InteractiveFly: GeneBrief

domeless : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The JAK/STAT signaling pathway plays important roles in vertebrate development and the regulation of complex cellular processes. Components of the pathway are conserved in Dictyostelium, Caenorhabditis, and Drosophila, yet the complete sequencing and annotation of the D. melanogaster and C. elegans genomes has failed to identify a receptor, raising the possibility that an alternative type of receptor exists for the invertebrate JAK/STAT pathway. domeless (dome) codes for a transmembrane protein required for all JAK/STAT functions in the Drosophila embryo. This includes its known requirement for embryonic segmentation and a newly discovered function in trachea specification. The Dome protein has an extracellular structure similar to the vertebrate cytokine class I receptors, although its sequence has greatly diverged. Like many interleukin receptors, Dome has a cytokine binding homology module (CBM) and three extracellular fibronectin-type-III domains (FnIII). Despite its low degree of overall similarity, key amino acids required for signaling in the vertebrate cytokine class I receptors are conserved in the CBM region. Dome is a signal-transducing receptor with most similarity to the IL-6 receptor family, but it also has characteristics found in the IL-3 receptor family. This suggests that the vertebrate families evolved from a single ancestral receptor which also gave rise to dome (Brown, 2001).

JAK/STAT signaling was first identified in vertebrates as mediating the response to some cytokines and growth factors. Ligand binding induces receptor homo- or hetero-dimerization and subsequent signal transduction. The receptors lack a tyrosine kinase domain but associate with cytoplasmic tyrosine kinases of the JAK family. After receptor dimerization, JAK phosphorylates a tyrosine residue on the receptor, and cytoplasmic STAT is recruited to the complex. JAK then phosphorylates STAT, which dimerizes, translocates to the nucleus, and induces gene transcription. In Drosophila, one JAK encoded by hopscotch (hop), one STAT encoded by STAT92E, and one ligand encoded by outstretched/unpaired (upd) have been identified, but no receptor has been found. Mutations for either hop, STAT92E, or upd result in an identical, characteristic segmentation phenotype (Brown, 2001).

Mutations in STAT92E affect the posterior spiracles, part of the respiratory apparatus of the larva. domeless was identified in a screen for P element insertion mutations that give a phenotype similar to STAT92E. The six alleles, three strong (dome²¹⁷, dome⁴⁴¹, and dome⁴⁶⁸) and three weak (dome³²¹, dome⁴⁰⁵, and dome³⁶⁷), all affect the shape of the posterior spiracles, with the strongest leading to a loss of the characteristic dome shape. Mobilization of the P element reverts both the lethality and the phenotype, confirming that the insertions cause the observed defects (Brown, 2001).

A database sequence search using DNA flanking the P elements identified an expressed-sequence tag (EST) encoding a putative transmembrane protein. The finding that expression of this cDNA rescues the dome spiracle phenotype confirms that this cDNA encodes the dome gene (Brown, 2001).

A second study identified domeless using a screen for suppressors of an eye phenotype caused by overexpression of unpaired. Overexpression of upd using a UAS-upd and GMR-Gal4 driver causes compound eye dramatic overgrowth in the adult eye because of an increase in the number of ommatidia. The average number of ommatidia in the compound eye of UAS-upd/GMR-Gal4 female flies is 978 ± 10 compared with 745 ± 7 in wild-type flies. Histological sections through the overgrown eyes reveal that most ommatidia have normal photoreceptor cells and regular cell size, indicating that Upd activity mainly regulates cell proliferation in the compound eye. However, the ommatidia look more crowded and have irregular space and arrangement, and several big vacuoles are integrated into the ommatidia lattice. The severity of eye morphology appears proportional to the strength of the Hop/Stat92E-mediated signaling, because removing one copy of hop partially suppresses the big eye phenotype; the average number of ommatidia is 854 ± 9). The advantage of this sensitized system lies in the possibility of conducting a screen for mutations that reduce (suppressors) or increase (enhancers) the degree of eye size. It was reasoned that a twofold reduction in the dose of a gene (by mutating one of its two copies) that functions downstream of Upd should dominantly alter signaling strength, which, in turn, should visibly modify the eye size. Based on this assumption, available X-chromosome P-element insertion mutations were screened and one complementation group of suppressors with four alleles was identified at the cytological location 18E. Based on its presumed role in the Hop/Stat92E signal transduction pathway, this novel gene was named master of marelle (mom). The relative strength of four mom alleles in suppressing the UAS-Upd/GMR-Gal4 fly big eye phenotype is mom¹ > mom² >  mom³ = mom⁴, and mom¹ is the strongest allele. mom is indeed the same gene as domeless (Chen, 2002).

Tests were performed to see whether dome and STAT92E interact genetically. Zygotic STAT92E homozygotes have a very mild spiracle phenotype due to the persistence of maternally expressed RNA. Despite this, the weak dome³⁶⁷ phenotype is strongly enhanced by STAT92E mutants, suggesting that both genes are in the same genetic pathway (Brown, 2001).

To determine the phenotype caused by eliminating domeless maternal and zygotic products, germ line clones were induced. Maternal and zygotic dome embryos have segmentation defects identical to those reported for mutations in the STAT92E and hop (JAK) mutants. Defects include the deletion of the A5 and most of the A4 denticle belts, partial or total fusion of A6 to A7, and a variable reduction of the thoracic and the A8 segments. These phenotypes are also observed in Df(1)osUE69, which deletes the ligand, upd. The segmentation defects in STAT92E, upd, and hop have been shown to be due to the abnormal expression of pair rule genes. In dome germ line clones, the expression of even-skipped is affected in stripes 3 and 5, as described for the other members of this pathway (Brown, 2001).

To further investigate whether dome has the genetic characteristics expected of the JAK/STAT receptor, dome interactions with upd, the known JAK/STAT ligand, were tested. To do this, advantage was taken of the fact that when the h-GAL4 line is used for ectopic expression of upd in the embryo, the result is abnormal head formation in 81% of the embryos. When upd is expressed ectopically in dome zygotic mutant embryos, this proportion is reduced to 16%. This result is consistent with dome being necessary to transduce the upd signal (Brown, 2001).

To find out if the intracellular domain of Dome is required for its function, UAS constructs were made in which the putative intracellular domain was deleted. One construct, UAS-dome^DeltaCYT, contains the extracellular and transmembrane portion of the protein and should be membrane bound. The other, UAS-dome^DeltaTMCYT, contains only the extracellular part and might be secreted. Neither of these proteins is able to rescue the dome zygotic phenotype, proving that the Dome intracellular domain is required for signal transduction. Because both forms still contain the cytokine binding domain, they must have the potential to titrate the ligand and act as signaling antagonists. This has been shown to be the case for the soluble form of gp130, the signal-transducing subunit of many vertebrate cytokine class I receptors. Consistent with this, when a maternal GAL4 was used for expression of UAS-dome^DeltaTMCYT or UAS-dome^DeltaCYT at early stages of development, approximately 50% of the larvae acquired segmentation defects. The most frequent defects were deletions and fusions of A4 and A5 segments, the segments more sensitive to loss of JAK/STAT function, but stronger defects were also observed. These phenotypes are increased if the mothers are also heterozygous for a hop allele, further proving the central role of dome in JAK/STAT signaling (Brown, 2001).

mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family

In mammals, the gp130 subfamily of receptors includes gp130, leukemia inhibitory factor receptor (LIFR), oncostatin M receptor (OSMR), ciliary neurotrophic factor receptor (CTNFR), cardiotrophin-1 receptor (CT-1R), granulocyte colony-stimulating factor receptor (G-CSFR), and the obesity/leptin gene receptor (OBR). The functional gp130 subfamily of receptors generally comprises multiple components of membrane proteins, and these multichain receptor complexes often share the common signal gp130 as a component critical for signal transduction (Hirano, 1998). Cytokines in the IL-6 family initiate intracellular signaling by binding to a specific ligand-binding subunit and then subsequently form a complex with the signal-transducing receptor component, gp130. IL-6 binds to IL-6R, and the IL-6/IL-6R complex then associates with gp130, allowing it to homodimerize. However, OSMR and G-CSFR are unique among the gp130 subfamily of receptors. In the case of OSMR, OSM binds directly to gp130 with low affinity. Upon binding by OSM, gp130 generates a high-affinity receptor complex by forming a heterodimer with either the LIFR- or the OSM-specific receptor subunit (LIFRß and OSMRß, respectively). The G-CSFR shows high homology with gp130; G-CSFR forms homodimers by itself in response to G-CSF (Chen, 2002 and references therein).

Upd has a very limited similarity to the IL-6 family of cytokines. It is difficult to predict whether Upd signaling is more like IL-6 or G-CSF. If it is more like IL-6, there should be another receptor subunit that would be functionally similar to IL-6R. Upd may first bind to the missing receptor and then form a functional complex with Mom. On the other hand, if Upd is more like G-CSF, Mom may be the only receptor. Upd binding should promote Mom to form a homodimer and start the signal transduction process. However, Upd may interact with its receptor in a different way. Upd structure is less consistent with a cytokine-type molecule. The Upd protein is extremely basic, with a predicted pI of nearly 12. In contrast to many soluble cytokines, Upd is associated with ECM (extracellular matrix), which may help it bind to the receptor and limit the range of activity of the ligand (Chen, 2002).

The gp130 subfamily of receptors has no intrinsic tyrosine kinase domain, but constitutively associates with tyrosine kinase JAKs. The family members possess conserved motifs in the cytoplasmic region, such as box 1, box 2, and box 3 in the membrane proximal-to-distal order. These conserved boxes are important for receptor-mediated signal transduction (Hirano, 1998). The box 1 motif in the cytoplasmic region of gp130 is important for the association with JAK kinases; the box 3 motif provides a docking site for the SH2 domain of STAT3 protein, which recruited to gp130 now serves as a substrate for JAKs. The activation of STAT3 is dependent on the phosphorylation of any one of the four tyrosines (Y767, Y814, Y905, Y915) in the C terminus of the gp130 that have a glutamine residue at the third position behind tyrosine (Y-X-X-Q). Mom has no visible box 1 and 2 motifs but has one tyrosine residue (Y966) fitting a YXXQ consensus motif. Mom binds Hop and Stat92E in biochemical experiments (Chen, 2002).

In addition to the JAK/STAT pathway, multiple signaling molecules are tyrosine-phosphorylated in response to the IL-6 family of cytokines. CNTF, LIF, OSM, and IL-6 induce tyrosine phosphorylation of phospholipase Cgamma and SHP-2 (a phosphotyrosine phosphatase, also called PTP1-D, SHPTP-2, PTP2C, and Syp), which is a mammalian homolog of Drosophila corkscrew (csw). The Ras-MAPK pathway is activated by the IL-6 cytokine family. The activation of the Ras-MAPK pathway is possibly mediated by SHP-2 and/or Shc, which bind a Grb2-SOS complex (Hirano, 1998). Tyrosine 759 of gp130 is required for the tyrosine phosphorylation of SHP-2 and its binding to gp130, the association between SHP-2 and Grb2, and the full activation of MAPK (Chen, 2002).

In flies, the phenotypes caused by loss of function of the Hop/Stat92E pathway and of the Ras-MAPK pathway are very distinct. In the embryo, loss-of-function mutations in the Hop/Stat92E pathway cause segmentation defects. The absence of additional phenotypes, such as terminal defects or poorly differentiated cuticle in germ-line clone-derived embryos, indicates that this pathway does not cross-talk with the Ras-MAPK pathway in either the Torso or Egfr RTK signaling pathways. In the eye, the Ras-MAPK pathway regulates photoreceptor formation. Changing the activity of the Hop/Stat92E pathway does not affect photoreceptor fate rather than affect cell proliferation (Chen, 2002).

However, there are some similarities in phenotypes between the Hop/Stat92E pathway and the Drosophila Ras-MAPK pathway. For example, overexpression of hop in the wing disc results in vein phenotypes similar to those seen in animals that express activated forms of D-raf. Loss-of-function D-raf mutations result in larval/pupal lethality with underproliferation of diploid tissues, similar to hop mutations. However, these similarities are based on overexpression phenotypes as well as most of the mammalian evidence for cross-talk. These results should be taken with caution just in case an artifactual phenomenon was created by these overexpressions (Chen, 2002).

The mammalian gp130 subfamily of receptors interacts with the receptors' respective ligands and has pleiotropic functions. G-CSF is the principal hematopoietic growth factor regulating the production of neutrophils, and it is widely used to treat neutropenia in a variety of clinical settings. The mouse obesity gene encodes a soluble protein (OB or leptin) that produces weight-reducing effects in mice when administrated in vivo. The structure of the receptor for this factor (OBR or leptin-R) is highly homologous to that of gp130. The IL-6 family of cytokines shares gp130 as a component critical for signal transduction in the cytokine receptor complexes and has redundant functions. Some of these biological activities of IL-6 are also often exerted by other cytokines, namely, IL-11, LIF, OSM, CNTF, and CT-1. The biological activity by which each cytokine was initially identified was growth promotion of myeloma and plasmacytoma for IL-11, growth inhibition of a mouse myeloid leukemia cell line for LIF, growth inhibition of a human melanoma cell line for OSM, promotion of survival of rat-cultured ciliary neurons for CNTF, and induction of cardiac hypertrophy in vitro for CT-1. These cytokines show structural similarity and, more importantly, have biological functions that overlap with those of IL-6 (Chen, 2002).

Mom is the only member of this family of receptors so far identified in Drosophila. The fly has also only one JAK (Hop) and one STAT (Stat92E). It remains to be seen whether this simple Mom/Hop/Stat92E pathway has the pleiotropic functions that are accomplished by a great complex of a homologous receptor family. This simple genetic model system will greatly enhance understanding of the gp130 subfamily of receptors' biological functions and mediated signal transductions (Chen, 2002).

Cytokine signaling through the JAK/STAT pathway is required for long-term memory in Drosophila

Cytokine signaling through the JAK/STAT pathway regulates multiple cellular responses, including cell survival, differentiation, and motility. Although significant attention has been focused on the role of cytokines during inflammation and immunity, it has become clear that they are also implicated in normal brain function. However, because of the large number of different genes encoding cytokines and their receptors in mammals, the precise role of cytokines in brain physiology has been difficult to decipher. This study took advantage of Drosophila's being a genetically simpler model system to address the function of cytokines in memory formation. Expression analysis showed that the cytokine Upd is enriched in the Drosophila memory center, the mushroom bodies. Using tissue- and adult-specific expression of RNAi and dominant-negative proteins, it was shown that not only is Upd specifically required in the mushroom bodies for olfactory aversive long-term memory but the Upd receptor Dome, as well as the Drosophila JAK and STAT homologs Hop and Stat92E, are also required, while being dispensable for less stable memory forms (Copf, 2011).

Using the Drosophila olfactory aversive learning paradigm in combination with a conditional tissue-specific expression system, this study has shown that cytokine signaling through the JAK/STAT pathway is necessary for protein synthesis-dependent LTM but is dispensable for less stable forms of memory. All four major components of this pathway -- the extracellular cytokine Upd, the cytokine receptor Dome, the tyrosine kinase Hop, and the transcription factor Stat92E -- are required within the MBs, the major olfactory learning and memory center for LTM processing (Copf, 2011).

Although cytokine signaling may be required for normal health and physiology of the MBs, this hypothesis is not favored because neither learning nor ARM formation are affected when this signaling pathway is compromised. Rather, it is suggested that the JAK/STAT pathway is specifically recruited for LTM processing. The requirement for de novo gene expression during LTM formation has been widely observed in a number of different model systems. Much attention has been focused on the role of transcription factor cAMP response element-binding protein (CREB) as an LTM-specific regulator of gene expression in Drosophila and other species. A number of other transcription factors have also been found to play an important role in LTM, including Adf-1 in Drosophila and CCAAT/enhancer-binding protein (C/EBP), Zif-268, AP-1, and NF-κB in mammals. Although the JAK/STAT pathway has been shown to be involved in diverse biological processes in flies, this study identifies a role in Drosophila adult brain physiology and behavioral plasticity. In addition, despite the plethora of studies examining the impact of cytokines in memory formation, the experiments presented in this study demonstrate that JAK/STAT signaling contributes to the transcriptional regulation thought to underlie synaptic plasticity and long-lasting memory (Copf, 2011).

To understand how Stat92E modulates memory, it will be necessary to identify its transcriptional targets in the adult MBs. Identification of such target genes could be approached by using bioinformatics and/or transcription profiling. Recent profiling studies have identified a number of putative Stat92E target genes in the Drosophila eye disk and hematopoietic system, some of which include Notch signaling pathway genes that have already been implicated in LTM. Another mode of action of JAK/STAT signaling in LTM could be through chromatin remodeling. Recent findings have identified a noncanonical mode of JAK/STAT signaling that directly regulates heterochromatin stability and cellular epigenetic status, affecting expression of genes beyond those under direct Stat92E transcriptional control. Finally, given that regulation of the actin cytoskeleton is central to both cell motility and neuronal structural plasticity, it will be interesting to determine whether some of the mechanisms by which JAK/STAT signaling drives border cell migration in the Drosophila germ line are also relevant to the formation of stable memories in the MBs (Copf, 2011).

These experiments demonstrate a clear positive role for signaling by the cytokine Upd in olfactory aversive memory, and, in doing so, they contribute to a lively debate as to the role played by cytokines in memory. Mammalian studies in which levels of proinflammatory cytokines are increased to pathogenic levels, either through direct injection or indirectly via induction of inflammation through injection of lipopolysaccharide or bacteria, tend to suggest that augmented cytokine signaling is detrimental for performance in a variety of learning and memory assays. This negative impact of cytokine signaling on memory is supported by studies that take a loss-of-function approach to address the physiological function of interleukins and their receptors in different cognitive tasks under nonpathological conditions. In contrast, several studies describe learning and memory defects attributed to loss of function of other cytokines or their receptors, using a variety of behavioral assays. Thus, despite significant efforts, understanding of the molecular and cellular basis for interactions between the cytokine network and learning and memory remains limited. The complexity of mammalian cytokine signaling, with its vast array of genes encoding ligands, receptors and downstream regulators, and the substantial degree of crosstalk between pathways, ensures that this task remains an enormous challenge. By using Drosophila, a simplified model system encoding single JAK and STAT genes, this study now shows that signaling through a cytokine-regulated JAK/STAT pathway is critical for LTM. In contrast to the mammalian gene-disruption studies, this study has been able to rule out the possibility that the observed memory impairments are attributable to defects in development because targeting of gene expression in this study was limited to adult flies. The crucial role of JAK/STAT signaling in memory, if conserved in vertebrates, may explain why inappropriate up-regulation of the pathway appears to disrupt memory, thus shedding light on the large number of diseases in which neuroinflammation is thought to drive pathogenesis (Copf, 2011).

REGULATION

The endocytic control of JAK/STAT signalling in Drosophila

Domeless (Dome) is an IL-6-related cytokine receptor that activates a conserved JAK/STAT signalling pathway during Drosophila development. Despite good knowledge of the signal transduction pathway in several models, the role of receptor endocytosis in JAK/STAT activation remains poorly understood. Using both in vivo genetic analysis and cell culture assays, it was shown that ligand binding of Unpaired 1 (Upd1) induces clathrin-dependent endocytosis of receptor-ligand complexes and their subsequent trafficking through the endosomal compartment towards the lysosome. Surprisingly, blocking trafficking in distinct endosomal compartments using mutants affecting either Clathrin heavy chain, Rab5, Hrs or deep orange led to an inhibition of the JAK/STAT pathway, whereas this pathway was unchanged when rab11 was affected. This suggests that internalization and trafficking are both required for JAK/STAT activity. The requirement for clathrin-dependent endocytosis to activate JAK/STAT signalling suggests a model in which the signalling 'on' state relies not only on ligand binding to the receptor at the cell surface, but also on the recruitment of the complex into endocytic vesicles on their way to lysozomes. Selective activation of the pool of receptors marked for degradation thus provides a way to tightly control JAK/STAT activity (Devergne, 2007).

Using genetic analysis this study shows that several regulators of the endocytic pathway are required for normal JAK/STAT signalling in vivo. The membrane-bound Dome receptors undergo ligand-dependent internalization in clathrin-coated vesicles, which are then targeted to the sorting endosome via Rab5. The function of Hrs is required for JAK/STAT activation and to direct most of the active receptors to the MVBs, targeting them to the lysosome for degradation (Devergne, 2007).

One important question is to know whether the trafficking of ligand-bound receptors has any effect on signalling. This question was addressed by looking at Stat nuclear localization, which represents a robust readout to assess JAK/STAT activity, and at pnt-lacZ expression (Devergne, 2007).

The effect of Hrs is opposite on the JAK/STAT pathway compared with its effect on other pathways. Indeed, in the egg chamber, Hrs plays a positive role on JAK/STAT activity, whereas it has been shown to downregulate the EGFR, Notch and TGF-β pathways in the same tissue. Interestingly, HRS has been shown to interact with STAM in the same mono-ubiquitylated recognition complex (Lohi, 2001). STAM is a known JAK/STAT activator (Pandey, 2000), suggesting that HRS could control STAT signalling through its interaction with STAM. So, Hrs could play two crucial roles: first, allowing the sequestration and the sorting of the receptor to the lysosome and, second, activating the ligand-receptor complex in collaboration with STAM (Devergne, 2007).

The data challenge the simple view whereby binding of the ligand to the receptor at the membrane would be sufficient to activate the pathway. Indeed, it was found that equally essential is the need of clathrin for the activation of JAK/STAT signalling. Thus, activation can occur only when the ligand-receptor complex is assembled into clathrin-coated vesicles. In this view, activation would proceed in two steps, requiring both binding of the ligand and interaction with clathrin. The role of clathrin could be to concentrate/cluster receptors and/or bring them together with other signal transducers in the endosomal compartment. This finding is in agreement with a recent work showing that, in mammals, clathrin is required to transduce JAK/STAT signals through the IFNα-receptor, but not the IFNγ-receptor, suggesting a conserved function for clathrin in JAK/STAT signalling (Marchetti, 2006). Interestingly, like in mammals, JAK/STAT signalling in Drosophila might be controlled in a cell-type-specific manner by Chc-dependent endocytosis. Indeed, in Drosophila eyes, Vps25 and TSG101 mutations lead to Upd upregulation and JAK/STAT activation in a Notch-dependent manner (Devergne, 2007).

What is the significance of clathrin function and, more generally, of the requirement for internalization, in JAK/STAT signalling? It has been shown for several signalling pathways that internalization brings together membrane receptors and intracellular pathway components in the endosomal compartment, which thus serves as a platform for signalling. The fact that Dome internalization and activation are coupled to degradation has important consequences. Making signalling complexes only active in the endosomal compartment is a powerful mechanism to control the number of active complexes in the cell. Their targeting to the lysosome allows the control of their lifetime as active receptors, providing a temporal -- hence quantitative -- control on signalling (Devergne, 2007).

Activation of JAK/STAT follows an off/on/off model in which two conditions are required for correct JAK/STAT activation: (1) formation of a ligand-receptor complex (as proposed in the classical model), followed by (2) the internalization of the complex via Chc-containing budding vesicles. The sole formation of the ligand-receptor interaction would lead to an inactive complex (off). However, interaction with Chc and subsequent internalization activate the complex (on), thus ensuring that only the complexes targeted for degradation are activated. Arrival of the complex in the MVB/lysosome turns it into the off state (Devergne, 2007).

Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut

Cells in intestinal epithelia turn over rapidly due to damage from digestion and toxins produced by the enteric microbiota. Gut homeostasis is maintained by intestinal stem cells (ISCs) that divide to replenish the intestinal epithelium, but little is known about how ISC division and differentiation are coordinated with epithelial cell loss. This study shows that when enterocytes (ECs) in the Drosophila midgut are subjected to apoptosis, enteric infection, or JNK-mediated stress signaling, they produce cytokines (Upd, Upd2, and Upd3) that activate Jak/Stat signaling in ISCs, promoting their rapid division. Upd/Jak/Stat activity also promotes progenitor cell differentiation, in part by stimulating Delta/Notch signaling, and is required for differentiation in both normal and regenerating midguts. Hence, cytokine-mediated feedback enables stem cells to replace spent progeny as they are lost, thereby establishing gut homeostasis (Jiang, 2009).

Rates of cell turnover in the intestine are likely to be in constant flux in response to varying stress from digestive acids and enzymes, chemical and mechanical damage, and toxins produced by both commensal and infectious enteric microbiota. This study shows feedback from differentiated cells in the gut epithelium to stem and progenitor cells is a key feature of this system. Genetically directed enterocyte ablation, JNK-mediated stress signaling, or enteric infection with P. entomophila all disrupt the Drosophila midgut epithelium and induce compensatory ISC division and differentiation, allowing a compromised intestine to rapidly regenerate. Other recent reports note a similar regenerative response following three additional types of stress: detergent (DSS)-induced damage (Amcheslavsky, 2009), oxidative stress by paraquat (Biteau, 2008), and enteric infection with another less pathogenic bacterium, Erwinia carotovora (Buchon, 2009). Remarkably, the fly midgut can recover not only from damage, but also from severe induced hyperplasia, such as that caused by ectopic cytokine (Upd) production. Thus, this system is robustly homeostatic (Jiang, 2009).

Each of the three stress conditions that were studied induced all three Upd cytokines, and genetic tests showed that Upd/Jak/Stat signaling was both required and sufficient for compensatory ISC division and gut renewal. Although JNK signaling was also activated in each instance, it was not required for the stem cell response to either EC apoptosis or infection, implying that other mechanisms can sense EC loss and trigger the cytokine and proliferative responses. JNK signaling may be important in specific contexts that were not tested, such as following oxidative stress, which occurs during some infections, activates JNK, and stimulates midgut DNA replication (Biteau, 2008; Jiang, 2009).

Following P. entomophila infection, virtually the entire midgut epithelium could be renewed in just 2-3 days, whereas comparable renewal took more than 3 weeks in healthy flies. Despite this radical acceleration of cell turnover, the relative proportions of the different gut cell types generated (ISC, EB, EE, and EC remained similar to those in midguts undergoing slow, basal turnover. These data suggested that de-differentiation did not occur, and little evidence was obtained of symmetric stem divisions (stem cell duplication) induced by enteric infection. Hence, it is suggested that asymmetric stem cell divisions as described for healthy animals, together with normal Delta/Notch-mediated differentiation, remain the rule during infection-induced regeneration. The results obtained using Reaper to ablate ECs are also consistent with this conclusion, as are those from detergent-induced midgut regeneration (Jiang, 2009).

Unlike infection, direct genetic activation of JNK or Jak/Stat signaling promoted large increases not only in midgut mitoses, but also in the pool of cells expressing the stem cell marker Delta. Cell type marker analysis discounted de-differentiation of EEs or ECs as the source of the new stem cells, but the reactivation of EBs as stem cells seems possible. For technical reasons, no tests were performed to whether stem cell duplications occur in response to Jak/Stat or JNK signaling, and this also remains possible. The ability of hyperplastic midguts to recover to normal following the silencing of cytokine expression suggests that excess stem cells are just as readily eliminated as they are generated. Further studies are required to understand how midgut stem cell pools can be expanded and contracted according to need (Jiang, 2009).

How the Upds are induced in the midgut by JNK, apoptosis, or infection remains an open question. Paradoxically, ISC divisions triggered by Reaper required EC apoptosis but not JNK activity, whereas ISC divisions triggered by JNK did not require apoptosis, and ISC divisions triggered by infection required neither apoptosis nor JNK activity. These incongruent results suggest that different varieties of gut epithelial stress may induce Upd cytokine expression via distinct mechanisms. In the case of EC ablation, physical loss of cells from the epithelium might drive the cytokine response. In the case of infection, it is expected the critical inputs to be the Toll and/or IMD innate immunity pathways, which signal via NF-kappaB transcription factors. Functional tests, however, indicated that the Toll and IMD pathways are required for neither Upd/Jak/Stat induction nor compensatory ISC mitoses following enteric infection by gram-negative bacteria. Hence, other unknown inputs likely trigger the Upd cytokine response to infection (Jiang, 2009).

Is the cytokine response to infection relevant to normal midgut homeostasis? This seems likely. Low levels of Upd3 expression and Stat signaling are observed in healthy animals, and midgut homeostasis required the IL-6R-like receptor Dome and Stat92E even without infection. Wild Drosophila subsist on a diet of rotting fruit, which is a good source of protein because it is teeming with bacteria and fungi. Given such a diet it seems likely that midgut cytokine signaling is constantly modulated by ever-present factors that impose dietary stress -- food composition and commensal microbiota -- even in healthy animals (Jiang, 2009).

Although studies in mammals have yet to unravel the details of a feedback mechanism underlying gut homeostasis, experimental evidence implies that such a mechanism exists and involves Cytokine/Jak/Stat signaling. As in Drosophila, damage to the mouse intestinal epithelium caused by detergents or infection can stimulate cell proliferation in the crypts, where stem and transient amplifying cells reside. In a mouse model of detergent (DSS)-induced colitis, colon epithelial damage caused by DSS allows exposure to commensal microbes, activating NF-kappaB signaling in resident macrophage-like Dentritic cells. These cells respond by expressing inflammation-associated cytokines, one of which (IL-6) activates Stat3 and is believed to promote cell proliferation and regeneration. Consistent with a functional role for Jak/Stat, disruption of the Stat inhibitor SOCS3 in the mouse gut increased the proliferative response to DSS and also increased DSS-associated colon tumorigenesis. Also pertinent is the presence of high levels of phospho-Stat3 in a majority of colon cancers, where it correlates with adverse outcome, and the observation that IL-6 can promote the growth of colon cancer cells, which are thought to derive from ISCs or transient amplifying cells. Increased colon cancer incidence is associated with gut inflammatory syndromes, such as inflammatory bowel disease (IBD) and Crohn's disease, which are likely to involve enhanced cytokine signaling. Whether cytokines mediate gut epithelial turnover in healthy people or only during inflammation is presently unclear, but it nevertheless seems likely that the mitogenic role of IL-6-like cytokines and Jak/Stat signaling in the intestine is conserved from insects to humans (Jiang, 2009).

The connection to inflammation suggests that these findings may also be relevant to the activity of nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, and celecoxib, as suppressors of colorectal carcinogenesis. These drugs target the cyclooxygenase activity of prostaglandin H synthases (PGHS, COX), which are rate-limiting for production of prostaglandin E2, a short-range lipid signal that promotes inflammation, wound healing, cell invasion, angiogenesis, and proliferation. Notably, COX-2 has been characterized as an immediate early gene that can be induced by signals associated with infection and inflammation, including the proinflammatory cytokines IL-1beta and IL-6, which activate NF-kappaB and STAT3, respectively. Whether prostaglandins mediate the effects of Jak/Stat signaling in the fly midgut remains to be tested, but insects do produce prostaglandins, and Drosophila has a functional COX homolog, pxt, whose activity can be suppressed by NSAIDs (Jiang, 2009).

DEVELOPMENTAL BIOLOGY

Embryonic

RNA in situ hybridization shows that, similar to hop (JAK) and STAT92E, dome is expressed maternally. At later stages dome expression appears ubiquitous, although there is some variation in the levels of expression. At stage 11 the tracheal pits show more intense expression, whereas at stage 14, higher expression is detected in the posterior spiracles, gut, and head (Brown, 2001).

Domeless dimerization is developmentally regulated

It is commonly accepted that activation of most signalling pathways is induced by ligand receptor dimerization. This belief has been challenged for some vertebrate cytokine receptors of the JAK/STAT pathway. This study addresses the question of whether DOME, the Drosophila receptor of the JAK/STAT pathway, can dimerize and whether the dimerization is ligand-dependent. To analyze DOME homo-dimerization, a ß-gal complementation technique was applied that allows the detection of protein interactions in situ. This technique has been used in cell culture but this is the first time that it has been applied to whole embryos. This technique, which has been rename ßlue-ßlau technique, can be used to detect DOME homo-dimerization in Drosophila developing embryos. Despite DOME being ubiquitously expressed, dimerization is developmentally regulated. The state of DOME dimerization was investigated in the presence or absence of ligand; DOME dimerization is not ligand-induced, indicating that ligand independent cytokine receptor dimerization is a conserved feature across phyla. The functional significance of ligand-independent receptor dimerization was further analyzed by comparing the effects of ectopic ligand expression in cells in which the receptor is, or is not, dimerized. Ligand expression can only activate STAT downstream targets or affect embryo development in cells in which the receptor is dimerized. These results suggest a model in which ligand-independent dimerization of the JAK/STAT receptor confers cells with competence to activate the pathway prior to ligand reception. Thus, competence to induce the JAK/STAT signalling pathway in Drosophila can be regulated by controlling receptor dimerization prior to ligand binding. These results reveal a novel level of JAK/STAT signalling regulation that could also apply to vertebrates (Brown, 2003).

The ßlue-ßlau ß-gal complementation technique allows the detection of protein-protein interactions. This technique uses two ß-gal mutants (Deltaalpha and Deltaomega) that are enzymatically inactive, but can complement if brought into proximity by fusing them to proteins that physically interact. Dimerization of the hybrid proteins does not depend on the ß-gal portion of the hybrid protein, but is directed by the proteins that have been fused to the ß-gal mutants. The Deltaalpha or the Deltaomega mutants were fused to the DOME carboxyl end and the hybrid receptors were expressed using the GAL4 system. The hybrid receptors are functional since they can rescue dome mutant phenotypes. To determine the sub-cellular localization of the fusion proteins, they were expressed in the large, polarized, ectodermal cells of the hindgut and the salivary gland using the h-Gal4 line. The hybrid receptors localize mainly to the apical membrane, although some protein can be detected in the cytoplasm (Brown, 2003).

Two different hypotheses have been proposed for the induction of dimerisation in vertebrates. The current results are not consistent with the view that JAK/STAT receptors become activated by ligand-induced dimerization, but suggest that the receptors dimerize prior to receptor activation. The patterned expression of X-gal shows that pre-dimerization is a developmentally regulated process that occurs mainly, but not exclusively, in cells that activate the pathway. Previous reports with the IL-2 alpha, ß and gamma subunits, the growth hormone and the erythropoietin receptors have presented evidence for preformed receptor complexes using three different techniques: FRET, immunoprecipitation and crystallography. Crystallographic evidence indicates that the erythropoietin receptor can dimerize in the absence of ligand. In this case, only the extracellular domains of the receptor are apposed. Ligand binding changes the extracellular conformation and, as a result, the intracellular domains come together allowing JAK phosphorylation. Although these results suggest that ligand binding induces an extracellular conformational change in the pre-associated receptor complex allowing receptor activation, they do not provide information about when and where pre-association occurs or whether it has a physiological role. The experiments described in this study allow visualization of receptor pre-association in situ and suggest that pre-association is essential for functionality (Brown, 2003 and references therein).

Ligand expression is not capable of inducing dimerization, and receptor dimerization does not require ligand expression. Since activation of the JAK/STAT pathway requires receptor dimerization, the discovery that DOME homo-dimerization is developmentally controlled reveals that there is yet another level of regulation within the JAK/STAT pathway. At least in Drosophila, in which all the effectors of the JAK/STAT signalling pathway are ubiquitously expressed in the embryo, not all cells are activated by ectopic ligand. Although other models are possible, based on the data it is proposed that signalling-competent cells are those that have the pre-associated receptor complex and it is only these cells that are able to respond to the extracellular signal (Brown, 2003).

Pre-association could be mediated by unknown ligands, membrane-spanning proteins or by intracellular proteins. Given that a deficiency deleting upd and three other homologous genes has an identical phenotype in the ectoderm to the inactivation of dome, stat92E or hop, it is unlikely that there are any other ectodermal ligands. This strongly indicates that other proteins play a role in the formation of the receptor pre-associated complexes. Recent results show that cytoplasmic scaffold proteins are fundamental for specificity of the signaling responses. It is proposed that cytoplasmic 'clamp' proteins expressed in a developmentally regulated pattern are responsible for cytokine receptor pre-association, and thus for deciding the competence of a cell to respond to a particular ligand (Brown, 2003).

It could be argued that the areas in which dimerization was observed are those where higher levels of hybrid protein are expressed. The experiments presented discard this possibility because not all strongly expressing areas lead to dimerization. For example, the head expresses high levels of hybrid protein but there is no dimerization in this area and the same can be said for the head and segmental grooves. Moreover, lines of very different origin give consistent results regardless of them being strong (da-Gal4) or weak ectodermal inducers (h-Gal4). The lack of correlation between high levels of hybrid protein expression and dimerization is further confirmed by the absence of dimerization when using strong mesodermal drivers. Finally, increasing the levels of expression by simultaneously expressing with 69B-Gal4 and da-Gal4 does not result in novel areas of dimerization (Brown, 2003).

The Drosophila lymph gland as a developmental model of hematopoiesis

Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from their precursors are defined. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis (Jung, 2005).

In the late embryo, the lymph gland consists of a single pair of lobes containing ~20 cells each. These express the transcription factors Srp and Odd skipped (Odd), and each cluster of hemocyte precursors is followed by a string of Odd-expressing pericardial cells that are proposed to have nephrocyte function. These lymph gland lobes are arranged bilaterally such that they flank the dorsal vessel, the simple aorta/heart tube of the open circulatory system, at the midline. By the second larval instar, lymph gland morphology is distinctly different in that two or three new pairs of posterior lobes have formed and the primary lobes have increased in size approximately tenfold (to ~200 cells. By the late third instar, the lymph gland has grown significantly in size (approximately another tenfold) but the arrangement of the lobes and pericardial cells has remained the same. The cells of the third instar lymph gland continue to express Srp (Jung, 2005).

The third instar lymph gland also exhibits a strong, branching network of extracellular matrix (ECM) throughout the primary lobe. This network was visualized using several GFP-trap lines in which GFP is fused to endogenous proteins. For example, line G454 represents an insertion into the viking locus, which encodes a Collagen IV component of the extracellular matrix. The hemocytes in the primary lobes of G454 (expressing Viking-GFP) appear to be clustered into small populations within pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as the uncharacterized GFP-trap line ZCL2867, also highlight this branching pattern. What role this intricate ECM network plays in hematopoiesis, as well as why multiple cells cluster within these ECM chambers, remains to be determined (Jung, 2005).

Careful examination of dissected, late third-instar lymph glands by differential interference contrast (DIC) microscopy revealed the presence of two structurally distinct regions within the primary lymph gland lobes that have not been previously described. The periphery of the primary lobe generally exhibits a granular appearance, whereas the medial region looks smooth and compact. These characteristics were examined further with confocal microscopy using a GFP-trap line G147, in which GFP is fused to a microtubule-associated protein. The G147 line is expressed throughout the lymph gland but, in contrast to nuclear markers such as Srp and Odd, distinguishes morphological differences among cells because the GFP-fusion protein is expressed in the cytoplasm in association with the microtubule network. Cells in the periphery of the lymph gland make relatively few cell-cell contacts, thereby giving rise to gaps and voids among the cells within this region. This cellular individualization is consistent with the granularity of the peripheral region observed by DIC microscopy. By contrast, cells in the medial region were relatively compact with minimal intercellular space, which is also consistent with the smoother appearance of this region by DIC microscopy. Thus, in the late third instar, the lymph gland primary lobes consist of two physically distinct regions: a medial region consisting of compactly arranged cells, which was termed the medullary zone; and a peripheral region of loosely arranged cells, termed the cortical zone (Jung, 2005).

Mature hemocytes have been shown to express several markers, including collagens, Hemolectin, Lozenge, Peroxidasin and P1 antigen. The expression of the reporter Collagen-gal4 (Cg-gal4), which is expressed by both plasmatocytes and crystal cells, is restricted to the periphery of the primary lymph gland lobe. Comparison of Cg-gal4 expression in G147 lymph glands, in which the medullary zone and cortical zone can be distinguished, reveals that maturing hemocytes are restricted to the cortical zone. In fact, the expression of each of the maturation markers mentioned above is found to be restricted to the cortical zone. The reporter hml-gal4 and Pxn, which are expressed by the plasmatocyte and crystal cell lineages, are extensively expressed in this region. Likewise, the expression of the crystal cell lineage marker Lozenge is restricted in this manner. The spatial restriction of maturing crystal cells to the cortical zone was verified by several means, including the distribution of melanized lymph gland crystal cells in the Black cells background and analysis of the terminal marker ProPOA1. The cortical zone is also the site of P1 antigen expression, a marker of the plasmatocyte lineage. The uncharacterized GFP fusion line ZCL2826 also exhibits preferential expression in the cortical zone. Last, it was found that the homeobox transcription factor Cut is preferentially expressed in the cortical zone of the primary lobe. Although the role of Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut are known to be regulators of the myeloid hematopoietic lineage in both mice and humans. Cells of the rare third cell type, lamellocytes, are also restricted to the cortical zone, based upon cell morphology and the expression of a msn-lacZ reporter (msn⁰⁶⁹⁴⁶). In summary, based on the expression patterns of several genetic markers that identify the three major blood cell lineages, it is proposed that the cortical zone is a specific site for hemocyte maturation (Jung, 2005).

The medullary zone was initially defined by structural characteristics and subsequently by the lack of expression of mature hemocyte markers. However, several markers have been identified that are exclusively expressed in the medullary zone at high levels but not the cortical zone. Consistent with the compact arrangement of cells in the medullary zone, it was found that Drosophila E-cadherin (DE-cadherin or Shotgun) is highly expressed in this region. No significant expression of DE-cadherin was observed among maturing cells in the cortical zone. E-cadherin, in both vertebrates and Drosophila, is a Ca²⁺-dependent, homotypic adhesion molecule often expressed by epithelial cells and is a crucial component of adherens junctions. Attempts to study DE-cadherin mutant clones in the medullary zone where the protein is expressed were unsuccessful since no clones were recoverable. The reporter lines domeless-gal4 and unpaired3-gal4 are preferentially expressed in the medullary zone. The gene domeless (dome) encodes a receptor molecule known to mediate the activation of the JAK/STAT pathway upon binding of the ligand Unpaired. The unpaired3 (upd3) gene encodes a protein with homology to Unpaired and has been associated with innate immune function. These gal4 lines are in this study only as markers that correlate with the medullary zone and, at the present time, there is no evidence that their associated proteins have a role in lymph gland hematopoiesis. Other markers of interest with preferential expression in the medullary zone include the molecularly uncharacterized GFP-trap line ZCL2897 and actin5C-GFP. Cells expressing hemocyte maturation markers are not seen in the medullary zone. It is therefore reasonable to propose that this zone is largely populated by prohemocytes that will later mature in the cortical zone. Prohemocytes are characterized by their lack of maturation markers, as well as their expression of several markers described as expressed in the medullary zone (Jung, 2005).

The posterior signaling center (PSC), a small cluster of cells at the posterior tip of each of the primary (anterior-most) lymph gland lobes, is defined by its expression of the Notch ligand Serrate and the transcription factor Collier. During this analysis, several additional markers were identified that exhibit specific or preferential expression in the PSC region. For example, it was found that the reporter Dorothy-gal4 is strongly expressed in this zone. The Dorothy gene encodes a UDP-glycosyltransferase, which belongs to a class of enzymes that function in the detoxification of metabolites. The upd3-gal4 reporter, which has preferential expression in the medullary zone, is also strongly expressed among cells of the PSC. Last, three uncharacterized GFP-gene trap lines, ZCL2375, ZCL2856 and ZCL0611 were found, that are preferentially expressed in the PSC. This analysis has made it clear that the PSC is a distinct zone of cells that can be defined by the expression of multiple gene products (Jung, 2005).

The PSC can be defined just as definitively by the characteristic absence of several markers. For example, the RTK receptor Pvr, which is expressed throughout the lymph gland, is notably absent from the PSC. Likewise, dome-gal4 is not expressed in the PSC, further suggesting that this population of cells is biased toward the production of ligands rather than receptor proteins. Maturation markers such as Cg-gal4, which are expressed throughout the cortical zone, are not expressed by PSC cells. Additionally, the expression levels of the hemocyte marker Hemese and the Friend-of-GATA protein U-shaped are dramatically reduced in the PSC when compared with other hemocytes of the lymph gland. Taken together, both the expression and lack of expression of a number of genetic markers defines the cells of the PSC as a unique hemocyte population (Jung, 2005).

In contrast to primary lobes of the third instar, maturing hemocytes are generally not seen in the secondary lobes. Correspondingly, secondary lobes often have a smooth and compact appearance, much like the medullary zone of the primary lobe. Consistent with this appearance, secondary lymph gland lobes also express high levels of DE-cadherin. The size of the secondary lobe, however, varies from animal to animal and this correlates with the presence or absence of maturation markers. Smaller secondary lobes contain a few or no cells expressing maturation markers, whereas larger secondary lobes usually exhibit groups of differentiating cells. Direct comparison of DE-cadherin expression in secondary lobes with that of Cg-gal4, hml-gal4 or Lz revealed that the expression of these maturation markers occurs only in areas in which DE-cadherin is downregulated. Therefore, although there is no apparent distinction between cortical and medullary zones in differentiating secondary lobes, there is a significant correlation between the expression of maturation markers and the downregulation of DE-cadherin, as is observed in primary lobes (Jung, 2005).

The relatively late 'snapshot' of lymph gland development in the third larval instar establishes the existence of spatial zones within the lymph gland that are characterized by differences in structure as well as gene expression. In order to understand how these zones form over time, lymph glands of second instar larvae, the earliest time at which it was possible to dissect and stain, were examined for the expression of hematopoietic markers. As expected, Srp and Odd are expressed throughout the lymph gland during the second instar since they are in the late embryo and third instar lymph gland. Likewise, the hemocyte-specific marker Hemese is expressed throughout the lymph gland at this stage, although it is not present in the embryonic lymph gland (Jung, 2005).

To determine whether the cortical zone is already formed or forming in second instar lymph glands, the expression of various maturation markers were examined in a pair-wise manner to establish their temporal order. Of the markers examined, hml-gal4 and Pxn are the earliest to be expressed. The majority of maturing cells were found to be double-positive for hml-gal4 and Pxn expression, although a few cells were found to express either hml-gal4 or Pxn alone. This indicates that the expression of these markers is initiated at approximately the same time, although probably independently, during lymph gland development. The marker Cg-gal4 is next to be expressed since it was found among a subpopulation of Pxn-expressing cells. Finally, P1 antigen expression is initiated late, usually in the early third instar. Interestingly, the early expression of each of these maturation markers is restricted to the periphery of the primary lymph gland lobe, indicating that the cortical zone begins to form in this position in the second instar. Whenever possible, each genetic marker was directly compared with other pertinent markers in double-labeling experiments, except in cases such as the comparison of two different gal4 reporter lines or when available antibodies were generated in the same animal. In such cases, the relationship between the two markers, for example dome-gal4 and hml-gal4, was inferred from independent comparison with a third marker such as Pxn (Jung, 2005).

By studying the temporal sequence of expression of hemocyte-specific markers, one can describe stages in the maturation of a hemocyte. It should be noted, however, that not all hemocytes of a particular lineage are identical. For example, in the late third instar lymph gland, the large majority of mature plasmatocytes (~80%) expresses both Pxn and hml-gal4, but the remainder express only Pxn (~15%) or hml-gal4 (~5%) alone. Thus, while plasmatocytes as a group can be characterized by the expression of representative markers, populations expressing subsets of these markers indeed exist. It remains unclear at this time whether this heterogeneity in the hemocyte population is reflective of specific functional differences (Jung, 2005).

In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar reveals an interesting developmental progression. A group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4, as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal. These cells resemble earlier precursors in the embryo, except they express the marker Hemese. These cells are called pre-prohemocytes. Interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively (Jung, 2005).

The cells of the PSC are already distinguishable in the late embryo by their expression of collier. It was found that the canonical PSC marker Ser-lacZ is not expressed in the embryonic lymph gland and is only expressed in a small number of cells in the second instar. This relatively late onset of expression is consistent with collier acting genetically upstream of Ser. Another finding was that the earliest expression of upd3-gal4 parallels the expression of Ser-lacZ and is restricted to the PSC region. Finally, Pvr and dome-gal4 are excluded from the PSC in the second instar, similar to what is seen in the third instar (Jung, 2005).

To determine whether maturing cortical zone cells are indeed derived from medullary zone prohemocytes, a lineage-tracing experiment was performed in which dome-gal4 was used to initiate the permanent marking of all daughter cell lineages. In this system, the dome-gal4 reporter expresses both UAS-GFP and UAS-FLP. The FLP recombinase excises an intervening FRT-flanked 'STOP cassette', allowing constitutive expression of lacZ under the control of the actin5C promoter. At any developmental time point, GFP is expressed in cells where dome-gal4 is active, while lacZ is expressed in all subsequent daughter cells regardless of whether they continue to express dome-gal4. In this experiment, cortical zone cells are permanently marked with ß-galactosidase despite not expressing dome-gal4 (as assessed by GFP), indicating that these cells are derived from a dome-gal4-positive precursor. This result is consistent with and further supports independent marker analysis that shows that dome-gal4-positive prohemocytes downregulate dome-gal4 expression as they initiate expression of maturation markers representative of cortical zone cells. As controls to the above experiment, the expression patterns of two other gal4 lines, twist-gal4 and Serrate-gal4 were determined. The reporter twist-gal4 is expressed throughout the embryonic mesoderm from which the lymph gland is derived. Accordingly, the entire lymph gland is permanently marked by ß-galactosidase despite a lack of twist-gal4 expression (GFP) in the third instar lymph gland. Analysis of Ser-gal4 reveals that PSC cells remain a distinct population of signaling cells that do not contribute to the cortical zone (Jung, 2005).

Genetic manipulation of Pvr function provides valuable insight into its involvement in the regulation of temporal events of lymph gland development. To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were generated in the lymph gland early in the first instar and then examined during the third instar for the expression of maturation markers. It was found that loss of Pvr function abolishes P1 antigen and Pxn expression, but not Hemese expression. The crystal cell markers Lz and ProPOA1 are also expressed normally in Pvr-mutant clones, consistent with the observation that mature crystal cells lack or downregulate Pvr. The fact that Pvr-mutant cells express Hemese and can differentiate into crystal cells suggests that Pvr specifically controls plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL positive but do express the hemocyte marker Hemese and can differentiate into crystal cells, all suggesting that the observed block in plasmatocyte differentiation within the mutant clone is not due to cell death. Additionally, Pvr-mutant clones were large and not significantly different in size from their wild-type twin spots. Thus, the primary role of Pvr is not in the control of cell proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same phenotypic features, confirming that Pvr controls the transition of Hemese-positive cells to plasmatocyte fate (Jung, 2005).

Entry into S phase was monitored using BrdU incorporation and distinct proliferative phases were identified that occur during lymph gland hematopoiesis. In the second instar, proliferating cells are evenly distributed throughout the lymph gland. By the third instar, however, the distribution of proliferating cells is no longer uniform; S-phase cells are largely restricted to the cortical zone. This is particularly evident when BrdU-labeled lymph glands are co-stained with Pxn. Medullary zone cells, which can be identified by the expression of dome-gal4, rarely incorporate BrdU. Therefore, the rapidly cycling prohemocytes of the second instar lymph gland quiesce as they populate the medullary zone of the third instar. As prohemocytes transition into hemocyte fates in the cortical zone, they once again begin to expand in number. This is supported by the observation that the medullary zone in white pre-pupae does not appear diminished in size, suggesting that the primary mechanism for the expansion of the cortical zone prior to this stage is through cell division within the zone. Proliferating cells in the secondary lobes continue to be distributed uniformly in the third instar, suggesting that secondary-lobe prohemocytes do not reach a state of quiescence as do the cells of the medullary zone. These results indicate that cells of the lymph gland go through distinct proliferative phases as hematopoietic development proceeds (Jung, 2005).

This analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, termed the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Jung, 2005).

Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. It is proposed that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types (Jung, 2005).

Based on this analysis a model is proposed by which hemocytes mature in the lymph gland. Hematopoietic precursors that populate the early lymph gland are first distinguishable as Srp+, Odd+ (S⁺O⁺) cells. These will eventually give rise to a primary lymph gland lobe where the steps of hemocyte maturation are most apparent. During the first or early second instar, these S⁺O⁺ cells begin to express the hemocyte-specific marker Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called pre-prohemocytes and, in the second instar, cells expressing only these markers occupy a narrow region near the dorsal vessel. Subsequently, a subset of these Srp+, Odd+, He+, Pvr+ (S⁺O⁺H⁺Pv⁺) pre-prohemocytes initiate the expression of dome-gal4 (d_g4), thereby maturing into prohemocytes. The prohemocyte population (S⁺O⁺H⁺Pv⁺d_g4⁺) can be subdivided into two developmental stages. Stage 1 prohemocytes, which are abundantly seen in the second instar, are proliferative, whereas stage 2 prohemocytes, exemplified by the cells of the medullary zone, are quiescent. As development continues, prohemocytes begin to downregulate dome-gal4 and express maturation markers (M; becoming S⁺O⁺H⁺Pv⁺d_g4^lowM⁺). Eventually, dome-gal4 expression is lost entirely in these cells (becoming S⁺O⁺H⁺Pv⁺d_g4^-M⁺), found generally in the cortical zone. Thus, the maturing hemocytes of the cortical zone are derived from prohemocytes previously belonging to the medullary zone. This is supported by lineage-tracing experiments that show cells expressing medullary zone markers can indeed give rise to cells of the cortical zone. In turn, the medullary zone is derived from the earlier, pre-prohemocytes. Early cortical zone cells continue to express successive maturation markers (M) as they proceed towards terminal differentiation. Depending on the hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1, msn-lacZ, etc. These studies have shown that differentiation of the plasmatocyte lineage requires Pvr, while previous work has shown that the Notch pathway is crucial for the crystal cell fate. Both the JAK/STAT and Notch pathways have been implicated in lamellocyte production (Jung, 2005).

Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis (Jung, 2005).

The adult Drosophila Malpighian tubules are maintained by multipotent stem cells

All animals must excrete the waste products of metabolism. Excretion is performed by the kidney in vertebrates and by the Malpighian tubules in Drosophila. The mammalian kidney has an inherent ability for recovery and regeneration after ischemic injury. Stem cells and progenitor cells have been proposed to be responsible for repair and regeneration of injured renal tissue. In Drosophila, the Malpighian tubules are thought to be very stable and no stem cells have been identified. This study has identified multipotent stem cells in the region of lower tubules and ureters of the Malpighian tubules. Using lineage tracing and molecular marker labeling, it was demonstrated that several differentiated cells in the Malpighian tubules arise from the stem cells and an autocrine JAK-STAT signaling regulates the stem cells' self-renewal. Identifying adult kidney stem cells in Drosophila may provide important clues for understanding mammalian kidney repair and regeneration during injury (Singh, 2008).

The regenerating renal cells may come from one of the three possible sources, based on previous studies. First, the circulating blood contains bone marrow-derived stem cells able to differentiate into non-haematopoietic cells, such as cells of the kidney. Second, the differentiated glomerular and tubular cells may also be able to dedifferentiate into stem-like cells to repair the damaged tissues. Third, large numbers of slowly cycling cells have recently been identified in the mouse renal papilla region; these cells may be adult kidney stem cells and may participate in renal regeneration after ischemic injury. Further, the ureter and the renal collecting ducts were formed from the epithelium originating from the ureteric bud, and the nephrons and glomeruli were formed from the metanephric mesoderm-derived portion during kidney development. Two distinguished stem cell types have been proposed as responsible for repairing the renal collecting tubules and the nephrons. This study identified a type of pluripotent stem cells (RNSCs) in the Drosophila renal organ. The stem cells are able to generate all cell types of the adult fly MTs. In the region of lower tubules and ureters, autocrine JAK-STAT signaling regulates the stem cell self-renewal. Weak JAK-STAT signaling may convert an RNSC into a renalblast (RB), which will differentiate into an RC in the region of lower tubules and ureters, and a type I or type II cell in the upper tubules. These data indicate that only one type of stem cell may be responsible for repair and regeneration of the whole damaged tissues in mammalian kidney (Singh, 2008).

The Drosophila RNSCs represent a unique model to study the molecular mechanisms that regulate stem cell or cancer stem cell behavior. In most of the stem cell systems that has been well characterized to date, stem cells always reside in a specialized microenvironment, called a niche. A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The stromal cells often secrete growth factors to regulate stem cell behavior. The stem cell niche plays an essential role in maintaining stem cells, and stem cells will lose stem cell status once they are detached from the niche. The niche often provides the balanced (proliferation-inhibiting and proliferation-stimulating) signals that keep the stem cells dividing slowly. The inhibitory signals keep the stem cell quiescent most of the time while the stimulating signals promote stem cell division, to replenish lost differentiated cells. Maintaining the balance between proliferation-inhibiting and proliferation-stimulating signals is the key to maintaining tissue homeostasis (Singh, 2008).

Drosophila RNSCs are controlled differently. This study has demonstrated that the JAK-STAT signaling regulates the stem cell self-renewal. Both the ligand Upd and the receptor Dome are expressed in the RNSCs and the autocrine JAK-STAT signaling regulates the stem cell self-renewal; thus, the self-sufficient stem cells control their self-renewal or differentiation and do not need to constrained to a fixed niche. However, the RNSCs are still confined to the region of lower tubules and ureters even in the Upd overexpressed flies, suggesting that some other factors besides the JAK-STAT signaling may restrict the RNSCs to the region of the lower tubules and ureters (Singh, 2008).

Recent studies also suggest that tumors may arise from small populations of so-called cancer stem cells (CSCs). The CSCs probably have arisen from mutations that dysregulate normal stem cell self-renewal. For example, mutations that block the proliferation-inhibiting signals or promote the proliferation-stimulating signals can convert the normal stem cells into CSCs. This study demonstrates that amplifying the JAK-STAT signaling by overexpressing its ligand Upd stimulates the RNSCs to proliferate and also to differentiate into RC, which results in tumorous overgrowth in the MT. Therefore, the Drosophila RNSC system may also be a valuable in vivo system in which to study CSC regulation (Singh, 2008).

The RNSCs are located in the region of the lower tubules and ureter of the MTs, while ISCs are located at the posterior midgut. The MTs' ureters connect to the posterior midgut. The two types of stem cells are at close anatomical locations in the adult fly digestion system and also share some properties. For example, both of them are small nuclear cells, Arm-positive, and express esg. However, RNSCs and ISCs produce distinctly different progenies. ISCs produce progenies that include either Su(H)GBE-lacZ- or Pros-positive cells, which are not among the progenies of RNSCs because Su(H)GBE-lacZ and Pros are not expressed in the MTs. RNSCs produce progenies that include Cut- or TSH-positive cells, which are not among the progenies of ISCs because Cut and TSH are not expressed in the posterior midgut. One possibility for this difference is that, although RNSCs and ISCs originate from the same stem cell pool, their particular environments restrict their differentiation patterns. Future experiments, such as transferring RNSCs to the posterior midgut and vice versa, should be able to test this model (Singh, 2008).

The JAK-STAT signaling regulates self-renewal of the male germline, the male somatic, female escort stem cells in fly. The signaling also regulates self-renewal and maintenance of mammalian embryonic stem cells. This study reports that the JAK-STAT signaling regulates self-renewal of RNSCs. The JAK-STAT signaling may be a general stem cell signaling and also regulate stem cell self-renewal in other, un-characterized stem cell systems (Singh, 2008).

esg has been used as a marker of both male germline stem cells. This study has demonstrated that the esg-Gal4. UAS-GFP transgene is specifically expressed in RNSCs. The function of the esg gene is to maintain cells as diploid in Drosophila imaginal cells. Stem cells may have to be diploid, and esg may be a general stem cell factor. Identifying a stem cell signaling pathway (such as the JAK-STAT signal transduction pathway) and a stem cell factor (such as esg) will provide useful tools for identifying stem cells in other systems and for understanding stem cell regulation in general (Singh, 2008).

EFFECTS OF MUTATION

In embryos lacking both maternal and zygotic dome function, the trachea does not develop. Because such an extreme tracheal defect has not been described for other mutants of the pathway, whether the trachea is similarly affected in STAT92E germ line clones was examined. In zygotic and maternal STAT92E mutants, the trachea is mostly absent. This cannot be a result of the abnormal segmentation because the trachea forms in paternally rescued embryos that still have segment defects. The tracheal system forms from ten pairs of tracheal pits arising in segments from T2-A8. The pits can be identified at early stages by the expression of three genes: trachealess (trh), ventral veinless (vvl; also known as drifter), and knirps (kni). These three genes are activated in the trachea independently of one another. trh and vvl are then required for the expression of all known tracheal genes (except kni) and for their own maintenance from stage 13 onward. To discover at what stage of tracheal development the JAK/STAT pathway is required, the expression of trh, kni, and vvl was studied in dome or STAT92E maternal and zygotic mutants. In both types of mutant embryos, neither trh nor kni is expressed, whereas early vvl expression is not affected. Because trh is essential for tracheal development, its loss from the tracheal pits is the likely cause for the tracheal defects observed. These results show that STAT92E is the earliest transcription factor required for trachea specification (Brown, 2001).

domeless, termed mom by Chen (2000) is required for zygotic viability because mutant animals homozygous for the mom allele die as early larvae. The dead larvae show a posterior spiracle defect. In addition to its zygotic function, mom is required maternally for normal embryonic segmentation because mom embryos derived from females lacking germ-line mom¹ activity die with segmentation defects that resemble the phenotype of hop and STAT92E embryos. As is the case with hop and STAT92E embryos, the severity of the defects observed in mom embryos is dependent on the paternal contribution. Both paternally rescued and unrescued mom embryos show a consistent deletion of the fifth abdominal segment and the posterior midventral portion of the fourth abdominal segment. Additional defects in the thoracic segments and the head and tail regions are observed in unrescued mom embryos. The maternal phenotypes associated with the mom¹ mutation are identical to those observed with complete loss of hop and STAT92E gene activities (Chen, 2002).

Whether mom operates upstream or downstream of hop was examined by testing genetic interaction between the mom loss-of-function mutation and a hyperactive hop allele. Hemizygous mom¹ zygotic mutant larvae show defects of the posterior spiracle, an organ that is connected to the tracheal system and used by the young larvae for gas exchange. If mom is required to transduce the Hop signal, then a hop gain-of-function mutation should have no effect on the posterior spiracle phenotype of the mom mutation. The dominant temperature-sensitive hop allele hop^Tum-l was used for this experiment. When grown above 25°C, flies heterozygous for hop^Tum-l evince increased tyrosine kinase activity. Hemizygous mom¹ and hop^Tum-l mutant larvae form wild-type posterior spiracles, suggesting that the hop gain-of-function mutation rescues the mom¹ mutant phenotype. This result further suggests that mom is a member of the Hop/Stat92E signal transduction pathway and functions upstream of the Hop tyrosine kinase (Chen, 2002).

In the mammalian cell culture system, the JAK/STAT pathway becomes activated when a ligand binds to its receptor, inducing tyrosine phosphorylation of the monomeric STAT molecule. Tyrosine phosphorylation causes the STAT protein to dimerize with another STAT molecule via reciprocal SH2 domain-phophotyrosine interactions, and the dimer translocates to the nucleus (Chen, 2002).

To explore the function of mom in activating the Hop/Stat92E signal transduction pathway, protein levels and distributions of Stat92E were compared in wild-type embryos and mutant embryos of Upd, mom, and hop genes. Embryos were stained using affinity-purified anti-Stat92E antibodies. Although strong Stat92E expression is detected as 15 clear stripes during stage 9 in the wild-type embryo, Stat92E expression is dramatically reduced in Upd, mom, and hop mutant embryos. As in wild-type embryos, the remaining Stat92E protein in mutant embryos is localized in both the nucleus and cytoplasm. These data suggest that Mom and the Upd/Mom/Hop signaling pathway regulate Stat92E protein expression (Chen, 2002).

The posterior spiracle defects of the mom mutation have led to an examination of functions of the Hop/Stat92E pathway in tracheal formation. Trachea form from 10 tracheal pits, 1 per hemisegment. The trachealess gene selects the tracheal primordia in the embryonic ectoderm and drives the conversion of these planar epithelial regions into tracheal pits. The tracheal pits then sprout successively finer branches and fuse together, forming the tracheal network. The trachea is further connected to the posterior spiracle, forming a functional tracheal system. Tracheal formation was examined in mom, hop, and STAT92E mutants by using an enhancer trap line in the trachealess gene (1-eve-1) and an antibody [(mAb)2A12] that stains tracheal branches and trunks. In hop null embryos, trachealess expression is completely abolished and tracheal formation is completely blocked. In paternally rescued embryos, a defective tracheal system forms, generally with several disruptions in the main trunk and several branches. Because all of the mom and STAT92E mutants examined were enhancer trap lines, trachealess gene expression could not be examined by directly using the 1-eve-1 enhancer trap line. However, in the paternally rescued STAT92E and mom mutant embryos, similar to the hop mutant embryos, a defective tracheal system formed, generally with several disruptions in the main trunk and several branches. These data suggest that Mom and the Hop/Stat92E signal transduction pathway play an indispensable role in tracheal formation (Chen, 2002).

Among the diverse cellular processes taking place during oogenesis, the delamination and migration of border cells (BCs), a group of anterior follicle cells, represent a powerful model to study cell invasion in a normal tissue. During stage 9 of oogenesis, BCs detach from the outer epithelium to invade the germline cyst compartment. The BC cluster contains two centrally located polar cells surrounded by approximately six outer border cells and undergoes a nearly 6-hour long posteriorward migration to reach the anterior part of the growing oocyte. Together with centripetal cells, they assemble the micropyle, a specialized structure required for sperm entry. domeless was isolated in a screen to identify genes essential in epithelial morphogenesis during oogenesis. The level of dome activity is critical for proper border cell migration and is controlled in part through a negative feedback loop. In addition to its essential role in border cells, dome is required in the germarium for the polarization of follicle cells during encapsulation of germline cells. In this process, dome controls the expression of the apical determinant Crumbs. In contrast to the ligand Upd, whose expression is limited to a pair of polar cells at both ends of the egg chamber, dome is expressed in all germline and follicle cells. However, Dome protein is specifically localized at apicolateral membranes and undergoes ligand-dependent internalization in the follicle cells. dome mutations interact genetically with JAK/STAT pathway genes in border cell migration and abolish the nuclear translocation of Stat92E in vivo. dome functions downstream of upd and both the extracellular and intracellular domains of Dome are required for JAK/STAT signaling. Altogether, the data indicate that Dome is an essential receptor molecule for Upd and JAK/STAT signaling during oogenesis (Ghiglione, 2002).

dome interacts genetically with the Stat92E and dpias ([a.k.a. Su(var)2-10 gene, Betz, 2001] a negative regulator of the JAK/STAT pathway) during BC migration, and dome phenotypes in ovaries are similar to those found in Stat92E and hop mutants. Furthermore, Stat92E nuclear localization is lost in dome mutant follicle cells, indicating that the mechanisms leading to Stat92E activation and subsequent nuclear translocation require dome. Since dome is epistatic to upd, the data indicate that dome is required downstream of upd and upstream of Stat92E for JAK/STAT signaling in egg chambers. Altogether, these results provide strong evidence that Dome is a receptor molecule for Upd during oogenesis (Ghiglione, 2002).

Dome is not uniformly distributed at the membrane but is restricted to apicolateral regions. Other receptor molecules have been shown to preferentially localize to apicolateral membranes, such as the EGF and Notch receptors, suggesting that the apical region is an active signaling interface for several receptors in follicle cells. Indeed, the apical localization of upd mRNA, membrane Dome and Dome-containing vesicles support a model in which ligand-receptor interactions take place apically in follicle cells, to activate the JAK/STAT pathway (Ghiglione, 2002).

Dome is a transmembrane protein with both extracellular and intracellular domains whose functions are unknown. The extracellular part contains a cytokine-binding module (CBM) and 3 fibronectin-type III domains likely participating in ligand binding, while the intracellular domain presumably interacts with Hop, through binding to one or several potentially phosphorylated tyrosines. Using truncated forms of Dome it has been shown that both the extracellular and intracellular domains are essential for BC migration and signal transduction. The dominant negative phenotypes that are observed are consistent with a model in which DomeDeltaCYT would titrate the ligand Upd, and DomeDeltaEXT titrates Hop, therefore inducing a dramatic reduction in signaling strength. Both constructs may also lead to the formation of non-functional Dome-Dome dimers by capturing the wild-type Dome protein in an inactive complex. Further biochemical work will be necessary to understand the molecular mechanisms underlying Dome signal transduction (Ghiglione, 2002).

Previous studies have shown that the migration of BCs is sensitive to gene dosage, making this migration a useful marker for genetic screens. The reduction or elevation of slbo, a gene encoding a C/EBP homolog, is sufficient to produce BC migration defects. Consistently, recent work has shown that Slbo protein levels are tightly regulated by the ubiquitination pathway. BCs are also sensitive to changes in Dome protein levels. Indeed, either a decrease or an increase of Dome causes BC migration defects. There are several mechanisms by which gene activity can be regulated, including post-translational regulation, as with the Slbo protein, or transcriptional regulation. The data suggest that dome expression is regulated in part by a transcriptional negative feedback loop. Two consensus STAT binding sites present in the promoter region of the dome gene may prove to be important for this regulation. Interestingly, it has been shown that vertebrate STAT proteins can have both positive and negative regulatory functions. Further work will be necessary to determine whether Stat92E is a direct repressor of dome (Ghiglione, 2002).

In addition to a transcriptional control of dome, there are also post-translational mechanisms regulating Dome function in follicle cells, through a dynamic pattern of intracellular vesicles. These Dome-containing vesicles are located at a relevant distance from Upd-producing cells, and Upd can promote the formation of de novo vesicles upon ectopic expression. These results, together with the presence of several tyrosine-based and di-leucine motifs known to sort proteins for internalization, are consistent with Dome-containing vesicles being the result of endocytosis upon ligand binding. Endocytosis is an important process controlling several signaling pathways during development, acting on the recycling or desensitization of ligand-receptor complexes. This work thus provides the first experimental evidence that JAK/STAT signaling in flies may be regulated by endocytosis (Ghiglione, 2002).

This analysis of dome reveals several important functions in follicle cells. During early oogenesis, dome is required for the encapsulation of germline cells into a functional egg chamber. dome mutant follicle cells made in the germarium are unable to assemble into the nascent follicular epithelium, thus leading to incompletely encapsulated egg chambers at stage 2-3. The fusion of some egg chambers suggests that follicle cells normally separating adjacent egg chambers in the germarium rapidly degenerate. This conclusion is supported by the fact that mutant cells cannot be observed in early egg chambers. This is an alternative to the model in which the formation of fused egg chamber would be associated to stalk cell defects (Ghiglione, 2002).

The dramatic, early follicle cell phenotype contrasts with the essentially normal phenotype of dome mutant cells observed in later stage egg chambers. In this case, follicle cells are viable and divide normally. A similar, dual phenotype has been reported in crumbs mutant chambers. After initial polarization of the follicle cells in the germarium, Crumbs is no longer required and its loss has no visible effects. Importantly, dome controls Crumbs expression in follicle cells, thus providing a novel link between the JAK/STAT signaling pathway and epithelial polarity (Ghiglione, 2002).

In addition to its early function in the germarium, dome is required for the normal expression of several follicle cell markers, including DE-cadherin and Fas3. It is important to note that despite a clear defect in the expression of these markers, dome mosaic egg chambers are morphologically normal. However, because completely mutant egg chambers cannot be obtained because of the early effect of dome in the germarium, one cannot rule out the possibility that large mutant clones would lead to abnormal development of egg chambers (Ghiglione, 2002).

The pattern of epithelial markers in dome mutant cells indicates that the JAK/STAT pathway is active in all follicle cells, a notion that is reinforced by the wide expression of nuclear Stat92E. How is Dome activated during egg chamber development and does this activation follow the same profile at all stages? Given the restricted pattern of upd expression in the egg chamber and its dramatic effect upon overexpression, it is unlikely that Upd is able to signal long distances in the follicular epithelium of late stage egg chambers. Rather, a model by which the JAK/STAT pathway plays a pre-patterning function is favored, acting early during egg chamber development to activate DE-cadherin and Crumbs expression. This view is consistent both with the expression pattern of upd and the distribution of Dome-containing vesicles described in this study. The formation of endogenous vesicles can be promoted by Upd, and a gradient of such vesicles is present around polar cells. Strikingly, these vesicles, which likely indicate active signaling through Dome, are widespread at early stages and become more restricted later on. It is proposed that during early development, the Upd signal produced by anterior and posterior polar cells contributes to the differentiation of all follicle cells. At this stage, Upd would be more diffusible than later, as suggested by the pattern of Dome intracellular vesicles. The study of the mechanisms controlling Dome activation and Upd activity will require additional tools to directly detect Upd, as, for example, Upd-GFP fusion proteins (Ghiglione, 2002).

In contrast to the situation in main body follicle cells, the role of dome in BCs is essential. dome and other JAK/STAT pathway components promote the differentiation of a selected group of follicle cells into a cohesive migratory cluster, a process requiring several other inputs. Mutations in dome induce phenotypes ranging from a complete absence of BCs to non-cohesive BC clusters. This suggests that dome may be required during migration itself, in addition to its role in the recruitment of BCs at stages 8-9. Although only conditional mutants could help to address this question, the pattern of Dome vesicles in the BCs before and during migration supports a sustained activation of the JAK/STAT pathway. Such a requirement could also explain the semi-dominant migration phenotype of dome heterozygous egg chambers (Ghiglione, 2002).

This study has revealed several new findings about the function of dome and the JAK/STAT pathway during oogenesis. Future work will help to understand how Upd and Dome initially interact at the cell surface and transduce the signal to downstream JAK/STAT pathway members (Ghiglione, 2002).

To characterize the features of JAK/STAT signaling in Drosophila immune response, totA was identified as a gene that is regulated by the JAK/STAT pathway in response to septic injury. Septic injury triggers the hemocyte-specific expression of upd3, a gene encoding a novel Upd-like cytokine that is necessary for the JAK/STAT-dependent activation of totA in the Drosophila counterpart of the mammalian liver, the fat body. In addition, totA activation is shown to require the NF-KB-like Relish pathway, indicating that fat body cells integrate the activity of NF-KB and JAK/STAT signaling pathways upon immune response. This study reveals that, in addition to the pattern recognition receptor-mediated NF-kappaB-dependent immune response, Drosophila undergoes a complex systemic response that is mediated by the production of cytokines in blood cells, a process that is similar to the acute phase response in mammals (Agaisse, 2003).

A Drosophila homolog of the vertebrate cytokine class I receptor, Dome (a.k.a. Mom), has been identified. Mutations in dome result in embryonic defects similar to the embryonic phenotype associated with mutation in the JAK/STAT pathway components. A truncated version of Dome, DomeΔCYT, has been generated by deletion of the intracellular region that is involved in signal transduction. This mutated receptor still contains the extracellular cytokine binding module and acts as a signaling antagonist, probably by titrating the ligand. Accordingly, DomeΔCYT overexpression during embryogenesis mimics the loss-of-function phenotype of dome mutants. To test whether Dome plays a role in totA expression, the GAL4/UAS system was used to express the dominant-negative form of Dome, DomeΔCYT, in adult fat body. Northern blot analysis has revealed that totA expression upon immune challenge is totally abolished in the corresponding animals. It is concluded that totA activation in response to bacterial infection is the result of a signaling event that is transduced by the Drosophila homolog of the vertebrate cytokine receptor, Dome, in the fat body (Agaisse, 2003).

Requirement for JAK/STAT signaling throughout border cell migration in Drosophila

The evolutionarily conserved JAK/STAT signaling pathway is essential for the proliferation, survival and differentiation of many cells, including cancer cells. Recent studies have implicated this transcriptional pathway in the process of cell migration in humans, mice, Drosophila and Dictyostelium. In the Drosophila ovary, JAK/STAT signaling is necessary and sufficient for the specification and migration of a group of cells called the border cells; however, it is not clear to what extent the requirement for cell fate is distinct from that for cell migration. It was found that STAT protein is enriched in the migrating border cells throughout their migration and is an indicator of cells with highest JAK/STAT activity. In addition, stat^ts mutants exhibit border cell migration defects after just 30 minutes at the non-permissive temperature, prior to any detectable change in the expression of cell fate markers. At later times, cell fate changes became evident, indicating that border cell fate is labile. JAK/STAT signaling was also required for organization of the border cell cluster. Finally, it is shown that both the accumulation of STAT protein and nuclear accumulation are positively regulated by JAK/STAT activity. The activity of the pathway is negatively regulated by overexpression of a suppressor of cytokine signaling (SOCS) protein and by blocking endocytosis. Together, these findings suggest that the requirement for STAT in border cells extends beyond the initial specification and delamination of cells from the epithelium (Silver, 2005).

Extra and ectopic border cells can be induced in at least two different ways. When ectopic polar cells form, for example in eyes absent or costal 2 (costa) mutant clones, or following mis-expresssion of activated Notch, they recruit surrounding cells into a cluster and these are capable of migration. Alternatively, ectopic expression of UPD, HOP or HOP^Tum is sufficient to induce large numbers of ectopic migrating border cells. These cells migrate in a variety of sizes of clusters, which lack polar cells, and can even migrate as individual cells. These findings indicate that UPD might be the only factor produced by polar cells that functions to recruit border cells and sustain their motility. To test this hypothesis, ectopic expression of UPD was induced in single anterior follicle cells, or in pairs of cells, to see whether UPD expression alone is as effective as polar cells in recruiting border cells. For comparison, the same method was also used to express a form of UPD that contains a transmembrane domain (UPD^TM). Expression of the wild-type form of UPD results in the recruitment of neighboring cells into a cluster, and these cells express high levels of STAT protein, like normal border cells. However, UPD alone is not as effective as a normal polar cell because, normally, two polar cells recruit four to eight cells to surround them, whereas UPD alone results in the recruitment of an average of only 1.1 border cell per UPD-expressing cell. Cells expressing the UAS-UPD^TM are actually more effective at recruiting border cells than cells expressing wild-type UPD. A single cell expressing the membrane-tethered form of UPD recruits an average of 3.25 cells to surround it, similar to normal or ectopic polar cells. In the case of the membrane-tethered form of UPD, ectopic migratory cells are observed only adjacent to the UPD-expressing cells. These findings suggested that JAK/STAT signaling contributes to the organization of the migrating border cell cluster. This was investigated further by analyzing the organization of migrating border cells following the reduction of JAK/STAT function (Silver, 2005).

Suppressors of cytokine signaling are thought to inhibit the JAK/STAT pathway, either by blocking JAK or STAT function via a SOCS SH2 domain, or by causing the destruction of these proteins by ubiquitination. There are three SOCS genes in Drosophila but, to date, no loss-of-function mutants have been reported. Using both slbo-GAL4,1310 and c306-GAL4 drivers, it was found that overexpression of wild-type SOCS36E inhibits border cell migration and recruitment, and overexpression of SOCS36E lacking the SOCS domain weakly inhibits migration. By contrast, overexpression of a SOCS36E protein that lacks the SH2 domain, or of slbo-GAL4,1310 or c306-GAL4 alone failed to disrupt border cell migration. Compared with an average border cell number of six for wild-type egg chambers, slbo-GAL4,1310;UAS-SOCS and c306-GAL4;UAS-SOCS egg chambers have an average number of border cells of 3.3 and 4.9, respectively. This is consistent with findings that reduction of JAK/STAT activity inhibits border cell recruitment. Those border cells that do form and migrate, frequently do so as single cells rather than as a cluster. In addition, the border cells had reduced levels of STAT (Silver, 2005).

Consistent with a requirement in cluster organization, when JAK/STAT signaling is reduced by overexpression of a dominant-negative form of dome lacking the cytoplasmic domain, border cells migrate slower than wild-type cells, and frequently as single cells. This is consistent with the finding that in dome mosaic egg chambers, border cells often fail to migrate in a cluster. Taken together, these results support the idea that signaling through the JAK/STAT pathway is responsible for the organization of the border cell cluster (Silver, 2005).

Normally border cells migrate as a cohesive cluster with the non-migratory, UPD-expressing cells in the center and the migratory cells surrounding them. These two cell types are dependent upon each other, since the central cells cannot migrate and are carried by the surrounding cells, and the migratory cells cannot move in the absence of the UPD signal from the central cells. Thus, the organization of the border cell cluster is crucial for normal migration. In addition to its function in border cell specification and motility, several lines of evidence demonstrate the role of UPD/JAK/STAT in organizing the border cell cluster. Ectopic expression of UPD in single anterior follicle cells, for example, is sufficient to recruit adjacent cells to form a cluster capable of migration. In addition, a variety of treatments that reduce STAT activity (dome mosaic clones, overexpression of dominant-negative Dome, and overexpression of SOCS) leads to disruptions of cluster formation. Disruption of the cluster is likely to affect migration through the egg chamber. For example, PAR6, an epithelial protein required for polarity and the migration of border cells, is disrupted in border cells in which dominant-negative Dome is overexpressed, lending support to the idea that JAK/STAT signaling helps to regulate the organization of cells within the cluster. Once the cluster is disrupted, the migratory cells become separated from the polar cells, presumably reducing STAT activity further and aggravating the migration defect. Thus, STAT activity promotes cluster organization, which feeds back to promote efficient UPD/DOME/JAK/STAT signaling (Silver, 2005).

Taken together, the results presented here demonstrate several inter-related properties of JAK/STAT signaling in the control of border cell migration and function. Both anatomical and biochemical mechanisms feed back upon each other to regulate the level of STAT activity precisely throughout the six hours of border cell migration. Positive-feedback mechanisms include maintaining close contact between UPD-expressing cells and the migratory cells, as well as stabilization and nuclear enrichment of STAT protein in response to signaling. One negative regulatory mechanism is the expression of SOCS36E (Silver, 2005).

The findings described here may also have relevance for understanding the requirement of STAT signaling in the progression of cancer. Constitutively activated STAT3 is associated with the aggressive clinical behavior of a number of cancers, including ovarian and renal cancers. Blocking STAT3 in pancreatic cancer cells inhibits tumor growth and metastases in mice, whereas expression of activated STAT3 promotes metastasis. Inhibiting STAT3 expression or activation in ovarian carcinoma cells impedes their motility in vitro. Thus, cancer cells too appear to require sustained activation of this pathway to survive, proliferate and migrate. The finding that JAK/STAT signaling appears to be tightly regulated by its own activity, by that of SOCS inhibitors and by endocytic processes suggests that these may provide points of clinical intervention in the treatment of STAT-dependent cancers (Silver, 2005).

Ken & barbie selectively regulates the expression of a subset of Jak/STAT pathway target genes

A limited number of evolutionarily conserved signal transduction pathways are repeatedly reused during development to regulate a wide range of processes. A new negative regulator of JAK/STAT signaling is described and a potential mechanism identified by which the pleiotropy of responses resulting from pathway activation is generated in vivo. As part of a genetic interaction screen, Ken & Barbie (Ken), which is an ortholog of the mammalian proto-oncogene BCL6, has been identified as a negative regulator of the JAK/STAT pathway. Ken genetically interacts with the pathway in vivo and recognizes a DNA consensus sequence overlapping that of STAT92E in vitro. Tissue culture-based assays demonstrate the existence of Ken-sensitive and Ken-insensitive STAT92E binding sites, while ectopically expressed Ken is sufficient to downregulate a subset of JAK/STAT pathway target genes in vivo. Finally, endogenous Ken is shown specifically represses JAK/STAT-dependent expression of ventral veins lacking (vvl) in the posterior spiracles. Ken therefore represents a novel regulator of JAK/STAT signaling whose dynamic spatial and temporal expression is capable of selectively modulating the transcriptional repertoire elicited by activated STAT92E in vivo (Arbouzova, 2006).

Analysis of phenotypes associated with mutations in Drosophila JAK/STAT pathway components have identified a wide variety of requirements for the pathway during embryonic development and in adults. What is less clear is how the repeated stimulation of a single pathway is able to generate this pleiotropy of developmental functions. In order to identify modulators of JAK/STAT signaling that may be involved in this process, a genetic screen was undertaken for modifiers of the dominant phenotype caused by the ectopic expression of the pathway ligand Unpaired (Upd) in the developing eye imaginal disc. Such misexpression by GMR-updΔ3′ results in overgrowth of the adult eye, a phenotype sensitive to the strength of pathway signaling activity. With this assay, one genomic region, defined by Df(2R)Chi^g320, was found to enhance the GMR-updΔ3′-induced eye overgrowth phenotype. Of the genes deleted by Df(2R)Chi^g320, only mutations in ken showed consistent and reproducible enhancement of the phenotype. In addition, other dominant phenotypes induced by transgene expression from the GMR promoter are not modulated by ken mutations, indicating that Ken is unlikely to interact with the misexpression construct used (Arbouzova, 2006).

The enhancement of the GMR-updΔ3′ phenotype after removal of one copy of ken implies that Ken normally functions antagonistically to JAK/STAT signaling. Therefore phenotypes associated with mutations in other pathway components were tested to establish the reliability of this initial observation. Consistent with this, genetic interaction assays between ken mutations and the hypomorphic loss-of-function allele stat92E^HJ show a reduction in the frequency of wing vein defects normally associated with this stat92E allele. Moreover, the degree of suppression is consistent with the strength of ken alleles tested. Similarly, the frequency of “strong” posterior spiracle phenotypes caused by the dome³⁶⁷ allele of the pathway receptor is also reduced when crossed to ken alleles or the Df(2R)Chi^g320 deficiency, with a concomitant increase in “weak” phenotypes (Arbouzova, 2006).

Thus, multiple independent ken alleles all modify diverse phenotypes caused by both gain- and loss-of-function mutations in multiple JAK/STAT pathway components. Each of these components acts at different levels of the signaling cascade and show interactions indicating that Ken consistently acts as an antagonist of the pathway (Arbouzova, 2006).

The ken locus contains three exons encoding a 601 aa protein. Ken possesses an N-terminal BTB/POZ domain between aa 17 and 131 and three C-terminal C2H2 zinc finger motifs from aa 502 to 590. Strikingly, a number of Zn finger-containing proteins that also contain BTB/POZ domains have also been shown to function as transcriptional repressors—often via the recruitment of corepressors such as SMRT, mSIN3A, N-CoR, and HDAC-1 (Arbouzova, 2006).

Searches for proteins similar to Ken identified homologs in Drosophila pseudoobscura and the mosquito Anopheles gambiae. In vertebrates, human B-Cell Lymphoma 6 (BCL6) was the closest full-length homolog. Drosophila Ken and human BCL6 share the same domain structure and show 20.3% overall identity. Proteins listed as potential vertebrate homologs of Ken in Flybase are more distantly related (Arbouzova, 2006).

Expression of ken was also examined during development, where it is detected in a dynamic pattern from newly laid eggs, throughout embryogenesis, and in imaginal discs. As such, endogenous Ken is present in all tissues and stages in which genetic interactions were observed (Arbouzova, 2006).

Given the presence of potentially DNA binding Zn finger domains and the nuclear localization of GFPKen, the DNA binding properties of Ken was determined by using an in vitro selection technique termed SELEX (systematic evolution of ligands by exponential enrichment). With a GST-tagged Ken Zn finger domain and a randomized oligonucleotide library, ten successive rounds of selection were undertaken. Sequencing of the resulting oligonucleotide pool and alignment of 43 independent clones showed that all recovered plasmids were unique and each contained one, or occasionally two, copies of the motif GNGAAAK (K = G/T) (Arbouzova, 2006).

To confirm the SELEX results, GFPKen was expressed in tissue culture cells and these were used for electromobility shift assays (EMSA). A radioactively labeled probe containing the wild-type (wt) consensus binding site GAGAAAG gives a specific band, which can be supershifted by an anti-GFP antibody and therefore represents a GFPKen/DNA complex. In order to identify positions essential for binding, a competition assay was used in which unlabeled oligonucleotides containing single substitutions in each position from 1 to 7 were added to binding reactions. 10-fold excess of unlabeled wild-type consensus oligonucleotide greatly diminished the intensity of the GFPKen band, while 50- and 100-fold excess totally blocked the original signal. By contrast, competition with unlabeled m3 oligonucleotides containing a G to A substitution at position 3 failed to significantly reduce the intensity of the band even at 100-fold excess. With this approach, the positions 1 and 7 are found dispensable for DNA binding, whereas the central GAAA core is absolutely required. Similar results were obtained with the converse experiment with labeled mutant probes, although in this case the wt probe produces a stronger signal than the m1 and m7 mutant oligonucleotides. Taken together, these experiments not only define the core sequence for Ken binding, but also demonstrate the specificity of Ken as a site-specific DNA binding molecule. Interestingly, the core consensus bound by Ken is very similar to that identified for human BCL6, with the Zn fingers of the latter binding to a DNA sequence containing a core GAAAG motif (Arbouzova, 2006).

One initial observation made is that the core GAAA essential for Ken binding overlaps the sequence recognized by STAT92E. Consistent with this overlap, a 100-fold excess of unlabeled oligonucleotide containing the STAT92E consensus is sufficient to fully compete for Ken in EMSA assays. Given this finding, it is hypothesized that the negative regulation of JAK/STAT signaling by Ken observed in genetic interaction assays may occur via a mechanism of competitive DNA binding site occupation. Due to the incomplete overlap between the STAT92E and Ken core sequences, this hypothesis also implies the existence of STAT92E DNA binding sites to which both STAT92E and Ken could bind (STAT⁺/Ken⁺) as well as sites with which Ken cannot associate (STAT⁺/Ken⁻) (Arbouzova, 2006).

To test this hypothesis, a cell culture-based assay was set up by using a luciferase-expressing reporter containing four STAT92E binding sites originally identified in the promoter of the Draf locus. In addition to this STAT⁺/Ken⁺ wild-type reporter, STAT⁺/Ken⁻ and STAT⁻/Ken⁻ variants identical but for the binding sequences were generated. When transfected into the hemocyte-like Kc₁₆₇ Drosophila cell line, both STAT⁺/Ken⁺ and STAT⁺/Ken⁻ reporters showed strong stimulation upon coexpression with the pathway ligand Upd, an assay previously shown to require an intact JAK/STAT cascade. When cotransfected with KenGFP, the activity of the STAT⁺/Ken⁺ reporter was reduced, an effect reproduced in three independent experiments with both KenGFP and Ken. While the reduction in reporter activity for the STAT⁺/Ken⁺ assay shown is statistically significant, the STAT⁺/Ken⁻ reporter was unaffected by the coexpression of Ken. Reporters containing binding sites mutated to prevent binding of both STAT92E and Ken (STAT⁻/Ken⁻) showed no activation after pathway stimulation and did not respond to Ken (Arbouzova, 2006).

These results indicate that Ken functions as a transcriptional repressor in this cell-culture system and shows that this effect is specific to the DNA sequence determined by SELEX and EMSA. This result is also consistent with a recent whole-genome RNAi-based screen, which used a reporter containing STAT⁺/Ken⁺ binding sites and includes Ken among the list of JAK/STAT regulators identified. In addition, recent reports have also demonstrated BCL6 binding to STAT6 sites in vitro and have shown that BCL6 can act as a repressor of STAT6-dependent target gene expression in cell culture. Although this repression is mediated by the binding to corepressors to the BTB/POZ domain of BCL6, no link between BCL6 and STAT activity has been demonstrated in vivo (Arbouzova, 2006).

Finally, it should also be noted that both the STAT⁺/Ken⁺ and STAT⁺/Ken⁻ reporters contain additional GAAA sequences that are not part of the characterized STAT92E binding sequences. However, despite the presence of these potential Ken binding sites within 15 bp of the STAT92E site, Ken expression did not affect the STAT⁺/Ken⁻ reporter, suggesting that Ken may require STAT92E to influence gene expression. Although no direct association between Ken and STAT92E has been demonstrated, this possibility cannot be excluded, and further analysis remains to be undertaken (Arbouzova, 2006).

Having established that Ken functions at the level of DNA binding in cell culture, it was asked whether Ken also acts as a transcriptional repressor of JAK/STAT pathway target genes in vivo. For this, the effect of ectopically expressed Ken on the expression of putative JAK/STAT pathway target genes was examined and, given the high levels of maternally loaded STAT92E present at blastoderm stage, focus was placed on targets expressed later in embryogenesis. These include the hindgut-specific expression of vvl, the expression of trachealess (trh) and knirps (kni) in the tracheal placodes, and the dynamic expression of socs36E throughout the embryo (Arbouzova, 2006).

First, the effect of Ken was addressed on trh, whose expression precedes the formation of the tracheal pits in the embryonic segments T2 to A8. Levels of trh are greatly reduced in embryos uniformly misexpressing Ken driven by the daughterless-GAL4 (da-GAL4) line. Many tracheal placodes express little or no trh, and tracheal pits fail to form even in the presence of residual trh. Similar effects are seen in upd^OS1A mutant embryos lacking all pathway activity. Likewise, downregulation of Kni expression is also observed in embryos misexpressing ken. These results show that both endogenous trh and kni are downregulated by ectopically expressed Ken (Arbouzova, 2006).

Whether Ken can modulate the expression of socs36E, a Drosophila homolog of mouse SOCS-5, was tested. socs36E expression closely mirrors that of upd, showing JAK/STAT pathway-dependent upregulation in segmentally repeated stripes, tracheal pits, and the hindgut. By contrast to trh and kni, ectopically expressed Ken does not affect any aspect of socs36E transcription. However, controls expressing a dominant-negative form of the pathway receptor DomeΔCyt, using the same Gal4 driver line, show a strong downregulation of socs36E, an effect reproduced by the complete removal of all JAK/STAT pathway activity by the upd^OS1A allele. Taken together, these results illustrate that ectopic expression of Ken during Drosophila development is sufficient to downregulate the expression of only a subset of putative JAK/STAT pathway target genes (Arbouzova, 2006).

As part of this analysis, modulation of vvl by Ken was tested. In wild-type embryos, vvl is expressed in the developing trachea and lateral ectoderm (in a JAK/STAT-independent manner) and in the hindgut of stage 12–14 embryos, where it requires JAK/STAT signaling. In upd^OS1A mutants, no vvl expression in the hindgut can be detected, indicating that this locus is a target of pathway activation. When Ken is uniformly misexpressed throughout the embryo, vvl expression is no longer detectable in the hindgut. Thus vvl, like trh and kni, can be a target of Ken-mediated repression (Arbouzova, 2006).

Having established that ectopic Ken is sufficient to downregulate vvl in the hindgut, whether endogenous Ken performs a similar role was determined. One overlap between ken expression and regions known to require JAK/STAT signaling are the developing posterior spiracles, structures in which both the pathway ligand upd and ken are simultaneously expressed. However, vvl is never detected in the posterior spiracle primordia in wild-type embryos, despite JAK/STAT pathway activity induced by upd expression in these tissues. Intriguingly, in a heteroallelic combination of the strongest ken^k11035 allele and Df(2R)Chi^g320, vvl transcript was detected not only in its normal expression domain within the hindgut but also in the posterior spiracles. This ectopic expression is initially detected from late stage 13 and rapidly strengthens during stage 14–15. When ken^k11035/Df(2R)Chi^g320 embryos simultaneously mutant for the amorphic upd^OS1A allele were analyzed, upregulation of vvl in the presumptive posterior spiracles was never observed at the stage by which ectopic vvl expression was first detected in the ken mutant embryos. At later stages, JAK/STAT pathway activity is required for posterior spiracle morphogenesis, posterior spiracles do not form, and upregulated vvl is not present (Arbouzova, 2006).

These results demonstrate that Ken is not only sufficient to downregulate the JAK/STAT pathway-dependent expression of vvl in the hindgut, but its endogenous expression is also necessary for vvl repression in the posterior spiracles. In ken mutants, ectopic vvl expression in the posterior spiracles results from a derepression of endogenous STAT92E activity (Arbouzova, 2006).

The overlap between the consensus sequences bound by STAT92E and Ken, together with the analysis of reporters containing STAT⁺/Ken⁺ and STAT⁺/Ken⁻ binding sites, indicate that Ken is likely to selectively regulate only a subset of JAK/STAT target genes. In this model, some target genes are regulated by binding sites compatible with both STAT92E and Ken, while others contain sequences to which only STAT92E can associate. While the DNA binding site is critical in cell-culture systems, similar proof is more difficult to establish in vivo. In particular, only a limited number of JAK/STAT pathway target genes have been rigorously demonstrated to require STAT92E binding in vivo (Arbouzova, 2006).

Although studied in some detail, the regulatory domains controlling vvl expression in the developing hindgut have not been identified. Therefore, although these results predict that such a domain would contain STAT⁺/Ken⁺ binding sequences, further analysis is required to confirm this hypothesis. By contrast, the regulatory domain of socs36E required to drive gene expression in the blastoderm, tracheal pits, and hindgut comprises a 350 bp region containing three STAT⁺/Ken⁺ and two STAT⁺/Ken⁻ binding sites. Although not conclusive, the presence of STAT92E-exclusive sites in this region may explain the inability of Ken to downregulate socs36E in vivo (Arbouzova, 2006).

The findings also draw a parallel between Drosophila Ken and BCL6. The data presented demonstrate that both proteins show similar abilities to bind DNA and to mediate transcriptional repression with some evidence also linking BCL6 to JAK/STAT signaling as described here. Taken together, these similarities suggest that Ken and BCL6 represent functional orthologs of one another. Given this evolutionary conservation, it is tempting to speculate that the selective regulation of JAK/STAT pathway target genes is also conserved and may represent a general mechanism by which the pathway is modulated to elicit diverse developmental roles in vivo. Although many STAT targets undoubtedly remain to be identified, it will be intriguing to see which may also be coregulated by Ken/BCL6-dependent mechanisms (Arbouzova, 2006).

Diverse tumor pathology due to distinctive patterns of JAK/STAT pathway activation caused by different Drosophila polyhomeotic alleles

Drosophila polyhomeotic (ph) is one of the important polycomb group genes that is linked to human cancer. In the mosaic eye imaginal discs, while ph^del, a null allele, causes only non-autonomous overgrowth, ph⁵⁰⁵, a hypomorphic allele, causes both autonomous and non-autonomous overgrowth. These allele-specific phenotypes stem from the different sensitivities of ph mutant cells to the Upd homologs that they secrete (Feng, 2012).

Different ph alleles cause tissue overgrowth in different ways. While a ph null allele, ph^del , causes only non-autonomous cell over-proliferation, a ph hypomorphic allele, ph⁵⁰⁵ , causes both autonomous and non-autonomous cell overproliferation. In mosaic tissues, overproliferation of mutant cells was defined as autonomous, whereas over-proliferation of genotypically wild type cells induced by mutant cells was defined as non-autonomous. The signaling pathway involved in ph^del induced non-autonomous cell over-proliferation. In summary, elevated Notch activity in ph cells up-regulates the expression of JAK/STAT pathway ligands Upd homologs, which in turn activate the JAK/STAT pathway in neighboring wild type cells and cause their over-proliferation. This study addressed why a ph null allele and a ph hypomorphic allele both cause tumors but in such different ways (Feng, 2012).

First whether the same signaling pathway underlay non-autonomous overproliferation induced by both ph^del and ph⁵⁰⁵ was tested. The functions of Notch and Upd homologs in the ph⁵⁰⁵ mosaic eyes were examined with the same strategy used for ph^del. A ph⁵⁰⁵ -Notch double mutant line was generated, and eyes mosaic for this line were essentially of the same size as wild type eyes. The mosaic eye discs had normal size and normal cell proliferation level, as shown by PH3 staining, which marks mitotic cells. Moreover, the size of ph⁵⁰⁵ -Notch clones was significantly reduced when compared to that of ph⁵⁰⁵ clones. These results indicated that Notch was required for both autonomous and non-autonomous overproliferation induced by ph⁵⁰⁵ (Feng, 2012).

Next ph⁵⁰⁵ was recombined with upd^Δ1-3, a deficiency line that lacks all three upd homologs in the Drosophila genome Mosaic analyses were then performed using this double mutant line. ph⁵⁰⁵ -upd^Δ1-3 mosaic eyes were significantly smaller than ph⁵⁰⁵ mosaic eyes and were comparable to wild type eyes, indicating that tissue overgrowth was largely suppressed. PH3 staining of the double mutant mosaic eye discs showed that these discs had relatively normal size and cell proliferation level. Importantly, ph⁵⁰⁵ -updd1-3 clones were also drastically reduced in size compared to ph⁵⁰⁵ clones. These results indicated that Upd homologs are required for not only non-autonomous but also autonomous cell over-proliferations induced by ph⁵⁰⁵ (Feng, 2012).

It is not surprising that the same signaling pathway is responsible for non-autonomous over-proliferation induced by both ph^del and ph⁵⁰⁵ , and it is not completely unexpected that Notch is also required for ph⁵⁰⁵ induced autonomous over-proliferation, as Notch is a transcription factor that has been shown to autonomously regulate cell proliferation. However, the three Upd proteins are secreted and are not expected to have any direct effect on autonomous cell proliferation. To interpret these observations, it was hypothesized that ph⁵⁰⁵ cells still respond to Upd ligands secreted by themselves in an autocrine or paracrine manner, and therefore over-proliferate. However, ph^del cells were thought to be no longer responsive to Upd ligands (Feng, 2012).

To functionally test this hypothesis, the double mutant strategy was applied, taking advantage of the fact that the genes domeless (dome, encoding the only transmembrane receptor of the Drosophila JAK/STAT pathway) and hopscotch (hop, encoding the only Drosophila JAK kinase) are also on X chromosome as is ph. First ph⁵⁰⁵ was recombined with two dome alleles to generate ph⁵⁰⁵ -dome double mutant lines. Eye discs mosaic for these lines were still significantly larger than wild type, but the size of double mutant clones was dramatically reduced, so that only a tiny portion of the disc was composed of mutant cells. PH3 staining indicated that non-autonomous proliferation level was still high, but autonomous proliferation largely disappeared. The adult eyes mosaic for such double mutant lines were further examinedm and these eyes were found to be still much larger than wild type and similar to ph⁵⁰⁵ mosaic eyes in size, but they generally were not folded as seen in ph⁵⁰⁵ mosaic eyes (Feng, 2012).

Next a ph⁵⁰⁵ -hop double mutant line was generated. Autonomous proliferation was found in mosaic eye discs of this double mutant that was also significantly suppressed, with mutant cells only accounted for a small portion of the whole disc. In contrast, non-autonomous cell over-proliferation was not affected and the overall size of these discs was still significantly larger than wild type. Adult eyes mosaic for this double mutant showed similar phenotypes as those of ph⁵⁰⁵ -dome mosaic eyes. These eyes were still significantly larger than wild type but they were generally not folded. Therefore, the removal of either dome or hop from ph⁵⁰⁵ cells only suppressed autonomous over-proliferation but did not affect non-autonomous overproliferation, making such double mutant mosaic discs phenotypically similar to ph^del mosaic discs (Feng, 2012).

As controls, ph^del -dome and ph^del -hop double mutant lines were also generated using the same dome and hop alleles. Mosaic analyses on eye discs showed that the removal of dome or hop from ph^del cells did not affect non-autonomous cell over proliferation. It did, however, mildly reduce the mutant clone size, suggesting that ph^del cells might still have a weak response to Upd ligands. Adult eyes mosaic for these double mutant lines were phenotypically indistinguishable from ph^del mosaic eyes, consistent with the above observations in mosaic eye discs (Feng, 2012).

Finally it was asked why ph^del and ph⁵⁰⁵ cells responds differently to the Upd ligands secreted by themselves. It was hypothesized that some of the JAK/STAT pathway modulators might be differentially expressed in ph^del and ph⁵⁰⁵ cells. To test this hypothesis, TU-Tagging, a technique that enables the purification of RNA from mutant cells without having to physically isolate such cells, was chosen. Briefly, Drosophila is unable to synthesize uridine from uracil due to the lack of phosphoribosyltransferase (UPRT). When exogenous UPRT is expressed in mutant cells by MARCM, such cells would acquire the ability to utilize uracil. If these larvae are fed with 4-thiouracil (4-TU), a uracil derivative that contains a thio group, only mutant cells would be able to use 4-TU and eventually incorporate thio- containing uridine into newly synthesized RNA. This treatment has little toxicity, and the thio-labeled RNA can be purified from total RNA using conventional biochemical methods (Feng, 2012).

TU-tagging was performed to isolate RNA from ph^del cells and ph⁵⁰⁵ cells, and qRTPCR was used to examine candidate gene expression. The expression of the JAK/STAT pathway receptor dome was significantly higher in ph⁵⁰⁵ cells than in ph^del cells. A higher receptor expression might sensitize ph⁵⁰⁵ cells to the Upd ligands. The levels of enok and socs42a, both negative regulators of the JAK/STAT pathway, were also significantly higher in ph⁵⁰⁵ cells compared to ph^del cells. This might represent feedback loops that negatively regulate the pathway activity. In fact, several such negative feedback loops, in which elevated pathway activity upregulates a negative pathway regulator, have been reported in JAK/STAT pathway (Feng, 2012).

Together, it is concluded that ph^del and ph⁵⁰⁵ both cause autonomous over-expression of Upd homologs in mutant cells, which represents the only driving force of cell overproliferation in ph^del and ph⁵⁰⁵ mosaic tissues and in essence acts non-autonomously to activate JAK/STAT pathway. The different phenotypes of these two types of mosaics are due to different sensitivity of mutant cells to Upd homologs. ph⁵⁰⁵ mutant cells robustly respond to Upd ligands that they secreted. Therefore, Upd ligands secreted by ph⁵⁰⁵ cells simultaneously induce over-proliferation in both mutant and wild type cells. In contrast, ph^del cells are largely insensitive to Upd ligands, so that Upd ligands secreted by ph^del cells only induce over-proliferation in wild type but not mutant cells. Furthermore, differential expression of the JAK/STAT pathway receptor dome might underlie the different sensitivity of ph^del and ph⁵⁰⁵ cells to Upd ligands (Feng, 2012).

EVOLUTIONARY HOMOLOGS

Heinrich, P. C., Behrmann, I., Muller-Newen, G., Schaper, F. and Graeve, L. (1998). Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem J. 334: 297-314. Online version

Hirano, T. (1998). Interleukin 6 and its receptor: ten years later. Int Rev Immunol. 16(3-4): 249-84. Online version

Kitamura, H., Ohno, Y., Toyoshima, Y., Ohtake, J., Homma, S., Kawamura, H., Takahashi, N. and Taketomi, A. (2017). Interleukin-6/STAT3 signaling as a promising target to improve the efficacy of cancer immunotherapy. Cancer Sci 108(10): 1947-1952. PubMed ID: 28749573

Tsukamoto, H., Fujieda, K., Senju, S., Ikeda, T., Oshiumi, H. and Nishimura, Y. (2017). Immune-suppressive effects of interleukin-6 on T-cell-mediated anti-tumor immunity. Cancer Sci. PubMed ID: 29090850

REFERENCES

Search PubMed for articles about Drosophila domeless

Agaisse, H., et al. (2003). Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury. Dev. Cell 5: 441-450. 12967563

Amcheslavsky, A., Jiang, J. and Ip, Y. T. (2009). Tissue damage-induced intestinal stem cell division in Drosophila. Cell Stem Cell 4: 49-61. PubMed Citation: 19128792

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Biteau, B., Hochmuth, C. E. and Jasper, H. (2008). JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut. Cell Stem Cell 3: 442-455. PubMed Citation: 18940735

Brown, S., Hu, N. and Castelli-Gair Hombria, J. (2001). Identification of the first invertebrate interleukin JAK/STAT receptor, the Drosophila gene domeless. Cur. Bio. 11: 1700-1705. 11696329

Brown, S., Hu, N. and Hombria, J. C.-G. (2003). Novel level of signalling control in the JAK/STAT pathway revealed by in situ visualisation of protein-protein interaction during Drosophila development. Development 130: 3077-3084. 12783781

Buchon, N., Broderick, N. A., Poidevin, M., Pradervand, S. and Lemaitre, B. (2009). Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation. Cell Host Microbe 5: 200-211. PubMed Citation: 19218090

Chen, H. W., Chen, X., Oh, S. W., Marinissen, M. J., Gutkind, J. S. and Hou, S. X. (2002). mom identifies a receptor for the Drosophila JAK/STAT signal transduction pathway and encodes a protein distantly related to the mammalian cytokine receptor family. Genes Dev. 16(3): 388-98. 11825879

Copf, T., et al. (2011). Cytokine signaling through the JAK/STAT pathway is required for long-term memory in Drosophila. Proc. Natl. Acad. Sci. 108(19): 8059-64. PubMed Citation: 21518857

Devergne, O., Ghiglione, C. and Noselli, S. (2007). The endocytic control of JAK/STAT signalling in Drosophila. J. Cell Sci. 120(Pt 19): 3457-64. Medline abstract: 17855388

Feng, S., Thomas, S. and Wang, J. (2012). Diverse tumor pathology due to distinctive patterns of JAK/STAT pathway activation caused by different Drosophila polyhomeotic alleles. Genetics 190(1): 279-82. PubMed Citation: 22048022

Ghiglione, C., et al. (2002). The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis Development 129: 5437-5447. 12403714

Hirano, T. (1998). Interleukin 6 and its receptor: ten years later. Int. Rev. Immunol. 16(3-4): 249-84. Online version

Hombria, J. C., Brown, S., Hader, S. and Zeidler, M. P. (2005). Characterisation of Upd2, a Drosophila JAK/STAT pathway ligand. Dev. Biol. 288(2): 420-33. 16277982

Jiang, H., et al. (2009). Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut. Cell 137(7): 1343-55. PubMed Citation: 19563763

Jung, S. H., Evans, C. J., Uemura, C. and Banerjee, U. (2005). The Drosophila lymph gland as a developmental model of hematopoiesis. Development 132(11): 2521-33. 15857916

Lohi, O. and Lehto, V. P. (2001). STAM/EAST/Hbp adapter proteins - integrators of signalling pathways. FEBS Lett. 508: 287-290. Medline abstract: 11728436

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Marchetti, M., Monier, M. N., Fradagrada, A., Mitchell, K., Baychelier, F., Eid, P., Johannes, L. and Lamaze, C. (2006). Stat-mediated signaling induced by type I and type II interferons (IFNs) is differentially controlled through lipid microdomain association and clathrin-dependent endocytosis of IFN receptors. Mol. Biol. Cell 17: 2896-2909. Medline abstract: 16624862

Silver, D. L., Geisbrecht, E. R. and Montell, D. J. (2005). Requirement for JAK/STAT signaling throughout border cell migration in Drosophila. Development 132(15): 3483-92. 16000386

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