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• W2009076560 abstract "Argos, a secreted antagonist of Drosophila epidermal growth factor receptor (dEGFR) signaling, acts by sequestering the activating ligand Spitz. To understand how different domains in Argos contribute to efficient Spitz sequestration, we performed a genetic screen aimed at uncovering modifiers of an Argos misexpression phenotype in the developing eye. We identified a series of suppressors mapping to the Argos transgene that affect its activity in multiple developmental contexts. These point mutations map to both the N- and C-terminal cysteine-rich regions, implicating both domains in Argos function. We show by surface plasmon resonance that these Argos mutants are deficient in their ability to bind Spitz in vitro. Our data indicate that a mere ∼2-fold decrease in KD is sufficient to compromise Argos activity in vivo. This effect could be recapitulated in a cell-based assay, where a higher molar concentration of mutant Argos was needed to inhibit Spitz-dependent dEGFR phosphorylation. In contrast, a ∼37-fold decrease in the binding constant nearly abolishes Argos activity in vivo and in cellular assays. In agreement with previously reported computational studies, our results define an affinity threshold for optimal Argos inhibition of dEGFR signaling during development. Argos, a secreted antagonist of Drosophila epidermal growth factor receptor (dEGFR) signaling, acts by sequestering the activating ligand Spitz. To understand how different domains in Argos contribute to efficient Spitz sequestration, we performed a genetic screen aimed at uncovering modifiers of an Argos misexpression phenotype in the developing eye. We identified a series of suppressors mapping to the Argos transgene that affect its activity in multiple developmental contexts. These point mutations map to both the N- and C-terminal cysteine-rich regions, implicating both domains in Argos function. We show by surface plasmon resonance that these Argos mutants are deficient in their ability to bind Spitz in vitro. Our data indicate that a mere ∼2-fold decrease in KD is sufficient to compromise Argos activity in vivo. This effect could be recapitulated in a cell-based assay, where a higher molar concentration of mutant Argos was needed to inhibit Spitz-dependent dEGFR phosphorylation. In contrast, a ∼37-fold decrease in the binding constant nearly abolishes Argos activity in vivo and in cellular assays. In agreement with previously reported computational studies, our results define an affinity threshold for optimal Argos inhibition of dEGFR signaling during development. The epidermal growth factor receptor belongs to a family of receptor tyrosine kinases that are well conserved from lower metazoans to humans (1Ben-Shlomo I. Yu Hsu S. Rauch R. Kowalski H.W. Hsueh A.J. Sci. STKE 2003. 2003; : RE9Google Scholar, 2Stein R.A. Staros J.V. J. Mol. Evol. 2000; 50: 397-412Crossref PubMed Scopus (76) Google Scholar). In humans, mutations or genetic alterations that alter receptor activity have been correlated with cancer progression and poor clinical outcome, validating the ErbB family as a target for therapeutic agents (3Arteaga C.L. Exp. Cell Res. 2003; 284: 122-130Crossref PubMed Scopus (200) Google Scholar, 4Graham J. Muhsin M. Kirkpatrick P. Nat. Rev. Drug Discov. 2004; 3: 549-550Crossref PubMed Scopus (189) Google Scholar, 5Holbro T. Civenni G. Hynes N.E. Exp. Cell Res. 2003; 284: 99-110Crossref PubMed Scopus (519) Google Scholar, 6Cohen M.H. Williams G.A. Sridhara R. Chen G. McGuinn Jr., W.D. Morse D. Abraham S. Rahman A. Liang C. Lostritto R. Baird A. Pazdur R. Clin. Cancer Res. 2004; 10: 1212-1218Crossref PubMed Scopus (451) Google Scholar, 7Gazdar A.F. Shigematsu H. Herz J. Minna J.D. Trends Mol. Med. 2004; 10: 481-486Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). In Drosophila melanogaster, EGF 5The abbreviations used are: EGF, epidermal growth factor; dEGFR, Drosophila EGF receptor; Aos, Argos; NCR, N-terminal cysteine-rich region; CCR, C-terminal cysteine-rich region; Spi, Spitz; GMR, glass multiple reporter; SPR, surface plasmon resonance; UAS, upstream activating sequence. 5The abbreviations used are: EGF, epidermal growth factor; dEGFR, Drosophila EGF receptor; Aos, Argos; NCR, N-terminal cysteine-rich region; CCR, C-terminal cysteine-rich region; Spi, Spitz; GMR, glass multiple reporter; SPR, surface plasmon resonance; UAS, upstream activating sequence. receptor signaling is utilized reiteratively throughout development to mediate a wide array of cellular decisions (8Shilo B.Z. Development. 2005; 132: 4017-4027Crossref PubMed Scopus (155) Google Scholar). Remarkably, this versatility is accomplished with a single receptor (dEGFR) and four activating ligands: Spitz (Spi), Gurken, Keren, and Vein. The functional diversity of dEGFR signaling has been partially attributed to the differential use of ligands throughout development (8Shilo B.Z. Development. 2005; 132: 4017-4027Crossref PubMed Scopus (155) Google Scholar). For instance, the ligand Gurken is produced exclusively in the germline to specify eggshell structures and the embryonic axes (9Gonzalez-Reyes A. St. Johnston D. Development. 1998; 125: 2837-2846PubMed Google Scholar, 10Nilson L.A. Schupbach T. Curr. Top Dev. Biol. 1999; 44: 203-243Crossref PubMed Scopus (178) Google Scholar). In contrast, Spi and Vein participate in numerous processes, sometimes sharing additive roles (such as in ventral ectoderm patterning) (11Chang J. Jeon S.H. Kim S.H. Mol. Cell. 2003; 15: 186-193Google Scholar, 12Chang J. Kim I.O. Ahn J.S. Kim S.H. Int. J. Dev. Biol. 2001; 45: 715-724PubMed Google Scholar, 13Golembo M. Raz E. Shilo B.Z. Development. 1996; 122: 3363-3370PubMed Google Scholar, 14Golembo M. Yarnitzky T. Volk T. Shilo B.Z. Genes Dev. 1999; 13: 158-162Crossref PubMed Scopus (52) Google Scholar) and in some instances acting as the main dEGFR ligand (such as in photoreceptor recruitment or wing vein differentiation, respectively) (15Guichard A. Biehs B. Sturtevant M.A. Wickline L. Chacko J. Howard K. Bier E. Development. 1999; 126: 2663-2676PubMed Google Scholar, 16Simcox A.A. Grumbling G. Schnepp B. Bennington-Mathias C. Hersperger E. Shearn A. Dev. Biol. 1996; 177: 475-489Crossref PubMed Scopus (87) Google Scholar, 17Wessells R.J. Grumbling G. Donaldson T. Wang S.H. Simcox A. Dev. Biol. 1999; 216: 243-259Crossref PubMed Scopus (40) Google Scholar, 18Tio M. Ma C. Moses K. Mech. Dev. 1994; 48: 13-23Crossref PubMed Scopus (98) Google Scholar, 19Tio M. Moses K. Development. 1997; 124: 343-351PubMed Google Scholar, 20Martin-Blanco E. Roch F. Noll E. Baonza A. Duffy J.B. Perrimon N. Development. 1999; 126: 5739-5747PubMed Google Scholar). Interestingly, no knock-out phenotype or expression pattern has been reported for the fourth ligand, Keren (21Reich A. Shilo B.Z. EMBO J. 2002; 21: 4287-4296Crossref PubMed Scopus (59) Google Scholar). In addition to the four agonists, the Drosophila EGF receptor signaling system includes two extracellular inhibitors, which function in negative feedback loops to antagonize dEGFR signaling. Kekkon 1 is a transmembrane molecule of the leucine-rich repeat-immunoglobulin (LIG) superfamily that attenuates dEGFR activity via a direct interaction (22Ghiglione C. Carraway 3rd, K.L. Amundadottir L.T. Boswell R.E. Perrimon N. Duffy J.B. Cell. 1999; 96: 847-856Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 23Alvarado D. Rice A.H. Duffy J.B. Genetics. 2004; 167: 187-202Crossref PubMed Scopus (14) Google Scholar). Argos (Aos) is a secreted molecule that was initially proposed to bind and inhibit dEGFR by virtue of its atypical EGF domain (24Freeman M. Klambt C. Goodman C.S. Rubin G.M. Cell. 1992; 69: 963-975Abstract Full Text PDF PubMed Scopus (211) Google Scholar). Recent work, however, has demonstrated that Aos instead exerts its antagonistic effect on dEGFR signaling by sequestering the activating ligand Spi, although its effect on the remaining three ligands has not yet been reported (25Klein D.E. Nappi V.M. Reeves G.T. Shvartsman S.Y. Lemmon M.A. Nature. 2004; 430: 1040-1044Crossref PubMed Scopus (114) Google Scholar). Aos was also shown to associate with the surface of cultured S2 cells in an interaction that is dissociable with excess soluble heparin (25Klein D.E. Nappi V.M. Reeves G.T. Shvartsman S.Y. Lemmon M.A. Nature. 2004; 430: 1040-1044Crossref PubMed Scopus (114) Google Scholar), suggesting a potential regulatory mechanism for Aos activity in vivo. However, the functional and physiological significance of this finding remains unclear. Structurally, Aos is composed of N- and C-terminal cysteine-rich regions (NCR and CCR, respectively), separated by a largely unconserved linker. The NCR, which contains 4 cysteines, has no known direct function, and misexpression of this domain alone displays no biological activity. The CCR includes 12 cysteines (see Fig. 1B) and contains a putative EGF-like domain (residues 363-424) (24Freeman M. Klambt C. Goodman C.S. Rubin G.M. Cell. 1992; 69: 963-975Abstract Full Text PDF PubMed Scopus (211) Google Scholar). Misexpression of the entire CCR from Aos (residues 225-444) displays partial activity in vivo and is sufficient for binding Spi in vitro (albeit with a ∼20-fold decreased affinity) (25Klein D.E. Nappi V.M. Reeves G.T. Shvartsman S.Y. Lemmon M.A. Nature. 2004; 430: 1040-1044Crossref PubMed Scopus (114) Google Scholar, 26Howes R. Wasserman J.D. Freeman M. J. Biol. Chem. 1998; 273: 4275-4281Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In contrast, misexpression of the putative EGF-like domain alone does not rescue Aos mutant phenotypes (26Howes R. Wasserman J.D. Freeman M. J. Biol. Chem. 1998; 273: 4275-4281Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), arguing that (if it does adopt an EGF-like fold) it is not sufficient for Aos function. Aos participates in multiple developmental processes where it is expressed as a “high threshold” gene, in response to high levels of Spi-induced dEGFR signaling (27Wasserman J.D. Freeman M. Cell. 1998; 95: 355-364Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 28Golembo M. Schweitzer R. Freeman M. Shilo B.Z. Development. 1996; 122: 223-230PubMed Google Scholar). Consistent with its role as an inhibitor of dEGFR signaling, Aos knock-outs exhibit phenotypes typical of dEGFR gain-of-function mutants, and Aos misexpression inhibits dEGFR signaling (26Howes R. Wasserman J.D. Freeman M. J. Biol. Chem. 1998; 273: 4275-4281Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 29Freeman M. Development. 1994; 120: 2297-2304PubMed Google Scholar). Aos contributes importantly to the spatio-temporal regulation of dEGFR signaling through its participation in a negative feedback loop as a result of Spi-dependent dEGFR signaling. For example, Aos is required for the proper timing of pulsations during oenocyte delamination (30Brodu V. Elstob P.R. Gould A.P. Dev. Cell. 2004; 7: 885-895Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Aos has also been described as a long range inhibitor during eye development (and other tissues), acting to ensure the formation of steep Spi gradients close to the source of Spi production (31Freeman M. Development. 1997; 124: 261-270Crossref PubMed Google Scholar). Recent computational studies have proposed two key roles for Aos in dEGFR signaling based on a model of the dEGFR/Spi/Aos module in embryonic ventral ectoderm patterning (32Reeves G.T. Kalifa R. Klein D.E. Lemmon M.A. Shvartsman S.Y. Dev. Biol. 2005; 284: 523-535Crossref PubMed Scopus (32) Google Scholar). First, sequestration by Aos limits the spatial range of Spi action. Second, the Aos negative feedback loop counteracts fluctuations in gene dosage (Spi secretion rate and dEGFR levels), imparting robustness to the system (32Reeves G.T. Kalifa R. Klein D.E. Lemmon M.A. Shvartsman S.Y. Dev. Biol. 2005; 284: 523-535Crossref PubMed Scopus (32) Google Scholar). One important prediction of the model was that Spi sequestration by Aos must be nearly irreversible (or of very high affinity) to provide a robust feedback loop. As such, an increase in the off rate would result in a loss of robustness, although this remains to be tested experimentally. To test the prediction that even small reductions in Spi/Aos affinity cannot be tolerated and to investigate the domain requirements for Aos activity, we screened for mutations in an Aos transgene that suppress the strong misexpression phenotype caused in the developing eye (29Freeman M. Development. 1994; 120: 2297-2304PubMed Google Scholar). We report the identification of a series of point mutations in Aos that reduce or impair its activity in multiple tissues. These lesions map to both the NCR and CCR regions, demonstrating that both modules are necessary for Aos function in vivo. We have also correlated reductions in the phenotypic strength of Aos mutants with decreases in their in vitro binding affinity for Spi. Whereas a mere ∼2-fold reduction in affinity appears to be sufficient to reduce the effectiveness of Aos as a dEGFR inhibitor, a ∼37-fold decrease in affinity greatly compromises its activity. We also show that these mutants are correspondingly less efficient in abolishing Spi-dependent dEGFR phosphorylation in cellular studies. Our data thus show that both the NCR and CCR in Aos are necessary for establishing a high affinity complex with Spi, which is critical for imparting dEGFR signaling robustness. Identification of Aos Orthologs—Publicly available genome sequences were searched using the tblastn algorithm for sequences related to Aos. Sequence alignments were performed with ClustalW1.8 (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and prepared for publication using the BOX-SHADE server (www.ch.embnet.org/software/BOX_form.html). Genetics—P{GAL4-ninaE.GMR}, P{UAS-aos}/CyO flies were generated by standard recombination methods from the individual P insertions. The males were mutagenized with 25 mm EMS (33Ashburner M. Drosophila: A Laboratory Handbook. Cold Spring Harbor, New York1989Google Scholar) and crossed to w; iso2; iso3 females at 27 °C. P{GAL4-ninaE.GMR}, P{UAS-aos} progeny were screened for suppression of the rough eye phenotype (see Fig. 2). Suppressors affecting activity of P{GAL4-ninaE.GMR} were identified by crossing them to P{UAS-egfrDN}/CyO and were subsequently discarded. Suppressors that retained GAL4 activity were balanced and characterized further by mapping, sequencing, Western analysis, and misexpression with embryonic and wing drivers (Tubulin-GAL4 and MS1096-GAL4). For embryonic activity, suppressor lines (UAS-aos*/CyO) were crossed to the Tubulin-GAL4/CyO strain, and percentage of viability was calculated as 100× (# flies with straight wing)/.5(# flies with curly wing). For rescue analysis, a sev-GAL4 driver was combined with each of the represented UAS-aos loss of function alleles (see Fig. 5) and then introduced into an aos null background (aosΔ7/Df(3L)Exel6129). For scanning electron microscopy of the adult eye, the females were dehydrated in an increasing ethanol: dH2O series, as described by Tio et al. (18Tio M. Ma C. Moses K. Mech. Dev. 1994; 48: 13-23Crossref PubMed Scopus (98) Google Scholar). Sequence and Western Blot Analysis of aos Alleles—For each putative loss of function allele genomic DNA was isolated from 10-20 adult flies with Qiagen DNeasy columns (Qiagen). The aos transgene was then amplified by PCR, purified by gel extraction (Qiagen), and sequenced using cycle sequencing according to the manufacturer's instructions (Applied Biosystems). At least two independent rounds of genomic DNA purification, PCR, and sequencing were carried out for each allele. For Western analysis, ovaries from four females misexpressing the suppressors during stages 9-11 in the follicle cells (CY2-GAL4, UAS-aos) were dissected in phosphate-buffered saline, transferred into 50 μl of phosphate-buffered saline +25 μl of 4× sample buffer on ice, and homogenized. 15 μl of each sample was loaded on an 8% SDS-PAGE gel and transferred to nitrocellulose. The blots were probed with anti-Aos (Developmental Studies Hybridoma Bank) at 1:100, stripped, and reprobed with anti-α-tubulin (12G10; Developmental Studies Hybridoma Bank) at 1:5000 as a loading control. Molecular Cloning and Protein Production—Full-length Aos was amplified by PCR, incorporating a SpeI restriction site in the 5′ primer and a His6 tag followed by a NotI restriction site in the 3′ primer, and subcloned into pFastbac (Invitrogen) giving pFbAosHis. Aos mutants were generated by QuikChange (Stratagene) using pFb-AosHis as a template. Baculoviruses encoding different Aos alleles were generated and amplified according to the manufacturer's instructions. For protein purification, 1 liter of Sf9 cells were infected with each corresponding baculovirus (except AosV146D, which required 2-2.5 liters) for 3 days. Conditioned medium was dialyzed against 12 volumes of 10 mm HEPES, pH 8, 150 mm NaCl, and flowed through a nickel-nitrilotriacetic acid column with a 2-ml bed volume (Qiagen). The column was washed with 25 ml of 20 mm imidazole in buffer A (25 mm Tris, pH 8, 100 mm NaCl), and protein was eluted in 5 ml of 300 mm imidazole/buffer A. The eluted protein was concentrated and further purified by gel filtration using a Superose 6 column (Amersham Biosciences) equilibrated with 25 mm HEPES, pH 8, 150 mm NaCl. Secreted His-tagged Spi (amino acids 1-128) was purified from transfected S2 cells as described previously (25Klein D.E. Nappi V.M. Reeves G.T. Shvartsman S.Y. Lemmon M.A. Nature. 2004; 430: 1040-1044Crossref PubMed Scopus (114) Google Scholar). The protein concentrations were determined using absorbance at 280 nm. Surface Plasmon Resonance (SPR)—SPR experiments were performed on a BIAcore 3000. Spi was immobilized onto a CM5 sensorchip by standard amine coupling using 10 mm acetate, pH 5.5, for preloading the CM-dextran surface. Purified Aos mutants were flowed over the Spi-containing sensorchip from lower to higher concentrations at 10 μl/min. The sensorchip was regenerated after running each sample with 10 mm glycine, pH 3, and 1 m NaCl. Binding curves were generated by normalizing the total response units at equilibrium against the maximal saturation response (Bmax) and plotting them as a function of protein concentration. Curves were fit to a single-site binding model using the program Prism, from which the KD values were derived. The experiments were done at least three times, generating error bars and standard error values. The experiments were also carried out by flowing protein from higher to lower concentration, yielding slightly higher KD values (likely because of sample aggregation and incomplete regeneration), but similar wild type to mutant ratios. Activation Studies— dEGFR activation assays in S2 cells were performed as described previously (25Klein D.E. Nappi V.M. Reeves G.T. Shvartsman S.Y. Lemmon M.A. Nature. 2004; 430: 1040-1044Crossref PubMed Scopus (114) Google Scholar). Briefly, dEGFR-expressing D2f cells (kindly provided by Benny Shilo) were serum-starved overnight, and dEGFR production was induced with 60 μm CuSO4 for 3 h. The cells were incubated on ice with purified Spi alone or in the presence of the indicated amount of purified Aos proteins for 10 min. The cells were lysed in radioimmune precipitation assay buffer (25 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS with phosphatase and protease inhibitors), and lysates were separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed with anti-pY20 (Santa Cruz), stripped, and reprobed with anti-dEGFR as a loading control (23Alvarado D. Rice A.H. Duffy J.B. Genetics. 2004; 167: 187-202Crossref PubMed Scopus (14) Google Scholar). Isolation of Mutations Affecting Aos Activity—Aos was first described in D. melanogaster as an antagonistic ligand of dEGFR with an atypical EGF motif that was hypothesized to direct its association with dEGFR and preclude Spi binding (24Freeman M. Klambt C. Goodman C.S. Rubin G.M. Cell. 1992; 69: 963-975Abstract Full Text PDF PubMed Scopus (211) Google Scholar, 34Schweitzer R. Howes R. Smith R. Shilo B.Z. Freeman M. Nature. 1995; 376: 699-702Crossref PubMed Scopus (223) Google Scholar). However, recent studies have shown that Aos inhibits dEGFR signaling by binding to the activating ligand Spi rather than associating with the receptor itself (25Klein D.E. Nappi V.M. Reeves G.T. Shvartsman S.Y. Lemmon M.A. Nature. 2004; 430: 1040-1044Crossref PubMed Scopus (114) Google Scholar), bringing the significance of the atypical EGF-like domain in Aos into question. As pointed out by Howes et al. (26Howes R. Wasserman J.D. Freeman M. J. Biol. Chem. 1998; 273: 4275-4281Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), sequences outside the putative EGF motif of Aos are strongly conserved in the house fly Musca domestica (representing an evolutionary distance of ∼100 million years) and are required for Aos function in vivo, further suggesting that more than just the putative EGF-like domain is required for Aos function. We have expanded upon these observations by identifying Aos orthologs outside the dipteran lineage (Fig. 1). We found Aos orthologs in two additional arthropods, the honeybee Apis mellifera and the beetle Tribolium castaneum, indicating conservation of Aos over a span of ∼500 million years. We were unable to identify Aos orthologs in vertebrates, suggesting that, like the dEGFR inhibitor Kekkon1, the existence of Aos is phylogenetically restricted (35MacLaren C.M. Evans T.A. Alvarado D. Duffy J.B. Dev. Genes Evol. 2004; 214: 360-366Crossref PubMed Scopus (19) Google Scholar, 36Gur G. Rubin C. Katz M. Amit I. Citri A. Nilsson J. Amariglio N. Henriksson R. Rechavi G. Hedman H. Wides R. Yarden Y. EMBO J. 2004; 23: 3270-3281Crossref PubMed Scopus (231) Google Scholar). Sequence conservation in Aos extends well beyond the boundaries of the putative EGF motif and reveals two strongly conserved regions (Fig. 1) that we term the NCR and CCR, which are separated by a region of variable length. The NCR and CCR are defined primarily by sets of 4 and 12 conserved cysteines, respectively. Previous studies have indicated that the CCR represents the primary ligand-binding domain and can provide a measure of activity in vivo (25Klein D.E. Nappi V.M. Reeves G.T. Shvartsman S.Y. Lemmon M.A. Nature. 2004; 430: 1040-1044Crossref PubMed Scopus (114) Google Scholar, 26Howes R. Wasserman J.D. Freeman M. J. Biol. Chem. 1998; 273: 4275-4281Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In contrast, no direct functionality has been ascribed to the NCR. Given these regions of strong sequence conservation and the reported function of Aos as a “Spi sink,” we sought to investigate the contributions of each domain to Aos function. We therefore carried out a screen for mutations that disrupt the ability of an Aos transgene to affect eye development upon misexpression, as diagrammed in Fig. 2A. During eye development Aos limits Spi availability, thus attenuating dEGFR signaling and preventing the specification of excess photoreceptors (24Freeman M. Klambt C. Goodman C.S. Rubin G.M. Cell. 1992; 69: 963-975Abstract Full Text PDF PubMed Scopus (211) Google Scholar, 29Freeman M. Development. 1994; 120: 2297-2304PubMed Google Scholar, 31Freeman M. Development. 1997; 124: 261-270Crossref PubMed Google Scholar). Alterations in the dosage of aos (and consequently dEGFR activity) are readily observed as disruptions in the highly organized array of facets in the adult compound eye. Misexpression of an Aos transgene in the developing eye (using GMR-GAL4) leads to inhibition of dEGFR signaling (presumably through sequestration of Spi) and causes a severe loss of photoreceptors and morphological defects in the adult eye (Figs. 2A and 3B) (37Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar, 38Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 39Taguchi A. Sawamoto K. Okano H. Genetics. 2000; 154: 1639-1648PubMed Google Scholar). Reducing transgene activity would restore normal morphology to the eye and thus provides a means to identify mutations in Aos that impair its activity in vivo. To carry out a screen for functionally defective aos transgenes, we utilized the GAL4/UAS system (37Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar, 40Duffy J.B. Genesis. 2002; 34: 1-15Crossref PubMed Scopus (711) Google Scholar). In this bipartite expression system, regulation of the transgene of interest, here aos, is controlled by UASs bound by the yeast transcriptional activator GAL4. Drosophila strains expressing GAL4 in specific spatio-temporal patterns are then used to direct UAS transgene expression in the tissue of interest. For this screen we generated a recombinant fly strain with the UAS-aos transgene under the control of a GAL4 line, P{GAL4-ninaE.GMR}, that drives GAL4 expression (and thus Aos misexpression) in the developing eye. This strain, referred to as GMR>aos+, was mutagenized and outcrossed, and the progeny were screened for suppression of the eye defects associated with Aos misexpression (Fig. 2A). In addition to recovering mutations in the aos transgene, the primary focus of this work, we anticipated the recovery of two additional classes of mutations that would also suppress the GMR>aos+ eye phenotype shown in Fig. 3B. Mutations in the GAL4 driver would prevent misexpression of the aos transgene and thus revert the associated eye phenotype. These mutations were identified as progeny that failed to show eye defects when combined with a distinct UAS responder transgene, here a dominant negative version of dEGFR (UAS-dEGFRDN), and were discarded (Fig. 2A). Progeny that retained an intact GMR-GAL4 driver (and thus Aos misexpression) were further characterized. Mutations in genes essential for Aos function might also be recovered and as such would be unlinked to the UAS-aos transgene (38Casci T. Vinos J. Freeman M. Cell. 1999; 96: 655-665Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 39Taguchi A. Sawamoto K. Okano H. Genetics. 2000; 154: 1639-1648PubMed Google Scholar). Here we report the isolation and characterization of 16 mutations linked to the aos transgene that resulted in suppression of the misexpression phenotype in the eye (Table 1 and Figs. 2B and 3). Of these 16 lines, 15 showed strong suppression in the eye, consistent with minimal Aos function. The remaining mutant (aos334, identified as AosP372S below) showed only partial suppression in the eye (Table 1 and Fig. 3C), suggesting that it retained moderate activity. In addition to its function in the eye, Aos has been implicated in regulating dEGFR signaling in other contexts, including wing development and embryogenesis (13Golembo M. Raz E. Shilo B.Z. Development. 1996; 122: 3363-3370PubMed Google Scholar, 34Schweitzer R. Howes R. Smith R. Shilo B.Z. Freeman M. Nature. 1995; 376: 699-702Crossref PubMed Scopus (223) Google Scholar). Suppressor activity was assessed in these two contexts as well, using wing-specific (MS1096-GAL4) and ubiquitous (Tubulin-GAL4) drivers. Misexpression of wild type aos with MS1096-GAL4 leads to a loss of wing veins, whereas embryonic misexpression with Tubulin-GAL4 results in lethality. All of the mutated aos transgenes showed similar activity profiles in other tissues (Table 1), indicating that the suppressor lines from which they were derived did not have tissue-specific alterations. The one exception to this is aosP372S, which showed only partial suppression in the eye (and in viability) but apparently strong suppression in the wing (Table 1).TABLE 1Molecular and genetic characterization of aos mutantsLineNucleotide changeAmino acid changeAllelic classActivityEyeaActivity based on the severity of the rough eye phenotype when the UAS-aos allele is combined with the GMR-GAL4 driver. ++ indicates a severe rough eye phenotype, whereas + and - indicate moderate or weak phenotypes, respectivelyWingbPercentage of wings with gap in the L4 vein when the UAS-aos allele is combined with the MS1096-GAL4 driver. The numbers refer to the percentages of times a gap in the L4 wing vein was observedViabilitycExpression of aos mutants was driven ubiquitously during embryogenesis using the Tubulin-GAL4 driver. The percentage of viability was determined as described under “Experimental Procedures.” The numbers in parentheses represent the total number of flies scored. ND, not determined%UAS-aos+Wild type++1000 (124)UAS-aos39C1112TS371FClass I–0111 (522)UAS-aos136T437AV146DClass I–0107 (556)UAS-aos334C1114TP372SClass I+410 (462)UAS-aos14T1021AC341SClass II–0NDUAS-aos97G1238AC413YClass II–0NDUAS-aos134G422AC141YClass II–0NDUAS-aos154G422AC141YClass II–0106 (410)UAS-aos182T1219AC407SClass II–0NDUAS-aos9A949TK317XClass III–0NDUAS-aos74T977AL326XClass III–0NDUAS-aos142C319TQ107XClass III–0NDUAS-aos160T1023AC341XClass III–093 (511)UAS-aos222C763TQ255XClass III–0NDUAS-aos233C613TR205XClass III–0NDUAS-aos270A664TK222XClass III–0NDUAS-aos325A1057TK353XClass III–0NDa Activity based on the severity of the rough eye phenotype when the UAS-aos allele is combined with the GMR-GAL4 driver. ++ indicates a severe rough eye phenotype, whereas + and - indicate moderate or weak phenotypes, respectivelyb Percentage of wings with gap in the L4 vein when the UAS-aos allele is combined with the MS1096-GAL4 driver. The numbers refer to the percentages of times a gap in the L4 wing vein was observedc Expression of aos mutants was driven ubiquitously during embryogenesis using the Tubulin-GAL4 driver. The percentage of viability was determined as described under “Experimental Proc" @default.
• W2009076560 created "2016-06-24" @default.
• W2009076560 creator A5028465682 @default.
• W2009076560 creator A5046726832 @default.
• W2009076560 creator A5062227404 @default.
• W2009076560 creator A5065067053 @default.
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• W2009076560 date "2006-09-01" @default.
• W2009076560 modified "2023-10-11" @default.
• W2009076560 title "Argos Mutants Define an Affinity Threshold for Spitz Inhibition in Vivo" @default.
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