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. Author manuscript; available in PMC: 2011 May 9.
Published in final edited form as: Mech Dev. 2003 Dec;120(12):1455–1468. doi: 10.1016/j.mod.2003.09.004

Patterns and functions of STAT activation during Drosophila embryogenesis

Jinghong Li a, Wenjun Li b, Healani C Calhoun a, Fan Xia a, Fen-Biao Gao b, Willis X Li a,*
PMCID: PMC3090291  NIHMSID: NIHMS288095  PMID: 14654218

Abstract

The JAK/STAT pathway mediates cytokine signaling in mammals and is involved in the function and development of the hematopoietic and immune systems. To investigate the biological functions of the JAK/STAT pathway during Drosophila development, we examined the tissue-specific localization of the tyrosine-phosphorylated, or activated form of Drosophila STAT, STAT92E. Here we show that during Drosophila embryonic development STAT92E activation is prominently detected in multiple tissues and in different developmental stages. These tissues include the tracheal pits, elongating intestinal tracks, and growing axons. We demonstrate that stat92E mutants are defective in tracheal formation, hindgut elongation, and nervous system development. Conversely, STAT92E overactivation caused premature development of the tracheal and nervous systems, and over-elongation of the hindgut. These results suggest that STAT activation is involved in proper differentiation and morphogenesis of multiple tissues during Drosophila embryogenesis.

Keywords: Drosophila, Janus kinase, Signal transducer and activator of transcription, Morphogenesis, Embryogenesis, Trachea, Gut, CNS, PNS

1. Introduction

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was initially described as mediating cytokine signaling in mammalian hematopoietic and immune systems (reviewed by Aaronson and Horvath, 2002; Ihle, 2001; Levy and Darnell, 2002). In the stereotypical model, activation of the JAK/STAT pathway is initiated by binding of extracellular ligands to the transmembrane cytokine receptor monomers, leading to juxtaposition and dimerization of the receptor. This results in the phosphorylation and activation of the receptor-associated tyrosine kinase JAKs. Subsequently, the cytoplasmic tail of the receptor is phosphorylated on Tyr residues, which provides docking sites for STAT binding through SH2 domain-phosphotyrosine interaction. STAT activation results from phosphorylation by JAK on a crucial C-terminal tyrosine residue. This phosphorylation event is necessary and sufficient for STAT dimerization and activation (Shuai et al., 1993). Upon activation, STAT dimers translocate to the nucleus where they bind to the cognate TTCNNNGAA sites and induce expression of target genes (reviewed by Aaronson and Horvath, 2002; Ihle, 2001; Levy and Darnell, 2002).

Although JAK/STAT signaling is primarily known to function in hematopoiesis and immune responses in mammals, the involvement of members of this pathway in cell proliferation, survival, and cancer progression has increasingly being recognized (reviewed by Bromberg, 2001). For instance, STAT3 appears to mediate v-src mediated cellular transformation and its constitutive activation is associated with many types of human cancers (Bromberg et al., 1998; Turkson et al., 1998). Furthermore, a constitutively activated STAT3, when expressed in cultured cells, behaves as an oncogene and confers on these cells the ability to form foci in soft agar and tumors in nude mice. Thus, STAT activation alone appears sufficient to promote tumorigenesis (Bromberg et al., 1999). Despite these observations, relatively little is known about the general biological functions of STAT activation during the development of a metazoan. Recent studies of genetically tractable organisms, such as Drosophila, have provided insights into developmental functions of this signaling pathway. These studies were aided by the fact that Drosophila has only a single JAK (Hopscotch, Hop) (Binari and Perrimon, 1994) and a single STAT protein (STAT92E; also known as Marelle) (Hou et al., 1996; Yan et al., 1996). In contrast, mammals have evolved multiple species of JAKs and STATs. The biological functions of STAT92E and Hop thus may represent those of the founding members of the ancestral JAK/STAT pathway.

The Hop/STAT92E pathway in Drosophila has been found to play roles in a variety of contexts (reviewed by Hou et al., 2002; Luo and Dearolf, 2001). The genes of this pathway, hop, stat92E, and unpaired (upd; encoding an extracellular ligand) (Harrison et al., 1998), were isolated based on their identical mutant larval cuticular phenotypes, characterized by the loss of the fourth and fifth ventral abdominal denticle belts (A4 and A5). In the early embryo, Hop and STAT92E are essential for the correct expression of a number of segmentation genes, including even-skipped (eve) and runt, that are expressed in alternating parasegments, forming seven stripes along the anteroposterior axis (Yan et al., 1996). A receptor for the HOP/STAT92E pathway has recently been identified in Drosophila. Domeless (also known as Master of Marelle; Mom) is related to mammalian cytokine receptors (Brown et al., 2001; Chen et al., 2002) and has been shown to mediate the activation of Hop by the extracellular ligand Upd in cultured cells (Chen et al., 2002). Similar to the mammalian system where the JAK/STAT pathway is crucial for hematopoiesis, Hop and STAT92E are required for the differentiation and proliferation of the fly blood lineage (reviewed by Hou et al., 2002; Luo and Dearolf, 2001). In addition to embryonic segmentation and hemolymph development, the Hop/STAT92E pathway has recently been shown to play roles in a number of tissues and processes. These include imaginal disc cell proliferation and the establishment of ommatidial polarity of the eye (Luo et al., 1999; Zeidler et al., 1999), male germ-line stem cell self renewal during spermatogenesis (Kiger et al., 2001; Tulina and Matunis, 2001), transcriptional regulation of the sex determination master switch gene Sex-lethal (Sxl) (Jinks et al., 2000; Sefton et al., 2000), and, more recently, ovarian border cell migration and follicle cell differentiation and fate determination (Baksa et al., 2002; Beccari et al., 2002; Silver and Montell, 2001; Xi et al., 2003). Thus, the Hope/STAT92E pathway appears to play diverse roles and is involved in many aspects of cellular functions including cell fate determination, proliferation, differentiation, and migration.

In order to gain a more comprehensive knowledge of the developmental functions of the JAK/STAT pathway in a multicellular organism, we examined the patterns of STAT activation during Drosophila development. We employed an antibody that was raised against the phosphorylated (or activated) STAT92E (pSTAT92E; see Section 4) to analyze the localization of STAT92E activation in whole-mount embryos of different developmental stages. Such immunohistochemical studies revealed patterns of STAT92E activation in diverse tissues that are undergoing morphogenetic movements during Drosophila embryogenesis. We have studied the functions of STAT92E in the primordial germ cells and demonstrated essential roles for STAT92E activation in the proliferation, migration, and adhesion of these cells during gonadogenesis (J. Li, F. Xia, and W. X. Li, submitted). Here, we report the patterns of STAT92E activation and the effects of stat92E mutations on the development of the trachea, hindgut, and embryonic nervous system. Taken together with the roles of STAT92E in directed cell migration during gonadogenesis and oogenesis, these studies suggest an essential role of STAT92E activation in morphogenesis and differetiation of multiple tissues during Drosophila embryogenesis.

2. Results

2.1. Detection of STAT92E activation in vivo

We confirmed the specificity of the anti-pSTAT92E antibody used in this study by the following experiments. First, we stained whole-mount embryos of different genetic backgrounds. In wild-type embryos, pSTAT92E signals were detected in the central region as well as terminal regions of the embryo (Fig. 1A). The central region staining resolved to weak pair rule-like stripes at cellular blastoderm stage (not shown). During gastrulation (at stage 8–10), STAT92E activation was detected in 14 stripes in the parasegments across all germ layers (Fig. 1B). These patterns were very similar to those of upd expression (Harrison et al., 1998), which is consistent with the expectation that Upd triggers STAT92E activation in these domains. Moreover, the pSTAT92E signals were completely absent in embryos lacking maternal stat92E product (referred to as stat92Emat− embryos; see Section 4; Fig. 1C). The detection of pSTAT92E signals at stage 8–10 was dependent on hop, as is evident by the absence of the 14 parasegmental stripes of pSTAT92E signals in embryos lacking maternal hop (referred to as hop mat− embryos; Fig. 1D). Second, in Western blots, a protein species corresponding to the phosphorylated STAT92E can be detected that is higher in intensity in protein extracts from embryos harboring a gain-of-function hop mutation, hop GOF (see Section 4), and is absent or much reduced in extracts from hopmat− embryos (Fig. 1E). Third, following an activation of STAT92E in Schneider 2 (S2) cells by vanadate/peroxide treatment (Li et al., 2002; Sweitzer et al., 1995) (see Section 4), we detected a strong increase in immunohistochemical staining with the anti-pSTAT92E antibody but not with an antibody to the unphosphorylated STAT92E (Fig. 1F). These results demonstrate that the anti-pSTAT92E antibody is specific for the activated STAT92E and is suitable for detecting STAT92E activation in whole-mount tissues.

Fig. 1.

Fig. 1

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Specificity of the anti-phospho-STAT92E antibody. (A) STAT92E activation was detected using an anti-pSTAT92E antibody (brown) in the central and terminal regions of stage 5 wild-type embryos. (B) pSTAT92E signals resolve into 14 parasegmental stripes as well as head and gut regions in stage 9 wild-type embryos. (C) This pattern was absent in stage 5 stat92E mat− embryos, and (D) was greatly diminished in hop mat− embryos. All embryos are arranged anterior to the left and dorsal up. (E). Western blot of protein extracts from hop GOF, wild-type, and hop mat− embryos. Note the band corresponding to pSTAT92E has a higher intensity in the hop GOF lane than wild type and is much reduced in the hop mat− lane. (F). S2 cells treated with vanadate/H2O2 for 0 min (untreated) or 30 min to stimulate STAT92E signaling were stained with the anti-pSTAT92E and anti-STAT92E antibodies, respectively. Note increased staining by anti-pSTAT92E, but not regular STAT92E, antibody following 30 min vanadate/H2O2 treatment.

2.2. STAT92E activation in tracheal formation

Following the disappearance of the parasegmental stripes, in stage 11–13 embryos, we detected persistent pSTAT92E staining in the tracheal pits (Fig. 2A,B), which are structures that mark the initial phase of tracheal development and are formed by the invagination of ten pairs of segmental ectodermal cell clusters that are fated to become trachea (Zelzer and Shilo, 2000). The tracheal cell fate is determined by the gene trachealess (trh), encoding a bHLH/PAS family transcription factor, which is expressed in these ectodermal cell clusters prior to their invagination (Wilk et al., 1996). Tracheal formation in Drosophila embryos takes place by the invagination, migration, and elongation of predetermined sets of epithelial cells without further cell division (reviewed by Zelzer and Shilo, 2000). The observation of pSTAT92E in the tracheal pits is consistent with the expression patterns of the ligand Upd (Harrison et al., 1998), suggesting that STAT92E activation may play a role in the invagination of the epidermis that leads to the formation of the trachea. Indeed, we found that in stat92E zygotic null (stat92Ezyg−) embryos, the tracheal pits formed later and remained smaller than in their wild-type siblings (cf Fig. 2C,E), and in late stages, they exhibited severe defects in the tracheal branches (cf Fig. 2D,F). Moreover, in embryos lacking both maternal and zygotic stat92E gene products (stat92Emat−zyg− embryos), all of the tracheal pits were missing and little or no tracheae were found in the later stage embryos (Fig. 2G,H). These results are consistent with the phenotypes of embryos mutant for stat92E and other components of this pathway (Brown et al., 2001; Chen et al., 2002). In addition, we found that in hop GOF embryos, the tracheal pits were larger than in wild-type embryos (Fig. 2I).

Fig. 2.

Fig. 2

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STAT92E activation in tracheal formation. Detection of STAT92E activation (brown in A,B) in tracheal pits of stage 11 (A) and stage 13 (B) wild-type embryos. Inset in (A) shows higher magnification of two tracheal pits. Note pSTAT92E staining surrounding the pits. The morphology of the tracheal pits was analyzed by an anti-Crumbs (Crb) antibody (Tepass et al., 1990) (brown in C,E,G,I), which is expressed in epithelia of ectodermal origin, including tracheal pits. The tracheal branches were visualized by staining with the monoclonal antibody mAb2A12 (brown in D,F,H) specific for the tracheal system. At stage 11, the tracheal pits were smaller in stat92E zyg− (E; cf C), nearly absent in stat92E mat− embryos (G), and larger in hop GOF embryos (I). The tracheal branches were broken in stat92E mat+zyg− (F; cf D) and nearly absent in stat92E mat− embryos (H). (J) A schematic illustration of STAT92E activation (red) during tracheal invagination. (K,L) Expression patterns of trh mRNA, detected by anti-sense RNA probes made from the 5’ UTR of the trh gene. Note trh expression in stat92E mat+zyg− embryos (L) was mostly normal compared with that of wild type (K).

The lack of trachea in stat92E mat−zyg− embryos may be explained by a failure in trachea specification, as trh is not expressed in the tracheal pits of these embryos (Brown et al., 2001). In addition, we propose that STAT92E activation may also be important for tracheal differentiation beyond cell fate specification based on the following observations. First, STAT92E activation persists long after the tracheal cell fate has been determined (see Fig. 2A,B). Second, the phenotypes of stat92E zyg− embryos were more consistent with defects in tracheal elongation and branching rather than cell fate specification. This is because we detected tracheal pit invagination, though smaller than wild type, and limited growth of dorsal truck in all segments of stat92E zyg− embryos, suggesting that these cells had been correctly specified (Fig. 2E,F). However, there appeared to be a failure in fusion of these rudimentary structures to form a continuous longitudinal dorsal chunk (Fig. 2F). A failure of tracheal cell fate determination would have resulted in missing tracheal pit invagination such as exhibited by trh or stat92E mat−zyg− embryos (Fig. 2G; Isaac and Andrew, 1996; Wilk et al., 1996). Consistent with this interpretation, we found normal trh expression in the tracheal pits of stat92E zyg− embryos (Fig. 2L). Taken together, these results suggest that STAT92E activation plays dual roles in tracheal development—specifying the tracheal cell fate and promoting the invagination, elongation, and migration of tracheal cells.

2.3. STAT92E activation in hindgut development

In stage 14 embryos, the activation of STAT92E was detected in parts of the foregut (not shown) and the anterior region of the elongating hindgut (Fig. 3A), suggesting that STAT92E may have a function in the development of the intestinal tract. During late embryogenesis, the hindgut elongates by cell rearrangement without changes of cell number (Lengyel and Iwaki, 2002). The region where pSTAT92E signals were detected at the highest levels in the hindgut corresponds to the small intestine, which leads hindgut elongation by cell rearrangement during late embryogenesis (reviewed by Lengyel and Iwaki, 2002). We measured the hindgut length and cell number of stage 16 embryos of different genetic backgrounds (see Section 4). We found that the hindgut of stat92E mat− zyg−embryos was shorter and had fewer cells than those of wild type (Table 1; also see examples in Fig. 3C,H–J), suggesting a failure of stat92E mutants in hindgut elongation and/or cell division. Conversely, in hop GOF embryos, the hindgut appeared significantly longer than wild type, although there were only slightly (statistically insignificant) more cells (Table 1; also see Fig. 3D,K–M), suggesting that STAT92E activation is sufficient for promoting hindgut elongation without changing cell number. Results from our loss of function experiments are consistent with a recent report by Johansen et al. (2003), who showed an essential requirement for localized JAK/STAT signaling in oriented cell rearrangement and hindgut elongation. However, Johansen et al. (2003) showed that expressing gain-of-function molecules of the Hop/STAT92E pathway components in the hindgut resulted in the same phenotypes as those of loss-of-function mutants, namely shorter and wider hindguts. This is in contrast to our results of gain-of-function experiments using a hop GOF allele (Luo et al., 1995) or by low-level uniform expression of UAS-hop using the hsp70-Gal4 at 25 °C (see Section 4). Both methods presumably resulted in moderately elevated levels of Hop signaling, as the majority of the embryos in these situations hatched and remained viable (not shown). The phenotypes of these hop GOF embryos, namely longer hindgut and some other internal tubule structures (see Fig. 3D), are the opposite of the loss-of-function mutants, which would be expected if higher levels of Hop/STAT92E signaling promotes the elongation of tubule structures.

Fig. 3.

Fig. 3

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STAT92E activation in hindgut elongation. (A) Detection of STAT92E activation (brown) in the anterior region of a stage 15 wild-type embryo (arrow). (B–D) Examples of stage 16 embryos of different genotypes stained by Anti-Crb (brown) antibody, which identifies the hindgut. Note the hindgut (arrow) is shorter and wider in stat92E mat−zyg− (C) and longer in hop GOF (D) embryos. stat92E mat−zyg− embryos exhibit defective midgut constriction and thus may appear younger. Hindguts of propidium iodide-stained stage 16 embryos of wild-type (E,F,G), stat92E mat−zyg− (H,I,J), and hop GOF (K,L,M) are shown. Note the nuclei in stat92E mat−zyg− hindgut are staggered, as if to form a double layer, while those in hop GOF hindgut are more stretched than wild type. (N) A proposed model for hindgut elongation without changing cell number.

Table 1.

Hindgut length and cell number in stage 16 wild-type and different mutant embryos

Genotype Hindgut length ± SD (μM) P-value Hindgur cell Number ± SD P-value
Wild type 223 ± 29 − 636 ± 62 −
stat92E mat− zyg− 92 ± 20 6.8 × 10−5 561 ± 109 3.2 × 10−4
hop GOF 318 ± 36 0.002 697 ± 78 0.22
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SD: standard deviations. P-value is calculated by Student’s t-Test, P < 0.05 indicates a significant difference when compared with wild type. More than 5 stage 16 embryos were counted for each genotype.

To further investigate the function of STAT92E activation in hindgut elongation, we examined in detail the relative positions of hindgut nuclei in propipium iodide-stained stage 16 embryos of different genetic backgrounds. The most significant difference exhibited by stat92E loss-and gain-of-function mutants appeared to be the position of hindgut nuclei relative to each other. Specifically, the nuclei in the hindgut of stat92E mat−zyg− embryos were staggered into double layers in the gut epithelium, making it thicker than that of wild type (Fig. 3H–J). In contrast, the hindgut nuclei in hop GOF embryos formed a single layer and were farther apart from each other, giving the appearance of being stretched (Fig. 3K–M). The wild-type hindgut nuclei, on the other hand, appeared to have an intermediate arrangement—slightly staggered while still forming a single layer of epithelium (Fig. 3E–G). Based on these observations, we propose that STAT92E activation-dependent hindgut elongation takes place by cell rearrangement in which a double-layered thicker epithelium stretches into a single-layered one (Fig. 3N). This model is in principle in agreement with but slightly different than that proposed by Lengyel and Iwaki (2002), who depicted an early hindgut as single cell-layered but with a much wider diameter. Consistent with our model, we noticed that in stage 11 wild-type embryos, the hindgut does have a thicker double-layered epithelium similar to that of late stage stat92E matzyg embryos (not shown; also see Fig. 4E in Lengyel and Iwaki, 2002). In summary, our results from the analysis of phenotypes of stat92E mutant and overactivation support the notion that the Hop/STAT92E pathway plays a crucial role in the cell rearrangement during hindgut elongation.

2.4. Roles of STAT92E in the development of the nervous system

We found that at stage 14, STAT92E activation was detectable in the embryonic nervous system, with prominent antibody labeling of the axon fibers of the ventral nerve cord (Fig. 4A). This staining was absent in stat92E mat− or hop mat− mutant embryos (Fig. 4B,C), suggesting that the staining was specific. To test the possibility that STAT92E activation might be involved in the development and/or function of the nervous system, we examined axon development in both the central and peripheral nervous system (CNS and PNS) of stat92E mutant embryos. We used the monoclonal antibody BP102 (Seeger et al., 1993) that specifically labels both longitudinal connectives and commissures of the CNS (Fig. 4D) and 22C10 that recognizes the microtubule associated Futsch protein in cell body and axons of all PNS (Fig. 5A) as well as a subset of CNS neurons (Hummel et al., 2000) (Fig. 4G,I). In stat92E mat−zyg− embryos, the organization of the CNS was grossly disrupted and some segments of the CNS were completely missing (Fig. 4E), Similar defects, though to a lesser extent, were also found in stat92E mat−zyg+ embryos, in which the segmentation defects were less severe (Fig. 4F). The CNS defects, namely, gaps in the longitudinal tracks and missing commissures, could be explained by an early requirement for STAT92E in segmentation and/or neuronal cell fate determination (Hou et al., 1996) and are consistent with a previous finding that hop mutant embryos exhibit gaps in the ventral nerve cord (Perrimon and Mahowald, 1986). However, since we detected STAT92E activation in axons, long after cell fate determination, we speculated that lack of STAT92E activation might additionally cause defects in the growth and organization of axonal projections.

Fig. 4.

Fig. 4

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STAT92E activation and requirement in CNS development. Anti-pSTAT92E staining (brown in A,B,C) is detected in the axon fibers of the CNS of wild-type (A) but not stat92E mat− (B) or hop mat− (C) embryos. Stage 15 embryos stained with the monoclonal antibody BP102 (D,E,F) that recognizes CNS axons, and with the monoclonal antibody 22C10 (G,H,I,J), which identifies a subset of CNS neurons and their axons. (D) A wild-type CNS showing the ventral nerve cord. Note the continuous longitudinal fibers along the midline and transversal fibers that form two commissures crossing the midline in each segment. CNS of stat92E mat−zyg− (E) and stat92E mat−zyg+ (F) embryos exhibited structural defects. Note the broken longitudinal tracks and missing commissures (arrow). CNS of stage 15 (G,H) and stage 13 (I,J) wild-type (G,I) and stat92E mat+zyg− (H) and stat92E mat−zyg+ (J) embryos stained with mAb22C10. Note the aCC and pCC motorneurons (arrowhead) project axons (arrows) in wild-type but fail to do so in stat92E mutant embryos. (K) A schematic illustration of neuronal projection patterns in wild-type and stat92E mutant CNS. Dotted lines indicate segmental borders. AC and PC indicate anterior and posterior commissures.

Fig. 5.

Fig. 5

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Requirement for STAT92E in PNS axonal growth. PNS neurons are detected by 22C10 staining. (A) A lateral view of a stage 14 wild-type embryo showing PNS. (B) Higher magnification of two adjacent dorsal and lateral PNS neuronal clusters. (C) A stat92E mat−zyg+ embryo of the same stage. Note that many neurons are missing axonal projections. (D) Higher magnification of two adjacent dorsal and lateral PNS neuronal clusters. The arrow indicates the absence of axons from the dorsal cluster PNS neurons in a stat92E mat−zyg− embryo. (E) In a small number of stat92E mat+zyg− embryos (5/92), one or two dorsal clusters failed to extend their axons (arrow). (F) A schematic illustration of the organization of a dorsal and a lateral PNS cluster. (G) A stage 12 hop GOF embryo, in which certain PNS neuronal clusters have prematurely extended their axons. (H) A higher magnification showing differentiated dorsal and lateral clusters. (I) No 22C10-positive PNS neurons were detectable in wild-type embryos at this stage.

To determine whether STAT92E activation plays a role in axonal growth, we examined a few identifiable mAb22C10-positive CNS neurons in stat92E mat−zyg+ embryos, in which the segmentation defects were less severe, presumably due to paternal rescue. The CNS neurons prominently stained by mAb22C10 include the anterior and posterior corner cells (aCC and pCC) that project axons laterally and the ventral unpaired median neurons (VUM), which send axons that bifurcate at the anterior commissure (Goodman et al., 1984) (Fig. 4G,I,K). In stage 15 stat92E mat−zyg+ and a small number of stat92E mat+zyg− embryos (Fig. 4H), these neurons were present, but often failed to grow axons (arrowheads in Fig. 4G,H). These neurons start to grow axons in stage 13 wild-type embryos (Fig. 4I). In contrast, in stage 13 stat92E matzyg embryos, we found that the aCC, pCC, and VUM neurons were born but failed or were delayed in sending axons (Fig. 4J). Therefore, we conclude that a failure of axonal growth contributed to the CNS defects exhibited by stat92E mutants.

We also studied the role of STAT92E in PNS development and found that, consistent with the defects exhibited by CNS neurons, many PNS neurons failed to extend their axons in stat92E mat− zyg+ embryos (Fig. 5C,D). The stat92E mat−zyg− embryos exhibited much more severe phenotypes and many neurons were absent, presumably resulting from a multitude of developmental defects (not shown). As some PNS neurons in the same embryos were able to extend their axons, it is unlikely that the failure of these neurons to extend axons was due to premature cessation of development of stat92E mat− zyg+ embryos (see Fig. 5D). Furthermore, in a small number of the stage 15 stat92E mat+zyg− embryos, which do not exhibit any segmentation defects, the neurons in one or two dorsal clusters did not grow axons (Fig. 5E). Conversely, when STAT92E was overactivated, such as in hop GOF embryos, PNS neurons prematurely differentiated and grew axons (Fig. 5G,H). In wild-type embryos, PNS neurons start to appear during stage 13 and will not fully differentiate until stage 15. In hop GOF mutants, in contrast, many of them (17/68) developed their PNS earlier. A few (as shown in Fig. 5G,H) exhibited nearly fully differentiated PNS as early as stage 12. No 22C10-positive PNS neurons were detectable in wild-type embryos at this stage (Fig. 5I). These results suggest that STAT92E activation is necessary for axonal extension and, when overactivated, it can also promote axonal growth.

In summary, it appears that STAT92E activation may play a role in axonal growth in both CNS and PNS. As with the tracheal system, it is possible that the Hop/STAT92E pathway is involved in both cell fate specification and morphogenetic movements or growth of particular cell types.

2.5. STAT92E activation in extra-embryonic cells and during gastrulation

Throughout embryogenesis, pSTAT92E staining was noticeably detected in certain extra-embryonic cells of unknown function. These were large cells with filopodia-like cytoplasmic extensions found outside of and attached to the epidermis of late-stage wild-type embryos (Fig. 6G) and were absent in stat92E mat−zyg− embryos (not shown). Such pSTAT92E-positive cells were first detectable in the amnioserosa at the start of germband retraction (Fig. 6A) and were often found next to the leading edge of the lateral epithelia during dorsal closure at stage 13–14 (Fig. 6B), suggesting that these extra-embryonic cells may be of amnioserosa origin and have become mesenchymal during germband movements and dorsal closure. This is confirmed by a detailed confocal analysis on the relative position of these cells during the closure of the dorsal epidermis (Fig. 6C–F). We therefore postulate that these extra-embryonic cells have detached from their original location, probably the amnioserosa membrane that originates from the dorsal epidermis, and become migratory. Interestingly, as for neuronal axons, pSTAT92E staining in these cells was confined to the membrane and cytoplasm. Moreover, a brief heat shock-induced expression of hop caused the appearance of many more of such pSTAT92E-positive cells with longer filopodial extensions (Fig. 6H), suggesting that STAT92E activation plays a role in the production of these extra-embryonic cells. Regardless of their function, the appearance of these cells is consistent with a role of STAT92E activation in their detachment from their original locations and transition to become migratory, a phenomenon analogous to the formation of the ovarian border cells from the follicle epithelium (Beccari et al., 2002; Silver and Montell, 2001).

Fig. 6.

Fig. 6

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STAT92E activation in extra-embryonic cells and during gastrulation. STAT92E activation (brown in A,G,H; green in B,D,E) was detected in large cells attached to the epidermis. These cells were initially detected in the amnioserosa at stage 11–12 embryos (A) and, at stage 13–14, next to the leading edge during dorsal closure (B). (C–E) An embryo with puckered-lacZ, a leading-edge marker, was double-stained with anti-βGal (red) and anti-pSTAT92E (green). Merged image (E) shows pSTAT92E large cells abutting the leading edge. (F) A schematic interpretation of (C)–(E), showing pSTAT92E amnioserosa cells escaping the closing epidermis. (G) A side-view of a stage-15 wild-type embryo showing about five pSTAT92E-positive extra-embryonic cells on the lateral epidermis. The ventral nerve cord (out of focus) is also stained by the anti-pSTAT92E antibody. Inset: higher magnification of an extra-embryonic cell. Note filopodia-like cellular extensions. (H) A ventral view of a stage-15 embryo carrying hsp70-Gal4 and UAS-hop transgenes following a brief heat-shock. Note there are more pSTAT92E-positive extra-embryonic cells and some of them extended long cellular processes (compare with G). Inset: higher magnification of a few extra-embryonic cells; one with a long extension. (I) A stage 8 wild-type embryo showing pSTAT92E signal in dorsal folds (df), cephalic furrow (cf), and the invaginating hindgut primordium (hg). (J). Detection of pSTAT92E was much reduced in hop mat− embryos.

Finally, consistent with a role of STAT92E activation in cell movements and shape changes, we detected elevated pSTAT92E signals in invaginating epithelial cells of the gastrulating embryo (Fig. 6I). These structures include the dorsal folds (df), cephalic furrow (cf), and invaginating hindgut primordium (hg). As expected, pSTAT92E signals in these structures were not detected in stat92E mat− embryos (not shown), although no obvious gastrulation defects were found in these embryos. In hop mat− embryos, STAT92E activation in these folding structures was much reduced but not completely eliminated (Fig. 6J), suggesting that Hop may not be totally responsible for activating STAT92E, at least at early stages of embryogenesis. Indeed, we have evidence that the RTK Torso is able to activate STAT92E in the absence of Hop (Li et al., 2002). It is thus possible that other tyrosine kinases are also able to activate STAT92E.

3. Discussion

The JAK/STAT pathway is best known for transducing cytokine signals and primarily functions in the hematopoietic and immune systems in mammals (reviewed by Aaronson and Horvath, 2002; Ihle, 2001; Levy and Darnell, 2002). However, the cellular biological functions of this pathway in animal development are less well understood. Here we report the use of an antibody specific for the activated STAT92E to detect tissue-specific STAT92E activation during Drosophila embryogenesis and further genetic analyses of the functional requirement for STAT92E in the development of these tissues. Such studies revealed an essential requirement for STAT92E in a number of developmental processes that involve cell shape changes and/or rearrangements. Results from these studies highlight a fundamental role of the JAK/STAT pathway in tissue development, differentiation, and morphogenesis.

A common denominator for the diverse tissues and processes in which we detected STAT92E activation appears to be cell movements. In tracheal development, the tracheal pits form by the invagination of a group of predetermined epidermal cells and the elongation and migration of these cells to form a network of tracheal branches in the absence of further cell division (Zelzer and Shilo, 2000). During hindgut elongation, cells rearrange without mitosis to form a thin, long tubule (Lengyel and Iwaki, 2002). Axon growth during the development of the nervous system represents a type of cell movement that involves a dramatic increase of cell membrane-based and actin-rich projections. Interestingly, the mysterious extra-embryonic migratory cells can extend long cellular projections (see Fig. 6), resembling neurons or fibroblasts in morphology. Finally, we also detected STAT92E activation in cells that are involved in shape changes during gastrulation, including those in the dorsal folds, cephalic furrows, invaginating posterior midgut rudiment and hindgut primordium. In addition to cell shape changes, guided and/or invasive cell migrations, as represented by the behaviors of the ovarian border cells and primordial germ cells, are also key features of morphogenetic movements essential for animal development. The invasive migration of both types of cells has been shown to require STAT92E activation (Beccari et al., 2002; Silver and Montell, 2001) (J. Li, F. Xia, and W.X. Li, submitted). Taken together, these observations seem to suggest that STAT activation may be fundamental to cell movements and shape changes. However, it is also noted that not all tissues that undergo morphogenesis exhibit prominent pSTAT92E staining, and therefore we did not investigate the requirement for STAT92E activation in these tissues. The tissues or biological processes that were not affected by the stat92E mutation or not investigated in this study include but not limited to dorsal closure, mesoderm formation and migration. Based on tissue and developmental stage-specific detection of STAT92E activation and our phenotypic analyses, we conclude that STAT92E activation is involved in at least a subset of morphogenetic movements during Drosophila embryogenesis. It would be interesting to investigate to what extent STAT activation is involved in morphogenesis in general and whether and how STAT activation collaborates with other signaling pathways to regulate morphogenetic movements.

Although STAT92E is prominently activated in a number of tissues undergoing morphogenetic changes and a mutation of stat92E exhibited developmental defects in these tissues, we cannot rule out a role of STAT92E activation in the initial specification of the cell types that constitute these tissues. This may be particularly true for the trachea and nervous system. Ideally, to distinguish the role of STAT92E activation in cell fate specification versus differentiation, mosaic analysis of marked mutant cells lacking STAT92E would be preferable. We have employed the ‘mosaic analysis with a repressible cell marker (MARCM)’ technique (Lee and Luo, 1999) to study the function of STAT92E in the embryonic PNS development by generating positively marked stat92E zygotic null neurons. Unfortunately, this effort is hindered by the abundant maternal contribution of the stat92E gene product and we could not find any defects in dendrites or axons of single stat92Ezyg− mutant PNS neurons in mosaic animals (not shown). Based on the finding that activated STAT92E is detected in postmitotic neurons, and that nearly all stat92Emat−zyg+ mutant embryos and a small number of neurons in stat92Emat+zyg− mutant embryos fail to extend their axons properly, we propose that the axonal phenotype is at least in part due to loss of STAT92E function in postmitotic neurons.

It has recently been shown that oriented cell rearrangement and hindgut elongation require localized JAK/STAT signaling (Johansen et al., 2003). Consistent with the results from our analysis of stat92E mutants, Johansen et al. (2003) demonstrated that mutants of a number of Hop/STAT92E pathway components exhibit shorter and wider hindgut, possibly as a result of defective cell rearrangement (Johansen et al., 2003). However, these authors showed that overactivating the Hop/STAT92E pathway in the hindgut had effects that were identical to a lack of STAT92E activation. This is in contrast to our results of gain-of-function experiments. We found that hop GOF embryos have phenotypes that are the opposite of that of the loss-of-function mutants, namely longer hindgut and other internal tubule structures (see Fig. 3D). The latter would be expected if overactivating the STAT92E pathway promotes hindgut elongation. Johansen et al. (2003) interpreted their results as to suggest that spatially restricted activation of the Hop/STAT92E pathway is necessary for hindgut elongation, whereas high-level uniform activation of this pathway is not compatible with oriented cell rearrangement. A plausible explanation to account for the different outcomes of the two sets of gain-of-function experiments is that the expression levels of these gain-of-function molecules are critical for the oriented cell rearrangement. Modestly higher levels of Hop/STAT92E signaling, while preserving the endogenous signaling gradient, may promote hindgut elongation, whereas too high levels of signaling could abolish the gradient along the gut and prevent oriented cell rearrangement.

Embryonic development involves a series of programmed morphogenetic movements that include bending, folding, and invaginating of epithelial tissues in order to form various organs as well as the final shape of the body. Cell migration, shape changes, and rearrangements underlie most of the morphogenetic movements essential for gastrulation and organogenesis during Drosophila embryonic development. Although it has been shown that actin-based cytoskeletal reorganization plays a crucial role in cell shape changes, little is known about the signaling pathways that trigger these morphogenetic movements during embryogenesis. The identification of STAT as a potential regulator of morphogenetic movements posed an interesting question regarding the involvement of STAT activation in the transcriptional regulation of genes required for these movements. Indeed, STAT92E has been shown to be involved in the transcriptional activation of many signaling molecules as well as key transcription factors (Hou et al., 2002; Li et al., 2002; Luo and Dearolf, 2001; Silver and Montell, 2001). A recent systematic search for STAT target genes has revealed a plethora of genes that might be directly activated by STAT92E, among which are those involved in the regulation of cytoskeletal movements and actin reorganization (F. Xia and W.X. Li, unpublished results). Elucidation of the target genes of STAT and the extracellular signals that lead to STAT activation should shed light on the molecular mechanisms that govern morphogenetic movements. It remains to be elucidated whether STAT92E directly activate these genes or whether it acts in collaboration with yet unidentified signaling pathways.

Finally, the JAK/STAT signaling pathway has also been extensively studied in model organisms other than Drosophila, and a general role of this pathway in morphogenesis and cell movement is beginning to emerge (Hou et al., 2002). For instance, in Dictyostelium discoideum, Dd-STATa is required for cell movement in the prestalk region in response to cAMP signals through a unique mechanism (Kawata et al., 1997). In Zebrafish, inhibition of JAK/STAT signaling slows cell intercalation movement during gastrulation (Conway et al., 1997). In the mouse, STAT3 deficiency results in early embryogenesis and gastrulation defects (Takeda et al., 1997) and compromised cell migration in keratinocytes (Kira et al., 2002). Therefore, it appears that the function of STAT activation in morphogenetic movements is not limited to Drosophila, but likely applies to animal development in general.

4. Materials and methods

4.1. Fly strains and genetics

The dominant female sterile (DFS) technique (Chou and Perrimon, 1992) was employed to generate embryos that lack the maternal product of stat92E and hop (referred to as stat92E mat− and hop mat− embryos, respectively) using the null alleles stat92E 6346 (or mrl 6346) (Hou et al., 1996) and hop C111 (Binari and Perrimon, 1994). For example, to produce stat92E mat− embryos, y w hs-Flp/w; FRT82B [ovo D1, w +]/TM3 males were crossed to FRT82B stat92E 6346/TM3 females. The resulting third instar larvae were subjected to heat-shock for 2 h daily. Adult y w hs-Flp/+; FRT82B stat92E 6346/FRT82B [ovo D1, w +] females were collected for production of stat92E mat− embryos. Heterozygous flies of stat92E 6346/TM3 were used to analyze the zygotic mutant phenotypes of stat92E 6346. When necessary, a ‘green balancer’ (Bloomington Stock Center) was used to recognize the wild-type chromosome in a collection of embryos. hop GOF embryos were collected by crossing hop Tum-l (Luo et al., 1995) heterozygous females to wild-type males (kept at 25 °C), or using the Gal4-UAS system (Brand and Perrimon, 1993), by crossing UAS-hop to a hs-Gal4 line. To stage embryos, eggs were collected for 2 hours and then were kept at 25 °C in the absence of the parents for a specified time before fixation.

4.2. Immunohistochemistry and Western blots

The following primary antibodies (dilutions) were used for whole-mount immunostaining of embryos by standard methods. The anti-pSTAT92E polyclonal antibodies were produced by Cell Signaling Technology, Inc. (Beverly, MA) following immunizing rabbits with a synthetic phospho-peptide corresponding to residues around Tyr704 (Hou et al., 1996; Yan et al., 1996). Affinity-purified anti-pSTAT92E rabbit serum was used at 1:1000. The following monoclonal antibodies were from the Developmental Studies Hybridoma Bank at the University of Iowa: anti-Crumbs (Crb) (1:100), mAb2A12 (1:10), mAb22C10 (1:100), and mAb BP102 (1:50). A rat anti-STAT92E antibody (1:500) was as described (Li et al., 2002).

For fluorescent immunostaining, embryos were dechorionated and fixed for 15 min in 5% paraformaldehyde/PB-S/heptane solution. Primary antibodies of appropriate dilution were incubated overnight at 4 °C. TRITC or FITC-conjugated secondary antibodies (Molecular Probes) at 1:250 dilution were used. Stained embryos were mounted in Mowiol mounting medium (Calbiochem) and analyzed using a Leica confocal microscope. For DIC microscopy, the primary antibody was detected by a biotinylated secondary antibody and the ABC Elite Kit (Vector Labs) according to manufacturer’s recommendations. Stained embryos were dehydrated with ethanol, mounted with Euparal (ASCO Laboratories, UK), and photographed with an Axiophot microscope.

For staining Schneider 2 (S2) cells, cells were treated with medium containing vanadate/peroxide (1 mM Vanadate, 2 mM H2O2) to activate the endogenous Hop/STAT92E signaling (Li et al., 2002; Sweitzer et al., 1995). Following treatment for a specified time, cells were immediately washed with PBS and fixed in 3.7% formaldehyde/PBS for 20 min, permeabilized by treating with 0.1% Triton X-100 and 3% BSA in PBS at room temperature for 20 min, and spread on a coverslip. The S2 cells fixed on coverslips were stained with antibodies according to standard procedures and analyzed with confocal microscopy.

To determine the hindgut cell number and length, stage-16 embryos stained with propidium iodide and images of the hindgut were acquired by a Leica confocal microscope. Measurements were made by using a Leica software. Nuclear number in the gut epithelium was counted on print-out pictures of multiple Z-section confocal images.

For Western blots, approximately 100 embryos of specified genotypes were collected, washed with 0.1% Triton X-100/H2O, homogenized in protein loading buffer, and immediately loaded into a mini protein gel. Western blots were performed according to standard procedure. Anti-pSTAT92E antibody was used at 1:500 dilution.

Acknowledgments

We especially thank Michael Melnick and Nicole Kelesoglu of Cell Signaling Technology, Inc. (Bervely, MA) for providing the anti-phospho-STAT92E antiserum. We thank Gavin Hickey and Russell LaFrance for technical assistance, Dr Norbert Perrimon, the Developmental Studies Hybridoma Bank at the University of Iowa, and the Bloomington Stock Center for providing various antibodies and fly strains. We thank Drs Dirk Bohmann, Hucky Land, Henri Jasper, and Vladic Mogila for comments on the manuscript. J.L. is a recipient of the Wilmot Cancer Research Fellowship from the James P. Wilmot Foundation. H.C.C. is a trainee of the NIH-sponsored PREP program. This study was supported by a Howard Hughes Medical Institute Research Resources Program (grant # 53000237) and a grant from the National Institutes of Health (R01 GM65774) to W.X.L., and grants from the Sloan Foundation, the Klingenstein Fund, and the McKnight Foundation to F-B.G.

References

  1. Aaronson DS, Horvath CM. A road map for those who know JAK–STAT. Science. 2002;296:1653–1655. doi: 10.1126/science.1071545. [DOI] [PubMed] [Google Scholar]
  2. Baksa K, Parke T, Dobens LL, Dearolf CR. The Drosophila STAT protein, stat92E, regulates follicle cell differentiation during oogenesis. Dev Biol. 2002;243:166–175. doi: 10.1006/dbio.2001.0539. [DOI] [PubMed] [Google Scholar]
  3. Beccari S, Teixeira L, Rorth P. The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis. Mech Dev. 2002;111:115–123. doi: 10.1016/s0925-4773(01)00615-3. [DOI] [PubMed] [Google Scholar]
  4. Binari R, Perrimon N. Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila. Genes Dev. 1994;8:300–312. doi: 10.1101/gad.8.3.300. [DOI] [PubMed] [Google Scholar]
  5. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–415. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
  6. Bromberg JF. Activation of STAT proteins and growth control. Bioessays. 2001;23:161–169. doi: 10.1002/1521-1878(200102)23:2<161::AID-BIES1023>3.0.CO;2-0. [DOI] [PubMed] [Google Scholar]
  7. Bromberg JF, Horvath CM, Besser D, Lathem WW, Darnell JE., Jr Stat3 activation is required for cellular transformation by v-src. Mol Cell Biol. 1998;18:2553–2558. doi: 10.1128/mcb.18.5.2553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, Albanese C, Darnell JE., Jr Stat3 as an oncogene. Cell. 1999;98:295–303. doi: 10.1016/s0092-8674(00)81959-5. [DOI] [PubMed] [Google Scholar]
  9. Brown S, Hu N, Hombria JC. Identification of the first invertebrate interleukin JAK/STAT receptor, the Drosophila gene domeless. Curr Biol. 2001;11:1700–1705. doi: 10.1016/s0960-9822(01)00524-3. [DOI] [PubMed] [Google Scholar]
  10. Chen HW, Chen X, Oh SW, Marinissen MJ, Gutkind JS, Hou SX. 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. 2002;16:388–398. doi: 10.1101/gad.955202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chou TB, Perrimon N. Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics. 1992;131:643–653. doi: 10.1093/genetics/131.3.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conway G, Margoliath A, Wong-Madden S, Roberts RJ, Gilbert W. Jak1 kinase is required for cell migrations and anterior specification in zebrafish embryos. Proc Natl Acad Sci USA. 1997;94:3082–3087. doi: 10.1073/pnas.94.7.3082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goodman CS, Bastiani MJ, Doe CQ, du Lac S, Helfand SL, Kuwada JY, Thomas JB. Cell recognition during neuronal development. Science. 1984;225:1271–1279. doi: 10.1126/science.6474176. [DOI] [PubMed] [Google Scholar]
  14. Harrison DA, McCoon PE, Binari R, Gilman M, Perrimon N. Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway. Genes Dev. 1998;12:3252–3263. doi: 10.1101/gad.12.20.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hou XS, Melnick MB, Perrimon N. Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs [published erratum appears in Cell 1996 Apr 19; 85(2):following 290] Cell. 1996;84:411–419. doi: 10.1016/s0092-8674(00)81286-6. [DOI] [PubMed] [Google Scholar]
  16. Hou SX, Zheng Z, Chen X, Perrimon N. The Jak/STAT pathway in model organisms. Emerging roles in cell movement. Dev Cell. 2002;3:765–778. doi: 10.1016/s1534-5807(02)00376-3. [DOI] [PubMed] [Google Scholar]
  17. Hummel T, Krukkert K, Roos J, Davis G, Klambt C. Drosophila Futsch/22C10 is a MAP1B-like protein required for dendritic and axonal development. Neuron. 2000;26:357–370. doi: 10.1016/s0896-6273(00)81169-1. [DOI] [PubMed] [Google Scholar]
  18. Ihle JN. The Stat family in cytokine signaling. Curr Opin Cell Biol. 2001;13:211–217. doi: 10.1016/s0955-0674(00)00199-x. [DOI] [PubMed] [Google Scholar]
  19. Isaac DD, Andrew DJ. Tubulogenesis in Drosophila: a requirement for the trachealess gene product. Genes Dev. 1996;10:103–117. doi: 10.1101/gad.10.1.103. [DOI] [PubMed] [Google Scholar]
  20. Jinks TM, Polydorides AD, Calhoun G, Schedl P. The JAK/ STAT signaling pathway is required for the initial choice of sexual identity in Drosophila melanogaster. Mol Cell. 2000;5:581–587. doi: 10.1016/s1097-2765(00)80451-7. [DOI] [PubMed] [Google Scholar]
  21. Johansen KA, Iwaki DD, Lengyel JA. Localized JAK/STAT signaling is required for oriented cell rearrangement in a tubular epithelium. Development. 2003;130:135–145. doi: 10.1242/dev.00202. [DOI] [PubMed] [Google Scholar]
  22. Kawata T, Shevchenko A, Fukuzawa M, Jermyn KA, Totty NF, Zhukovskaya NV, Sterling AE, Mann M, Williams JG. SH2 signaling in a lower eukaryote: a STAT protein that regulates stalk cell differentiation in dictyostelium. Cell. 1997;89:909–916. doi: 10.1016/s0092-8674(00)80276-7. [DOI] [PubMed] [Google Scholar]
  23. Kiger AA, Jones DL, Schulz C, Rogers MB, Fuller MT. Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue. Science. 2001;294:2542–2545. doi: 10.1126/science.1066707. [DOI] [PubMed] [Google Scholar]
  24. Kira M, Sano S, Takagi S, Yoshikawa K, Takeda J, Itami S. STAT3 deficiency in keratinocytes leads to compromised cell migration through hyperphosphorylation of p130(cas) J Biol Chem. 2002;277:12931–12936. doi: 10.1074/jbc.M110795200. [DOI] [PubMed] [Google Scholar]
  25. Lee T, Luo L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron. 1999;22:451–461. doi: 10.1016/s0896-6273(00)80701-1. [DOI] [PubMed] [Google Scholar]
  26. Lengyel JA, Iwaki DD. It takes guts: the Drosophila hindgut as a model system for organogenesis. Dev Biol. 2002;243:1–19. doi: 10.1006/dbio.2002.0577. [DOI] [PubMed] [Google Scholar]
  27. Levy DE, Darnell JE., Jr Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 2002;3:651–662. doi: 10.1038/nrm909. [DOI] [PubMed] [Google Scholar]
  28. Li WX, Agaisse H, Mathey-Prevot B, Perrimon N. Differential requirement for STAT by gain-of-function and wild-type receptor tyrosine kinase Torso in Drosophila. Development. 2002;129:4241–4248. doi: 10.1242/dev.129.18.4241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Luo H, Dearolf CR. The JAK/STAT pathway and Drosophila development. Bioessays. 2001;23:1138–1147. doi: 10.1002/bies.10016. [DOI] [PubMed] [Google Scholar]
  30. Luo H, Hanratty WP, Dearolf CR. An amino acid substitution in the Drosophila hopTum-l Jak kinase causes leukemia-like hematopoietic defects. Eur Mol Biol Org J. 1995;14:1412–1420. doi: 10.1002/j.1460-2075.1995.tb07127.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Luo H, Asha H, Kockel L, Parke T, Mlodzik M, Dearolf CR. The Drosophila Jak kinase hopscotch is required for multiple developmental processes in the eye. Dev Biol. 1999;213:432–441. doi: 10.1006/dbio.1999.9390. [DOI] [PubMed] [Google Scholar]
  32. Perrimon N, Mahowald AP. l(1)hopscotch, a larval–pupal zygotic lethal with a specific maternal effect on segmentation in Drosophila. Dev Biol. 1986;118:28–41. doi: 10.1016/0012-1606(86)90070-9. [DOI] [PubMed] [Google Scholar]
  33. Seeger M, Tear G, Ferres-Marco D, Goodman CS. Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline. Neuron. 1993;10:409–426. doi: 10.1016/0896-6273(93)90330-t. [DOI] [PubMed] [Google Scholar]
  34. Sefton L, Timmer JR, Zhang Y, Beranger F, Cline TW. An extracellular activator of the Drosophila JAK/STAT pathway is a sex-determination signal element. Nature. 2000;405:970–973. doi: 10.1038/35016119. [DOI] [PubMed] [Google Scholar]
  35. Shuai K, Stark GR, Kerr IM, Darnell JE., Jr A single phosphotyrosine residue of Stat91 required for gene activation by interferon-gamma [see comments] Science. 1993;261:1744–1746. doi: 10.1126/science.7690989. [DOI] [PubMed] [Google Scholar]
  36. Silver DL, Montell DJ. Paracrine signaling through the JAK/ STAT pathway activates invasive behavior of ovarian epithelial cells in Drosophila. Cell. 2001;107:831–841. doi: 10.1016/s0092-8674(01)00607-9. [DOI] [PubMed] [Google Scholar]
  37. Sweitzer SM, Calvo S, Kraus MH, Finbloom DS, Larner AC. Characterization of a Stat-like DNA binding activity in Drosophila melanogaster. J Biol Chem. 1995;270:16510–16513. doi: 10.1074/jbc.270.28.16510. [DOI] [PubMed] [Google Scholar]
  38. Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, Kishimoto T, Akira S. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci USA. 1997;94:3801–3804. doi: 10.1073/pnas.94.8.3801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tepass U, Theres C, Knust E. crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell. 1990;61:787–799. doi: 10.1016/0092-8674(90)90189-l. [DOI] [PubMed] [Google Scholar]
  40. Tulina N, Matunis E. Control of stem cell self-renewal in Drosophila spermatogenesis by JAK–STAT signaling. Science. 2001;294:2546–2549. doi: 10.1126/science.1066700. [DOI] [PubMed] [Google Scholar]
  41. Turkson J, Bowman T, Garcia R, Caldenhoven E, De Groot RP, Jove R. Stat3 activation by Src induces specific gene regulation and is required for cell transformation. Mol Cell Biol. 1998;18:2545–2552. doi: 10.1128/mcb.18.5.2545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wilk R, Weizman I, Shilo BZ. trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila. Genes Dev. 1996;10:93–102. doi: 10.1101/gad.10.1.93. [DOI] [PubMed] [Google Scholar]
  43. Xi R, McGregor JR, Harrison DA. A gradient of JAK pathway activity patterns the anterior-posterior axis of the follicular epithelium. Dev Cell. 2003;4:167–177. doi: 10.1016/s1534-5807(02)00412-4. [DOI] [PubMed] [Google Scholar]
  44. Yan R, Small S, Desplan C, Dearolf CR, Darnell JE., Jr Identification of a Stat gene that functions in Drosophila development. Cell. 1996;84:421–430. doi: 10.1016/s0092-8674(00)81287-8. [DOI] [PubMed] [Google Scholar]
  45. Zeidler MP, Perrimon N, Strutt DI. Polarity determination in the Drosophila eye: a novel role for unpaired and JAK/STAT signaling. Genes Dev. 1999;13:1342–1353. doi: 10.1101/gad.13.10.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zelzer E, Shilo BZ. Cell fate choices in Drosophila tracheal morphogenesis. Bioessays. 2000;22:219–226. doi: 10.1002/(SICI)1521-1878(200003)22:3<219::AID-BIES3>3.0.CO;2-A. [DOI] [PubMed] [Google Scholar]

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