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. 2016 May 16;203(3):1283–1295. doi: 10.1534/genetics.115.185918

Retinal Axon Guidance Requires Integration of Eya and the Jak/Stat Pathway into Phosphotyrosine-Based Signaling Circuitries in Drosophila

Charlene S L Hoi *,†, Wenjun Xiong *,‡,1, Ilaria Rebay *,2
PMCID: PMC4937463  PMID: 27194748

Abstract

The transcriptional coactivator and phosphatase eyes absent (Eya) is dynamically compartmentalized between the nucleus and cytoplasm. Although the nuclear transcriptional circuits within which Eya operates have been extensively characterized, understanding of its cytoplasmic functions and interactions remains limited. Our previous work showed that phosphorylation of Drosophila Eya by the Abelson tyrosine kinase can recruit Eya to the cytoplasm and that eya–abelson interactions are required for photoreceptor axons to project to correct layers in the brain. Based on these observations, we postulated that photoreceptor axon targeting might provide a suitable context for identifying the cytoplasmic signaling cascades with which Eya interacts. Using a dose-sensitive eya misexpression background, we performed an RNA interference-based genetic screen to identify suppressors. Included among the top 10 hits were nonreceptor tyrosine kinases and multiple members of the Jak/Stat signaling network (hop, Stat92E, Socs36E, and Socs44A), a pathway not previously implicated in axon targeting. Individual loss-of-function phenotypes combined with analysis of axonal projections in Stat92E null clones confirmed the importance of photoreceptor autonomous Jak/Stat signaling. Experiments in cultured cells detected cytoplasmic complexes between Eya and Hop, Socs36E and Socs44A; the latter interaction required both the Src homology 2 motif in Socs44A and tyrosine phosphorylated Eya, suggesting direct binding and validating the premise of the screen. Taken together, our data provide new insight into the cytoplasmic phosphotyrosine signaling networks that operate during photoreceptor axon guidance and suggest specific points of interaction with Eya.

Keywords: SH2, genetic screen, retinal determination network, eye development, Socs44A

A remarkable feature of multicellular animal development is that the complexity and diversity in tissue type and patterning is achieved using fewer than a dozen signaling pathways, including Notch, receptor tyrosine kinase, Wnt, Hedgehog, TGFβ, Jak/Stat, and Hippo. These cascades are repeatedly deployed in different contexts to regulate the majority of the critical proliferation, survival, specification, and differentiation decisions (reviewed in Voas and Rebay 2004; Housden and Perrimon 2014). One strategy to achieve context-specific developmental regulation is for proteins and pathways to function together in interconnected networks. Further, individual proteins within these networks may encode multiple independent functions that can be executed in distinct parts of the cell, cell types, or stages of development. In this way, even a modest number of individual proteins and core pathways can create vast combinatorial possibilities.

Eyes absent (Eya), a protein conserved from plants to humans, presents an ideal model to study integration because its multifunctionality and dynamic subcellular localization provide opportunities for interaction with many signaling pathways (reviewed in Jemc and Rebay 2007 and Tadjuidje and Hegde 2013). In metazoans, Eya has been studied extensively as a transcriptional coactivator and core member of the retinal determination gene network (reviewed in Silver and Rebay 2005 and Kumar 2009). In this context, Eya participates in nuclear transcriptional complexes to regulate programs of gene expression that direct cell proliferation, differentiation, and survival in a variety of tissues and organs, including the Drosophila eye (Bonini et al. 1993, 1998; Pignoni et al. 1997; Heanue et al. 1999; Ohto et al. 1999; Zou et al. 2004; Nica et al. 2006; Hirose et al. 2010). Eya also carries two different phosphatase domains, one with specificity for phosphotyrosine and a second directed toward phosphothreonine (Li et al. 2003; Rayapureddi et al. 2003; Tootle et al. 2003; Okabe et al. 2009). Although Eya’s tyrosine phosphatase is not required for normal development in Drosophila (Jin et al. 2013), in mammals it dephosphorylates H2AX to promote repair and survival in response to DNA damage (Cook et al. 2009; Krishnan et al. 2009) and aPKCζ to direct lung epithelial polarity and morphogenesis (El-Hashash et al. 2012). Eya’s threonine phosphatase is less well characterized, but appears to act both cytoplasmically to regulate innate immunity and nuclearly to provide transactivation and regulate the activity of other transcription factors (Okabe et al. 2009; Liu et al. 2012; Xu et al. 2014; Jin and Mardon 2016).

Despite increased awareness from mammalian studies of the requirement for Eya function in the cytoplasm, understanding of the specific signaling cascades with which it interacts remains limited. To increase knowledge of the signaling networks with which Eya interfaces, we took advantage of our observation that Drosophila Eya can be sequestered in the cytoplasm upon tyrosine phosphorylation (pY) by the Abelson (Abl) kinase (Xiong et al. 2009) by performing a genetic modifier screen designed to uncover interactions with genes encoding phosphotyrosine binding proteins. The photoreceptor axon mistargeting phenotypes associated with Eya misexpression provided sensitized backgrounds, and secondary genetic tests allowed us to distinguish interactions likely to impact Eya’s cytoplasmic vs. nuclear functions. Prominent among the final list of candidate cytoplasmic interactors were several nonreceptor tyrosine kinases and multiple members of the Jak/Stat pathway. Follow-up genetic experiments, including mosaic analysis with a repressible cell marker (MARCM) of Stat92E clones, confirmed the pathway is required in the differentiating photoreceptors for axon targeting. To our knowledge, these results provide the first demonstration, either in invertebrates or vertebrates, of a requirement for the Jak/Stat pathway in axon guidance. Focusing on the Jak/Stat pathway factors, we validated the premise of our screen by showing that Eya can be recruited to cytoplasmic complexes via pY–SH2-mediated interactions. Together our results uncover novel requirements for phosphotyrosine-based signaling networks in photoreceptor axon targeting and provide a strong impetus for future mechanistic explorations of how cytoplasmic Eya interfaces with these pathways in both Drosophila and mammals.

Materials and Methods

Fly strains

The screen was carried out by crossing RNA interference (RNAi) lines acquired from the Vienna Drosophila RNAi Center (VDRC) to five different recombinant stocks containing GMR-Gal4 and Ro-LacZtau: (1) GMR-Gal4/CyO-actGFP;Ro-lacZtau; (2) GMR > EyaWT/CyO-actGFP;Ro-lacZtau; (3) GMR > EyaRNAi/CyO-actGFP;Ro-lacZtau; (4) GMR-Gal4/CyO-actGFP;Uas-NLS-EyaWT,Ro-lacZtau/TM6; and (5) GMR-Gal4/CyO-actGFP;Uas-Myr-EyaWT,Ro-lacZtau/TM6 (Xiong et al. 2009).

UAS-Stat92E and stat85c9 flies were generously provided by E. Bach. MARCM clones were generated by heat shocking hsFLP, Elav-Gal4, UAS-mCD8::GFP;;FRT82B, stat85c9/FRT82B, tub-Gal80 flies 48 hr after egg laying for 2 hr at 37°.

Socs36E and Socs44A RNAi recombinants were confirmed by wing vein phenotypes using the engrailed driver (Rawlings et al. 2004b). upd and dome RNAi lines were acquired from the Bloomington Drosophila Stock Center. UAS-Socs36E flies were a gift from B. Mathey-Prevot and UAS-Socs44A flies were shared by D. Harrison.

Co-immunoprecipitation, immunostaining, and antibodies

Drosophila S2 cells were cultured at 25° in Schneider’s medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% insect medium supplement (Sigma-Aldrich) and penicillin and streptomycin (Invitrogen, Carlsbad, CA). Cells were transfected with 1.0 μg of each plasmid using dimethyldioctadecyl-ammonium bromide (DDAB) (Sigma-Aldrich) transfection and induced with CuSO4. Expression constructs included pMT-Myc-Abl (Xiong et al. 2009) Eya B isoform complementary DNA (cDNA) cloned into the gateway pAFW vector; Hop, Stat92E, Socs36E, and Socs44A cDNA cloned into the gateway pAHW vector; and SOCS mutants “QuikChanged” from pAHW-SOCSWT plasmids. Oligos used were as follows: (1) Socs36EΔNT, 5′-GGTACCAACACCATG AGCAGCTTCTACTGGGGC-3′ and 5′-GCCCCAGTAGAAGCTGCTCATGGTGTTGGTA CC-3′; (2) Socs36EΔSH2, 5′-CTCGAGAAGATCACGAACAGCGGTCACAAGTTCAG-3′ and 5′-CTGAACTTGTGACCGCTGTTCGTGATCTTCTCGAG-3′; (3) Socs36ESH2* (R500K), 5′-GGCACGTTCCTGCTGAAAGACTCCGCCCAGGAGG-3′ and 5′-CCTCCTGGGCGG AGTCTTTCAGCAGGAACGTGCC-3′; (4) Socs36EΔSB, 5′-CTGCACAGAAGGCAGACCT AGGAATTCGCGGCCGCA-3′ and 5′-TGCGGCCGCGAATTCCTAGGTCTGCCTTCTG TGCA G-3′; (5) Socs44AΔNT, 5′-AACACCATGTACTGGGGTGAG-3′ and 5′-CTCACCCC AGTACATGGTGTT-3′; (6) Socs44AΔSH2, 5′-AACCAAGTGTGGATCTTGCACCGC-3′ and 5′-G CGGTGCAAGATCCACACTTGGTT-3′; (7) Socs44ASH2* (R214K), 5′-TTTCTAGTC AAAGACTCGGAA-3′ and 5′-TTCCGAGTCTTTGACTAGAAA-3′; and (8) Socs44AΔSB, 5′-CGGTACTCCAACTAGCTCGAG-3′ and 5′-CTCGAGCTAGTTGGAGTACCG-3′. For transfections containing Abl or Hop, cells were treated with 100 μM pervanadate and 200 μM H2O2 prior to fixation or lysis. Co-immunoprecipitation assays were performed by lysing cells in lysis buffer (50 mM Hepes, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.9 M glycerol) supplemented with protease inhibitors (Roche), 0.5 mM DTT, and 0.1% Triton X-100, incubating the cell lysate with anti-Flag agarose beads (Sigma-Aldrich) overnight at 4°, washing three times with lysis buffer, and resolving the proteins on 8% SDS-PAGE gels. Proteins were visualized by immunoblotting, using either mouse or rabbit anti-Flag (1:1000; Sigma-Aldrich) and rabbit or mouse anti-HA (1:1000; Rockland or 12CA5 University of Chicago Frank W. Fitch Monoclonal Antibody Facility, respectively) and secondary antibodies (1:2000; Li-COR Biosciences).

Eye imaginal discs and brains were dissected from prepupal third instar larvae and stained as previously described (Xiong et al. 2009) with rabbit anti-β-gal (1:20,000; Promega, Madison, WI). Transfected S2 cells were settled onto poly-L-lysine-treated slides, fixed with 4% paraformaldehyde for 10 min, and stained with anti-Flag and anti-HA as described above, followed by secondary antibodies (1:2000; Jackson ImmunoResearch). Samples were mounted with N-propyl-gallate solution in 90% glycerol.

Images were taken using a Zeiss LSM 510 confocal microscope. All axon images were taken at 0.5- to 1.5-μm steps for the entire depth of the brain and then projected maximally in ImageJ. All single cell images are single scans through the middle of the cell. Unless otherwise stated, statistical P-values were calculated by performing Student’s t-test between control crosses with no RNAi transgene and the cross of interest. * P-value <0.05, ** P-value < 0.01, *** P-value < 0.001.

Fly strains and expression constructs are available upon request.

Data availability

The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.

Results and Discussion

A genetic screen for SH2/PTB family genes that synergize with eya during photoreceptor axon targeting

We previously showed that Eya shuttles between the nucleus and cytoplasm and that Abl-mediated tyrosine phosphorylation (pY) can sequester it in the cytoplasm (Xiong et al. 2009). These observations led us to hypothesize that pY–Eya might be recruited to cytoplasmic signaling complexes through interactions with Src homology 2 (SH2) or phosphotyrosine binding (PTB) domain-containing proteins. To test this idea, we leveraged our prior demonstration of genetic synergy between eya and abl during axon guidance (Xiong et al. 2009) to design an RNAi-based genetic interaction screen aimed at identifying SH2/PTB factors relevant to Eya function in differentiating photoreceptors.

Our prior work showed that in viable hypomorphic eya mutants, photoreceptor axons fasciculate aberrantly and frequently overshoot the lamina (Xiong et al. 2009). MARCM clones ruled out the possibility that these phenotypes stemmed from defects in the target layers of the brain, and GMR–Gal4-driven eya knockdown further confirmed its requirement in the photoreceptors (Xiong et al. 2009). Although the GMR > EyaRNAi mistargeting phenotype was modifiable, as evidenced by its enhancement upon abl knockdown (Xiong et al. 2009), we opted against using it as the primary background for our screen. Briefly, because we predicted positive genetic interactions between eya and relevant SH2/PTB genes, and therefore enhancement of the GMR > EyaRNAi phenotype, we were concerned that the difficulty in distinguishing a relevant enhancement from a nonspecific additive interaction would lead to a high false positive rate.

As an alternative, we explored the suitability of an eya overexpression phenotype for the screen. Using the membrane marker Ro-lacZtau (RTL) to label photoreceptors R2–R5 (Garrity et al. 1999), we quantified the average number of axon bundles that mistarget past the lamina to the medulla in GMR-Gal4 > UAS–EyaWT animals as 25.7 ± 6.1 (mean ± SD), a significant increase over the driver-alone baseline of 8.1 ± 2.2 (Figure 1, B vs. A and D) (Xiong et al. 2009). Reducing endogenous eya dosage with heterozygosity strongly suppressed the mistargeting (9.2 ± 4.4), confirming that the GMR > EyaWT phenotype resulted from increased Eya activity rather than from a dominant negative effect of eya overexpression (Figure 1, C and D).

Figure 1.

Figure 1

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Eya overexpression axon targeting phenotypes provide dose-sensitive, suppressible genetic backgrounds. (A–C) The Ro-lacZtau (RTL) marker labels R2–R5 and is visualized by staining for β-galactosidase in third instar larval brains. (A) Wild-type RTL pattern shows most R2–R5 axons terminate correctly at the lamina, whose edges are marked by yellow arrowheads; a few axon bundles overshoot to the medulla, blue arrowhead. (B) eya overexpression causes many photoreceptor axon bundles to overshoot the lamina, marked by yellow arrowheads. (C) eya heterozygosity suppresses eya overexpression defects. (D) Quantification of genetic interaction tests, demonstrating eya overexpression phenotypes are dose sensitive and suppressible. N values are shown in parentheses. (E) Thirty-six SH2/PTB domain-containing genes in Drosophila, grouped into known categories of signaling, that were targeted in our RNAi-based genetic screen.

Although the similarity between eya loss- and gain-of-function phenotypes may seem surprising, there is precedent for biological processes, including photoreceptor axon guidance, being sensitive to precisely balanced gene dosage such that both loss and gain produce similar phenotypes. For example, analogous to our observations with eya, either increased or decreased activity of the p21-activated kinase Pak (Hing et al. 1999) or of the transcription factor hindsight (Oliva and Sierralta 2010) causes photoreceptor axon mistargeting. It is also important to note that our system of counting the number of RTL-labeled axons that overshoot their laminar target, although highly effective as a first-pass quantification of a phenotype or interaction, ignores qualitative differences. For example, eya knockdown tends to produce thicker fascicles and greater disruptions to the lamina plexus than eya overexpression (Figure 1B, Supplemental Material, Figure S1) (Xiong et al. 2009). Future detailed mechanistic studies will be required to understand the requirement for balanced Eya levels in photoreceptor axon guidance, and the eya interactors identified in our screen should provide useful tools in that endeavor.

Given the general caveats associated with overexpression, it was critical to confirm the GMR > EyaWT mistargeting phenotype could be appropriately modified by a verified eya interacting gene. Building on our prior work (Xiong et al. 2009), we showed that reduced abl dose, either by heterozygosity or by expression of a weak UAS–AblRNAi transgene that on its own did not produce targeting defects, dominantly suppressed GMR > EyaWT (13.4 ± 3.5 and 13.5 ± 5.4, respectively; Figure 1D shows data for AblRNAi). Thus reduced abl dominantly enhances eya loss-of-function and dominantly suppresses eya gain-of-function-induced axon mistargeting. We conclude that despite both overexpression and knockdown of eya causing axons to overshoot the lamina, these genetic backgrounds produce genetically distinct and biologically informative phenotypes and interactions.

We also tested sensitivity of the GMR > EyaWT background to enhancement by coexpressing a second UAS–EyaWT transgene that had a comparably strong phenotype as the first (24.0 ± 5.4), but found only a modest increase to 30.3 ± 4.1 mistargeted axon bundles per brain. Thus the overexpression phenotype was dose sensitive toward suppressive interactions, but was not easily enhanced (Figure 1D). Because we predicted that reducing the dosage of an SH2/PTB-containing factor relevant to cytoplasmic Eya functions would suppress rather than enhance, the GMR > EyaWT background seemed ideal for the screen.

We screened a collection of 63 RNAi transgenes that targeted 36 SH2/PTB family genes for their ability to suppress the GMR > EyaWT axon targeting phenotype (Figure 1E). This panel included two independent RNAi transgenes per gene whenever possible to minimize the likelihood of false positives from off-target effects and false negatives from insufficient knockdown. Our use of GMR–Gal4 restricted knockdown to retinal cells posterior to the morphogenetic furrow, thereby bypassing any earlier requirements for the SH2/PTB genes to be tested. The screen was performed blind as to SH2/PTB gene identity, and ∼10–20 third larval instar eye–brain complexes were dissected, stained, and scored for each interaction test.

To prioritize further study, suppressors were binned into strong, moderate, or mild interest groups based on P-value. A total of 11 strong (P < 0.001), 6 moderate (P < 0.01), and 10 mild (P < 0.05) hits were recovered, identifying 21 potential eya interacting genes (Table 1 shows genes with one strong or moderate hit; full screen results are in Table S1). The rather high hit rate from the primary screen may reflect extensive connectivity between phosphotyrosine signaling modules (Koytiger et al. 2013). Inclusion of dab, a positive factor in the Abl signaling network (Song et al. 2010), in this initial list provided proof of principle for the screen by highlighting the importance of eya–abl interactions to axon guidance and thereby further validating the biological relevance of the eya overexpression phenotype. To narrow the scope of the follow-up experiments to a technically manageable level, we focused only on the strong and moderate suppressors, with one exception, Stat92E.

Table 1. Short list of candidate Eya-interacting SH2/PTB genes after primary and secondary genetic interaction tests.

RNAi element GMR > EyaWT;RTL GMR > EyaRNAi;RTL GMR > Myr-EyaWT;RTL GMR > NLS-EyaWT;RTL
None (w1118) 25.7 ± 6.1 18.8 ± 2.5 18.9 ± 5.0 26.6 ± 4.9
Stat92E43866 19.8 ± 2.6* Inline graphic 21.1 ± 3.3* Inline graphic 13.6 ± 4.5** Inline graphic 31.6 ± 4.9** Inline graphic
Stat92E106980 19.3 ± 6.1* Inline graphic 25.0 ± 3.7*** Inline graphic 12.9 ± 4.8*** Inline graphic 42.3 ± 7.3*** Inline graphic
Socs36E51821 22.3 ± 3.8
Socs36E52182 17.7 ± 4.4** Inline graphic 22.0 ± 4.4* Inline graphic 11.2 ± 4.1*** Inline graphic 27.5 ± 7.6
Socs44A33489 25.4 ± 3.8
Socs44A102764 14.3 ± 5.3*** Inline graphic 21.0 ± 4.0* Inline graphic 12.1 ± 7.3** Inline graphic 40.7 ± 3.5*** Inline graphic
hop40037 22.4 ± 4.8
hop102830 16.6 ± 6.8*** Inline graphic 23.2 ± 5.1* Inline graphic 14.3 ± 6.2* Inline graphic 39.8 ± 6.6*** Inline graphic
Src42A100708 12.7 ± 4.3*** Inline graphic 21.3 ± 4.0* Inline graphic 10.6 ± 5.1*** Inline graphic 36.1 ± 5.4*** Inline graphic
Src64B35252 15.5 ± 2.9*** Inline graphic 22.8 ± 3.8*** Inline graphic 9.8 ± 3.2*** Inline graphic 20.2 ± 4.9*** Inline graphic
Csk32877 16.7 ± 4.0*** Inline graphic 24.5 ± 4.7*** Inline graphic 12.1 ± 5.2*** Inline graphic 41.0 ± 6.5** Inline graphic
shark25304 17.2 ± 4.7*** Inline graphic 23.3 ± 2.3*** Inline graphic 10.3 ± 3.8*** Inline graphic 36.8 ± 4.5*** Inline graphic
shark105706 18.4 ± 3.1** Inline graphic 16.1 ± 3.4*** Inline graphic
Lnk32892 22.6 ± 3.3
Lnk103646 13.4 ± 6.5*** Inline graphic 23.3 ± 4.6* Inline graphic 12.6 ± 5.3*** Inline graphic 45.3 ± 6.0*** Inline graphic
vap44638 20.2 ± 5.7* Inline graphic
vap107341 17.9 ± 5.9** Inline graphic 17.1 ± 2.9* Inline graphic
csw21756 18.6 ± 4.7** Inline graphic 24.5 ± 3.4*** Inline graphic 19.9 ± 6.2 20.9 ± 4.4*** Inline graphic
csw21757 22.7 ± 2.9
CG1552950228 21.8 ± 7.7
CG15529100438 17.9 ± 2.8** Inline graphic 18.8 ± 2.6
Dab13005 18.1 ± 6.7** Inline graphic 23.6 ± 2.9*** Inline graphic 14.8 ± 3.5** Inline graphic 13.7 ± 3.6*** Inline graphic
Dab14008 13.0 ± 4.3*** Inline graphic 24.0 ± 2.7*** Inline graphic 11.8 ± 2.7*** Inline graphic 25.7 ± 5.0
plx27335 6.54 ± 3.1*** Inline graphic 19.8 ± 3.3
plx106969 26.6 ± 4.5
X11L27479 13.5 ± 4.4*** Inline graphic 24.7 ± 5.1* Inline graphic 11.1 ± 4.4*** Inline graphic 34.8 ± 4.6*** Inline graphic
X11Lβ8309 10.4 ± 5.1*** Inline graphic 21.8 ± 3.9* Inline graphic 19.8 ± 6.0 33.4 ± 4.2*** Inline graphic
X11Lβ14872 27.4 ± 5.8
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A short list of RNAi lines identified in the primary screen as suppressors were crossed to GMR > EyaRNAi;RTL, GMR > Myr-EyaWT;RTL, and GMR > NLS-EyaWT;RTL backgrounds as secondary tests. Numbers shown are averages of overshooting axons per brain with SD. Superscripted numbers for each RNAi element indicate the VDRC stock line used. Solid down arrows mark significant suppressive interactions; open up arrows mark enhancement. Full table of results for all RNAi lines screened along with N values can be found in Table S1. * P-value <0.05, ** P-value <0.01, *** P-value <0.001.

Genetic triage highlights potential links between Eya and both the Jak/Stat signaling pathway and nonreceptor tyrosine kinases

Three secondary genetic tests were used to verify these hits as bona fide genetic interactors and to determine which genes may be relevant to cytoplasmic Eya function. First, we asked whether lines that suppressed GMR > EyaWT conversely enhanced eya loss-of-function axon targeting phenotypes (Table 1). All but three genes (CG15529, plx, and vap) enhanced GMR > EyaRNAi, while shark yielded mixed results, as one shark RNAi line enhanced and the other suppressed. Next, we used membrane-tethered myristoylated Eya (Myr–Eya) and nuclearly localized Eya (NLS–Eya) (Xiong et al. 2009) to distinguish interactions specific to Eya’s cytoplasmic and nuclear functions, respectively. GMR–Gal4-driven expression of both Myr–Eya and NLS–Eya produce axon guidance defects that can be suppressed by eya heterozygosity (Figure 1D), indicating gain-of-function effects. However, we hypothesized that Myr–Eya and NLS–Eya perturb axon targeting via distinct mechanisms given their restricted subcellular localizations. Consistent with this idea, abl knockdown suppressed the axon mistargeting phenotypes associated with Myr–Eya but not NLS–Eya (Figure 1D). Of the candidate lines tested, all except csw and X11Lβ significantly suppressed GMR > Myr–Eya mistargeting defects (Table 1). When tested against GMR > NLS–Eya, only Src64B and csw suppressed, while the remaining 10 lines enhanced. Because relevant interactions with eya gain-of-function phenotypes should manifest as suppression, we suspected the enhancement of GMR > NLS–Eya reflected additive effects rather than real interactions. Consistent with this prediction, GMR–Gal4 knockdown of these SH2/PTB genes produced significant targeting defects (Figure 2A). In sum, the results of our secondary screens whittled the number of candidates to 10 genes: Csk, hop, Lnk, shark, Socs36E, Socs44A, Src42A, Src64B, Stat92E, and X11L. Among these, Src64B was unique in its potential to influence both cytoplasmic and nuclear Eya functions.

Figure 2.

Figure 2

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Eya-interacting SH2/PTB genes are required for photoreceptor axon guidance. (A) Quantification of axon mistargeting phenotypes resulting from RNAi knockdown of 10 SH2/PTB genes from Table 1. The RNAi lines indicated were crossed to GMR;RTL. Superscripted numbers indicate the VDRC stock used. Numbers shown are averages of overshooting axons per brain with SD. * P-value <0.05, ** P-value < 0.01, *** P-value < 0.001. N values are shown on the right. (B–F) Representative brains of the indicated genotype stained with β-galactosidase.

Two groups of genes were notably enriched in this short list: the Jak/Stat pathway (hop, Stat92E, Socs36E, and Socs44A) and nonreceptor tyrosine kinases (Csk, hop, shark, Src42A, and Src64B). In the follow-up studies described below, we focused on uncovering the mechanistic links that may underlie the Jak/Stat signature. Two reasons drove this decision. First, because the Jak/Stat pathway had not been previously implicated in axon guidance in any system, discovery of a novel context for Jak/Stat signaling in neuronal development had the potential for the broadest impact. Second, with the exception of Stat92E, the interactions that produced our 10-gene short list were based on a single RNAi transgene, either because only one line was available (Csk, Src42A, Src64B, and X11L) or because only one of the two lines tested showed interaction in the primary (hop, Lnk, Socs36E, and Socs44A) or secondary (shark) screens (Table 1, Table S1). Independent identification of four different Jak/Stat pathway members provided collective validation, and so mitigated the risk of pursuing spurious off-target effects of single hits. In the future, comparison of target gene messenger RNA levels between pairs of lines and addition of Dicer2 to the line that did not interact could be performed to determine whether such discrepancies reflect false positives due to off-target effects or false negatives due to either poor RNAi efficiency or additive interactions. We consider the latter to be the most likely explanation, given that our screen required suppression of GMR > EyaWT by RNAi knockdowns that on their own often produced strong mistargeting defects (Figure 2A). Thus a “hit” in the primary screen often reflects not only suppression of eya overexpression phenotypes by SH2/PTB knockdown, but also mutual suppression of SH2/PTB knockdown phenotypes by increased eya dose. In cases where the additive effects were sufficiently strong as to mask such mutual suppression, inconsistent results between independent lines would be expected.

The second category of interactors, the nonreceptor tyrosine kinases, underscores the importance of phosphotyrosine-based signaling for photoreceptor axon targeting. Previous work hinted at this, as a number of tyrosine phosphatases were found to be critical for regulating axon targeting (Garrity et al. 1999; Schindelholz et al. 2001; Jeon et al. 2008). Our results identify a set of candidate tyrosine kinases that could provide the opposing catalytic activity. The remaining two genes on the short list, Lnk and X11L, encode adapter proteins. X11L has been implicated in amyloid precursor protein (APP) regulation in both Drosophila and mammalian cells (Hase et al. 2002; Gross et al. 2008), with no reported connections to either Jak/Stat or tyrosine kinase signaling. Lnk negatively feeds back on Jak/Stat signaling in mammalian cells, although in Drosophila it has been studied as a positive regulator of insulin/insulin-like growth factor signaling (Werz et al. 2009; Oh et al. 2010; Slack et al. 2010; Almudi et al. 2013). The Jak/Stat ligands unpaired (Upd) 2 and 3 influence insulin signaling in Drosophila (Rajan and Perrimon 2012; Woodcock et al. 2015), thus potentially implicating Lnk in Jak/Stat signaling in flies.

Jak/Stat signaling is necessary for photoreceptor axon guidance

Jak/Stat signaling in Drosophila is transduced via three ligands, Upd1–3, one receptor, Dome, one Janus kinase (JAK), Hopscotch (Hop), and one signal transducer and activator of transcription (STAT), Stat92E (Stat). Signaling can be attenuated by a variety of negative regulators, including the suppressor of cytokine signaling (SOCS) proteins encoded by Socs36E and Socs44A (Rawlings et al. 2004a,b; Arbouzova and Zeidler 2006). Jak/Stat signaling directs a diverse spectrum of developmental events including cell migration, proliferation, patterning, and morphogenesis (reviewed in Arbouzova and Zeidler 2006 and Li 2008). During eye development, the pathway promotes growth of the early eye field, contributes to initiation and movement of the morphogenetic furrow and to regional eye specification, transduces information from the dorsal–ventral axis to direct ommatidial rotation, and regulates specification and maintenance of the optic lobe neuroepithelium (Luo et al. 1999; Zeidler et al. 1999; Chao et al. 2004; Tsai and Sun 2004; Ekas et al. 2006; Gutierrez-Aviño et al. 2009; Ngo et al. 2010; Wang et al. 2011).

Identification of hop, Stat92E, Socs36E, and Socs44A in our genetic screen (Table 1) and finding that GMR-driven knockdown of these genes produces axon mistargeting phenotypes (Figure 2, A–E) suggested that the Jak/Stat pathway is also active in postmitotic photoreceptors. In support of this, expression of the 10xStat–eGFP pathway activity reporter suggests that early expression of the ligand, upd, which is absent by the third instar, enables sustained Jak/Stat signaling in the photoreceptors at the time they are sending out their axons (Bach et al. 2007). As predicted, GMR–Gal4-driven knockdown of the receptor dome, but not of the upd ligands, produced significant mistargeting defects (Figure 3, A–C).

Figure 3.

Figure 3

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Jak/Stat signaling is required cell autonomously in photoreceptors for axon targeting. (A) Quantification of axon mistargeting phenotypes, resulting from RNAi knockdown of upd and dome. The RNAi lines indicated were crossed to GMR;RTL. Superscripted numbers indicate the Bloomington stock used. Numbers shown are averages of overshooting axons per brain with SD. * P-value <0.05, ** P-value < 0.01, *** P-value < 0.001. N values are shown to the right. (B and C) Representative brains of the indicated genotype stained with β-galactosidase. (B) upd knockdown does not perturb axon targeting. (C) Knockdown of dome leads to significant overshooting of the lamina. (D) GFP+ (green) MARCM clone showing that the axons of Stat92E null photoreceptors overshoot the lamina. Chaoptin staining (red) marks all photoreceptors in the eye disc (top right) and highlights their axonal projections through the optic stalk and into the brain. (E) Representative brain stained with β-galactosidase, showing that Socs36E knockdown suppresses the axon mistargeting phenotypes of GMR > hopRNAi (compare to Figure 2B) (F) Quantification and statistics of axon overshooting phenotypes in double knockdown interaction assays show that Socs genes function as Jak/Stat pathway antagonists. GMR;RTL (blue), GMR > Socs36ERNAi;RTL (red), and GMR > Socs44ARNAi;RTL (green) were crossed to the hopRNAi line indicated on the x-axis. N values per experiment from left to right are 10, 7, 10, 13, 13, and 14. P-values were calculated by performing Student’s t-test between the experimental cross and driver-alone control (i.e., red/green vs. blue). * P-value <0.05, ** P-value <0.01, *** P-value <0.001. Superscripted numbers refer to VDRC RNAi line.

To confirm further the requirement for Jak/Stat signaling in photoreceptor axon guidance, we generated MARCM clones of a Stat92E null allele, stat85c9. Axons originating in photoreceptors that lacked Stat92E overshot the lamina into wild-type brain tissue (Figure 3D), while clones within the brain did not cause mistargeting of wild-type photoreceptors (data not shown). This demonstrates that correct axon targeting requires Stat92E in the photoreceptors. The result also eliminates the possibility that RNAi-induced phenotypes resulted from either leaky GMR–Gal4 expression in the brain (Li et al. 2012) or off-target effects. Together, our results indicate a cell autonomous role for Jak/Stat signaling in postmitotic photoreceptors that directs axon projections to the correct layers of the brain.

One apparent inconsistency between this conclusion and our genetic screen results is that SOCS factors typically antagonize Jak/Stat signaling (Chen et al. 2000; Callus and Mathey-Prevot 2002; Rawlings et al. 2004b) yet knockdown of Socs, hop, or Stat92E all suppressed GMR > EyaWT. Since axon targeting is a novel context for Jak/Stat signaling, one possibility is that the SOCS factors do not antagonize the pathway in postmitotic photoreceptors as they do in other situations (Callus and Mathey-Prevot 2002; Rawlings et al. 2004b). Arguing against this, knockdown of either Socs36E or Socs44A suppressed the GMR > hopRNAi mistargeting phenotypes (Figure 3, E and F). In addition to their roles as negative regulators of Jak/Stat signaling, SOCS factors also influence a variety of growth factor signaling cascades (De Sepulveda et al. 1999; Chen et al. 2000; Bayle et al. 2004; Rawlings et al. 2004b; Kazi et al. 2012; Trengove and Ward 2013). Thus the eya–Socs synergy observed in our screen could reflect interactions relevant to those other pathways. Mechanistically, although SOCS proteins belong to the family of Cullin–Ring E3 ubiquitin ligases (reviewed in Piessevaux et al. 2008), a model in which they target Eya for degradation seemed unlikely because the predicted increase in Eya levels upon Socs knockdown should enhance, rather than suppress, GMR > EyaWT phenotypes. Consistent with such reasoning, Socs knockdown did not alter Eya protein levels in the GMR > EyaWT background (data not shown).

Given the sensitivity of photoreceptor axon targeting to both loss and overexpression of eya (Figure 1, Table 1, Figure S1), we asked whether the same might hold true for hop, Stat92E, Socs36E, and Socs44A. GMR–Gal4-driven overexpression of hop was lethal prior to the third instar, and Stat92E overexpression did not produce targeting defects (data not shown and Figure 4, A and C). Stat92E overexpression also did not modify GMR > EyaRNAi (Figure 4, B and F), suggesting endogenous signaling is limited and insufficient to activate the extra Stat protein. Consistent with this interpretation, previous reports have shown that although UAS–Stat can rescue Stat92E null phenotypes, its overexpression in an otherwise wild-type background does not perturb eye development (Ekas et al. 2006).

Figure 4.

Figure 4

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Eya and SOCS synergize in axon targeting. (A) Quantification of axon phenotypes resulting from overexpression of Stat92E, Socs36E, and Socs44A. (B) Quantification of axon phenotypes from genotypes in (A) in a GMR > EyaRNAi background. (C–H) Representative images of the indicated genotypes stained with anti-β-galactosidase. P-values were calculated by performing Student’s t-test between the experimental cross and the control of that group. In B, the first is relative to GMR > EyaRNAi and the second is relative to GMR > SOCSWT, revealing mutual suppression. * P-value <0.05, ** P-value <0.01, *** P-value <0.001.

In contrast, GMR–Gal4-driven overexpression of either Socs36E or Socs44A significantly perturbed axon targeting (Figure 4, A, D, and E). We took advantage of this result to validate independently the Socs–eya synergy predicted by our genetic screen interactions. Just as Socs knockdown suppressed eya overexpression phenotypes (Table 1), Socs overexpression suppressed eya knockdown phenotypes (Figure 4, B, G, and H). Thus cooperative eya–Socs interactions appear integral to photoreceptor axon targeting.

Eya co-immunoprecipitates and colocalizes with Socs44A via an SH2–pY-mediated interaction

Given that the impetus for our genetic screen was the possibility that SH2/PTB domains interact with pY–Eya, we next asked whether Eya could co-immunoprecipitate (CoIP) Jak/Stat proteins from lysates of Drosophila S2 cultured cells transiently transfected with epitope-tagged expression constructs. CoIPs were performed both with and without cotransfection of Abl. We found that Flag-tagged Eya can CoIP HA-tagged Hop, Socs36E, and Socs44A, but not Stat, and that Abl improved Eya’s CoIP of Socs36E and Socs44A, but not that of Hop or Stat (Figure 5, A–D). Abl stimulates Eya tyrosine phosphorylation and cytoplasmic accumulation (Xiong et al. 2009 and Figure 6, A vs. B, quantified in N) and thus should enhance SH2/PTB–pY–Eya interactions. Abl is expressed endogenously in S2 cells (Cherbas et al. 2011), and some tyrosine phosphorylation of Eya can be detected in cells transfected with Eya alone (Tootle et al. 2003), perhaps explaining the low levels of Socs36E and Socs44A CoIP detected when Abl was not supplied by cotransfection (Figure 5, C and D). The Abl independence of the Eya–Hop CoIP (Figure 5A) suggests that either Hop phosphorylates Eya at different tyrosine residues, or that pY–SH2 interactions do not drive the CoIP.

Figure 5.

Figure 5

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pY–SH2 dependence of Eya CoIP with Hop, Socs36E, and Socs44A. (A–F) Western blots showing CoIP results from transfected Drosophila S2 cells. Top panels, blotted with anti-HA, show input levels for the HA-tagged Jak/Stat pathway factor. Middle panels, blotted with anti-Flag, show IP of Flag–Eya. Bottom panels show CoIP of the HA-tagged Jak/Stat pathway protein. Key below indicates factors transfected in each experiment. (A–D) Eya CoIPs full-length Hop, Socs36E, and Socs44A but not Stat. Broad smeary band in lane 4, middle panels, reflects the extensive tyrosine phosphorylation of Eya that occurs in the presence of Abl (Xiong et al. 2009). (A) Flag–Eya CoIPs HA–Hop in the presence or absence of Abl. (B) Flag–Eya does not CoIP HA–Stat. (C and D) Flag–Eya CoIP of Socs36E (C) and Socs44A (D) is increased in the presence of Abl. (E and F) Eya–Socs44A but not Eya–Socs36E requires the SH2 domain of that SOCS factor. (E) Flag–Eya CoIPs all HA–Socs36E mutant constructs. (F) Flag–Eya CoIPs all HA–Socs44A deletion constructs except that lacking the SH2 domain. Black triangles mark IgG bands. Full-length Socs44A (lanes 2 and 4) runs below IgG heavy chain, while Socs44AΔNT runs coincident with IgG light chain (lane 3).

Figure 6.

Figure 6

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Eya localizes to cytoplasmic punctate structures when cotransfected with Abl and Socs44A. (A–M) Individual transfected S2 cells showing representative subcellular distribution of Flag–Eya and HA–Socs44A. Nuclei are marked with DAPI. (A) Flag–Eya is predominantly nuclear. (B) Cotransfection of Abl causes cytoplasmic redistribution of Flag–Eya. (C) Full-length HA–Socs44A appears cytoplasmic and membrane associated. (D) Myr–Flag–Eya is tightly membrane associated, with some punctate structures apparent. (E) Cotransfection of Abl does not alter Myr–Flag–Eya distribution. (F and G) Two representative cells showing that cotransfection of Socs44A does not induce a major redistribution of Eya (F), although in a small fraction of cells, bright membrane proximal punctate structures are seen (G). Socs44A appears more tightly membrane associated in cells in which Eya puncta form (G′), with reduced general cytoplasmic accumulation (compare to F′). (H and I) Two representative cells showing Eya cytoplasmic (H) and punctate (I) localization increases in response to cotransfection of Abl and Socs44A. In cells with Eya puncta, Socs44A tends to be strongly membrane associated (I′). (J and K) Redistribution of Eya to membrane-associated puncta requires the SH2 domain of Socs44A. Socs44AΔSH2 localizes diffusely throughout the cytoplasm and is not strongly membrane associated (Figure S3L), and localization does not change with cotransfection of Eya (J) or Eya and Abl (K). (L) Socs44A only slightly increases the punctate distribution of Myr–Eya. (M) Cotransfection of Abl and Socs44A results in punctate Myr–Eya distribution in 75% of cells. (N) Quantification of nuclear vs. cytoplasmic distribution of Flag–Eya in experiments A, B, and F–I. N values are shown across the top of the graph. (O) Quantification of cytoplasmic Eya distribution into punctate structures in experiments A, B, and D–M. Punctate classification is a subset of membrane associated (i.e., some cells categorized as having punctate staining also have membrane-associated staining). N values are shown across the top of the graph.

Because Abl increased Eya’s CoIP with Socs36E and Socs44A, we focused on them as the top candidates for forming SH2–pY–Eya complexes. To test this, we generated three deletion mutants that removed either the N terminus (ΔNT), the SH2 domain (ΔSH2), or the Socs Box (ΔSB) and a point mutant (SH2*, Socs36ER500K, or Socs44AR214K) that compromises SH2 function in homologous mammalian SOCS proteins (Nicholson et al. 1999). Consistent with expectations, the Socs44A SH2 domain was required for CoIP, while the SH2* point mutant weakened the interaction (Figure 5F, Figure S2). Similar experiments with Socs36E did not identify any single domain as being required for CoIP (Figure 5E), suggesting that the Eya–Socs36E interaction is more complicated, either requiring multiple Socs36E domains to assemble the complex and/or additional proteins to bridge the interactions. Socs44A and Socs36E are both endogenously expressed in S2 cells (Callus and Mathey-Prevot 2002; Zhu et al. 2013) and can CoIP each other (Figure S3). Thus, Socs44A is an appealing candidate to bridge the Eya–Socs36E interaction, as its association with both proteins would confer the sensitivity to Abl seen in the Eya–Socs36E CoIPs (Figure 5C).

The pY–SH2-mediated CoIP of Eya and Socs44A predicted cytoplasmic colocalization. Our previous work has shown that Eya is predominantly nuclear but can be relocalized to the cytoplasm upon phosphorylation by Abl (Xiong et al. 2009 and Figure 6, A and B). Socs44A accumulated in the cytoplasm, with strong association to the plasma membrane (Figure 6, C and F–I; localization of Hop, Stat, and Socs36E is shown in Figure S4). In Eya–Socs44A cotransfected cells, we observed a subtle increase in the proportion of cells with cytoplasmic Eya, while Socs44A localization was not changed (Figure 6, F, G, and N). Occasionally, membrane-associated Eya puncta were observed and these tended to colocalize with Socs44A+ spots (Figure 6G). The frequency of cells displaying these punctate structures dramatically increased when Abl was cotransfected (Figure 6, H, I, and O). Confirming the SH2 requirement for Eya recruitment, we did not observe Eya-containing puncta when Socs44AΔSH2 was expressed (Figure 6, J, K, and O). To separate Abl’s role in phosphorylating Eya from its role in recruiting Eya to the cytoplasm, we examined the response of Myr–Eya, which is constitutively targeted to the cytoplasmic membrane (Figure 6, D and E). Only when Abl was cotransfected did Socs44A promote Eya punctate localization (Figure 6, L, M, and O). We speculate that these puncta represent Eya–Socs44A complexes and that they are mediated by pY–SH2 interactions.

Concluding remarks

Our genetic screen was initially motivated by the idea that the cytoplasm was a likely site for Eya’s phosphotyrosine phosphatase activity (Xiong et al. 2009), a model which was subsequently validated in mammals (El-Hashash et al. 2012). Recent work has shown that this catalytic function of Eya is dispensable for Drosophila development (Jin et al. 2013). What then might be the role of cytoplasmic Eya? One possibility is that Eya might act as a scaffolding factor that nucleates the formation of specific protein complexes in response to different upstream signals. Because our biochemical experiments relied on interactions between overexpressed proteins in cultured cells, it will be important to confirm the results using endogenous proteins from Drosophila tissues. Another possibility is that Eya might modulate the composition or function of such complexes via its phosphothreonine phosphatase activity. Eya’s threonine phosphatase activity has been implicated in innate immunity in both mammals and Drosophila, and although substrates have not been identified, cytoplasmic activity was implied by the identified interactions (Okabe et al. 2009; Liu et al. 2012). Intriguingly, the Jak/Stat pathway is relevant to innate immunity in both Drosophila and mammals (reviewed in Agaisse and Perrimon 2004 and O’Shea and Plenge 2012). In Drosophila, Jak/Stat signaling regulates hemocyte (blood cell) development and release of Upd cytokines in response to infection (Agaisse et al. 2003; Copf et al. 2011; Minakhina et al. 2011; Woodcock et al. 2015). The tyrosine kinases from our short list of candidates also have potential links to immunity. shark and Src42A are important for Draper-mediated glial phagocytosis of axonal debris (Ziegenfuss et al. 2008) which, if left uncleared, stimulates an immune response, and Src64B overexpression has been shown to be sufficient for inducing an immune response in larva (Williams 2009). Even if Eya’s threonine phosphatase activity is not involved, extending exploration of the interactions we have uncovered in the context of axon guidance to other biological contexts in which the candidate interacting partner is known to function may provide new insight into the cytoplasmic signaling networks in which Eya participates during development and disease.

Acknowledgments

We thank Fangfang Jiang for being integral to this work by providing some data included in Figure 2A and for performing the experiments shown in Figure 5, E and F and Figure S3; J. F. Boisclair-Lachance, T. Davis, M. Hope, N. Sanchez, and J. Webber for comments on the manuscript; R. Fehon, E. Ferguson, M. Glotzer, R. Jones, and V. Prince for discussions; and E. Bach for reagents. C.S.L.H. was supported by National Institutes of Health (NIH) T32-GM07197 and by NIH R01-EY12549 (to I.R.). W.X. was supported by a Women’s Board fellowship of the University of Chicago and by an American Heart Association predoctoral fellowship. Sequencing and monoclonal antibody costs were partially subsidized by the University of Chicago Cancer Center support grant P30CA014599.

Footnotes

Communicating editor: M. F. Wolfner

Supplemental material is available online at www.genetics.org/lookup/suppl/doi:10.1534/genetics.115.185918/-/DC1.

Literature Cited

  1. Agaisse H., Perrimon N., 2004.  The roles of JAK/STAT signaling in Drosophila immune responses. Immunol. Rev. 198: 72–82. [DOI] [PubMed] [Google Scholar]
  2. Agaisse H., Petersen U. M., Boutros M., Mathey-Prevot B., Perrimon N., 2003.  Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury. Dev. Cell 5: 441–450. [DOI] [PubMed] [Google Scholar]
  3. Almudi I., Poernbacher I., Hafen E., Stocker H., 2013.  The Lnk/SH2B adaptor provides a fail-safe mechanism to establish the Insulin receptor-Chico interaction. Cell Commun. Signal. 11: 26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Arbouzova N. I., Zeidler M. P., 2006.  JAK/STAT signalling in Drosophila: insights into conserved regulatory and cellular functions. Development 133: 2605–2616. [DOI] [PubMed] [Google Scholar]
  5. Bach E. A., L. A. Ekas, A. Ayala-Camargo, M. S. Flaherty, H. Lee et al, 2007.  GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo. Gene Expr. Patterns 7: 323–331. [DOI] [PubMed] [Google Scholar]
  6. Bayle J., Letard S., Frank R., Dubreuil P., De Sepulveda P., 2004.  Suppressor of cytokine signaling 6 associates with KIT and regulates KIT receptor signaling. J. Biol. Chem. 279: 12249–12259. [DOI] [PubMed] [Google Scholar]
  7. Bonini N. M., Leiserson W. M., Senzer S., 1993.  The eyes absent gene : genetic control of cell survival and differentiation in the developing Drosophila eye. Cell 72: 379–395. [DOI] [PubMed] [Google Scholar]
  8. Bonini N. M., Leiserson W. M., Benzer S., 1998.  Multiple roles of the eyes absent gene in Drosophila. Dev. Biol. 196: 42–57. [DOI] [PubMed] [Google Scholar]
  9. Callus B. A., Mathey-Prevot B., 2002.  SOCS36E, a novel Drosophila SOCS protein, suppresses JAK/STAT and EGF-R signalling in the imaginal wing disc. Oncogene 21: 4812–4821. [DOI] [PubMed] [Google Scholar]
  10. Chao J.-L., Tsai Y.-C., Chiu S.-J., Sun Y. H., 2004.  Localized Notch signal acts through eyg and upd to promote global growth in Drosophila eye. Development 131: 3839–3847. [DOI] [PubMed] [Google Scholar]
  11. Chen X. P., Losman J. A, and P. Rothman, 2000.  SOCS proteins, regulators of intracellular signaling. Immunity 13: 287–290. [DOI] [PubMed] [Google Scholar]
  12. Cherbas L., AWillingham, DZhang, LYang, YZou et al, 2011.  The transcriptional diversity of 25 Drosophila cell lines. Genome Res. 21: 301–314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cook P. J., Ju B. G., Telese F., Wang X., Glass C. K., et al. , 2009.  Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions. Nature 458: 591–596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Copf T., Goguel V., Lampin-Saint-Amaux A., Scaplehorn N., Preat T., 2011.  Cytokine signaling through the JAK/STAT pathway is required for long-term memory in Drosophila. Proc. Natl. Acad. Sci. USA 108: 8059–8064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ekas L. A., G. -H. Baeg, M. S. Flaherty, A. Ayala-Camargo, and E. A. Bach, 2006.  JAK/STAT signaling promotes regional specification by negatively regulating wingless expression in Drosophila. Development 133: 4721–4729. [DOI] [PubMed] [Google Scholar]
  16. El-Hashash A. H., Turcatel G., Varma S., M. BerikaAl Alam D. et al, 2012.  Eya1 protein phosphatase regulates tight junction formation in lung distal epithelium. J. Cell Sci. 125: 4036–4048. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  17. Garrity P. A., Lee C.-H., Salecker I., Robertson H. C., Desai C. J., et al. , 1999.  Retinal axon target selection in Drosophila is regulated by a receptor protein tyrosine phosphatase. Neuron 22: 707–717. [DOI] [PubMed] [Google Scholar]
  18. Gross G. G., Feldman R. M. R., Ganguly A., Wang J., Yu H., et al. , 2008.  Role of X11 and ubiquilin as in vivo regulators of the amyloid precursor protein in Drosophila. PLoS One 3: e2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gutierrez-Aviño F. J., Ferres-Marco D., Dominguez M., 2009.  The position and function of the Notch-mediated eye growth organizer: the roles of JAK/STAT and four-jointed. EMBO Rep. 10: 1051–1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hase M., Yagi Y., Taru H., Tomita S., Sumioka A., et al. , 2002.  Expression and characterization of the Drosophila X11-like/Mint protein during neural development. J. Neurochem. 81: 1223–1232. [DOI] [PubMed] [Google Scholar]
  21. Heanue T. A., Reshef R., Davis R. J., Mardon G., Oliver G., et al. , 1999.  Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation. Genes Dev. 13: 3231–3243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hing H., Xiao J., Harden N., Lim L., Zipursky S. L., 1999.  Pak functions downstream of dock to regulate photoreceptor axon guidance in Drosophila. Cell 97: 853–863. [DOI] [PubMed] [Google Scholar]
  23. Hirose T., Galvin B. D., Horvitz H. R., 2010.  Six and Eya promote apoptosis through direct transcriptional activation of the proapoptotic BH3-only gene egl-1 in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 107: 15479–15484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Housden B. E., Perrimon N., 2014.  Spatial and temporal organization of signaling pathways. Trends Biochem. Sci. 39: 457–464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jemc J., Rebay I., 2007.  The eyes absent family of phosphotyrosine phosphatases: properties and roles in developmental regulation of transcription. Annu. Rev. Biochem. 76: 513–538. [DOI] [PubMed] [Google Scholar]
  26. Jeon M., Nguyen H., Bahri S., Zinn K., 2008.  Redundancy and compensation in axon guidance: genetic analysis of the Drosophila Ptp10D/Ptp4E receptor tyrosine phosphatase subfamily. Neural Dev. 3: 3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jin M., Mardon G., 2016.  Distinct biochemical activities of eyes absent during Drosophila eye development. Sci. Rep. 6: 23228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jin M., Jusiak B., Bai Z., Mardon G., 2013.  Eyes absent tyrosine phosphatase activity is not required for Drosophila development or survival. PLoS One 8: e58818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kazi J. U., Sun J., Phung B., Zadjali F., Flores-Morales A., et al. , 2012.  Suppressor of cytokine signaling 6 (SOCS6) negatively regulates Flt3 signal transduction through direct binding to phosphorylated tyrosines 591 and 919 of Flt3. J. Biol. Chem. 287: 36509–36517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Koytiger G., Kaushansky A., Gordus A., Rush J., Sorger P. K., et al. , 2013.  Phosphotyrosine signaling proteins that drive oncogenesis tend to be highly interconnected. Mol. Cell. Proteomics 12: 1204–1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Krishnan N., Jeong D. G., Jung S.-K., Ryu S. E., Xiao A., et al. , 2009.  Dephosphorylation of the C-terminal tyrosyl residue of the DNA damage-related histone H2A.X is mediated by the protein phosphatase eyes absent. J. Biol. Chem. 284: 16066–16070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kumar J. P., 2009.  The molecular circuitry governing retinal determination. Biochim. Biophys. Acta 1789: 306–314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Li W. X., 2008.  Canonical and non-canonical JAK-STAT signaling. Trends Cell Biol. 18: 545–551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Li W.-Z., Li S.-L., Zheng H. Y., Zhang S.-P., Xue L., 2012.  A broad expression profile of the GMR-GAL4 driver in Drosophila melanogaster. Genet. Mol. Res. 11: 1997–2002. [DOI] [PubMed] [Google Scholar]
  35. Li X., K. AOghi, J. Zhang, A. Krones, K. T. Bush et al, 2003.  Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature 426: 247–254. [DOI] [PubMed] [Google Scholar]
  36. Liu X., Sano T., Guan Y., Nagata S., Hoffmann J. A., et al. , 2012.  Drosophila EYA regulates the immune response against DNA through an evolutionarily conserved threonine phosphatase motif. PLoS One 7: e42725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Luo H., Asha H., Kockel L., Parke T., Mlodzik M., et al. , 1999.  The Drosophila Jak kinase hopscotch is required for multiple developmental processes in the eye. Dev. Biol. 213: 432–441. [DOI] [PubMed] [Google Scholar]
  38. Minakhina S., Tan W., Steward R., 2011.  JAK/STAT and the GATA factor Pannier control hemocyte maturation and differentiation in Drosophila. Dev. Biol. 352: 308–316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ngo K. T., Wang J., Junker M., Kriz S., Vo G., et al. , 2010.  Concomitant requirement for Notch and Jak/Stat signaling during neuro-epithelial differentiation in the Drosophila optic lobe. Dev. Biol. 346: 284–295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nica G., Herzog W., Sonntag C., Nowak M., Schwarz H., et al. , 2006.  Eya1 is required for lineage-specific differentiation, but not for cell survival in the zebrafish adenohypophysis. Dev. Biol. 292: 189–204. [DOI] [PubMed] [Google Scholar]
  41. Nicholson S. E., Willson T. A., Farley A., Starr R., Zhang J. G., et al. , 1999.  Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. EMBO J. 18: 375–385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. O’Shea J. J., Plenge R., 2012.  JAK and STAT signaling molecules in immunoregulation and immune-mediated disease. Immunity 36: 542–550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Oh S. T., Simonds E. F., Jones C., Hale M. B., Goltsev Y., et al. , 2010.  Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms. Blood 116: 988–992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ohto H., Kamada S., Tago K., Ozaki H., Sato S., 1999.  Cooperation of Six and Eya in activation of their target genes through nuclear translocation of Eya. Mol. Cell. Biol. 19: 6815–6824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Okabe Y., Sano T., Nagata S., 2009.  Regulation of the innate immune response by threonine-phosphatase of Eyes absent. Nature 460: 520–524. [DOI] [PubMed] [Google Scholar]
  46. Oliva C., Sierralta J., 2010.  Regulation of axonal development by the nuclear protein hindsight (pebbled) in the Drosophila visual system. Dev. Biol. 344: 911–921. [DOI] [PubMed] [Google Scholar]
  47. Piessevaux J., Lavens D., Peelman F., Tavernier J., 2008.  The many faces of the SOCS box. Cytokine Growth Factor Rev. 19: 371–381. [DOI] [PubMed] [Google Scholar]
  48. Pignoni F., BHu, K. HZavitz, JXiao, P. AGarrity et al, 1997.  The eye-specification proteins So and Eya form a complex and regulate multiple steps in Drosophila eye development. Cell 91: 881–891. [DOI] [PubMed] [Google Scholar]
  49. Rajan A., Perrimon N., 2012.  Drosophila cytokine unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion. Cell 151: 123–137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rawlings J. S., K. MRosler, and D. AHarrison, 2004a The JAK/STAT signaling pathway. J. Cell Sci. 117: 1281–1283. [DOI] [PubMed] [Google Scholar]
  51. Rawlings J. S., GRennebeck, S. M. WHarrison, RXi, and D. AHarrison, 2004b Two Drosophila suppressors of cytokine signaling (SOCS) differentially regulate JAK and EGFR pathway activities. BMC Cell Biol. 5: 38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Rayapureddi J. P., Kattamuri C., Steinmetz B. D., Frankfort B. J., Ostrin E. J., et al. , 2003.  Eyes absent represents a class of protein tyrosine phosphatases. Nature 426: 295–298. [DOI] [PubMed] [Google Scholar]
  53. Schindelholz B., Knirr M., Warrior R., Zinn K., 2001.  Regulation of CNS and motor axon guidance in Drosophila by the receptor tyrosine phosphatase DPTP52F. Development 128: 4371–4382. [DOI] [PubMed] [Google Scholar]
  54. De Sepulveda P., Okkenhaug K., Rose J. L., Hawley R. G., Dubreuil P., et al. , 1999.  Socs1 binds to multiple signalling proteins and suppresses steel factor-dependent proliferation. EMBO J. 18: 904–915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Silver S. J., Rebay I., 2005.  Signaling circuitries in development: insights from the retinal determination gene network. Development 132: 3–13. [DOI] [PubMed] [Google Scholar]
  56. Slack, C., C. Werz, D. Wieser, N. Alic, A. Foley et al., 2010 Regulation of lifespan, metabolism, and stress responses by the Drosophila SH2B protein, Lnk. PLoS Genet. 6: e1000881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Song J. K., Kannan R., Merdes G., Singh J., Mlodzik M., et al. , 2010.  Disabled is a bona fide component of the Abl signaling network. Development 137: 3719–3727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tadjuidje E., Hegde R. S., 2013.  The Eyes Absent proteins in development and disease. Cell. Mol. Life Sci. 70: 1897–1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Tootle T. L., Silver S. J., E. LDavies, VNewman, R. RLatek et al, 2003.  The transcription factor Eyes absent is a protein tyrosine phosphatase. Nature 426: 299–302. [DOI] [PubMed] [Google Scholar]
  60. Trengove M. C., Ward A. C., 2013.  SOCS proteins in development and disease. Am. J. Clin. Exp. Immunol. 2: 1–29. [PMC free article] [PubMed] [Google Scholar]
  61. Tsai Y.-C., Sun Y. H., 2004.  Long-range effect of upd, a ligand for Jak/STAT pathway, on cell cycle in Drosophila eye development. Genesis 39: 141–153. [DOI] [PubMed] [Google Scholar]
  62. Voas M. G., Rebay I., 2004.  Signal integration during development: insights from the Drosophila eye. Dev. Dyn. 229: 162–175. [DOI] [PubMed] [Google Scholar]
  63. Wang W., Li Y., Zhou L., Yue H., Luo H., 2011.  Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe. Biochem. Biophys. Res. Commun. 410: 714–720. [DOI] [PubMed] [Google Scholar]
  64. Werz C., Köhler K., Hafen E., Stocker H., 2009.  The Drosophila SH2B family adaptor Lnk acts in parallel to chico in the insulin signaling pathway. PLoS Genet. 5: e1000596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Williams M. J., 2009.  The c-src homologue Src64B is sufficient to activate the Drosophila cellular immune response. J. Innate Immun. 1: 335–339. [DOI] [PubMed] [Google Scholar]
  66. Woodcock K. J., Kierdorf K., Pouchelon C. A., Vivancos V., Dionne M. S., et al. , 2015.  Macrophage-derived upd3 cytokine causes impaired glucose homeostasis and reduced lifespan in Drosophila fed a lipid-rich diet. Immunity 42: 133–144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Xiong W., Dabbouseh N. M., Rebay I., 2009.  Interactions with the Abelson tyrosine kinase reveal compartmentalization of eyes absent function between nucleus and cytoplasm. Dev. Cell 16: 271–279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Xu J., Wong E. Y. M., Cheng C., Li J., Sharkar M. T. K., et al. , 2014.  Eya1 interacts with Six2 and Myc to regulate expansion of the nephron progenitor pool during nephrogenesis. Dev. Cell 31: 434–447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zeidler M. P., Perrimon N., Strutt D. I., 1999.  Polarity determination in the Drosophila eye: a novel role for unpaired and JAK/STAT signaling. Genes Dev. 13: 1342–1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Zhu F., Ding H., Zhu B., 2013.  Transcriptional profiling of Drosophila S2 cells in early response to Drosophila C virus. Virol. J. 10: 210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Ziegenfuss J. S., Biswas R., Avery M. A., Hong K., Sheehan A. E., et al. , 2008.  Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase signalling. Nature 453: 935–939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Zou D., Silvius D., Fritzsch B., Xu P.-X., 2004.  Eya1 and Six1 are essential for early steps of sensory neurogenesis in mammalian cranial placodes. Development 131: 5561–5572. [DOI] [PMC free article] [PubMed] [Google Scholar]

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