during syncytial mitoses relates to the machinery driving PM invagination. It was found that the PM was organized into highly convoluted microvillous membrane buds over interphase nuclei and these flattened out as soon as nuclei entered mitosis before reorganizing again into microvillous buds upon re-entry into the next interphase. Furthermore, the rate at which PM invaginated (~1.5-2 μm/min) was twice as fast as during the fast phase of cellularization, which involves de novo membrane delivery. Although endocytosis was recently shown to accompany metaphase furrow ingression, the current observations support a mechanism for PM invagination in mitosis that involves contractile machinery which transiently redistributes PM from microvilli caps into transient furrows surrounding mitotic units rather than an internal membrane source (Mavrakis, 2008).

A final implication of these findings relates to cellularization, which produces the primary epithelial cells of the embryo. Polarization of the invaginating PM during cellularization has been reported, and it is during cellularization that PM polarity is first thought to be achieved in early fly embryogenesis. Because the data demonstrate that the PM is already polarized prior to cellularization, it is likely that the embryo uses this organization to initiate and organize the cellularization process. Consistent with this, it was found that the junctional proteins E-cadherin and DPatj, the septin protein Peanut, and Toll are all highly enriched in the PM at sites between adjacent nuclei during syncytial interphases, which reflects the PM organization between nuclei right at the onset of cellularization (first few minutes of interphase 14). Indeed, these are precisely the PM sites that become further differentiated within the first 5 min into cellularization, with the formation of an invaginating membrane front that contains Peanut and DPatj, basal adherens junctions directly adjacent to the invaginating front that contain E-cadherin, and the extension of the lateral membranes that are positive for Toll. The epithelial polarization occurring during cellularization is thus already reflected in the organization of the syncytial blastoderm PM (Mavrakis, 2008).

In summary, these findings that the syncytial blastoderm PM exhibits an epithelial-like polarization prior to cellularization, and that distinct PM domains do not significantly exchange membrane components, point to an as yet unexplored mechanism for how the embryo maintains and generates morphogen gradients at this stage. By preventing activation zones of membrane receptors on the PM from spreading, robust downstream signaling events within the cytoplasm and nuclei of the embryo can be established. This mechanism would work in conjunction with nuclear-cytoplasmic shuttling of transcription factors, and a compartmentalized secretory pathway, to generate the dorsal-ventral and terminal patterning systems of the blastoderm fly embryo (Mavrakis, 2008).

Effects of Mutation: Toll and the immune response

Cactus and Dorsal play a critical role in a hemocyte-dependent function in Drosophila. In invertebrates such as Drosophila, hematopoietic stem cells are located in the lymph gland. They give rise to progenitors of at least two lineages, plasmatocytes and crystal cells. Plasmatocytes are the predominant form of hemocytes in the wild-type larval hemolymph and, like mammalian macrophages or neutrophils, they perform phagocytic functions. Plasmatocytes are small, spherical and non-adhesive, and engulf bacteria and cell debris. Plasmatocytes also secrete extracellular matrix components. When a larva experiences an immune challenge, plasmatocytes become stimulated, increase in number and, depending on the nature of infection, engage in phagocytosis or differentiate into discoidal and adhesive lamellocytes. Lamellocytes do not show any capacity for phagocytosis. Instead, they form multilayered capsules around foreign invaders or objects that are too large for phagocytosis. These capsules get melanized by the activities of crystal cells. Crystal cells house the substrates and enzymes for melanization reactions. In the absence of an immune challenge, plasmatocytes of a normal larva differentiate into lamellocytes at the onset of pupariation. However, in certain Drosophila mutants, lamellocytes form melanotic capsules around self tissue, even in the absence of an immune challenge. The mechanisms that control the production, differentiation and functions of these cells in wild-type and mutant Drosophila are poorly understood but they appear to represent constitutive activation of the immune system (Qiu, 1998).

Dominant mutations in Toll or constitutive expression of dorsal can induce lamellocyte differentiation and cause the formation of melanotic capsules. The hemocyte density of mutant Toll, tube or pelle hemolymph is significantly lower than that of the wild type. Lethality of mutant cactus animals can be rescued either by the selective expression of wild-type Cactus protein in the larval lymph gland or by the introduction of mutations in Toll, tube or pelle. Cactus, Toll, Tube and Pelle proteins are expressed in the nascent hemocytes of the larval lymph gland. These results suggest that the Toll/Cactus signal transduction pathway plays a significant role in regulating hemocyte proliferation and hemocyte density in the Drosophila larva (Qui, 1998).

Since effects of the Toll/Cactus pathway mutants appear to be confined to the regulation of hemocyte density in the larva, other basal and regulatory signals must contribute more directly to lineage specification, hemocyte turnover and differentiation. Phenotypes of other hematopoietic mutants in Drosophila range from the absence of lymph glands to the presence of massively overgrown lymph glands. Analysis of some of these mutants suggests that these genes play specific roles in hemocyte specification, differentiation or turnover. For example, recent studies on the constitutive JAK mutant hop Tum-l (which results in lymph gland overgrowth and hematopoietic neoplasm) suggest that, like the Toll/Cactus pathway, the JAK/STAT signaling pathway also regulates hematopoiesis and hemocyte density in the Drosophila larva. It is possible that multiple regulatory inputs, such as the Toll/Cactus and JAK/STAT signals, are integrated with, or superimposed on one another, to ensure that hematopoietic precursors survive and divide, and progress normally through their developmental program to give rise to mature hemocytes in a consistently controlled manner (Qiu, 1998 and references).

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. This pathway is required in the epidermis for proper muscle development. Because Toll mutations affect the development of all 30 muscle fibers in each hemisegment, and not just the several that express Toll, or those closest to the CNS, it semed likely that the epidermal expression is most relevant to muscle development. In the epidermis, Toll expression is highest in the epidermal muscle attachment (EMA) cells, aligned along the segment border; these cells are known to play an important role in muscle patterning. The zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, 1998).

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

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

The Dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults

There are a number of different controls on the expression of the antifungal polypeptide gene drosomycin in adults: 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. Mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. In Tl-deficient adults, the cecropin A and, to a lesser extent, attacin, drosomycin and defensin genes are only minimally inducible, in contrast with the diptericin and drosocin genes, which remain fully inducible in this context. The drosomycin gene induction is not affected in mutants deficient in gastrulation defective, snake and easter, all upstream of spätzle in the dorsoventral pathway. The involvement of Spätzle 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 Dorsal-related immunity factor (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).

Constitutive activation of Toll-mediated antifungal defense in serpin-deficient Drosophila

The antifungal defense of Drosophila is controlled by the spaetzle/Toll/cactus gene cassette. A loss-of-function mutation in the gene encoding a blood serine protease inhibitor, Spn43Ac, has been shown to lead to constitutive expression of the antifungal peptide drosomycin, and this effect is mediated by the spaetzle and Toll gene products. Spaetzle is cleaved by proteolytic enzymes to its active ligand form shortly after immune challenge; cleaved Spaetzle is constitutively present in Spn43Ac-deficient flies. Hence, Spn43Ac negatively regulates the Toll signaling pathway, and Toll does not function as a pattern recognition receptor in the Drosophila host defense (Levashina, 1999).

Flies carrying ethylmethane sulfonate-induced mutations in the necrotic (nec) locus were used. The locus, which maps at position 43A, generates three transcripts encoding putative serine protease inhibitors of the serpin family. The nec mutants exhibit brown spots along the body and the leg joints, corresponding to necrotic areas in the epidermis. This mutant phenotype is rescued by a single transgenic copy of one of the serpin genes, Spn43Ac. Because the absence of a functional Spn43Ac serpin may affect proteolytic cascades involved in the host defense of Drosophila, the level of expression of the antimicrobial peptide genes were examined in nec mutants. All genes were induced 6 hours after challenge in wild-type (WT) flies; however, in nec mutants the gene encoding drosomycin is strongly expressed in the absence of immune challenge. The expression is further enhanced by immune challenge. The gene encoding the peptide metchnikowin, which has both antibacterial and antifungal activities, also exhibits constitutive expression in nec mutants, although the response is less marked than for drosomycin. In contrast, no constitutive expression is observed by genes encoding diptericin and cecropin A1, whose expression is either independent of the Toll signaling pathway or requires a signal from an additional pathway, depending on the immune deficiency (imd) gene (Levashina, 1999).

Overexpression of the Spn43Ac gene in nec flies abolishes the constitutive expression of drosomycin, whereas overexpression of a different serpin gene from the same cluster, Spn43Aa, has no effect on this phenotype. In a Tl or spz loss-of-function background, the nec-mediated constitutive expression of drosomycin is abolished, indicating that Spn43Ac acts upstream of spz and Tl. However, when the nec mutation is combined with gastrulation defective (gd) or snake (snk) loss-of-function mutations, constitutive expression of drosomycin is still observed, confirming that these proteases are not necessary for the Toll-controlled antifungal response. Furthermore, the constitutive expression of drosomycin is not affected when the nec mutation is in an imd mutant background, suggesting that the imd-mediated expression of the antibacterial peptide genes is independent of the proteolytic cascade controlled by Spn43Ac (Levashina, 1999).

The expression of the Tl gene and that of the downstream genes in the signaling cascade is up-regulated by immune challenge. The transcription of the Spn43Ac gene is up-regulated by immune challenge. This up-regulation is not observed in a Tl loss-of-function background. Conversely, Tl gain-of-function mutants exhibit a constitutive expression of Spn43Ac. In imd mutants, the up-regulation of Spn43Ac by immune challenge is similar to that in wild-type flies. Thus, Spn43Ac is an immune-responsive gene, and its expression is under the positive control of the Toll pathway. This could represent a negative feedback mechanism to shut down the activation of Toll by inhibiting the upstream proteolytic cascade (Levashina, 1999).

Constitutive expression of a complement-like protein in Toll and JAK gain-of-function mutants of Drosophila

It has been proposed that germ-line-encoded pattern recognition receptors bind microbial cell wall determinants (such as lipopolysaccharides, mannans, and peptidoglycans) and initiate an immune response, either by activating associated proteases in circulation or by directly triggering intracellular signaling pathways in immune responsive cells. To date, no pattern recognition receptor has been firmly identified in Drosophila and shown to activate an immune response. A primitive complement-like system, evocative of the alternative or the lectin pathways of complement, could be involved in the activation of some of the Drosophila host defense mechanisms. This hypothesis was made attractive by the recent reports that invertebrates such as sea urchins and tunicates have a complement-like system, and produce proteins with structural similarities to vertebrate complement C3 proteins, containing an intrachain thiolester bond. Similar proteins have also been described in the horseshoe crab, a member of the class of arthropods to which Drosophila also belongs (Lagueux, 2000 and references therein).

Drosophila expresses four genes encoding proteins with significant similarities with the thiolester-containing proteins of the complement C3/alpha2-macroglobulin superfamily. The genes are transcribed at a low level during all stages of development, and their expression is markedly up-regulated after an immune challenge. For one of these genes, which is predominantly expressed in the larval fat body, a constitutive expression was observed in gain-of-function mutants of the Janus kinase (JAK) hop and a reduced inducibility in loss-of-function hop mutants. A constitutive expression was observed in gain-of-function Toll mutants. These novel complement-like proteins are likely to play roles in the Drosophila host defense (Lagueux, 2000).

In higher vertebrates, the complement system consists of about 30 serum and cell surface proteins and mediates inflammatory reactions, opsonization of microorganisms for phagocytosis, and direct killing of some pathogens. Activation can occur via the classical antibody-dependent pathway, the alternative pathway, and the lectin pathway, which all converge on the central complement C3 protein. The presence in Drosophila of several proteins with basic structural characteristics similar to complement C3 makes attractive the working hypothesis that an ancient equivalent of the alternative pathway and/or the lectin pathway exists in this species. In vertebrates, the activation of the alternative pathway is initiated by spontaneous hydrolysis of the thiolester bond of complement C3, resulting, through association with other proteins of the complement system, in an active C3 convertase that is normally inactivated by regulatory proteins present on self tissue, but absent from non-self, providing for a relative primitive mode of discriminating self from non-self. In turn, active C3 convertase activates complement C3 and, through an amplification loop, triggers the conventional effector mechanisms of complement. Activation of the lectin pathway is initiated when various sugars present on the surface of microorganisms bind to a collectin, the mannan-binding lectin (MBL), thereby inducing proteolytic cascades that activate complement C3 and the downstream events common to all three activation pathways (Lagueux, 2000).

In the mid-nineties, it became apparent that the complement system is not a unique property of the host defense armatarium of vertebrates. ESTs from cDNA libraries of sea urchin coelomocytes were found to encode a protein with structural similarities to vertebrate complement C3, including an intrachain thiolester motif, plus a homolog of vertebrate factor B, which participates in the activation of complement C3 through the alternative pathway. More recently, an ascidian species was reported to possess homologs of complement C3 and of two mannose-binding lectin-associated proteases (MASPs), plus a homolog of factor B, raising the possibility that equivalents of both the lectin and the alternative activation pathways are present in these deuterostome invertebrates. Experiments with ascidian coelomocytes further indicate that the complement C3-like molecules act as opsonic factors and are activated through a complement-like cascade (Lagueux, 2000 and references therein).

TEPs are also structurally close to alpha₂-macroglobulins, which are evolutionary ancient protease inhibitors, from which complement C3 has been proposed to have arisen by gene duplication. Protease inhibitors related to alpha₂-macroglobulin have been described in several invertebrates and have been particularly well studied in the horseshoe crab Limulus. Indeed, Limulus alpha₂-macroglobulin has been proposed to function as a protease inhibitor, particularly of proteases released by tissue damage caused by injury or pathogens and of soluble or surface bound proteases produced by invading microorganisms. It has been suggested that the first opsonic system could have required no specific recognition or activation mechanism other than the presence of exogenous proteases causing alpha₂-macroglobulin to bind directly to the protease-producing organism (Lagueux, 2000 and references therein).

Because Drosophila has four expressed genes encoding proteins with structural similarities to the superfamily of complement C3/alpha₂-macroglobulin, significant functional versatilities may be expected, all the more so, because one of the Tep genes, Tep2, gives rise to five different transcripts. Interestingly, the Tep2 transcripts are identical except for a short region of 30 aa. This region is encoded by alternatively spliced exons corresponding to the hypervariable region of the TEPs; it is located in a relative position similar to the bait domain in alpha₂-macroglobulins or the anaphylatoxins in complement C3. Alternative splicing has not been reported in vertebrates for members of the complement C3/alpha₂-macroglobulin superfamily. By increasing the number of putative recognition motifs for microorganisms or proteases, it may contribute to the fine-tuning of recognition of noxious structural patterns in the absence of the large repertoire of receptors of the adaptive immune response in vertebrates (Lagueux, 2000 and references therein).

The Drosophila plasmatocytes are macrophage-like blood cells that readily engulf bacteria or fungal spores, as well as various cellular debris resulting from injury or apoptosis. Nothing is known about possible opsonization in this model, and a tempting working hypothesis is that the complement-like proteins described here precisely fulfill such a role in the host defense. Future efforts will be directed toward experimentally testing this hypothesis, and it is anticipated that the generation of mutants of the various Tep genes, which all map to the left arm of the second chromosome, will be invaluable in this endeavor (Lagueux, 2000).

TEP1 is produced mainly in the fat body, and its expression is up-regulated by immune challenge. It is hypothesized that, as is the case for immune-induction of antimicrobial peptides in this tissue, the up-regulation would be dependent on either the Toll or the imd pathway. This control is strongly dependent on the JAK hopscotch. Hopscotch is the only JAK identified in Drosophila, and in the gain-of-function mutant hop^Tum-l, Tep1 is constitutively expressed, whereas its immune-inducibility is dramatically reduced in the loss-of-function mutant hop^M38. Gain-of-function mutations of hop have remarkable effects on hemopoiesis in Drosophila and result in overproliferation of blood cells, increased differentiation of lamellocytes, and aggregation of blood cells into masses, which tend to become melanized, a process referred to as melanotic tumor formation. The data thus show that these events are concomitant with an increased transcription of the Tep1 gene. Whether they are causally related remains an open question, but it will be worthwhile investigating whether the TEP1 protein can affect the aggregation of blood cells and the localized induction of melanization (Lagueux, 2000).

In addition to its established role in the control of synthesis of the antifungal peptide drosomycin, the Toll pathway has been proposed to be implicated in the control of hemocyte density. Hemocyte numbers are increased in Toll gain-of-function mutants. Of potential interest in the present context is the observation that, in these mutants, melanotic tumors develop that are similar to those seen in JAK gain-of-function mutants. In Toll gain-of-function mutants, Tep1 is strongly expressed in the absence of immune challenge. However, this effect must be indirect, because immune challenge can up-regulate Tep1 expression in spaetzle and Toll loss-of-function mutants. Toll gain-of-function mutants are known to produce a large number of peptides or polypeptides, referred to as Drosophila immune-induced molecules, which are absent from wild-type nonchallenged insects. These and/or other modifications in the hemolymph induced by melanotic tumors might account for the constitutive expression of Tep1 in Toll gain-of-function mutants (Lagueux, 2000 and references therein).

The Toll and Imd pathways are the major regulators of the immune response in Drosophila

Microarray studies have shown recently that microbial infection leads to extensive changes in the Drosophila gene expression program. However, little is known about the control of most of the fly immune-responsive genes, except for the antimicrobial peptide (AMP)-encoding genes, which are regulated by the Toll and Imd pathways. Oligonucleotide microarrays have been used to monitor the effect of mutations affecting the Toll and Imd pathways on the expression program induced by septic injury in Drosophila adults. Toll and Imd cascades were found to control the majority of the genes regulated by microbial infection in addition to AMP genes and are involved in nearly all known Drosophila innate immune reactions. However, some genes controlled by septic injury were identified that are not affected in double mutant flies where both Toll and Imd pathways are defective, suggesting that other unidentified signaling cascades are activated by infection. Interestingly, it was observed that some Drosophila immune-responsive genes are located in gene clusters, which often are transcriptionally co-regulated (De Gregorio, 2002).

To identify the target genes of the Toll and Imd pathways in response to microbial infection, the gene expression programs induced by septic injury have been compared in wild-type and mutant adult male flies using oligonucleotide microarrays. In parallel, the survival rate and the expression level of various AMP genes have been monitored after infection by various microorganisms. For the Toll pathway, a strong homozygous viable allele of spz (rm7) was selected. The spz, Tl and pll mutations, alone or in combination with rel, have similar effects on both the survival rate and pattern of AMP gene expression after microbial infection. These findings suggest that the effects of spz mutation on the transcription program induced by infection reflect the role of the entire Toll pathway in the immune response. For the Imd pathway, a null viable allele of relish (E20) was selected. Similarly to the Toll pathway, previous comparative studies did not reveal any striking difference between mutations in relish and null mutations in the genes encoding the other members of the Imd pathway such as kenny, ird5 and dredd, with the sole exception of mutations in dTAK1, which have a slightly weaker phenotype. Again, these data suggest that the effects of rel mutation on the immune response reflect the role of the whole Imd pathway. However, other pathways, including Toll, cannot be excluded from having a minor role in Relish activation (De Gregorio, 2002).

The septic injury experiments were performed using a mixture of Gram-positive and Gram-negative bacteria. This type of infection activates a wide immune response and allows the simultaneous analysis of several categories of immune-responsive genes. However, it has been shown that Toll and Imd pathways are activated selectively by different classes of microorganisms; thus, the use of a bacterial mixture might increase the redundancy of the two pathways in the control of common target genes (De Gregorio, 2002).

The microarray analysis demonstrates that the functions of Toll and Imd pathways in Drosophila immunity can be extended beyond the regulation of AMP genes. The majority of the Drosophila immune-regulated genes (DIRGs) are affected by the mutations in the Toll or Imd pathways. Many of these genes are unknown (see www.fruitfly.org/expression/immunity/ for a complete list); others can be assigned to several immune functions. The susceptibility of the Imd and Toll pathway mutants to different types of microbial infection suggested a dual aspect to the control of the antifungal response by the Toll pathway: a major role for the Toll pathway for the response to Gram-positive bacteria with a minor contribution of Imd, and a predominant role of Imd with a minor contribution of Toll to the resistance against Gram-negative bacteria. In agreement, microarray analysis shows that the Toll pathway controls most of the late genes induced by fungal infection and cooperates with the Imd pathway for the control of genes implicated in several immune reactions such as coagulation, AMP production, opsonization, iron sequestration and wound healing. Interestingly, defensin, which encodes the most effective antimicrobial peptide directed against Gram-positive bacteria, is co-regulated by both the Imd and Toll pathways. The hierarchical cluster analysis of the expression profiles combining the effect of the mutations after septic injury with the response to fungal infection provides a wealth of information that may help to elucidate the function of some of the uncharacterized DIRGs. Until now, the increased susceptibility to infection of Imd- or Toll-deficient flies has been attributed to the lack of expression of AMP genes, and it has been shown recently that the constitutive expression of single AMP genes in imd;spz double mutant flies can increase the survival rate of some types of bacterial infection. The finding that the Toll and Imd pathways are the major regulators of the Drosophila immune response now suggests that other immune defence mechanisms might contribute to the increased susceptibility to infection displayed by mutant flies (De Gregorio, 2002).

The interactions between the Toll and Imd pathways are more complex than merely regulating the same target genes. In agreement with Northern blot analysis, it has been shown that the transcriptional control of relish in response to infection receives a modest input from the Toll pathway, revealing an additional level of interaction between the two cascades. The activation of Toll may increase the level of Relish to allow a more efficient response to bacterial infection. This finding is in agreement with previous observations showing that in mutants where the Toll pathway is constitutively active (Tl^10b), all the antibacterial peptides genes, including diptericin, are induced with more rapid kinetics than in wild-type flies. Furthermore, the higher susceptibility to E.coli infection of the rel,spz double mutant compared with the rel single mutants flies indicates that Toll also has a direct, Relish-independent effect on the resistance to infection by Gram-negative bacteria. Northern blot analysis shows that relish induction in response to infection is significantly reduced in dTAK1 and dredd mutants, indicating that the Imd pathway undergoes autoregulation. Interestingly, the Imd pathway can influence the Toll pathway through the control of PGRP-SA, which encodes a recognition protein essential for the activation of the Toll pathway by Gram-positive bacteria. Again, it is interesting to notice that this interaction between the Toll and Imd pathways correlates with the contribution of both pathways to fight infection with Gram-positive bacteria. Interestingly, all the genes encoding components of the Toll pathway required for both antibacterial and antifungal responses (necrotic, spaetzle, Toll, pelle, cactus and Dif) are not controlled by the Imd pathway and are subjected to autoregulation (De Gregorio, 2002).

The Rel/NF-kappaB proteins Dif, Dorsal and Relish, which are the transactivators induced by the Toll and Imd pathways, bind to the kappaB sites present in the promoters of target genes, such as AMP genes, regulating their expression. Therefore, the analysis of the promoters of the DIRGs controlled by Toll or Imd pathways could help to identify all the direct NF-kappaB targets during infection. However, some of the effects of mutations affecting the Toll or Imd pathways that were monitored by microarray analysis might be mediated by the regulation of other transcription factors or signaling cascades. It has been shown recently in larvae that the Tep1 gene is regulated by the JAK-STAT pathway and can be activated by the Toll pathway, suggesting that Toll can control, at least partially, the JAK-STAT cascade. Two genes encoding components of the JNK pathway (puc and d-Jun) are partially regulated by Toll and Imd in response to septic injury (De Gregorio, 2002).

The presence of DIRGs independent of or only partially dependent on both the Imd and Toll pathways suggests the presence of other signaling cascades activated after septic injury. Potential candidates are MAPK and JAK-STAT pathways. Beside their developmental functions, the MAPK pathways have been implicated in wound healing (JNK) and the stress response (MEKK). The JAK-STAT pathway controls the Drosophila complement-like gene TepI. The stimuli that trigger these cascades are not known and it is not clear if these cascades are activated by exogenous or host factors. Interestingly, in vertebrates, the JAK-STAT pathway is activated by cytokines during the immune response. The microarray analysis of mutants in these pathways might help to reveal their exact contribution to the Drosophila immune response. The observation that Toll and Imd pathways control most of the DIRGs raises the question of whether these two pathways are the sole signaling cascades directly activated by microbial elictors, while the other signaling pathways are triggered by other stimuli associated with infection such as wound, stress, cytokine-like factors and Toll and Imd activities (De Gregorio, 2002).

In vertebrates, many genes involved in the immune response are grouped in large chromosomal complexes. The recent completion of the Drosophila genome did not reveal any striking chromosomal organization beside clustering of genes belonging to the same family, probably reflecting recent duplication events. In this study, it was observed that some of the genes responding to microbial infection are located in the same cytological region or are associated in transcriptionally co-regulated genomic clusters. Interestingly, microarray analysis of circadian gene expression in Drosophila has led to the identification of similar clusters of genes. Other microarray analyses might reveal the importance of the genome organization in the definition of adequate transcription programs in response to a variety of stimuli (De Gregorio, 2002).

Sequential activation of signaling pathways during innate immune responses in Drosophila

Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. These transcriptional patterns can be dissected into distinct groups. In addition to signaling through the Toll and Imd pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, the results may imply a close link between the control of tissue repair and antimicrobial processes (Boutros, 2002).

Lipopolysaccharides (LPS) are the principal cell wall components of gram-negative bacteria. In mammals, exposure to LPS causes septic shock through a Toll-like receptor TLR4-dependent signaling pathway. LPS treatment of Drosophila SL2 cells leads to rapid expression of antimicrobial peptides, such as Cecropins (Cec). SL2 cells resemble embryonic hemocytes and have also been used as a model system to study JNK and other signaling pathways. LPS-responsive induction of the antimicrobial peptides AttacinA (AttA), Diptericin (Dipt), and Cec relies on IKK and Relish. In order to obtain a broad overview on the transcriptional response to LPS in Drosophila, genome-wide expression profiles of SL2 cells were generated at different time points following LPS treatment. Altered expression of 238 genes was detected (Boutros, 2002).

In time-course experiments, a complex pattern of gene expression was observed that can be separated into different temporal clusters. A first group, with peak expression at 60 min after LPS, primarily consists of cytoskeletal regulators, signaling, and proapoptotic factors. This group includes cytoskeletal and cell adhesion modulators such as Matrix metalloprotease-1, WASp, Myosin, and Ninjurin, proapoptotic factors such as Reaper, and signaling proteins such as Puckered and VEGF-2. A second group, with peak expression at 120 min, includes many known defense and immunity genes, such as Cec, Mtk, and AttA, but not the gram-positive-induced peptide Drs. Interestingly, this cluster also includes PGRP-SA, which is a gram-positive pattern recognition receptor in vivo, suggesting possible crossregulation between gram-positive- and gram-negative-induced factors. A third group is transiently downregulated upon LPS stimulation. This cluster includes genes that play a role in cell cycle control, such as String and Rca1. Altogether, these results show that, in response to LPS, a defined gram-negative stimulus, cells elicit a complex transcriptional response (Boutros, 2002).

The signaling requirements for differentially expressed transcripts were examined in mutant alleles of known Toll and Imd/Rel pathway components, reasoning that additional pathways might be uncovered by analyzing patterns that cannot be reconciled with expected signaling patterns. Flies homozygous for loss-of-function mutations in tube, key, or rel were infected with gram-negative and gram-positive bacteria, and expression profiles were generated for a 6 hr time point after infection. In addition, noninfected Tl^10b, a gain-of-function allele of the receptor, and cact, a homolog of the inhibitory factor IkappaB, were used to monitor transcripts that are constitutively expressed in gain-of-function signaling mutants. The antimicrobial peptides dipt and drosomycin (drs) are representative targets for the Toll and Imd/Rel pathways, respectively. dipt induction is not detectable in the expression profiles in either a rel or key mutant background, whereas its expression is not affected in tube mutants. In contrast, drs relies on Tube to convey a Toll-dependent signal. Consistently, the expression profiles show that, in a tube mutant background, drs expression is diminished. These experiments showed that the analysis of mutant expression profiles can be used to deduce signaling requirements for distinct target groups (Boutros, 2002).

Toward a computational annotation of signaling pathways, a pattern-matching strategy was employed to rank transcripts by similarity to bona fide Toll or Imd/Rel pathway targets, such as dipt and drs. A set of 91 transcripts that matched the filtering criteria was analyzed for differential expression at a 6 hr time point after septic injury. To determine their dependence on known immunity signaling pathways, the correlation coefficients were calculated of the individual gene expression level in mutant backgrounds to binary Toll or Imd/Rel patterns. Genes were subsequently ordered according to their correlation coefficients for each pathway signature. Using this strategy, transcripts were separated that primarily belong to either the Toll or Imd/Rel pathway groups. For example, genes that show a high correlation coefficient for a Toll pathway pattern include drs, transferrin, a secreted iron binding protein, IM2, and a cluster of homologous secreted peptides at 55C9. These genes have a low correlation coefficient for an Imd/Rel pattern, indicating that they are primarily dependent on Toll pathway signaling in response to microbial infection. In contrast, a group of genes score low for a Toll pathway pattern but have high correlation coefficients for an Imd/Rel pattern. This group includes known gram-negative antimicrobial peptides, such as cec and dipt, peptidoglycan receptor-like genes (PGRP-SD, PGRP-SB1), other small transcripts (CG10332), and genes coding for putative transmembrane proteins, such as CG3615 (Boutros, 2002).

Interestingly, some genes do not fit either pattern, suggesting that they are regulated by other pathways. One group of genes, including cytoskeletal factors such as actin88F, flightin, and tpnC41C, is induced in Tl^10b, but not in cact, mutants. In contrast, totM and CG11501 are expressed at high levels in cact mutant flies but are not expressed in Tl^10b mutant flies. In addition, these transcripts are highly inducible in a tube genetic background, but they are not inducible in key or rel. This may suggest that Toll, Tube, and Cact do not act in a linear pathway under all circumstances. Moreover, rel shows an expression pattern suggesting that it is regulated by both the Imd/Rel and Toll pathways. Thus, these results indicate that, in addition to the canonical Toll and Imd pathways, other signaling events and possibly signaling pathway branching contribute to the complex expression patterns after septic injury. Finally, there is a strong correlation between pathway requirement and temporal expression pattern. Whereas Toll targets are exclusively found in the sustained cluster, Imd/Rel targets are expressed early and transiently after septic injury. The two additional clusters with noncanonical patterns show temporal patterns distinct from either Toll or Imd pathways (Boutros, 2002).

It was reasoned that the patterns observed in the mutant analysis might reflect the contributions of additional signaling pathways. Also, these noncanonical clusters show distinct temporal expression patterns, suggesting that they are separately controlled. One group of genes consists primarily of cytoskeletal regulators and structural proteins that are expressed early on, with peak expression at 3 hr. These include several muscle-specific proteins, thus possibly reflecting the organ that is injured during injection. For example, flightin encodes a cytoskeletal structural protein expressed in the indirect flight muscle (Boutros, 2002).

Since the expression of cytoskeletal genes after LPS stimulation is dependent on a JNK cascade, whether removing JNK activity in vivo affects the induction of fln was examined. In Drosophila, JNK signaling pathways have been previously implicated in epithelial sheet movements during embryonic and pupal development, a process that has been likened to wound-healing responses. hep¹ (JNKK) mutants, which are impaired in JNK signaling, the induction of fln is diminished, whereas the expression of the antimicrobial peptide dipt is not affected. A test was performed to see whether fln induction in Tl loss-of-function alleles is affected. These experiments show that fln expression is lost in Tl mutants, suggesting that Toll acts upstream of a JNK pathway to induce septic injury-induced target genes (Boutros, 2002).

NFkappaB pathways play a central role for innate and adaptive immune response in mammals. In innate immune responses, TLRs on dendritic cells recognize microbial agents and activate NFkappaB, leading to the expression of proinflammatory cytokines and other costimulatory factors required to initiate an adaptive immune response. Additionally, other signaling pathways have been implicated at later stages during immune responses in mammals, but their physiological role in innate immunity remains rather poorly understood. For example, several cytokines, such as IL-6 and IL-11, signal through a JAK/STAT pathway to induce the expression of acute phase proteins. Similarly, JNK pathways are activated in response to TNF and IL-1, may lead to the expression of immune modulators, and are required for T cell differentiation. In Drosophila, studies have investigated two distinct NFkappaB-pathways --Toll and Imd/Rel -- that have been shown to mediate gram-positive/fungal and gram-negative responses. Both pathways induce specific antimicrobial peptides and thereby focus the response on the invading microbial agent. Genetic analysis has shown that functions of the NFkappaB-pathways are separable; flies that are mutant for only one of these pathways are susceptible to subgroups of pathogens. Could the contribution of NFkappaB-dependent and, possibly, other signaling pathways be identified by examining global expression profiles? The obtained data set demonstrates that NFkappaB-independent signaling pathways contribute to the transcriptional patterns observed after microbial infection. Both in cells and in vivo, JNK-dependent targets precede the peak expression of antimicrobial peptides that require NFkappaB. JAK/STAT targets are induced with a distinct temporal pattern that shows late, but only transient, expression characteristics. The stereotyped pathway patterns after microbial challenge suggest that the correct temporal execution of signaling events, similar to signaling during development, may play an important role in the regulation of homeostasis (Boutros, 2002).

The signaling requirements for these differentially expressed transcripts were examined in mutant alleles of known Toll and Imd/Rel pathway components, reasoning that additional pathways might be uncovered by analyzing patterns that cannot be reconciled with expected signaling patterns. Flies homozygous for loss-of-function mutations in tube, key, or rel were infected with gram-negative and gram-positive bacteria, and expression profiles were generated for a 6 hr time point after infection. In addition, noninfected Tl^10b, a gain-of-function allele of the receptor, and cact, a homolog of the inhibitory factor IkappaB, were used to monitor transcripts that are constitutively expressed in gain-of-function signaling mutants. The antimicrobial peptides dipt and drosomycin (drs) are representative targets for the Toll and Imd/Rel pathways, respectively. dipt induction is not detectable in the expression profiles in either a rel or key mutant background, whereas its expression is not affected in tube mutants. In contrast, drs relies on Tube to convey a Toll-dependent signal. Consistently, the expression profiles show that, in a tube mutant background, drs expression is diminished. These experiments showed that the analysis of mutant expression profiles can be used to deduce signaling requirements for distinct target groups (Boutros, 2002).

Toward a computational annotation of signaling pathways, a pattern-matching strategy was employed to rank transcripts by similarity to bona fide Toll or Imd/Rel pathway targets, such as dipt and drs. A set of 91 transcripts that matched the filtering criteria was analyzed for differential expression at a 6 hr time point after septic injury. To determine their dependence on known immunity signaling pathways, the correlation coefficients were calculated of the individual gene expression level in mutant backgrounds to binary Toll or Imd/Rel patterns. Genes were subsequently ordered according to their correlation coefficients for each pathway signature. Using this strategy, transcripts were separated that primarily belong to either the Toll or Imd/Rel pathway groups. For example, genes that show a high correlation coefficient for a Toll pathway pattern include drs, transferrin, a secreted iron binding protein, IM2, and a cluster of homologous secreted peptides at 55C9. These genes have a low correlation coefficient for an Imd/Rel pattern, indicating that they are primarily dependent on Toll pathway signaling in response to microbial infection. In contrast, a group of genes score low for a Toll pathway pattern but have high correlation coefficients for an Imd/Rel pattern. This group includes known gram-negative antimicrobial peptides, such as cec and dipt, peptidoglycan receptor-like genes (PGRP-SD, PGRP-SB1), other small transcripts (CG10332), and genes coding for putative transmembrane proteins, such as CG3615 (Boutros, 2002).

Interestingly, some genes do not fit either pattern, suggesting that they are regulated by other pathways. One group of genes, including cytoskeletal factors such as actin88F, flightin, and tpnC41C, is induced in Tl^10b, but not in cact, mutants. In contrast, totM and CG11501 are expressed at high levels in cact mutant flies but are not expressed in Tl^10b mutant flies. In addition, these transcripts are highly inducible in a tube genetic background, but they are not inducible in key or rel. This may suggest that Toll, Tube, and Cact do not act in a linear pathway under all circumstances. Moreover, rel shows an expression pattern suggesting that it is regulated by both the Imd/Rel and Toll pathways. Thus, these results indicate that, in addition to the canonical Toll and Imd pathways, other signaling events and possibly signaling pathway branching contribute to the complex expression patterns after septic injury. Finally, there is a strong correlation between pathway requirement and temporal expression pattern. Whereas Toll targets are exclusively found in the sustained cluster, Imd/Rel targets are expressed early and transiently after septic injury. The two additional clusters with noncanonical patterns show temporal patterns distinct from either Toll or Imd pathways (Boutros, 2002).

It was reasoned that the patterns observed in the mutant analysis might reflect the contributions of additional signaling pathways. Also, these noncanonical clusters show distinct temporal expression patterns, suggesting that they are separately controlled. One group of genes consists primarily of cytoskeletal regulators and structural proteins that are expressed early on, with peak expression at 3 hr. These include several muscle-specific proteins, thus possibly reflecting the organ that is injured during injection. For example, flightin encodes a cytoskeletal structural protein expressed in the indirect flight muscle (Boutros, 2002).

Since the expression of cytoskeletal genes after LPS stimulation is dependent on a JNK cascade, whether removing JNK activity in vivo affects the induction of fln was examined. In Drosophila, JNK signaling pathways have been previously implicated in epithelial sheet movements during embryonic and pupal development, a process that has been likened to wound-healing responses. hep¹ (JNKK) mutants, which are impaired in JNK signaling, the induction of fln is diminished, whereas the expression of the antimicrobial peptide dipt is not affected. A test was performed to see whether fln induction in Tl loss-of-function alleles is affected. These experiments show that fln expression is lost in Tl mutants, suggesting that Toll acts upstream of a JNK pathway to induce septic injury-induced target genes (Boutros, 2002).

NFkappaB pathways play a central role for innate and adaptive immune response in mammals. In innate immune responses, TLRs on dendritic cells recognize microbial agents and activate NFkappaB, leading to the expression of proinflammatory cytokines and other costimulatory factors required to initiate an adaptive immune response. Additionally, other signaling pathways have been implicated at later stages during immune responses in mammals, but their physiological role in innate immunity remains rather poorly understood. For example, several cytokines, such as IL-6 and IL-11, signal through a JAK/STAT pathway to induce the expression of acute phase proteins. Similarly, JNK pathways are activated in response to TNF and IL-1, may lead to the expression of immune modulators, and are required for T cell differentiation. In Drosophila, studies have investigated two distinct NFkappaB-pathways --Toll and Imd/Rel -- that have been shown to mediate gram-positive/fungal and gram-negative responses. Both pathways induce specific antimicrobial peptides and thereby focus the response on the invading microbial agent. Genetic analysis has shown that functions of the NFkappaB-pathways are separable; flies that are mutant for only one of these pathways are susceptible to subgroups of pathogens. Could the contribution of NFkappaB-dependent and, possibly, other signaling pathways be identified by examining global expression profiles? The obtained data set demonstrates that NFkappaB-independent signaling pathways contribute to the transcriptional patterns observed after microbial infection. Both in cells and in vivo, JNK-dependent targets precede the peak expression of antimicrobial peptides that require NFkappaB. JAK/STAT targets are induced with a distinct temporal pattern that shows late, but only transient, expression characteristics. The stereotyped pathway patterns after microbial challenge suggest that the correct temporal execution of signaling events, similar to signaling during development, may play an important role in the regulation of homeostasis (Boutros, 2002).

In conclusion, genome-wide expression profiling was employed to examine the contribution of different signaling pathways in complex tissues and to assign targets to candidate pathways. Both a cell culture model system and an in vivo analysis were used to show the temporal order of NFkappaB-dependent and -independent pathways after septic injury. An interesting question that remains is, how do the extracellular events leading to pathway activation reflect the nature of the pathogen? Clean injury experiments induce a largely overlapping set of induced genes, but to a lower extent than septic injury. This is consistent with experiments showing that septic injury with only gram-negative E. coli induces both anti-gram-negative and anti-gram-positive responses. These results can be interpreted to suggest that wounding, in itself, might be sufficient to induce a transient (and unspecific) innate immune response. However, further studies are needed to understand the nature of the inducing agent (Boutros, 2002).

Genetic analysis of contributions of dorsal group and JAK-Stat92E pathway genes to larval hemocyte concentration and the egg encapsulation response in Drosophila

Drosophila larvae defend themselves against parasitoid wasps by completely surrounding the egg with layers of specialized hemocytes called lamellocytes. Similar capsules of lamellocytes, called melanotic capsules, are also formed around 'self' tissues in larvae carrying gain-of-function mutations in Toll and hopscotch. Constitutive differentiation of lamellocytes in larvae carrying these mutations is accompanied by high concentrations of plasmatocytes, the major hemocyte class in uninfected control larvae. The relative contributions of hemocyte concentration vs. lamellocyte differentiation to wasp egg encapsulation are not known. To address this question, Leptopilina boulardi was used to infect more than a dozen strains of host larvae harboring a wide range of hemocyte densities. A significant correlation exists between hemocyte concentration and encapsulation capacity among wild-type larvae and larvae heterozygous for mutations in the Hopscotch-Stat92E and Toll-Dorsal pathways. Larvae carrying loss-of-function mutations in Hopscotch, Stat92E, or dorsal group genes exhibit significant reduction in encapsulation capacity. Larvae carrying loss-of-function mutations in dorsal group genes (including Toll and tube) have reduced hemocyte concentrations, whereas larvae deficient in Hopscotch-Stat92E signaling do not. Surprisingly, unlike hopscotch mutants, Toll and tube mutants are not compromised in their ability to generate lamellocytes. These results suggest that circulating hemocyte concentration and lamellocyte differentiation constitute two distinct physiological requirements of wasp egg encapsulation and Toll and Hopscotch proteins serve distinct roles in this process (Sorrentino, 2004).

These results suggest that the suppression of encapsulation capacity by loss of function of hop, Tl, or tub is likely to be due to distinct requirements of these genes. The suppression of lymph gland response to parasitization in the hop^M4 background is consistent with the observed reduction in hop^M4/Y encapsulation capacity and suggests that the Hopscotch protein is necessary for a parasite-induced signal for lamellocyte differentiation. This signal for lamellocyte differentiation is most likely mediated by the transcription factor Stat92E: Loss of function of one copy of Stat92E suppresses the penetrance of the hop^Tum-l-induced melanotic tumor phenotype and Stat92E is constitutively activated in Drosophila cell cultures that overexpress Hop^Tum-l. These results are consistent with the proposed Stat92E-dependent lamellocyte signal: stat92E larvae are immune compromised and are unable to mount an efficient egg encapsulation response despite exhibiting control circulating hemocyte concentration levels. Additionally, mean circulating lamellocyte percentage in hop^Tum-l/Y; stat92E^HJ/stat92E^HJ larvae that are tumor-free is ~1%, which is indistinguishable from the control value (Sorrentino, 2004).

In contrast to Hop and Stat92E, Toll and Tube appear not to play a role in lamellocyte differentiation; rather, loss-of-function mutations in Toll or tube probably suppress encapsulation via other mechanisms. Since Toll and tube larvae have very few circulating hemocytes, reduction in encapsulation in Tl and tub mutants might be due to defects in wasp egg recognition or a reduction in hemocyte proliferation that normally follows parasitization. The effect of these mutations on crystal cells is unclear. While the possibility that these mutations reduce encapsulation capacity by reducing the crystal cell population cannot be ruled out, this is unlikely, since Black cells mutant larvae without functional crystal cells are immune competent and can still successfully encapsulate wasp eggs. The fact that lymph glands of loss-of-function Tl and tub mutant larvae can support lamellocyte differentiation suggests that the low circulating hemocyte concentration in Tl and tub larvae in itself does not hinder lamellocyte differentiation or the ability of the lymph gland to disperse after the wasp egg is introduced into the hemocoel. Given that gain-of-function Tl alleles induce lamellocyte differentiation, the lack of effect of Tl^- on lamellocyte differentiation is somewhat unexpected, and it is possible that lamellocyte differentiation is in some way secondarily activated in the Tl^10b background. Thus, the wasp egg encapsulation assay is a useful tool for evaluating the genetic requirements for lamellocyte differentiation (Sorrentino, 2004).

In conclusion, this study shows that while there is substantial variation in hemocyte concentration in control larvae, this variation is consistent with a log-normal distribution. Such a distribution could be a result of the inherently logarithmic process of cell division. Using this quantitative method of circulating hemocyte concentration data analysis, it was found that previously reported circulating hemocyte concentration values for mutant larvae that exhibit reduced or increased hemocyte densities are also log-normally distributed and that approximately half of each of these mutant distributions lie beyond the limits of the control distribution, allowing ranges of circulating hemocyte concentration values to be defined as being low, control, and high. In addition, encapsulation capacity in control and DV mutant larvae correlates with circulating hemocyte concentration. Evidence for biological significance of this correlation also comes from observations that D. melanogaster larvae selected for higher resistance against A. tabida have twice as many circulating hemocytes as compared to control larvae. These observations support the notion that circulating hemocytes, possibly plasmatocytes, contribute to the efficiency of the egg encapsulation response. However, high circulating hemocyte concentration alone is insufficient to trigger encapsulation; lamellocytes must be present. For example, massive 20- to 300-fold increases in circulating hemocyte concentration involving plasmatocytes and crystal cells, but not lamellocytes, are insufficient to trigger constitutive encapsulation of self tissue in the larva. The combined use of genetic and immune approaches used in this study demonstrates that different developmental signals independently contribute to the maintenance of the steady-state hemocyte concentration in circulation and the ability to differentiate lamellocytes. Together, these physiological parameters enable larval hosts to efficiently defend themselves against wasp infections (Sorrentino, 2004).

Functional Evolution of the Vertebrate Myb Gene Family: B-Myb, but neither A-Myb nor c-Myb, complements Drosophila Myb in Hemocytes: Genetic interaction with Toll

The duplication of genes and genomes is believed to be a major force in the evolution of eukaryotic organisms. However, different models have been presented about how duplicated genes are preserved from elimination by purifying selection. Preservation of one of the gene copies due to rare mutational events that result in a new gene function (neo-functionalization) necessitates that the other gene copy retain its ancestral function. Alternatively, preservation of both gene copies due to rapid divergence of coding and non-coding regions such that neither retains the complete function of the ancestral gene (sub-functionalization) may result in a requirement for both gene copies for organismal survival. The duplication and divergence of the tandemly arrayed homeotic clusters have been studied in considerable detail and have provided evidence in support of the sub-functionalization model. However, the vast majority of duplicated genes are not clustered tandemly, but instead are dispersed in syntenic regions on different chromosomes, most likely as a result of genome-wide duplications and rearrangements. The Myb oncogene family provides an interesting opportunity to study a dispersed multigene family because invertebrates possess a single Myb gene, whereas all vertebrate genomes examined thus far contain three different Myb genes (A-Myb, B-Myb and c-Myb). A-Myb and c-Myb appear to have arisen by a second round of gene duplication, which was preceded by the acquisition of a transcriptional activation domain in the ancestral A-Myb/c-Myb gene generated from the initial duplication of an ancestral B-Myb-like gene. B-Myb appears to be essential in all dividing cells, whereas A-Myb and c-Myb display tissue-specific requirements during spermatogenesis and hematopoiesis, respectively. The absence of Drosophila Myb (Dm-Myb) causes a failure of larval hemocyte proliferation and lymph gland development, while Dm-Myb(-/-) hemocytes from mosaic larvae reveal a phagocytosis defect. Vertebrate B-Myb, but neither vertebrate A-Myb nor c-Myb, can complement these hemocyte proliferation defects in Drosophila. Indeed, vertebrate A-Myb and c-Myb cause lethality in the presence or absence of endogenous Dm-Myb. These results are consistent with a neomorphic origin of an ancestral A-Myb/c-Myb gene from a duplicated B-Myb-like gene. In addition, these results suggest that B-Myb and Dm-Myb share essential conserved functions that are required for cell proliferation. Finally, these experiments demonstrate the utility of genetic complementation in Drosophila to explore the functional evolution of duplicated genes in vertebrates (Davidson, 2004).

To establish whether Dm-Myb is generally required for proliferation of hemocytes, epistasis experiments were conducted using a Dm-Myb null mutation and dominant substitution mutations of the Toll receptor (Tl^10b) and the Jak kinase, hopscotch (hop^Tuml); dominant gain-of-function mutations in these genes result in hyperactivation of their respective pathways leading to hemocyte overproliferation and abnormal lamellocyte differentiation. To determine whether Dm-Myb is required for the dysregulated overproliferation and differentiation phenotypes of Tl^10b and hop^Tuml mutants, double mutant larvae lacking Dm-Myb in conjunction with these dominant alleles of Toll and hopscotch were generated. It was found that, in addition to an overproliferation of plasmatocytes in the primary lymph gland lobes, the secondary lymph gland lobe hemocytes aberrantly differentiate into lamellocytes in hop^Tuml mutants. It is thought that the normally smaller secondary lymph gland lobes serve as a reservoir of undifferentiated prohemocytes, however, in hop^Tuml larvae the secondary lobes enlarge with concomitant abnormal differentiation of lamellocytes. While hemocytes in the secondary lymph gland lobes of hop^Tuml, Dm-Myb^-/- double mutants show an increased expression of the lamellocyte enhancer-trap marker, these ß-gal positive cells fail to overproliferate and do not adopt the flattened shape characteristic of differentiated lamellocytes. In summary, an activated JAK/STAT pathway cannot drive the proliferation of hemocytes in the absence of Dm-Myb. In addition, an activated Toll pathway cannot drive the proliferation of hemocytes in the absence of Dm-Myb (Davidson, 2004).

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).

Comprehensive identification of Drosophila dorsal-ventral patterning genes using a whole-genome tiling array

Dorsal-ventral (DV) patterning of the Drosophila embryo is initiated by Dorsal, a sequence-specific transcription factor distributed in a broad nuclear gradient in the precellular embryo. Previous studies have identified as many as 70 protein-coding genes and one microRNA (miRNA) gene that are directly or indirectly regulated by this gradient. A gene regulation network, or circuit diagram, including the functional interconnections among 40 Dorsal (Dl) target genes and 20 associated tissue-specific enhancers, has been determined for the initial stages of gastrulation. This study attempts to extend this analysis by identifying additional DV patterning genes using a recently developed whole-genome tiling array. This analysis led to the identification of another 30 protein-coding genes, including the Drosophila homolog of Idax, an inhibitor of Wnt signaling. In addition, remote 5' exons were identified for at least 10 of the ~100 protein-coding genes that were missed in earlier annotations. As many as nine intergenic uncharacterized transcription units (TUs) were identified, including two that contain known microRNAs, miR-1 and -9a. The potential functions of these recently identified genes are discussed and it is suggested that intronic enhancers are a common feature of the DV gene network (Biemar, 2006).

The Dl nuclear gradient differentially regulates a variety of target genes in a concentration-dependent manner. The gradient generates as many as five different thresholds of gene activity, which define distinct cell types within the presumptive mesoderm, neuroectoderm, and dorsal ectoderm. Total RNA was extracted from embryos produced by three different maternal mutants: pipe^–/pipe^–, Toll^rm9/Toll^rm10, and Toll^10B. pipe^–/pipe^– mutants completely lack Dl nuclear protein and, as a result, overexpress genes that are normally repressed by Dl and restricted to the dorsal ectoderm. For example, the decapentaplegic (dpp) TU is strongly "lit up" by total RNA extracted from pipe^–/pipe^– mutant embryos. The intron-exon structure of the transcribed region is clearly delineated by the hybridization signal, most likely because the processed mRNA sequences are more stable than the intronic sequences present in the primary transcript. There is little or no signal detected with RNAs extracted from Toll^rm9/Toll^rm10 (neuroectoderm) and Toll^10B (mesoderm) mutants. Instead, these other mutants overexpress different subsets of the Dl target genes. For example, Toll^rm9/Toll^rm10 mutants contain low levels of Dl protein in all nuclei in ventral, lateral, and dorsal regions. These low levels are sufficient to activate target genes such as intermediate neuroblasts defective (ind), ventral neuroblasts defective (vnd), rhomboid (rho), and short gastrulation (sog) but insufficient to activate snail (sna). In contrast, Toll^10B mutants overexpress genes (e.g., sna) normally activated by peak levels of the Dl gradient in ventral regions constituting the presumptive mesoderm (Biemar, 2006).

To identify potential Dl targets, ranking scores were assigned for the six possible comparisons of the various mutant backgrounds, pipe vs. Toll^rm9/Toll^rm10, pipe vs. Toll^10B, Toll^rm9/Toll^rm10 vs. Toll^10B, Toll^rm9/Toll^rm10 vs. pipe, Toll^10B vs. Toll^rm9/Toll^rm10, and Toll^10B vs. pipe, using the TiMAT software package. As a first approximation, only hits with a median fold difference of 1.5 and above were considered. For further analysis, the top 100 TUs were selected for each of the comparisons, with the exception of Toll^rm9/Toll^rm10 vs. pipe for which the TiMAT analysis returned only 43 hits that meet the cutoff. To refine the search for TUs specifically expressed in the mesoderm, where levels of nuclear Dl are highest, only those present in the Toll^10B vs. Toll^rm9/Toll^rm10 and Toll^10B vs. pipe, but not pipe vs. Toll^rm9/Toll^rm10 comparisons were selected. For TUs induced by intermediate and low levels of nuclear Dl in the neuroectoderm, those present in both the Toll^rm9/Toll^rm10 vs. Toll^10B and Toll^rm9/Toll^rm10 vs. pipe, but not pipe vs. Toll^10B comparisons were selected. For TUs restricted to the dorsal ectoderm, only those present in the pipe vs. Toll^rm9/Toll^rm10 and pipe vs. Toll^10B, but not Toll^rm9/Toll^rm10 vs. Toll^10B, were selected. Finally, the TUs corresponding to annotated genes already identified in the previous screen were eliminated to focus on annotated genes not previously considered as potential Dorsal targets, as well as transcribed fragments (transfrags) not previously characterized. Using these criteria, 45 previously annotated protein-coding genes were identified, along with 23 uncharacterized transfrags. Of the 45 protein-coding genes, 29 exhibited localized patterns of gene expression across the DV axis, whereas the remaining 16 were not tested (Biemar, 2006).

The previous microarray screen relied on high cutoff values for the identification of authentic DV genes. For example, only genes exhibiting 6-fold up-regulation in pipe^–/pipe^– mutant embryos were tested by in situ hybridization for localized expression in the dorsal ectoderm. Many other genes displayed >2-fold up-regulation but were not explicitly tested for localized expression. The whole-genome tiling array permitted the use of much lower cutoff values. For example, CG13800, which was identified by conventional microarray screens, falls just below the original cutoff value but displays 5-fold up-regulation in pipe^–/pipe^– mutants in the analysis. In situ hybridization assays reveal localized expression in the dorsal ectoderm. This pattern is greatly expanded in embryos derived from pipe^–/pipe^– mutant females, as expected for a gene that is either directly or indirectly repressed by the Dl gradient. Genes exhibiting even lower cutoff values were also found to display localized expression. Among these genes is a Wnt homologue, Wnt2, which is augmented only 2.25-fold in mutant embryos lacking the Dl nuclear gradient (Biemar, 2006).

The 4-fold cutoff value used in the previous screen for candidate protein-coding genes expressed in the neuroectoderm also excluded genes expressed in this tissue. The Trim9 gene exhibits just a 2-fold increase in mutant embryos derived from Toll^rm9/Toll^rm10 females. Nonetheless, in situ hybridization assays reveal localized expression in the neuroectoderm of WT embryos. As expected, expression is expanded in Toll^rm9/Toll^rm10 mutant embryos. Another gene, CG9973, displays just 1.8-fold up-regulation but is selectively expressed in the neuroectoderm. CG9973 encodes a putative protein related to Idax, an inhibitor of the Wnt signaling pathway. Idax inhibits signaling by interacting with the PDZ domain of Dishevelled (Dsh), a critical mediator of the pathway. A Wnt2 homologue is selectively expressed in the dorsal ectoderm. Recent studies identified a second Wnt gene, WntD, which is expressed in the mesoderm. Thus, the CG9973/Idax inhibitor might be important for excluding Wnt signaling from the neuroectoderm. Such a function is suggested by the analysis of Idax activity in vertebrate embryos (Biemar, 2006).

Additional genes were also identified that are specifically expressed in the mesoderm. Among these is CG9005, which encodes an unknown protein that is highly conserved in different animals, including frogs, chicks, mice, rats, and humans. It displays <2-fold up-regulation in Toll^10B embryos but is selectively expressed in the ventral mesoderm of WT embryos. Expression is expanded in embryos derived from Toll^10B mutant females (Biemar, 2006).

Other protein-coding genes were missed in the previous screen because they were not represented on the Drosophila Genome Array used at the time. These include, for instance, CG8147 in the dorsal ectoderm and CG32372 in the mesoderm (Biemar, 2006).

An interesting example of the use of tiling arrays to identify tissue-specific isoforms is seen for the bunched (bun) TU. bun encodes a putative sequence-specific transcription factor related to mammalian TSC-22, which is activated by TGFβ signaling. It was shown to inhibit Notch signaling in the follicular epithelium of the Drosophila egg chamber. Three transcripts are expressed from alternative promoters in bun, but it appears that only the short isoform (bun-RC) is specifically expressed in the dorsal ectoderm. A number of bun exons are ubiquitously transcribed at low levels in the mesoderm, neuroectoderm, and dorsal ectoderm. However, the 3'-most exons are selectively up-regulated in pipe^–/pipe^– mutants. It is conceivable that Dpp signaling augments the expression of this isoform, which in turn, participates in the patterning of the dorsal ectoderm (Biemar, 2006).

In addition to protein-coding genes, the tiling array also identified uncharacterized TUs not previously annotated. Some of them are associated with ESTs, providing independent evidence for transcriptional activity in these regions. For 14 of these transfrags (61%), visual inspection of neighboring loci using the Integrated Genome Browser suggested coordinate expression of a neighboring protein-coding region (i.e., overexpressed in the same mutant background). The N-Cadherin gene (CadN) has a complex intron-exon structure consisting of ~20 different exons. The strongest hybridization signals are detected within the limits of exons, but an unexpected signal was detected ~10 kb upstream of the 5'-most exon. It is specifically expressed in the mesoderm, suggesting that it represents a previously unidentified 5' exon of the CadN gene. Support for this contention stems from two lines of evidence: (1) in situ hybridization using a probe against the 5' exon detects transcription in the presumptive mesoderm, the initial site of CadN expression; (2) using primers anchored in the 5' transfrag as well as the first exon of CadN, confirmation was obtained by RT-PCR that the recently identified TU is part of the CadN transcript. This recently identified 5' exon appears to contribute to the 5' leader of the CadN mRNA. It is possible that this extended leader sequence influences translational efficiency as seen in yeast. Because there seems to be a considerable lag between the time when CadN is first transcribed and the first appearance of the protein, it is suggested that this extended leader sequence might inhibit translation. An interesting possibility is that it does so through short upstream ORFs, as has been shown for several oncogenes in vertebrates (Biemar, 2006).

A 5' exon was also identified for crossveinless-2 (cv-2), a component of the Dpp bone morphogenetic protein (BMP) signaling pathway. cv-2 binds BMPs and functions as both an activator and inhibitor of BMP signaling. It is specifically required in the developing wing disk to generate peak Dpp signaling in the presumptive crossveins. cv-2 is also expressed in the dorsal ectoderm of early embryos, but its role during embryonic development has not been investigated. The whole-genome tiling array identified a 5' exon located ~10 kb 5' of the transcription start site of the cv-2 TU. Using RT-PCR and in situ hybridization assays, it was confirmed that the exon is part of the cv-2 transcript. It is possible that the exon resides near an embryonic promoter that is inactive in the developing wing discs. Future studies will determine whether this 5' exon influences the timing or levels of Cv-2 protein synthesis (Biemar, 2006).

In addition to the identification of 10 5' exons associated with previously annotated genes such as CadN and cv-2, three other transfrags appear to correspond to 3' exons, and nine of the RNAs seem to arise from autonomous TUs. Three of these represent annotated computational RNA (CR) genes: CR32777, CR31972, and CR32957. CR32777 corresponds to roX1, which is ubiquitously expressed at the blastoderm stage, hence it represents a false positive. The other two potential noncoding RNAs were recently identified independently in two other studies, and although the expression of CR32957 could not be detected by in situ hybridization, CR31972 transcripts are detected in the mesoderm. There is no evidence that these transcripts are processed into miRNAs, but noncoding genes corresponding to known miRNA loci were also identified in the screen. Transfrag 22 corresponds to the miR-9a primary transcript (pri-mir9a) and is detected in both the dorsal- and neuroectoderm. Expression of pri-mir9a is ubiquitous in embryos derived from pipe^–/pipe^– or Toll^rm9/Toll^rm10 females. Transfrag 8 corresponds to pri-mir1, which is present in the mesoderm (Biemar, 2006).

A third noncoding transcript (Transfrag 12) maps next to a known miRNA, miR-184. It is selectively expressed in the mesoderm and overexpressed in Toll^10B mutants. The mesodermal expression of miR-184 has been reported. It is possible that Transfrag 12 corresponds to pri-mir-184, and that secondary structures in the miRNA region preclude detection on the array. This is seen for several other miRNA precursors expressed at various stages during embryogenesis. Alternatively, Transfrag 12 might represent the fragment resulting from Drosha cleavage of the pri-mir-184 to produce the miR-184 precursor hairpin (pre-miR-184). A similar situation has been observed for the iab4 locus. Like miR-1, miR-184 is selectively expressed in the ventral mesoderm. It will be interesting to determine whether the two miRNAs jointly regulate some of the same target mRNAs (Biemar, 2006).

The identity of the last three transfrags is less clear. Visual inspection using the Integrated Genome Browser suggests expression of Transfrag 10 in the mesoderm, Transfrag 21 in the neuroectoderm, and Transfrag 11 in both the dorsal ectoderm and neuroectoderm. However, in situ hybridization assays confirm the predicted expression pattern only for Transfrag 11. Computational analyses designed to estimate the likelihood of translation suggest a protein-coding potential for Transfrag 10 [Likelihood Ratio Test (LRT) P < 0.001] and possibly Transfrag 11 (LRT P < 0.01), whereas Transfrag 21 could not be analyzed because of lack of conservation in other Drosophila species (Biemar, 2006).

This work has attempted to identify nonprotein coding genes involved in patterning the DV axis of the Drosophila embryo using an unbiased approach to survey the entire genome. This study, along with earlier analyses, identified as many as 100 protein-coding genes and five to seven noncoding genes that are differentially expressed across the DV axis of the early Drosophila embryo. Roughly half of the noncoding RNAs correspond to miRNAs, although <1% of the annotated genes in the Drosophila genome encode miRNAs. Future studies will determine how these RNAs impinge on the DV regulatory network (Biemar, 2006).

Recent studies have identified large numbers of noncoding transcripts in the mouse and human genomes. If the present study is predictive, less than one-fourth of the transcripts correspond to novel noncoding RNAs of unknown function, akin to CR31972 and Transfrag 11 expressed in the mesoderm and ectoderm, respectively. Most of the noncoding transcripts are likely to derive from intronic sequences because of the occurrence of cryptic remote 5' exons as seen for the CadN and cv-2 genes. At least 10% of the DV protein-coding genes were found to contain such exons. As a result, these genes contain large tracts of intronic sequences that might encompass regulatory DNAs such as tissue-specific enhancers. The FGF8-related gene, thisbe (ths), represents such a case. A neurogenic-specific enhancer that was initially thought to reside 5' of the TU actually maps within a large intron because of the occurrence of a remote 5' exon. It is suggested that such exons are responsible for the evolutionary "bundling" of genes and their associated regulatory DNAs. Gene duplication events are more likely to retain this linkage when regulatory DNAs map within the TU. In contrast, enhancers mapping in flanking regions can be uncoupled from their normal target gene by chromosomal rearrangements (Biemar, 2006).

Control of Drosophila blood cell activation via Toll signaling in the fat body

The Toll signaling pathway, first discovered in Drosophila, has a well-established role in immune responses in insects as well as in mammals. In Drosophila, the Toll-dependent induction of antimicrobial peptide production has been intensely studied as a model for innate immune responses in general. Besides this humoral immune response, Toll signaling is also known to activate blood cells in a reaction that is similar to the cellular immune response to parasite infections, but the mechanisms of this response are poorly understood. This paper describes a study of this response in detail and found that Toll signaling in several different tissues, activated by Toll^10b gain-of-function mutant can activate a cellular immune defense and that this response does not require Toll signaling in the blood cells themselves. As in the humoral immune response, Toll signaling in the fat body (analogous to the liver in vertebrates) is of major importance in the Toll-dependent activation of blood cells. However, this Toll-dependent mechanism of blood cell activation contributes very little to the immune response against the parasitoid wasp, Leptopilina boulardi, probably because the wasp is able to suppress Toll induction. Other redundant pathways may be more important in the defense against this pathogen (Schmid, 2014: PubMed).

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).

A single-cell atlas and lineage analysis of the adult Drosophila ovary

The Drosophila ovary is a widely used model for germ cell and somatic tissue biology. This study used single-cell RNA-sequencing (scRNA-seq) to build a comprehensive cell atlas of the adult Drosophila ovary that contains transcriptional profiles for every major cell type in the ovary, including the germline stem cells and their niche cells, follicle stem cells, and previously undescribed subpopulations of escort cells. In addition, Gal4 lines were identified with specific expression patterns, and lineage tracing of subpopulations of escort cells and follicle cells was performed. A distinct subpopulation of escort cells was capable of converting to follicle stem cells in response to starvation or upon genetic manipulation, including knockdown of escargot, or overactivation of mTor or Toll signalling (Rust, 2020).

In summary, this study has generated a detailed atlas of the cells in the adult Drosophila ovary. This atlas consists of 26 clusters that each correspond to a distinct population in the ovary. Through experimental validation and referencing well-characterized markers in the literature, the identity of each cluster was identified, and all of the major cell types in the ovariole were found to be represented. Several transcriptionally distinct subpopulations were identified within these major cell types, such as the anterior, central, and posterior Escort cell (EC) populations. Both the GSCs and the FSCs were identified in the dataset, which revealed several genes that are predicted to be specific for each of these stem cell populations. In addition, several Gal4 drivers, including Pdk1-Gal4, fax-Gal4, and stl-Gal4, were identified with unique expression patterns that make it possible to target transgene expression to the subsets of cells marked by these drivers. Lastly, although this study primarily focused on the most uniquely expressed genes for each cluster in this study, the transcriptional profile of each cluster is a rich dataset that can be mined to identify populations of cells that are relevant for a topic of interest. For example, gene expression profile of each cluster was compared to a list of human disease genes that are well-suited for analysis in Drosophila. It was found that germ cells are enriched for cells expressing major drivers of cancer, and ECs and follicle cells are enriched for genes involved in cardiac dysfunction, suggesting that these cell types may be good starting points for studies into the genetic interactions that underlie these human diseases (Rust, 2020).

This study also demonstrates the utility of using CellFindR9 in combination with monocle320 to identify unique populations of cells within a dataset. Because CellFindR produces clusters in a structured, iterative fashion, it was possible to construct a hierarchical tree that corresponds to a transcriptome relationship between clusters, and this outperformed other clustering methods. The tree built by CellFindR aligns well with expectations and provides some interesting new insights. For example, it was expected that germ cells would cluster apart from somatic cells in Tier 1 because these populations are substantially different from each other, arising at different times during development and from completely different lineages. However, it was surprising that the FSC, pFCs, polar cells, and stalk cells clustered more closely to escort cells than to the follicle cells of budded follicles. This suggests that many cell types in the germarium, which are often studied separately, have biologically relevant similarities (Rust, 2020).

The use of G-TRACE to assess the lineage potential of somatic cells in the germarium led to the surprising finding that ECs can convert to FSCs under starvation conditions. Recent studies have described similar forms of cellular plasticity in other tissues, suggesting that the ability of non-stem cells to convert to stem cells may be a more general feature of adult stem cell niches. However, this aspect of tissue homeostasis remains poorly understood. The finding that the conversion of ECs to FSCs can be induced by perturbations of mTor or Toll signaling is consistent with a role for these pathways in responding to starvation and cellular stress in other tissues, and provides a new opportunity to investigate the mechanisms of cellular responses to physiological stress in an adult stem cell niche (Rust, 2020).

Overall, this study provides a resource that will be valuable for a wide range of studies that use the Drosophila ovary as an experimental model. Additional scRNA-seq datasets provided by other studies will further increase the accuracy and resolution of the ovary cell atlas, and it will be important to follow up on the predictions of the atlas with detailed studies that focus on specific populations of cells. Collectively, these efforts will help drive discovery forward by providing a deeper understanding of the cellular composition of the Drosophila ovary (Rust, 2020).

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