Interactions with the Abelson Tyrosine Kinase Reveal Compartmentalization of Eyes Absent Function Between Nucleus and Cytoplasm

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Interactions with the Abelson Tyrosine Kinase Reveal Compartmentalization of Eyes Absent Function between Nucleus and Cytoplasm

Wenjun Xiong,1 Noura M. Dabbouseh,1 and Ilaria Rebay1,* 1Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637, USA *Correspondence: [email protected] DOI 10.1016/j.devcel.2008.12.005

SUMMARY The ED also possesses intrinsic protein tyrosine phosphatase activity (Li et al., 2003; Rayapureddi et al., 2003; Tootle et al., Eyes absent (Eya), named for its role in Drosophila eye 2003). Although no physiological substrates have yet been iden- development but broadly conserved in metazoa, tified, the observation that Eya can be tyrosine phosphorylated in possesses dual functions as a transcriptional coacti- cultured cells and can dephosphorylate itself in vitro suggests vator and protein tyrosine phosphatase. Although that it is a target of phosphotyrosine signaling pathways and Eya’s transcriptional activity has been extensively may have autocatalytic activity (Tootle et al., 2003). Impaired characterized, the physiological requirements for its phosphatase activity has been associated with defects in both Drosophila and human development (Rayapureddi et al., 2003; phosphatase activity remain obscure. In this study, Tootle et al., 2003; Mutsuddi et al., 2005; Rayapureddi and we provide insight into Eya’s participation in phos- Hegde, 2006), indicating an essential contribution to Eya function. photyrosine-mediated signaling networks by demon- Given Eya’s well established role within the RD network, we strating cooperative interactions between Eya and and others proposed that phosphatase activity might directly the Abelson (Abl) tyrosine kinase during development influence Eya-So transcriptional output (Li et al., 2003; Tootle of the Drosophila larval visual system. Mechanisti- et al., 2003; Rebay et al., 2005). However, a recent systems level cally, Abl-mediated phosphorylation recruits Eya to analysis of Eya-So regulation of gene expression found that loss the cytoplasm, where in vivo studies reveal a require- of Eya phosphatase function did not globally impair transcrip- ment for its phosphatase function. Thus, we propose tional output, suggesting an alternate model in which Eya phos- a model in which, in addition to its role as a transcrip- phatase and transcriptional activities make independent and tion factor, Eya functions as a cytoplasmic protein distinct contributions to developmental processes requiring Eya function (Jemc and Rebay, 2007a, 2007b). tyrosine phosphatase. Here, we describe the results of a set of experiments designed to identify the phosphotyrosine signaling pathways in which Eya INTRODUCTION participates and to test the hypothesis that Eya phosphatase function can operate independently of its nuclear transcriptional Organogenesis requires coordinated cell proliferation, differenti- activities. Our findings reveal a requirement for Eya phosphatase ation, and morphogenesis. The Retinal Determination (RD) gene activity in the cytoplasm and demonstrate that full Eya function network, a conserved collection of transcription factors named can be reconstituted by coexpression of nuclearly and cytoplas- for their key roles in Drosophila eye specification but that partic- mically restricted protein pools. Mechanistically, we describe an ipate in the development of numerous organ systems in both flies enzyme-substrate relationship between the Abelson (Abl) nonre- and mammals (reviewed by Wawersik and Maas, 2000; Pappu ceptor tyrosine kinase and Eya such that Abl-mediated phos- and Mardon, 2004), provides a tractable model for investigating phorylation relocates Eya from the nucleus to the cytoplasm. how signaling pathways interact with tissue-specific transcrip- Genetic synergy between eya and abl contributes to multiple tional networks to coordinate developmental programs. developmental programs, including axon pathfinding in the Eyes absent (Eya), an RD network member, was first character- embryonic central nervous system (CNS) and the larval visual ized as a novel nuclear factor required for Drosophila eye devel- system. Together, our data support a model in which Eya function opment. Thus eye-specific loss of eya leads to an ‘‘eyeless’’ is partitioned between two independent subcellular sites: the phenotype, whereas misexpression can induce the formation of nucleus, where it fulfills its well-established role as a transcription ectopic eye tissue (Bonini et al., 1993, 1997; Pignoni et al., factor; and the cytoplasm, where it participates in phosphotyro- 1997). Eya family members are identified by a conserved sine signaling mechanisms. C-terminal Eya Domain (ED) that mediates its interaction with another RD protein, Sine oculis (So; Six in vertebrates) (Bonini RESULTS et al., 1993; Pignoni et al., 1997). The Eya-So complex functions as a bipartite transcriptional factor, with Eya providing transacti- Genetic Cooperativity between eya and abl vation and So contributing DNA-binding specificity (Ohto et al., The discoveries that Eya possesses protein tyrosine phosphatase 1999; Silver et al., 2003). activity and is tyrosine phosphorylated in cultured cells (Li et al.,

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A B Figure 1. Genetic Interactions Reveal Cooperativity between eya and abl (A) Altered abl dosage dominantly modifies Eya’s ectopic eye induction efficiency. Lines 1–4 are independent transgenic lines; lof, loss of function; gof, gain of function; kd, kinase dead; n, number scored; NR, none recovered; %EE, percent of flies of geno- typekinase/+;dpp-Gal4 > UAS-Eya withectopic eye tissue on head. (B) Reduced eya dosage impairs viability of abl mutant embryos. N, number of animals scored. (C–G) Dissected ventral nerve cords from stage-16 embryos stained with BP102 to reveal the pattern of the axon scaffold. (C) Wild- type. (D) abl2 homozygotes have intact commissures. (E) eyaA188 homozygotes are indistinguishable from wild-type. (F) eyaA188/+, abl2 mutant embryos have discontinuities along the longitudinal axon bundles with 20% of commissures lost or defective. (G) In C D E F G eyaA188;abl2 double homozygotes, 77% of commissures are lost, and the longitudinal tracts show severe disruptions.

Interactions with abl Reveal a Role for eya in Embryonic CNS Axonogenesis To elucidate the developmental processes that require eya-abl genetic cooperativity, we asked whether the embryonic lethality of eya/+; abl1/abl2 animals might 2003; Rayapureddi et al., 2003; Tootle et al., 2003) imply participa- be caused by defects in the CNS, a tissue in which both Abl and tion in phosphotyrosine-mediated signaling. To identify the Eya are expressed (Gertler et al., 1989; Bennett and Hoffmann, relevant pathways, we asked whether an altered dosage of any 1992; Bonini et al., 1998) and in which Abl function has been tyrosine kinase could dominantly modify the frequency with which characterized (Gertler et al., 1989; Fogerty et al., 1999; Moresco Eya overexpression induces ectopic eye formation. The rationale and Koleske, 2003). In wild-type embryos, the CNS axon was that if activity of a particular kinase is important for Eya func- scaffold consists of two longitudinal axon bundles connected tion, then a two-fold alteration in dose might be sufficient to change by segmentally repeated pairs of commissural tracts (Figure 1C); the level of Eya activity and alter ectopic eye induction efficiency. zygotic loss of abl results in mild discontinuities in the longitu- Although full details of the screen will be reported elsewhere, dinal tracts (Wills et al., 1999; Grevengoed et al., 2001) most striking among the results were interactions with alleles (Figure 1D). Removal of both maternal and zygotic abl leads of the abl nonreceptor tyrosine kinase, the Drosophila homolog to severe disruption of the axon scaffold and fully penetrant of the mammalian c-abl oncogene (Figure 1A). Heterozygosity embryonic lethality (Grevengoed et al., 2001). Thus, if for abl loss-of-function mutations or coexpression of a kinase- the eya and abl gene products function cooperatively, then dead Abl transgene previously shown to function as a dominant reducing eya dose in a zygotic abl background might compro- negative (Hsouna et al., 2003) dominantly suppressed Eya’s mise the function of maternally provided Abl, thereby resulting ectopic eye induction ability. Conversely, whereas overexpres- in exacerbated axonal defects and penetrant embryonic sion of Abl alone resulted in minimal phenotypic perturbation, lethality. co-overexpression of Eya and Abl led to synthetic lethality. Although the CNS of eyaA188 homozygous, eyaA188/+ hetero- To confirm the eya-abl synergy predicted by the ectopic eye zygous, or eyaA188; abl2/+ mutant embryos were indistinguish- induction results, we first asked whether expression of kinase- able from wild-type, reduction or loss of eya enhanced the abl dead Abl could interfere with the ability of an Eya transgene axon patterning defects such that more pronounced gaps to rescue the ‘‘eyeless’’ phenotype associated with the eye- were seen along the longitudinal tracts and the commissures specific loss-of-function allele eya2. Whereas Eya transgenes were lost at high penetrance (Figures 1E–1G). eyaA188; abl2 alone restore eye tissue to 100% of eya2 flies (Tootle et al., embryos phenocopied maternal and zygotic abl mutants (Gre- 2003; Mutsuddi et al., 2005), coexpression of kinase-dead Abl vengoed et al., 2001) in that the distance between the two longi- reduced rescue efficiency to 40%. Second, we asked whether tudinal axon bundles became irregular, with increased separa- a reduced eya dosage could alter the pupal lethality associated tion apparent in segments in which commissures were lost with zygotic loss of abl. Using two different recessive eya alleles (Figure 1G). Together, these results suggest that eya and abl (eyaA188 and eyaG130)(Rebay et al., 2000), we found that whereas work cooperatively to control axon targeting in the CNS. In this 90% of abl1/abl2 transheterozygotes survived until the pupal context, eya must function redundantly with other pathway stage, only 20% of eya/+; abl1/abl2 embryos hatched to the larval components given that eya single mutants lack obvious axonal stage (Figure 1B); eya/+ animals were indistinguishable from +/+ defects; the lack of eya expression in the female germline (Bonini controls in this assay. Together, these genetic results suggest et al., 1998) rules out the possibility that maternal rescue masks a cooperative interaction between eya and abl. a zygotic phenotype.

272 Developmental Cell 16, 271–279, February 17, 2009 ª2009 Elsevier Inc. Developmental Cell Nuclear-Cytoplasmic Partitioning of Eya Function

eya and abl Are Required for Photoreceptor Axon Targeting We next asked whether eya and abl also control photoreceptor axon targeting. Eya and Abl are both expressed in retinal neurons (Bennett and Hoffmann, 1992; Bonini et al., 1998), yet potential roles in photoreceptor morphogenesis have not been explored. During normal development, axonal projections from the differentiating photoreceptor neurons in each ommatidium of the third-instar eye imaginal disc travel together through the optic stalk into the optic lobes of the larval brain, where R1–R6 growth cones target the lamina while R7 and R8 axons travel deeper to the medulla. Larval eye-brain complexes dissected from viable hypomor- phic allelic combinations for each gene were examined for photoreceptor axon targeting defects by staining with anti-chaoptin (24B10) to highlight all photoreceptor projections or with anti- b-galactosidase to follow the Ro-lacZtau marker in R2–R5 projections (Garrity et al., 1999). Instead of forming an even plexus of growth cones at the lamina, as seen in wild-type, in eya or abl mutant larvae the axons fasciculated aberrantly to produce an irregular pattern of gaps and thickenings in the lamina, with a significant percentage of axon bundles failing to terminate properly at the lamina (Figures 2A–2C and 2E–2G). To determine whether eya and abl might function synergistically during photoreceptor axon targeting, we investigated dose-sensitive genetic interactions. Although eya2/eyaA188; abl2/+ or eya2/eyaA188; abl1/abl2 were synthetic lethal prior to the third instar stage, we were able to analyze eyaA188/+; abl1/abl2 animals. Although eyaA188/+ larvae appeared to be wild-type, heterozygosity for eya dominantly enhanced the abl mistargeting phenotype, result- Figure 2. Eya and Abl Are Required for Photoreceptor Axon Target- ing in a highly disorganized lamina plexus (Figures 2D and 2H). ing in the Brain In order to determine if defects observed in eya and abl (A–L) Dissected eye-brain complexes from third-instar larvae stained with (A–D mutants resulted from loss of gene function in the photorecep- and I–L) anti-chaoptin 24B10 or with (E–H) anti-b-galactosidase to visualize the tors, the brain, or in both tissues, we used the mosaic analysis R2–R5-specific Ro-lacZtau marker. (A and E) In wild-type, R1–R6 axons form with a repressible cell marker (MARCM) approach (Lee and a normal lamina plexus, and R7 and R8 axons are arranged in regular stag- Luo, 2001) to generate mutant clones positively marked with gered rows within the medulla. R2–R5 axons mostly stop in the lamina. (B 1 2 a membrane-tethered GFP. Whereas both wild-type control and F) In abl /abl trans-heterozygotes, the lamina plexus is discontinuous, 2 with gaps in the plexus, and thicker axon bundles beneath. A subset of R2– and small abl clones exhibited normal axon targeting, larger R5 axons fail to stop in the lamina and extend into the medulla. (C and G) 2 abl clones exhibited aberrant fasciculation and targeting to eya2/eyaA188 mutants phenocopy abl mutants. (D and H) eya A188/+; abl wild-type brain tissue (Figures 2I–2K); eya mutant photoreceptor mutants have a highly disorganized lamina, with thick bundles of R2–R5 axons clones were unrecoverable due to apoptosis (Bonini et al., 1993). failing to stop in the lamina. (I–L) MARCM clones of eya and abl labeled with Wild-type photoreceptors exhibited normal targeting to either 24B10 (red). GFP, green, marks the mutant axons. (I–I00) Photoreceptor axons 00 eya or abl mutant brain tissue (Figure 2J0 insets; Figure 2L), of large wild-type clones ([I], inset) exhibit normal targeting to the brain. (J–J ) Axonal projections appear normal in small abl clones. Targeting of wild-type suggesting that both abl and eya are required autonomously in axons to mutant brain tissue appears normal ([J0], insets). (K–K00) Larger abl the photoreceptors for proper terminal differentiation. clones show fasciculation defects and laminar gaps. (L–L00) Wild-type axons Whereas abl loss does not perturb retinal induction or photore- exhibit normal targeting to eya mutant brain tissue. ceptor specification (see Figure S1A available online), eya has well established roles in these processes (Bonini et al., 1993). To investigate whether eya axon targeting defects resulted from ally thicker than those in the control, suggesting that the scoring earlier disruptions in eye induction and photoreceptor specifica- scheme underestimates the severity of the eya defects. tion, we used GMR-Gal4-driven expression of an RNAi transgene Confirming cooperative cell-autonomous interactions in the to knock down eya posterior to the morphogenetic furrow. A differentiating photoreceptors, GMR-Gal4-driven coexpression significant reduction of Eya protein levels was observed without of a weak abl RNAi transgene that on its own has no phenotype apparent defects in photoreceptor specification and patterning enhanced the EyaRNAi targeting defects (21.8 axon bundles/brain, (Figure S1). Using the Ro-lacZtau marker to assess targeting compared to 18.2 in EyaRNAi alone) (Figures 3B–3D and 3J). defects, EyaRNAi discs showed a clear ‘‘shoot-through’’ pheno- Finally, RNAi-mediated abl knockdown dominantly suppressed type, with an average of 18.2 overshooting axon bundles per the overshooting phenotypes observed upon GMR-Gal4-driven brain, compared to 8.4 in control discs (Figures 3A, 3B, and 3J). overexpression of UAS-EyaWT (12.4 overshooting axon bundles/ The mistargeted axon bundles in the eya knockdown were gener- brain versus 25.7 for EyaWT alone) (Figures 3E, 3F, and 3J).

Developmental Cell 16, 271–279, February 17, 2009 ª2009 Elsevier Inc. 273 Developmental Cell Nuclear-Cytoplasmic Partitioning of Eya Function

B A C A Abl - WT KD B Eya - + + + + Eya Abl -- + + + λ-PTP -- -10’30’ pY Flag IP

EyaRNAi AblRNAi GMR-Gal4 merge D F E C D AblWT -+- -+- -+- AblWT -+ -+-+ AblKD +-- +-- +--

100 Eya pY merge Eya pY merge EyaRNAi+AblRNAi EyaWT EyaWT+AblRNAi

G H I E F -Abl +Abl PST: 23Ys, 9 in D2

Eya 6YsD2 ED:10Ys 1: Full- Length 100 * * 75 2:Δ D2 = Δ318-353 * Myr-EyaWT Myr-EyaK699Q Myr-EyaWT+AblRNAi Δ Δ 3: PST = 223-438 50 * J ΔΔ Transgene Average # bundles/brain 4: ED = 438-760 * 5: ED =Δ 1-438 Control 8.4 ± 2 (n=29) MW1234512345 Abl RNAi 6.5 ± 3 (n=12)† Eya RNAi 18.2 ± 3 (n=33)

ST ST

PST P P PST p=4.4E-5 ED ED Eya RNAi + Abl RNAi 21.8 ± 3 (n=38) G H

Eya Eya Eya Eya

Eya Eya

ED WT P

- - - - Eya 25.7 ± 6 (n=11) - - RNAi T T WT p=4.5E-8 ya

Eya

E Eya + Abl 12.4 ± 4 (n=17) Eya Abl WT KD

MW MW -

GST GST GST GST -

GST GS GS GST

- Myr-Eya K699Q 11.8 ± 3 (n=20) p=3.7E-9 cAbl +++- +++- T EyaPST

GST

GST WT ± 4 (n=30) GST Myr-Eya 19.0 Myc-Abl+ + + + + + P32 WT RNAi p=1.1E-10 75 Myr-Eya + Abl 8.7 ± 5 (n=31) 45 1 50 Abl 5.5 1 Figure 3. Eya and Abl Interact in Postmitotic Photoreceptor Cells to 25 Regulate Axon Targeting CoomassieP32 Coomassie P32 (A–I) Dissected eye-brain complexes from third-instar larvae stained with tau anti-b-galactosidase to visualize the Ro-LacZ marker. (A) GMR-Gal4/+; Figure 4. Eya Is a Substrate of Abl Ro-lacZtau/+ controls show a few thin overshooting bundles. (B) UAS- (A–D and F) Immunoblots of immunoprecipitated Flag-Eya double labeled with EyaRNAi/+; GMR-Gal4/+; Ro-lacZtau/+ larvae have significant mistargeting anti-Flag (red) and anti-phosphotyrosine (anti-pY) (green). Reduced sensitivity defects. (C) UAS-AblRNAi/+; GMR-Gal4/+; Ro-lacZtau/+ was similar to control. of anti-Flag relative to anti-pY may explain the lack of complete overlap (D) Double knockdown of abl and eya (UAS-EyaRNAi, UAS-AblRNAi/+; GMR- (yellow) of the two signals. (A) In transiently transfected S2 cells, the Eya pY Gal4/+; Ro-lacZtau/+) causes multiple thick axon bundles to overshoot the signal increases in the presence of Abl. (B) Treatment with l phosphatase lamina. (E) Increased Eya expression (UAS-Eya/GMR-Gal4; Ro-lacZtau/+) removes the pY signal. (C) Actin-Gal4-driven coexpression of AblWT, but not causes targeting defects. (F) Reduced abl expression suppresses Eya overex- kinase-dead AblKD, increases tyrosine phosphorylation of Flag-Eya in pression phenotypes (UAS-AblRNAi/+; UAS-Eya/GMR-Gal4; Ro-lacZtau/+). (G) embryos. (D) GMR-Gal4-driven coexpression of Abl increases tyrosine phos- Increased cytoplasmic Eya perturbs axon targeting (GMR-Gal4/+; Ro-lacZtau/ phorylation of Flag-Eya in third-instar eye discs. (E) Schematic of Eya deletion UAS-Myr-EyaWT). (H) Expression of phosphatase-dead Myr-tagged Eya constructs. (F) Abl primarily targets the PST-rich region of Eya. Lane (GMR-Gal4/+; Ro-lacZtau/UAS-Myr-EyaK699Q) shows only mild targeting numbering matches the construct number in (E). Molecular weight (MW) stan- defects. (I) Reduced abl expression suppresses Myr-EyaWT phenotypes dards are indicated in Kd on the left. Arrowheads on the right point out IgG (UAS-AblRNAi/+; UAS-Myr-EyaWT/GMR-Gal4; Ro-lacZtau/+). (J) Summary of bands. Asterisks indicate constructs run with expected mobility in the absence the average number of overshooting axon bundles per brain for each genotype of Abl. (G) In vitro kinase assay with recombinant mammalian c-Abl. Coomas- ± standard deviation. Scoring was performed blind to the genotype. n, number sie staining in the left panel shows fusion protein amounts. The right panel of brains scored. Statistical significance (p values) were calculated by using shows the phosphoimager exposure of the same blot showing the signal in Excel’s built-in TTEST function after performing an unpaired, one-tailed the GST-EyaPST lane. (H) In vitro kinase assays with immunoprecipitated t test for each pair of genotypes. All phenotypes were significant compared RNAi Myc-tagged Drosophila Abl from transfected S2 cells phosphorylates GST- to control (p < 0.001) except that of Abl (y). P values for other relevant PST Eya . Left panel, Coomassie stain; middle panel, phosphoimager exposure comparison pairs are indicated next to brackets. of same blot. Right panels show that immunoprecipitated kinase-dead Abl (KD) does not phosphorylate GST-EyaPST: top, Coomassie; middle, P32 expo- Abl Directly Phosphorylates Eya sure; bottom, anti-Myc immunoblot; the quantitation of relative intensity of To elucidate the molecular mechanisms underlying eya-abl signals is indicated. genetic synergy, we asked whether Eya might be a substrate of the Abl tyrosine kinase. Using anti-phosphotyrosine immuno- To determine which region of Eya becomes tyrosine phosphor- blotting of Eya immunoprecipitated from transfected S2 cells, we ylated, we transfected S2 cells with a set of Eya deletion constructs found that coexpression of wild-type Abl, but not kinase-dead (Silver et al., 2003)(Figure 4E) in the presence or absence of Abl. Abl, increased Eya tyrosine phosphorylation (Figure 4A). Treat- Deletion of the ED increased the phosphotyrosine signal, whereas ment with lambda phosphatase removed the phosphotyrosine deletion of the PST-rich region reduced it (Figure 4F, lanes 4 and 3, signal (Figure 4B). Co-overexpression of Eya and Abl transgenes respectively). Comparable results were obtained by using either in embryos and eye imaginal discs confirmed that expression of wild-type or phosphatase-dead Eya constructs, indicating that Abl, but not kinase-dead Abl, increased the phosphotyrosine the changes do not simply reflect loss or hyperactivity of Eya signal on Eya in Drosophila tissues (Figures 4C and 4D). phosphatase activity. Examination of smaller deletions within the

274 Developmental Cell 16, 271–279, February 17, 2009 ª2009 Elsevier Inc. Developmental Cell Nuclear-Cytoplasmic Partitioning of Eya Function

Eya+Abl E Cytoplasmic Figure 5. Abl Expression Relocalizes Eya to Membrane-associated the Cytoplasm A C D 25 (A–D0) Indirect immunoﬂuorescence of transfected S2 cells stained with anti-Flag to recognize Flag-

ya 20 Eya and/or anti-Myc to detect Abl-Myc. (A) Eya is Eya E 15 predominantly nuclear, although cytoplasmic staining can be seen. (B) Abl localizes to the cyto- B C’ D’ 10 plasm with enrichment at the plasma membrane. (C–D) Cells coexpressing Eya and Abl double 5 0 0

Abl Abl labeled with (C and D) anti-Flag and (C and D ) anti-Myc. (C and C0) Example of an Abl-expressing

% cells with extranuclear 0 Eya Eya+Abl Eya+AblKD cell with uniform distribution of Eya. (D–D0) Example of an Abl-expressing cell withexclusive cytoplasmic Eya+Abl Eya+Abl Eya+AblKD Eya localization and membrane enrichment. F G H (E) Quantitation of Eya localization. Approximately 300 cells were counted for each condition. (F–H) Anti-Eya staining of wing imaginal discs coexpressing Eya and Abl transgenes with Ptc-

Eya localization Gal4. (F–G) Different optical sections of the same disc coexpressing Eya and Abl reveals expression of Eya in both (F) cytoplasm and (G) nucleus. (H) No cytoplasmic localization was detected upon coexpression of Eya and kinase-dead AblKD.

215 amino acid PST-rich domain demonstrated that Abl can phos- tissues, Eya/Abl coexpression in the wing imaginal disc resulted phorylate multiple residues across the region (data not shown). in significant cytoplasmic Eya accumulation (Figures 5F and 5G), To ask whether Eya can be a direct substrate of Abl, in vitro whereas Eya expressed alone or in combination with kinase- kinase assays were performed with recombinant c-Abl and dead Abl appeared to be exclusively nuclear (Figure 5H). GST-Eya fusion proteins expressing either the PST-rich region, PST ED the ED, or GST alone (GST-Eya , GST-Eya , and GST; Extranuclear Eya Localization Is Required for Function GST-EyaED was inactive as a phosphatase in the assay condi- The discovery that Abl promotes the cytoplasmic accumulation tions). Consistent with the S2 cell deletion analysis results, of Eya suggests that extranuclear Eya localization might be phys- GST-EyaPST was directly phosphorylated by c-Abl, whereas iologically relevant; if so, then nuclear restriction of Eya should GST-EyaED and GST were not (Figure 4G). Comparable results compromise function. To test this hypothesis, we inserted the were obtained with Drosophila Abl isolated by immunoprecipita- SV40 nuclear localization sequence (NLS) (Kalderon et al., tion from transfected S2 cells, but not with kinase-dead 1984) into Eya and tested its efficacy by examining NLS-Eya Drosophila Abl (Figure 4H). Thus Abl-mediated tyrosine phos- localization in S2 cells. Expressed alone or with Abl, NLS-Eya phorylation of the PST-rich region of Eya is likely direct. was exclusively nuclear, whereas the control protein containing a nonfunctional mutant NLS (NLSMUT-Eya) relocalized to the Abl-Mediated Tyrosine Phosphorylation Relocalizes Eya cytosol as efficiently as untagged Eya in response to Abl activa- to the Cytoplasm tion (Figure S3). NLS-Eya retained wild-type transactivation ability (Figure S4), indicating that the NLS insertion does not Eya has been extensively characterized as a nuclear transcription compromise nuclear function. factor, whereas Drosophila Abl is a cytoplasmic/membrane- To determine the functional consequences of nuclear restric- associated tyrosine kinase (Bennett and Hoffmann, 1992; Bonini tion during eye development, transgenic lines were generated, et al., 1998; Fox and Peifer, 2007). To investigate how the two expressed under control of the dpp-Gal4 driver, and assessed proteins might interact and the potential consequences of Abl- in both ectopic eye induction and genetic rescue assays. NLS- mediated phosphorylation of Eya, we examined the subcellular EyaWT transgenes exhibited an average 20% frequency of localization of Eya and Abl in cotransfected S2 cells. Although ectopic eye induction and 48% rescue efficiency, significantly Eya is predominantly nuclear, a cytoplasmic signal is often lower than the 49% and 100% respective averages obtained apparent (Figures 5A and 5E). Treatment with Leptomycin B, an from comparable analysis of untagged EyaWT (Figures 6A–6D) inhibitor of Crm-1-dependent nuclear export, resulted in exclu- (Hsiao et al., 2001; Tootle et al., 2003). Ruling out the possibility sively nuclear Eya localization (Figure S2), suggesting a dynamic that reduced activity might be attributed solely to insertion of the nuclear export/import cycle. In Eya/Abl cotransfected cells, tag, NLSMUT-EyaWT lines exhibited almost twice the activity of whereas Abl’s subcellular distribution remained constant, Eya NLS-EyaWT (Figures 6A and 6B). showed a marked increase in cytoplasmic accumulation (Figures 5B–5E). Kinase-dead Abl did not alter Eya subcellular distribution (Figure 5E), consistent with Abl-mediated phosphorylation of Eya Coexpression of Membrane-Tethered and Nuclearly triggering this effect. Cotransfection of So blocked Abl-induced Restricted Eya Reconstitutes Full Function relocation of Eya to the cytosol (Figure S3). Finally, indicating Our finding that nuclear tethering of Eya compromised its activity that Abl can trigger a comparable localization shift in Drosophila led us to consider how cytoplasmic Eya might contribute to

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nucleus both in S2 cells and in Drosophila tissues (Figures S5B and S6B). When tested in either the ectopic eye or genetic rescue assays, Myr-EyaWT transgenes lacked activity, consistent with the essential requirement for Eya transcriptional activity during retinal specification (Tables S1 and S2; Figure S7). We then asked whether coexpression of Myr-EyaWT and NLS- EyaWT, which results in two distinct protein pools targeted and restricted to different subcellular compartments (Figures S5C and S6), could reconstitute full Eya function. Four NLS-EyaWT transgenes selected based on an ectopic eye induction frequency that approximated the average value (20%) were recombined with Myr-EyaWT transgenes that expressed comparable levels of cytosolic Eya protein (Figure S8). When tested in the ectopic eye induction assay, a consistent trend emerged in which coexpression of NLS-EyaWT and Myr-EyaWT resulted in an approximate two-fold increase in activity relative to the expression of NLS-EyaWT alone (Figure 6G; Table S3). Increased activity was also apparent in the genetic rescue assay, which showed that the size of rescued eye tissue was markedly enhanced (Figures 6D and 6E). Because Myr-EyaWT alone lacked activity in these assays (Tables S1 and S2), the activity increase obtained from coexpressing NLS- and Myr-tethered Eya proteins reflects strong synergy rather than additivity. Excluding the possibility that the increased activity might result from NLS-EyaWT recruiting Myr-EyaWT to the nucleus, immunostaining revealed both nuclear and cytoplasmic Eya localization in the NLS-EyaWT + Myr-EyaWT-expressing cells, with the expres- Figure 6. Coexpression of Membrane-Tethered and Nuclearly sion level in each compartment comparable to that observed Restricted Eya Reveals a Cytoplasmic Requirement for Eya Phos- phatase Activity when either transgene was expressed alone (Figure S6). Taken (A) Average activities of Eya transgenes in the ectopic eye induction assay (see together, these results support a model in which cytoplasmic Table S1 for details). n, number of transgenic lines tested; N, total number of Eya and nuclear Eya have separate roles such that full Eya func- flies scored. tion can be reconstituted from coexpression of two spatially (B) Average activities of Eya transgenes in the genetic rescue assay (see Table restricted protein pools. S2 for details). n, number of transgenic lines tested; N, total number of flies scored. (C–F) Adult heads representative of eyeless phenotype of eya2, modest rescue Eya Phosphatase Activity Is Required in the Cytoplasm by NLS-EyaWT, strong rescue by coexpressed NLS-EyaWT + Myr-EyaWT, and The complementation assay described above provides an ideal modest rescue by coexpressed NLS-EyaWT + Myr-EyaK699Q. system by which to test whether Eya phosphatase activity might (G) Average ectopic eye induction efficiency of recombinant lines obtained by be important for cytoplasmic function. We therefore generated WT WT systematic crossing of four NLS-Eya lines with multiple Myr-Eya and Myr- additional Myr-Eya transgenes carrying the K699Q missense K699Q Eya transgenes (see Table S3 for details). n, number of independent mutation that ablates in vitro phosphatase activity, retains recombinant lines tested; N, total number of flies scored; the Student’s t test was applied to determine the p values between groups. No significant dif- productive interactions with So, and exhibits reduced activity in ference is determined between NLS-EyaWT and NLS-EyaWT + Myr-EyaK699Q both ectopic eye and genetic rescue assays (Tootle et al., 2003). (p = 0.48). As previously shown for the untagged versions (Tootle et al., 2003), Myr-EyaWT and Myr-EyaK699Q lines expressed comparable overall function. In one scenario, nucleocytoplasmic shuttling protein levels (Figure S8). Like Myr-EyaWT, Myr-EyaK699Q trans- might allow Eya to be ‘‘activated’’ in the cytoplasm in a manner genes alone had no activity in ectopic eye or genetic rescue critical for proper nuclear function. Alternatively, nucleocytoplas- assays (Tables S1 and S2). However, in contrast to Myr-EyaWT, mic shuttling might establish two physically and functionally Myr-EyaK699Q showed very limited synergy when coexpressed separate pools of Eya protein: a nuclear pool devoted to tran- with NLS-EyaWT, with only an 10% increase in ectopic eye scriptional regulation and a cytosolic pool that participates in induction relative to NLS-EyaWT alone (Figure 6G). Similarly, in cytoplasmic signaling events. It should be noted that, to date, the genetic rescue experiment, the NLS-EyaWT + Myr-EyaK699Q only nuclear Eya has been visualized in developing tissues recombinants restored smaller eye fields compared to the NLS- (Bonini et al., 1998). Accordingly, we predict that low endoge- EyaWT + Myr-EyaWT recombinants (Figures 6E and 6F). nous cytoplasmic Eya concentrations will prove sufficient for To begin to investigate whether cytoplasmic Eya activity might its cytoplasmic functions. be relevant to photoreceptor morphogenesis, we asked whether To generate the cytoplasmically restricted pool of Eya needed GMR-Gal4-driven expression of Myr-EyaWT could perturb axon to distinguish between these models, we inserted the Src myris- targeting. Expression of Myr-EyaWT caused an average of toylation tag (Cross et al., 1984) at the Eya N terminus and 19.0 axon bundles/brain to overshoot the lamina (Figure 3G), showed that Myr-EyaWT was effectively excluded from the significantly higher than the 8.4 average obtained in controls.

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In contrast, expression of phosphatase-dead Myr-EyaK699Q the presence of its binding partner Six, such that in its absence resulted in only 11.8 axon bundles/brain overshooting the lamina Eya localizes to the cytosol (Ohto et al., 1999; Zhang et al., (Figure 3H; Figure S9). Reduction in abl dose dominantly 2004). Second, protein-protein interactions with several suppressed the targeting defects associated with expression membrane-associated and cytoplasmic proteins have been of Myr-EyaWT, further implicating Eya-Abl synergy in this context demonstrated in two-hybrid screens, although only one interac- (Figure 3I). tion has been further investigated (Fan et al., 2000; Embry et al., 2004; Li et al., 2004). In this example, interactions between Eya DISCUSSION and the G protein Gai can recruit Eya to the cytoplasm of cultured cells, and a balance between binding to G proteins The discovery that Eya possesses intrinsic protein tyrosine and Six has been proposed to regulate Eya distribution and func- phosphatase activity suggests that prior studies of its nuclear tion (Fan et al., 2000; Embry et al., 2004). transcriptional functions within the RD network may have To what aspects of eye development might cytosolic Eya revealed only a partial picture of the signaling pathways and activity contribute? Although identification of Eya substrates developmental contexts in which it operates. The data presented and elucidation of the specific signaling events regulated by in this paper lead us to propose a model in which Eya, in addition cytoplasmic Eya activity will be required to answer this question to operating as a nuclear transcription factor, participates inde- definitively, several intriguing models are worth considering. pendently as a phosphatase in cytoplasmic signaling events First, the ectopic eye induction and genetic rescue assays used important for eye development. to characterize the complementation between Myr-Eya and Our analysis of the subcellular compartmentalization of Eya NLS-Eya transgenes imply a requirement for extranuclear Eya function has revealed a requirement for Eya activity in the cyto- in retinal specification. In considering this context, it is important plasm. Specifically, although nuclearly restricted NLS-Eya to note that whereas a great deal is understood about how tran- appears to be fully competent as a coactivator, as judged by scriptional hierarchies such as the RD network drive retinal induc- cultured cell transcriptional reporter assays, it exhibits a reduced tion, much less is known about how specific differentiation ability to induce eye tissue in either wild-type or eya loss-of-func- programs are coordinated with the morphogenetic events that tion backgrounds. Coexpression of cytoplasmically restricted pattern the tissue. In Drosophila, specification of retinal fates is Myr-Eya restores a wild-type level of eye inducing activity to immediately preceded by adhesive and morphological changes the NLS-Eya background, supporting the interpretation that in and posterior to the morphogenetic furrow (reviewed by NLS-Eya is fully competent with respect to transcription, but Carthew, 2007). Although phosphotyrosine signaling at the cannot perform the essential function normally provided by cyto- morphogenetic furrow has not been extensively studied, its plasmic Eya. Eya phosphatase activity appears to contribute to importance to cell adhesion and epithelial morphogenesis in cytosolic function, as phosphatase-dead versions of cytoplasmi- other contexts is well documented. For example, recent work cally restricted Eya transgenes fail to complement the NLS-Eya studying the invagination of the ventral furrow during Drosophila background effectively. Thus, we propose that whereas regula- gastrulation demonstrated that Abl signaling acting in parallel to tion of gene expression by the core RD network relies primarily the Rho activator RhoGEF2 regulates actin organization to drive on nuclear Eya function, other signaling events important for apical cell constriction (Fox and Peifer, 2007). Given the impor- retinal development may rely on transcription-independent func- tance of cell constriction in the retinal furrow, it will be interesting tions of the cytoplasmic Eya phosphatase. to investigate whether similar signaling mechanisms operate in Mechanistically, we propose that Eya traffics dynamically this context and whether cytoplasmic Eya phosphatase activity between nuclear and cytoplasmic compartments, with its final is involved. Encouragingly, loss of eya impairs morphogenetic localization determined by its phosphorylation state and interac- furrow propagation (Pignoni et al., 1997), suggesting that investi- tions with specific signaling partners. Thus, in contexts in which gation of defects in epithelial remodeling and reorganization of Abl signaling is activated, Abl-mediated phosphorylation may cell-cell contacts at the furrow in eya mutants could be fruitful. provide a cytoplasmic retention signal that targets Eya to its Another possibility is that cytoplasmic Eya phosphatase func- appropriate site of action, presumably through interactions with tion might provide critical feedback regulation on other signaling specific phosphotyrosine-binding proteins. Autocatalytic Eya pathways during retinal specification. Indeed, a complex web of phosphatase activity (Tootle et al., 2003) would play a critical interactions between multiple signaling networks including the positive role with respect to overall Eya function by returning Wingless, Notch, Hedgehog, and EGFR pathways has been Eya to the nucleus to prevent depletion of the nuclear pool shown to be critical for RD network function and retinal induction needed to carry out essential transcriptional programs. Although (reviewed by Pappu and Mardon, 2004; Silver and Raby, 2005). cytosolic Eya substrates have not yet been identified, the fact that Thus, if cytosolic Eya phosphatase activity were absent or mis- phosphatase-dead cytoplasmically restricted Eya was less localized, the resulting signaling imbalances could potentially active than the wild-type version in an assay in which dynamic compromise eye specification and development. shuttling between nuclear and cytoplasmic compartments was Finally, cytoplasmic Eya function could be important for not relevant suggests that Eya-mediated dephosphorylation of neuronal morphogenesis, perhaps through involvement in Abl- substrates other than itself is likely important. mediated signaling events. Because Abl signaling has not yet Although Eya has been primarily characterized as a nuclear been explored in the retina, determining which downstream protein, several previous observations are consistent with our branches of the pathway operate in this developmental context proposed model of extranuclear function. First, in mammalian will be important for elucidating the molecular and cellular cultured cells, Eya nuclear localization and/or retention requires defects underlying the phenotypes and interactions we have

Developmental Cell 16, 271–279, February 17, 2009 ª2009 Elsevier Inc. 277 Developmental Cell Nuclear-Cytoplasmic Partitioning of Eya Function

reported. For example, the photoreceptor axon targeting defects transfection and were induced after 24 hr with 0.7 mM CuS04. Pervanadate WT observed in eya or abl mutants, or upon Myr-Eya expression, treatment with 100 mM NaVO3 and 200 mMH2O2 was performed for 15 min could reflect impaired receiving or processing of attractive prior to fixation or lysis. For immunoprecipitation studies, cells were lysed in whole-cell lysis buffer signals from either brain cells or adjacent retinal neurons, weak- (100 mM NaCl, 50 mM Tris [pH 7.5], 2 mM EDTA, 2 mM EGTA, 1% NP-40) ening of repulsive signaling between axons, or strengthening of with protease inhibitors (Roche), incubated with 20 ml anti-Flag agarose adhesive properties between the axons such that they fail to (Sigma) for 1 hr at 4C, washed twice with buffer and twice with TBS, boiled spread properly as they exit the optic stalk. in SDS loading buffer, and resolved on 8% SDS-PAGE gels. In considering the mechanistic possibilities whereby Eya might Proteins were visualized by immunoblotting by using mouse anti-Flag (1:1000, interact with the Abl signaling network, it is important to reiterate Sigma), rabbit anti-phosphotyrosine (1:400, Upstate), mouse anti-Myc (1:500, Santa Cruz Biotechnology), and IRDye secondary antibodies (1:5000, Li-COR that although our genetic analyses indicate eya and abl function Biosciences) with the Odyssey Infrared Imaging System (Li-COR Biosciences). cooperatively, the two genes encode proteins with opposing Dephosphorylation of immunoprecipitated Eya was achieved by incubation catalytic functions. Thus, a simple relationship whereby Eya with 400 units of Lambda phosphatase (New England Biolab). dephosphorylates Abl or its substrates is unlikely to offer a suit- In vitro kinase assays were performed by using mammalian c-Abl (NEB able explanation since this would most likely be reflected as P6050S) and purified recombinant GST-EyaPST, GST-EyaED, and GST as antagonism rather than synergy. Instead, Abl phosphorylation substrates. Reactions (20 ml) containing 1 mg GST fusion protein, kinase buffer and recruitment of Eya to the cytoplasm may facilitate formation (NEB: 50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, 0.01% Brij 35 [pH 7.5] at 25C, supplemented with 20 M cold ATP), 1 ml[g-32P]ATP (Perkin of protein complexes important for Abl signaling and/or promote Elmer BLU502H, SA 3000 Ci), and either 0.5–1 ml NEB Abl or immunoprecipitated interactions with components of other phosphotyrosine signaling Drosophila Abl were incubated for 30 min at 30C, mixed with 20 ml23 SDS gel pathways, which together would target Eya phosphatase activity loading buffer, and boiled before loading onto polyacrylamide gels. Gels were toward appropriate substrates. Finally, our results do not pre- either Coomassie stained or transferred to nitrocellulose for exposure on a Storm clude Eya’s nuclear transcriptional activities from also contrib- phosphoimager. uting to Abl signaling; thus, it will be important to investigate further the complex spatiotemporal requirements for Eya, other Immunostaining and Antibodies members of the RD network, and the Abl signaling pathway Imaginal discs dissected from late third-instar larvae were fixed in 4% paraformaldehyde in PBT (0.1% Triton X-100 in PBS) for 10 min. Genotyped stage-16 during retinal development. embryos (GFP negative) were dechorionated in 50% bleach and fixed in a 1:1 mix of heptane and 4% paraformaldehyde in PBS for 20 min. Tissues EXPERIMENTAL PROCEDURES were blocked in 1% normal goat serum PBS, incubated with primary antibodies at 4C overnight, washed three times in PBT, and incubated with Fly Strains Cy3- or FITC-conjugated secondary antibodies (Jackson Immunoresearch, We used the following 16 fly stocks: (1) hsFLP,Elav-Gal4,UAS- 1:2000) for 1 hr at room temperature, washed, and mounted in Prolong anti- mCD8GFP;tub-Gal80FRT80B/TM6Tb, (2) hsFLP,Elav-Gal4,UAS-mCD8GFP; fade (Invitrogen). Fluorescent images were taken with a Zeiss 510 confocal tub-Gal80FRT40A/Cyo, (3) Ptc-Gal4, (4) Apt-Gal4,(5)Act-Gal4, (6) GMR- microscope. Antibodies used were guinea pig anti-Eya (1:10000); mouse Gal4(Zipursky), (7) dpp-Gal440C6, (8) Ro-lacZtau, (9) eyaA188,(10)eyaG130,(11) anti-Myc (1:1000, Santa Cruz Biotechnology); mouse anti-Elav, anti-lamin, eya2, (12) eyaClift, (13) abl1, (14) abl2, (15) abl4, and (16) abl04674. anti-BP102, anti-24B10 (1:10, Developmental Studies Hybridoma Bank); and eya and abl MARCM clones were generated by heat shocking hsFLP, rabbit anti-b-gal (1:10000, Promega). Elav-Gal4,UAS-mCD8GFP; eyaCliftFRT40A/tub-Gal80FRT40A animals and hsFLP,Elav-Gal4, UAS-mCD8GFP; abl2FRT80B/tub-Gal80FRT80B animals SUPPLEMENTAL DATA at 37 C for 2 hr on the second day after embryos were laid. For CNS studies, abl2/TM3TwiGFP, eyaA188/CyoTwiGFP, and eyaA188/ Supplemental Data include Supplemental Experimental Procedures, nine CyoTwiGFP; abl2/TM3TwiGFP flies were crossed as appropriate. Non-GFP- figures, and three tables and can be found with this article online at http:// expressing embryos were selected for immunostaining. www.cell.com/developmental-cell/supplemental/S1534-5807(08)00515-7. UAS-EyaWT, UAS-NLS-EyaWT, UAS-Myr-EyaWT, UAS-Myr-EyaK699Q, UAS- NLSMUT-EyaWT, and UAS-MyrMUT-EyaWT transgenics were generated by standard P element-mediated transformation. In the ectopic eye assay, 200 flies ACKNOWLEDGMENTS of the genotype dpp-Gal4 > UAS-Eya were examined for each line for the presence of pigmented eye-like tissue under the antennae. For ease of genetic We thank J. Jemc, C. Wrobel, R. Fehon, and E. Ferguson for comments on the manipulation, only lines with third chromosome inserts were tested in the manuscript; I.R. and Fehon laboratory members for discussions; K. Nyberg rescue assay, and 100 flies of the genotype eya2; dpp-Gal4/UAS-Eya were and M. DiMarco for confocal assistance; J. Weinberg for help with genetics; scored for recovery of eye tissue. and M. Seeger, M. Peifer, and D. Van Vactor for reagents. We acknowledge Recombinants carrying two different Eya transgenes were confirmed by the Bloomington and Vienna Drosophila stock centers for flies, and the Devel- immunostaining. opmental Studies Hybridoma Bank for antibodies. This research was sup- To examine the R2–R5 overshooting phenotype, GMR-Gal4/CyoGFP; ported by National Institutes of Health grant R01 EY12549 to I.R., a Women’s Ro-lacZtau flies were crossed to GFP-balanced lines of UAS-EyaWT, UAS- Board Fellowship of the University of Chicago to W.X., and the Medical Scien- EyaRNAi/y, UAS-AblRNAi/y, UAS-EyaRNAi,UAS-AblRNAi/y, UAS-AblRNAi/y; tist National Research Service Award 5T32 GM07281 to N.D. UAS-EyaWT, UAS-Myr-EyaWT, UAS-Myr-EyaK699Q or UAS-AblRNAi/y; UAS- Myr-EyaWT. Quantitation of mistargeted axon bundles was performed blind Received: May 19, 2008 as to genotype. 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