The roles of the Drosophila JAK/STAT pathway

Martin P Zeidler1, Erika A Bach1 and Norbert Perrimon*,1

1Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, 200 Longwood Avenue, Boston,Massachusetts, MA 02115, USA

The JAK/STAT signal transduction pathway has beenconserved throughout evolution such that true structuraland functional homologues of components originallyidenti®ed in vertebrate systems are also present in themodel genetic system Drosophila melanogaster. Inaddition to roles during larval hematopoiesis reminis-cent of the requirement for this pathway in mammaliansystems, the JAK/STAT pathway in Drosophila is alsoinvolved in a number of other developmental events.Recent data has demonstrated further roles for theJAK/STAT pathway in the establishment of sexualidentity via the early embryonic expression of Sexlethal, the segmentation of the embryo via the controlof pair rule genes including even skipped and theestablishment of polarity within the adult compoundeye via a mechanism that includes the four jointedgene. Use of the powerful genetics in the modelorganism Drosophila may identify new components ofthe JAK/STAT pathway, de®ne new roles for thispathway, and provide insights into the function of thissignal transduction system. Here we review the roles ofSTAT and its associated signaling pathway during bothembryonic and adult stages of Drosophila developmentand discuss future prospects for the identi®cation andcharacterization of novel pathway components andtargets. Oncogene (2000) 19, 2598 ± 2606.

Keywords: hopscotch; unpaired; stat92E; ommatidia;segmentation; polarity

Introduction

The JAK/STAT signal transduction cascade was ®rstidenti®ed in mammalian systems because of its role inthe transduction of a variety of cytokines and growthfactor signals (reviewed in Darnell, 1997). Extensivestudies in mammalian systems have led to thedevelopment of a canonical model in which the non-receptor tyrosine kinase JAK is associated with theintracellular portion of transmembrane cytokine recep-tors (Figure 1a). Following ligand binding, receptorsdimerize bringing two JAK molecules into juxtaposi-tion where they trans-phosphorylate one another.JAKs activated in this manner then tyrosine phosphor-ylate their associated receptors causing normallycytosolic STAT molecules to bind to the receptorcomplex via their SRC homology 2 (SH2) domains.The STAT molecules recruited in this manner arethemselves activated by JAK-mediated phosphoryla-tion of an invariant tyrosine residue in their C-terminalregion and then either homo- or hetero-dimerize prior

to nuclear translocation. Once in the nucleus activatedSTAT dimers bind to consensus DNA target siteswhere they act as activators of transcription (Figure 1aand other reviews in this issues).

Interestingly the JAK/STAT pathway and STAT-like molecules in particular are present throughoutevolution. To date one (or possibly two) STATs havebeen found in the nematode C. elegans (Lui et al.,1999), and three STAT-like molecules have beenidenti®ed in the slime mould Dictyostelium (MFukazawa and JG Williams, unpublished data reportedin Williams, 1999). However, neither organism appearsto have JAK-like protein kinases.

In the Drosophila system a `complete' JAK/STATpathway that consists of one JAK (called hopscotch)and one STAT (called stat92E) has been identi®ed, instark contrast to the multitude of JAK and STAThomologues present in mammals. The presence of arelatively simple JAK/STAT pathway, together withthe wealth of sequence data and genetic techniquesavailable, make Drosophila a very powerful system withwhich to study this pathway. These advantages,especially with respect to developmental requirementsfor JAK/STAT signaling, are particularly relevant forthe ¯y model system and hold much promise for thefuture elucidation of interactions between the JAK/STAT pathway and other signal transduction cascadesinvolved in developmental decisions.

In this review we discuss recent developments intoour understanding of the components and roles of theJAK/STAT pathway during Drosophila developmentand address potential future directions in the ®eld.

Characterization of Drosophila JAK/STAT pathwaycomponents

With the recent cloning of unpaired (upd) and itsidenti®cation as a potential ligand for the DrosophilaJAK/STAT pathway (Harrison et al., 1998), threepathway components have been identi®ed on thebasis of their biochemical, genetic and mutantphenotypes.

The hopscotch (hop) locus encodes a 1177 amino acidprotein that shares all the characteristics of mammalianJAK family non-receptor tyrosine kinases. Hop is mostsimilar to human JAK2 (27% identity), with higherlevels of homology in kinase and kinase-like domains(Figure 1b, Binari and Perrimon, 1994). The DrosophilaSTAT homologue Stat92E is also highly homologousto human STAT5 (37% identity). Stat92E includes aSH2 domain, DNA binding domain and the singletyrosine residue around position 700 found in allSTAT-like genes (Figure 1b, Hou et al., 1996; Yan etal., 1996a). Indeed this residue in Stat92E (Y711) hasbeen shown to be phosphorylated in response to

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*Correspondence: N Perrimon

pathway activation in tissue culture systems (Yan et al.,1996a).

It has previously been reported that a second, non-Stat92E derived STAT-like activity is detectable inDrosophila cell extracts (Yan et al., 1996a; Sweitzer etal., 1995). A series of searches of the Drosophilagenome sequencing project data were therefore con-ducted on a complete 106 coverage database toaddress this question. Using Stat92E protein sequencesas `probes' for tblastn search programs no candidateSTAT-like molecules, other than stat92E itself, wereidenti®ed (E Spana, personal communication). While,it remains possible that a STAT-like protein, which hasdiverged su�ciently far from Stat92E to be undetect-able with sequence based searches, is in fact present, itseems that the complex situation in vertebrate modelsystems is not mirrored in Drosophila. Therefore bothHopscotch and Stat92E probably represent the sole

examples of canonical JAK- and STAT-like proteinspresent in ¯ies.

While mutations in the unpaired locus have beenknown for over 75 years, only recently has theassociated gene been identi®ed as a ligand for theDrosophila JAK/STAT pathway (Venderosa andMuller, 1954; Harrison et al., 1998). The unpairedlocus encodes a predicted 47 kDa secreted protein withan N-terminal signal sequence and several potential N-linked glycosylation sites (Figure 1b). While a proteinsimilar to Upd called Om1E has been identi®ed in theclosely related fruit ¯y Drosophila ananassae, no knownvertebrate homologues have been found in publiclyavailable databases to date (Harrison et al., 1998; Juniet al., 1996). Biochemical analysis of Upd revealed thatthe protein has an apparent molecular weight of65 kDa, is indeed glycosylated and is localized in theextracellular matrix. Furthermore, tissue culture experi-

ss

Figure 1 (a) The JAK/STAT pathway in which receptor/JAK (red) complexes dimerize on binding of ligand (blue) (1). Followingreceptor and JAK phosphorylation (2) cytosolic STATs (green) are recruited to the complex (3). Phosphorylated STAT moleculesdimerize (4) and translocate to the nucleus where they bind DNA and activate transcription (5). (b) Schematic representation of theDrosophila Unpaired (blue), Hopscotch (red) and Stat92E (green) proteins. Gray bars in Unpaired represent potential amino-linkedglycosylation sites. The activating mutations in Hopscotch are indicated (see text for details) as is the invariant tyrosine residue inStat92E. SS=signal sequence, DB=DNA binding domain and SH2=Src Homology 2 domain. Each protein is drawn to scale (scalebar is 100 amino acids) and numbers represent amino acid position

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ments have shown that Upd is capable of inducingHop phosphorylation and activation in Clone 8 tissueculture cells. Furthermore, activation of the JAK/STAT pathway by Upd does not require co-expressionof Upd in the signaling cell as Upd supplied by either aphysically separate, but co-cultured, (S2) cell type, oreven from previously conditioned media, is su�cient toactivate Hop (Harrison et al., 1998). Taken togetherwith in vivo evidence showing that Upd functions as anactivator of JAK/STAT signaling in the eye (Zeidler etal., 1999b), it is clear that Unpaired represents a bona®de JAK/STAT pathway ligand.

Although the identity of Drosophila JAK and STAThomologues and a pathway ligand are now known,the identity of a pathway receptor(s) remains unclear.Vertebrate studies have shown that the JAK/STATpathway is downstream of cytokine receptors andreceptor tyrosine kinases such as EGFR and PDGFR(Darnell, 1997). While a similar situation may also bethe case in Drosophila no evidence had emerged todate regarding the nature of a potential pathwayreceptor.

Fly homologues of other components of themammalian JAK/STAT pathway, speci®cally PIAS,SOCS, and STAM, have also been identi®ed. Two ofthese are homologues of previously identi®ed negativeregulators of the mammalian JAK/STAT pathway;Protein Inhibitor of Activated STAT (PIAS) andSuppressor Of Cytokine Signaling (SOCS). PIASproteins contain a putative zinc-®nger domain, andspeci®c PIAS proteins bind to and inhibit the activityof speci®c STATs (Chung et al., 1997). In Drosophila,a PIAS homologue named zimp, has been identi®ed bysequence homology and although mutations are lethal,no further functional data has been published. It istherefore not yet clear whether this protein inhibits theactivity of Stat92E (Mohr and Boswell, 1999). TheSOCS family of proteins are characterized by thepresence of a highly conserved 40-amino acid C-terminal SOCS domain preceded by an SH2 domain(Nicholson et al., 1999). Studies indicate that SOCSproteins can inhibit cytokine signaling either directlyby inhibition of JAKs or indirectly by competitionwith STAT for a phospho-tyrosine binding site on thereceptor. Interestingly a ¯y homologue of SOCS-5 hasrecently been identi®ed, that retains sequence identityin both the SH2 domain and regions previouslyidenti®ed as being required for SOCS activity(Nicholson et al., 1999). Currently there are nomutations in the Drosophila SOCS gene, and thusthe physiological function of this gene is presently notknown. The ®nal Drosophila homologue to humanpathway components is the Signal TransducingAdaptor Molecule (STAM). STAMs were originallyidenti®ed as positive regulators of cytokine-dependentsignal transduction, contain a SRC homology 3 (SH3)domain and associate with Jak2 and Jak3 (Takeshitaet al., 1997). The ¯y homologue of STAM, also calledstam, was cloned by homology, and again nofunctional data is available for this gene (Mesilaty-Gross et al., 1999). Future genetic and biochemicalcharacterization of these Drosophila homologues mayestablish that Drosophila PIAS, SOCS and STAM aretrue orthologues and represent additional levels ofJAK/STAT pathway complexity in the ¯y modelsystem.

Sex determination

The sisterless C (sisC) gene has been known for sometime as a component of the Drosophila sex determina-tion system (Cline, 1993) and the recent identi®cationof sisC as an allele of Upd has implicated the JAK/STAT pathway in one of the earliest processes ofembryonic development.

The process of sexual identity determination inDrosophila is based on very early mechanisms whichdetect the X chromosome to autosome ratio of thenewly fertilized embryo (with XY individuals becomingmales and XX developing into females). The cellularmechanism which determines the number of Xchromosomes establishes the expression state of theSex lethal gene which acts as a `master switch' todetermine sexual identity and X chromosome dosagecompensation. The dose of X chromosomes present ina newly fertilized embryo is relayed into this sexualdetermination machinery via the activity of a numberof X-linked signal element (XSE) genes which areencoded on the X chromosome and include sisA, sisBand sisC (see Cline, 1993 for review). The two copies ofthese XSE genes produced in future females is su�cientto activate the `Sex lethal establishment' promoter andcause Sex lethal expression which, via a positiveautoregulatory feedback loop, is then maintainedthroughout the life of the ¯y. In future males theXSE genes present on the single X chromosome areexpressed at insu�cient levels to activate the Sex lethalpromoter.

A recent report has identi®ed the XSE gene sisC asbeing allelic to upd. While previously identi®ed XSEgenes, sisA and sisB represent transcription factors thatcan bind directly to the Sex lethal promoter, sisC/updobviously represents an extracellular JAK/STAT path-way ligand. This ®nding suggests that the JAK/STATpathway is required for the transduction of the SisC/Upd signal and, either directly or indirectly, controlsSex lethal expression (Thomas Cline, personal commu-nication).

Although a similar role for the JAK/STAT pathwayin sexual identity determination has not been pre-viously described in other systems, it is clear that themechanisms by which sexual identity is determined inother animals are very diverse. As a result it is possiblethat research into a potential role for the JAK/STATpathway in sex determination will provide insights notonly into the rapid evolutionary development of sexualidentity, but also the mechanisms whereby establishedsignal transduction pathways are co-opted into newdevelopmental roles.

Embryonic segmentation

Both hopscotch and stat92E are deposited maternally inthe developing oocyte during oogenesis. Hop isexpressed essentially uniformly throughout the variousstages of embryonic development, while Stat92E isexpressed uniformly during early stages but subse-quently resolves initially into seven and then 14segmental stripes (Binari and Perrimon, 1994; Hou etal., 1996; Yan et al., 1996a). Upd, in contrast, is notmaternally supplied but is expressed from the zygoticgenome in a highly dynamic pattern: broadly in thetrunk and in a stripe in the head before cellularization,

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seven stripes during cellularization, 14 stripes duringgastrulation and later in the tracheal pits (Harrison etal., 1998). Despite these very di�erent patterns ofexpression the three known components of the JAK/STAT pathway exhibit very similar, loss-of-functionphenotypes in the embryo. However because of thematernal contributions, the roles of hop and stat92E inthe embryo can be only assessed in embryos that lackthis contribution and that are derived from femaleswith homozygous mutant germlines. Using a geneticmethod, the `dominant female sterile technique', whichallows the generation of females that have a homo-zygous mutant germ-line in an otherwise wild type ¯y(Chou and Perrimon, 1996), embryos that lackmaternally contributed hop or stat92E can be generated(hereafter referred to as hop and stat92E mutantembryos). It should however be noted that maternaldeposition of hop and stat92E does not appear to beabsolutely essential. The embryonic phenotypes asso-ciated with removal of maternally supplied hop orstat92E can be partially rescued by a wild type copy ofthe gene supplied paternally and also by the injectionof wild type stat92E mRNA into pre-blastodermstat92E mutant embryos (Binari and Perrimon, 1994;Hou et al., 1996).

All embryos mutant for hop, stat92E or zygotic updexhibit a characteristic segmentation defect. Thesedefects are readily identi®able as a disruption in thenormal pattern of hairs or denticles that make up partof the external cuticle secreted by the embryo shortlybefore hatching. While wild type embryos have astereotyped arrangement of denticle belts that corre-spond to each thoracic and abdominal segment (Figure2a), JAK/STAT mutant embryos typically exhibit aloss of the ®fth segment with variable deletion of thefourth and eighth segments, as well as occasionalfusion of the sixth and seventh segments (Figure 2b;

Binari and Perrimon, 1994; Harrison et al., 1998; Houet al., 1996; Yan et al., 1996a).

Proper segmentation of the embryo is established byseveral sets of temporally expressed genes. Maternalgenes establish the anteroposterior and dorsoventralaxes of the embryo, and along the anteroposterior axisthe sequential expression and activity of zygoticsegmentation genes (gap, pair-rule, and segment-polarity) progressively re®ne the number and orienta-tion of the segments. Mutations in the gap genes resultin deletion of multiple adjacent segments, those in thepair-rule genes in deletion of alternative segments, andthose in the segment polarity genes result in loss ofpart of each segment. However the distinctive cuticlephenotypes of upd, hop and stat92E mutant embryosare shared only by mutations now identi®ed as beingcomponents of the JAK/STAT pathway, and weretherefore classi®ed as a separate class of segmentationmutants. In upd, hop and stat92E mutant embryos, thegap-genes are expressed normally. However, there aredefects in the expression of the pair-rule genes even-skipped (eve), fushi tarazu and runt (Binari andPerrimon, 1994; Hou et al., 1996).

The e�ect of mutations in the JAK/STAT pathwayon pair rule gene expression is quite variable anddepends on the stripe in question. Levels of endogen-ous eve stripes 3 and 5 are signi®cantly reduced in upd,hop and stat92E mutant embryos (Binari and Perri-mon, 1994; Harrison et al., 1998; Hou et al., 1996; Yanet al., 1996a). Although the regulatory domainscontrolling stripe 5 have not been identi®ed, a 500 bpregulatory sequence in the eve promoter has beende®ned that controls expression of eve stripe 2, 3 and 7(Binari and Perrimon, 1994; Small et al., 1996). Whenthis enhancer is fused to a lacZ reporter andintroduced into wild type embryos b-galactosidaseactivity is detected in three stripes corresponding to

Figure 2 (a) The characteristic pattern of denticle belts visible in a wild type embryonic cuticle showing abdominal (A1-A8)denticle bands). (b) The characteristic pattern of a Stat92E mutant embryo lacking any maternal contribution. As is characteristicfor all JAK/STAT mutant embryos, bands A1-3 are normal. However A4 is partially deleted and A5 is absent. The occasionalreduction or loss of A6-A8 in stat92E mutant embryos is not shown in this example. (c) The pattern of lacZ driven by the 500 bpeve promoter fragment in a wild type embryo. Bands that correspond to the positions of eve stripes 2,3 and 7 are visible. (d) Thepattern of lacZ driven by the 500 bp eve promoter fragment in a stat92E mutant embryo. The band corresponding to stripe 3 isgreatly reduced in intensity (arrow head). A similar reduction in stripe 3 expression is also produced in upd and hop mutant embryosand if the Stat92E binding sites in the 500 bp eve promoter are mutated

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the endogenous eve stripes 2, 3 and 7 (Figure 2c).When this enhancer, is introduced into upd, hop orstat92E mutant embryos, expression of stripe 3 isalmost completely abolished (Figure 2d; Binari andPerrimon, 1994; Hou et al., 1996; Harrison et al.,1998). This enhancer contains two sequences thatclosely resemble the mammalian consensus STATbinding site, and in vitro assays indicate that,tyrosine-phosphorylated, activated stat92E can bindto these sites. Moreover, when these STAT-bindingsites are mutated, the 500 bp enhancer no longer driveseve stripe 3 expression. Taken together these resultsindicate that the JAK/STAT pathway is directlyrequired for expression of eve stripe 3 (Hou et al.,1996; Yan et al., 1996a).

However, the control of endogenous eve stripe 3expression is not solely a consequence of JAK/STATactivity. Additional levels of regulation must exist asendogenous eve stripe 3 is not completely abolished inJAK/STAT pathway mutant embryos. Therefore it hasbeen suggested that at least one additional unidenti®edfactor co-operates with Stat92E to activate stripe 3expression in the embryo. Similarly, because theexpression of stripe 7, as reported by the 500 bpenhancer, is not diminished in upd, hop or stat92Emutant embryos, another unidenti®ed co-activator thatacts within the enhancer element to control stripe 7expression must also exist.

The analysis of eve stripe 3 regulation by JAK/STATpathway has led to the model that this pathway plays apermissive, and not an instructive, role in embryonicpatterning at this stage. In this model, the pathway actsto potentiate and amplify an unknown factor thatdrives eve stripe 3 expression rather than beingresponsible for the generation of the primary signalthat positions eve stripe 3 directly. Genetic analysissupports this view and has shown that the anterior andposterior domains of eve stripe 3 expression arecontrolled by the Hunchback and Knirps proteins,both of which are transcriptional repressors. Removalof Hunchback expands the domain of stripe 3anteriorly while removal of Knirps expands it poster-iorly. When both repressors are removed, stripe 3expands in both directions and is expressed throughoutthe embryo. Therefore, because the borders of evestripe 3 expression are already tightly controlled byHunchback and Knirps, the JAK/STAT pathway doesnot have to be activated in a spatially restricteddomain. Consistent with this model, precise spatialactivation of the JAK/STAT pathway by the ligandUpd does not appear to be essential for propersegmentation.

The JAK/STAT pathway and larval hematopoiesis

As is also the case in mammalian systems, arequirement for the JAK/STAT pathway duringDrosophila hematopoiesis has also been established.Although there are many unresolved issues about theontogeny of the cells in Drosophila hemolymph, it isthought that the larval hematopoietic organ, the lymphgland, gives rise to plasmatocytes which make upapproximately 90% of the circulating cells in the larva.Plasmatocyes are phagocytic cells that play animportant role in immune defense and cell-scavenging,and it is these cells which then terminally di�erentiate

into encapsulating lamellocytes, at the end of larvaldevelopment (see Mathey-Prevot and Perrimon, 1998,for recent review).

One of the ®rst indications that the JAK/STATpathway is critical to the development of thesecirculating cells was the identi®cation of dominantgain-of-function alleles of hop. Two temperaturesensitive mutations in the hop locus have beenidenti®ed that result in proteins with constitutivelyhyperactivated kinase activity. The hopTum-l allele is apoint mutation (G431E, see Figure 1b) of a residueotherwise unconserved in other JAKs (Hanratty andDearolf, 1993). The other, hopT42, is slightly strongerthan hopTum-l and also contains a single amino acidsubstitution (E695K, see Figure 1b) present in thekinase-like domain and represents the mutation of aresidue conserved in all known JAK homologues (Luoet al., 1997). These substitutions represent the onlyhyper-activating mutations currently identi®ed in anyJAK kinase. An equivalent substitution to the hopTum-l

allele engineered into murine JAK2 results in similaroveractive molecules (Luo et al., 1997). hopTum-l

individuals form 5 ± 20-fold more plasmatocytes whenraised above the restrictive temperature. Many of theseprematurely di�erentiate into lamellocytes, form largeaggregates and are encapsulated to form melanotictumors. When transplanted into a wild type host,hopTum-l hypertrophied larval lymph glands retain theability to cause overproliferation of plasmatocytes andmelanotic tumors (Hanratty and Dearolf, 1993;Harrison et al., 1995; Luo et al., 1995).

Overexpression experiments have also shown thatwild type hop misexpression can also produce amelanotic blood cell tumor phenotype similar tohopTum-l. Furthermore, both hop overexpression andhopTum-l-induced melanotic blood cell tumor phenotypescan be suppressed by the removal of a copy of thestat92E locus, and the resultant reduction in the levelof Stat92E activity. However, while Stat92E isobviously important for the melanization of tumors,it has been reported that the blood cell overprolifera-tion phenotype is not e�ected by changes in Stat92E. Itwas therefore suggested that the pathway maybifurcate downstream of hop, proliferation being aStat92E-independent process (Luo et al., 1995). Whilethis may be the case, it is also possible thatproliferation is simply not as easily suppressed as themelanization by removal of a single copy of stat92E inthis context.

JAK/STAT functions at other developmental stages

While Hop misexpression of HopTum-l activity in thelymph gland leads to blood cell overproliferation, thesituation in imaginal discs, tissue destined to form theadult ¯y, is not as straightforward. Over- or mis-activation of the pathway in imaginal tissue by ectopicexpression of Hop or HopTum-l does not generallyproduce an overproliferation of target tissue. Rathersuch ectopic activation results in a range of as yetlargely uncharacterized fate changes and developmentaldefects (Harrison et al., 1995). While these e�ects areintriguing and indicative of uncharacterized roles forthe JAK/STAT pathway in adult development, it doesnot at present appear that the JAK/STAT pathway islinked to tissue neoplasia in imaginal tissue.

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Loss of Hop activity in hetero-allelic mutantcombinations result in `small' or `no' disc phenotype(Perrimon and Mahowald, 1986). In addition theremoval of the hop locus from imaginal cells resultsin a mutant clone that is often far smaller thanexpected given the size of the `twin spot' WT cloneproduced during mitotic recombination. This indicatesthat these mutant cells are at a proliferative disadvan-tage (Luo et al., 1999, D Strutt and MP Zeidler,unpublished observations). However it is not clear thatstat92E mutant clones show a similar `undergrowth'and both hop and stat92E mutant tissue can surviveand proliferate to some degree. Therefore it wouldseem that the JAK/STAT pathway is not absolutelyrequired for cellular proliferation in the ¯y.

While the hopTum-l gain-of-function mutation discussedabove indicates an important role for the JAK/STATpathway during hematopoiesis, further functions for thepathway during adult development have been indicatedby a range of putative regulatory and partial loss offunction alleles, of pathway components, recovered byvirtue of their adult phenotypes (Perrimon and Maho-wald, 1986; Venderosa and Muller, 1954). Of these,alleles of upd were originally identi®ed (and named) onthe basis of their distinctive `outstretched' wing pheno-type in which wings are held out from the body of theadult ¯y and a `small eye' phenotype characterized by asmall roughened adult eye or both `outstretched-smalleye' defects (Venderosa and Muller, 1954). All of theseappear to represent regulatory alleles of the unpairedlocus in which protein expression domains or levels arealtered (Harrison et al., 1998). In addition to these updalleles a number of weak loss-of-function alleles of hophave also been identi®ed (Perrimon and Mahowald,1986; Luo et al., 1999). In di�erent hetero-alleliccombinations hop alleles can give rise to both small orno disc phenotypes as well as adult animals with held outwings and rough or disrupted eye phenotypes (Luo et al.,1999). Finally a weak stat92E allele, called stat92Ehi-jak,has also been described and shows a range of adultphenotypes, including a subtle but clearly reproducibleectopic wing vein formation (Yan et al., 1996b).

Taken together it is clear that the components of theDrosophila JAK/STAT signaling pathway are involvedin multiple aspects of adult development, includingwing vein, wing hinge and eye development. However,the precise roles of the pathway in the development ofthese tissues remain to be characterized.

Ommatidial polarity

One adult tissue in which the role of the JAK/STATpathway has been studied in detail is the eye. TheDrosophila compound eye consists of approximatelyeight hundred 20-cell subunits called ommatidia thatform the individual facets of the adult eye. Theseommatidia are formed in the eye imaginal disc androtate during development to assume dorsal andventral rotational fates (shown as green and redclusters in Figure 3a) in each hemisphere of the futureeye separated by a central line of mirror imagesymmetry known as the equator (Figure 3a andreviewed in Wol� and Ready, 1993). It has recentlybeen shown that there is a requirement for the JAK/STAT pathway for the process of ommatidial rotation(Zeidler et al., 1999b). Moreover the process of

ommatidial rotation requires not only JAK/STATactivity, but also both Wingless (Heberlein et al.,1998; Wehrli and Tomlinson, 1998), and Notchfunction (Cho and Choi, 1998; DomõÂ nguez and deCelis, 1998; Papayannopoulos et al., 1998).

While clones lacking either hop or stat92E generatedin the developing eye primordia are generally smallerthan expected, they can proliferate and give rise tonormal ommatidial clusters. Ommatidia within JAK/STAT mutant clones contain all cell types and appearto di�erentiate correctly and most rotate as antici-pated. However, ommatidia situated close to themargin of large or broad mutant regions furthest fromthe equator often assume a 1808 inverted orientation(note red clusters in Figure 3b). Consequently dorsallyoriented ommatidia can be associated with hop clonesin the ventral hemisphere of the eye and ventrallyrotated ommatidia can be associated with dorsal clones(as shown in Figure 3b). Interestingly, invertedommatidia present near the margins of hop mutantclones do not always lack JAK/STAT signaling but aresometimes comprised exclusively of wild type cells(arrows in Figure 3b). The fact that ommatidialclusters at this boundary composed entirely of wildtype cells can still be inverted suggests that the JAK/STAT pathway, which molecular evidence suggestsshould be acting autonomously (within a single cell), isactually acting in a non-autonomous manner (onneighboring cells). This unexpected non-autonomouse�ect of hop and stat92E mutant clones is thought toresult from a second, genetically downstream di�usiblemolecule that is controlled by the JAK/STAT pathwayand is able to act non-autonomously on the juxtaposedwild type tissue.

A second signal and ommatidial polarity determination

The ®nding that clones of autonomously acting JAK/STAT pathway components can give rise to non-autonomous ommatidial inversion phenotypes, whileunexpected, is not entirely without precedent. Similarresults have also been reported for the Winglesssignaling pathway (Wehrli and Tomlinson, 1998). Asis the case for hop and stat92E, autonomously actingmutants of Wingless pathway components can alsoresult in the inversion of entirely wild type ommatidiaadjacent to mutant clonal areas. The existence of non-autonomous ommatidial inversion phenotypes has beeninterpreted to suggest that both the JAK/STAT andWingless pathways, cannot be acting directly, butrather must be exerting their e�ects via the productionof one or more intermediate molecules of signalingpathways capable of acting at a distance. These factorshave been given various names including, the `secondsignal' (the term we shall use in this review), factor Xand Wnt X (Papayannopoulos et al., 1998; Wehrli andTomlinson, 1998; Zeidler et al., 1999b). The identity ofa putative second signal has remained elusive despitethe fact that a number of predictions regarding themolecular nature and expression pattern of the secondsignal can be made (Zeidler et al., 1999b). However, arecent report has described the identi®cation of such amolecule (Zeidler et al., 1999a). Four jointed (Fj) is aputative secreted type II trans-membrane molecule thatis expressed in a broad gradient across the developingeye (Figure 3c; Brodsky and Steller, 1996; Villano and

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Katz, 1995). Both fj gain- and loss-of-function clonesproduce ommatidial inversion phenotypes as predictedfor a di�usible second signal proposed to be down-stream of the Wingless and JAK/STAT pathways(Zeidler et al., 1999a). Interestingly, fj expression isregulated by both Upd and the JAK/STAT pathway aswould be expected of a bona ®de second signal. fj isdown regulated in loss-of-function clones lacking hopand is upregulated in and around clones of cells thatmisexpress Upd (Figure 3d,e). Further research isrequired to determine whether this e�ect is a directconsequence of Stat92E mediated expression as thepromoter region of four jointed has not been analysed,and details regarding the mechanism of activation bythe Upd and JAK/STAT pathway are as yet unknown.However, fj appears to ful®l the requirements for asecond signal and may well represent another devel-opmentally relevant, in vivo target of the JAK/STATpathway.

A gradient of JAK/STAT signaling activity across the eye

Analysis of an enhancer detector P-element insertionin the stat92E locus has shown this insertion (knownas STAT-lacZ) to act as an in vivo reporter of JAK/STAT pathway activity. The activity of the reporter isinversely proportional to the actual level of JAK/STAT pathway activity. Clones of cells mutant forhop result in up-regulation of STAT-lacZ while clonesof cells ectopically expressing upd strongly down-regulate STAT-lacZ, not only within the region ofUpd mis-expression, but also in surrounding tissues(Zeidler et al., 1999b). Based on these ®ndings, it isclear that the pattern of STAT-lacZ in the wild typesituation can be used to indicate the level of JAK/STAT pathway signaling. Such an analysis shows thata clear gradient of JAK/STAT pathway activity existsacross the developing eye primordia with highestactivity at the midline of the eye imaginal disc and

Equator

Figure 3 (a) A schematic cartoon illustrating the stages of ommatidial development and rotation in the eye imaginal disc. Afterbeginning their development in the morphogenetic furrow (vertical gray line) the future ommatidia grow by recruiting surroundingcells to the cluster. Dorsal (green) and ventral (red) clusters then begin to rotate and form a line of mirror image symmetry along themidline of the disc known as the equator (blue line). An example of dorsal and ventral clusters that contain all eight photoreceptorcells is shown (insert). (b) A schematic representation of an eye imaginal disc that contains a mutant clone lacking all hopscotchactivity (white background outlined in yellow) in an otherwise wild type background (gray background). While the clone is in thedorsal hemisphere of the eye and most mutant ommatidia have adopted the dorsal fate (green), ventrally oriented ommatidia (red)are present at the margin of the clone. Furthermore ommatidia entirely within the wild type (gray) region have also adopted aventral (red) fate (indicated by arrows). (c) The expression pattern of the second signal molecule four jointed in the developing eyeimaginal disc. Highest levels of expression are present at the midline of the disc. (d, e) Ectopic expression of Unpaired in a smallregion within the developing eye disc (green spot in d) causes the up-regulation of four jointed expression both in and around theUnpaired expressing clone (red in d and gray in e). (e) shows only the red channel of (d) for clarity. Arrow indicates the position ofthe Unpaired expressing clone

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lowest at the dorsal and ventral poles. The pattern ofJAK/STAT pathway activity implied by STAT-lacZ isconsistent with the pattern of Unpaired expression.Both immunological and enhancer detector basedassays show expression of Upd from an early stageat the posterior midline of the developing eye (Zeidleret al., 1999b; H Sun, personal communication). This®nding represents one of the clearest correlationsbetween the pattern of Upd and the resultant activityof the JAK/STAT pathway in vivo, further strengthen-ing the link between Upd and its role in the activationof the JAK/STAT pathway. The fact that the gradientof Upd induced JAK/STAT pathway activity (asdemonstrated by STAT-lacZ) is central to the correctorientation of ommatidia is supported by experimentsin which Upd is mis-expressed. Experiments in whichUpd is mis-expressed in domains other than themidline of the disc (where endogenous Upd is present)change the local gradient of JAK/STAT activity andhave been shown to result in ommatidial inversionphenotypes (Zeidler et al., 1999b), presumably via theresultant ectopic expression of the second signal fourjointed (Figure 3d,e). This is the ®rst case in which agradient of JAK/STAT pathway signaling activity hasbeen shown to exist, to be required for normaldevelopment, and to be the result of localizedexpression of a pathway ligand.

Redundancy in JAK/STAT signaling

Although both hop and stat92E loss of functionclones can generate ommatidial inversion phenotypes,the relative strength (or expressivity) of thesephenotypes are very di�erent. Alleles of stat92Ethought to be amorphic (total loss of function)produce e�ects at a much lower frequency thanamorphic alleles of hop (Zeidler et al., 1999b). It wasoriginally suggested that this di�erence in phenotypicstrength could be due to partial redundancy betweenstat92E and other as yet undiscovered DrosophilaSTAT molecules. However, as discussed above, thatexplanation now seems increasingly unlikely. Asstat92E probably represents the only DrosophilaSTAT, it is possible that another as yet unidenti®edmechanism exists downstream of Hop, and parallel toStat92E. While a potential explanation is simply thatan alternative STAT like molecule is too divergent tobe identi®ed by sequence searches, the precise natureof the mechanisms that would partially mediate thetransduction of Upd initiated Hop signaling remainunidenti®ed.

Future directions

Our understanding of the roles of the JAK/STATpathway during Drosophila development has advancedconsiderably in recent years and the study of thepathway is sure to be a source of new and unexpected®ndings in the years to come. However, a number ofoutstanding questions remain in our understanding ofboth the pathway itself, its functions during develop-ment and its interactions with other signal transductioncascades. The answers to these questions are likely tolie in a number of directions. While a number ofputative additional JAK/STAT pathway componentshave been identi®ed by homology, many components

of this pathway have yet to be identi®ed genetically in¯ies. Given the genetic nature of the Drosophila system,the generation and identi®cation of mutations in thesecomponents will be essential. It is conceivable thatscreens to identify mutations in these componentscould be based on a number of di�erent aspects of theJAK/STAT pathway. These include further screensdesigned to identify the characteristic JAK/STAT-likeembryonic cuticle phenotype in germline clones,suppression of the dominant phenotypes associatedwith hopTum-l or hopT42 mutations or sensitized systemsthat select candidate mutations by their interactionwith an engineered over-activation phenotype. Indeedeach of these possibilities either has been, or is being,pursued in a number of laboratories and the next fewyears should lead to a signi®cant increase in thenumber of identi®ed, genetically de®ned, JAK/STATpathway components.

A further major advance in our understanding ofJAK/STAT signaling will undoubtedly come from theanalysis of other aspects of both embryonic and adultdevelopment that require the pathway. These mayinclude other stages and tissues of embryonic develop-ment such as the trachaeal pits in which Upd isstrongly expressed (Harrison et al., 1998), larval bloodcell development and the insect immune response(Barillas et al., 1999). Moreover, the analysis of otherstages of adult development such as wing vein andwing hinge defects observed to be part of existingpartial loss-of-function pathway mutant phenotypesmay also prove fruitful.

In addition to these developmental roles, it isbecoming increasingly clear that the JAK/STAT path-way cannot function in isolation. Indeed analysis of eveexpression in mutant embryos indicates that while theJAK/STAT pathway is undoubtedly important in theregulation of eve stripe 3 expression, additional signalsfeed in to maintain low levels of eve expression in thisthird stripe even in the total absence of JAK/STATactivity (Hou et al., 1996; Yan et al., 1996a). In additionthe proper expression of fj in the developing eye is notsimply a consequence of Upd/JAK/STAT signaling butrather appears to require the additional involvement ofWingless signaling, Notch activity and an as yetuncharacterized component of fj autocrine feedback(Zeidler et al., 1999a). No doubt similar complicationswill arise with regard not only to JAK/STAT signaling,but a wide range of signal transduction responses as thetools available and our resultant understanding of thesystems being analysed continue to improve.

One such set of tools which would facilitate ourunderstanding of JAK/STAT signaling would bereagents with which to visualize the activity of thepathway in vivo. While STAT-lacZ is able to reportJAK/STAT activity in imaginal discs this does notrepresent an assay suitable for individual cells or tissueculture based systems and additional techniques wouldalso be useful. Given the recent development and useof antibodies speci®c for activated forms of DrosophilaERK (Gabay et al., 1997) and the commercialavailability of phospho-speci®c forms of mammalianSTATs, it seems plausible that similar antibodies thatrecognize activated JAK or STAT could be developed.Alternatively the subcellular translocation of STATinto the nucleus following activation may also providethe basis of such techniques.

Oncogene

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2605

AcknowledgmentsThe authors would like to thank Thomas Cline, Henry Sunand David Strutt for sharing results prior to publication,Eric Spana for Drosophila genome project sequencesearches, and Susan Smith for comments on the manu-

script. MP Zeidler is a Leukemia Society of AmericaSpecial Fellow, EA Bach is a Fellow of The Jane Co�nChilds Fund for Medical Research and N Perrimon is anInvestigator of the Howard Hughes Medical Institute.

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