stall Encodes an ADAMTS Metalloprotease and Interacts ... · stall Encodes an ADAMTS...

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Copyright Ó 2009 by the Genetics Society of America DOI: 10.1534/genetics.109.107367 stall Encodes an ADAMTS Metalloprotease and Interacts Genetically With Delta in Drosophila Ovarian Follicle Formation Emily F. Ozdowski,* ,† Yvonne M. Mowery* and Claire Cronmiller* ,1 *Department of Biology, University of Virginia, Charlottesville, Virginia 22904-4328 and Institute for Genome Sciences and Policy/Department of Biology, Duke University Medical Center, Durham, North Carolina 27710 Manuscript received July 15, 2009 Accepted for publication September 8, 2009 ABSTRACT Ovarian follicle formation in Drosophila melanogaster requires stall (stl) gene function, both within and outside the ovary, for follicle individualization, stalk cell intercalation, and oocyte localization. We have identified the stl transcript as CG3622 and confirmed the presence of three alternatively spliced isoforms, contrary to current genome annotation. Here we show that the gene is expressed in both ovarian and brain tissues, which is consistent with previous evidence of an ovary nonautonomous function. On the basis of amino acid sequence, stl encodes a metalloprotease similar to the ‘‘adisintegrin and metalloprotease with thrombospondin’’ (ADAMTS) family. Although stl mutant ovaries fail to maintain the branched structure of the fusome and periodically show improperly localized oocytes, stl mutants do not alter oocyte determination. Within the ovary, stl is expressed in pupal basal stalks and in adult somatic cells of the posterior germarium and the follicular poles. Genetically, stl exhibits a strong mutant interaction with Delta (Dl), and Dl mutant ovaries show altered stl expression patterns. Additionally, a previously described genetic interactor, daughterless, also modulates stl expression in the somatic ovary and may do so directly in its capacity as a basic helix-loop-helix (bHLH) transcription factor. We propose a complex model of long-range extraovarian signaling through secretion or extracellular domain shedding, together with local intraovarian protein modification, to explain the dual sites of Stl metalloprotease function in oogenesis. A N emerging picture of the regulation of oogenesis in Drosophila includes multiple, diverse molecular and cellular mechanisms that take place in the ovary itself, as well as a growing number of regulatory pro- cesses that act from outside the ovary to coordinate the external/internal environmental conditions with the founding and development of the oocyte. In the ovary this process requires molecular communication between soma and germline for proper cell fate determination, adhesion, and migration and organization of follicular structure; it begins in the germarium, at the anterior end of each of the ovary’s 15–20 oocyte assembly line structures (called ovarioles) (Figure 1A) (for reviews, see King 1970; Spradling 1993). Here, 2–3 germline stem cells (GSCs) divide asymmetrically to produce daughter cystoblasts, while maintaining stem cells within the molecular niche. A specialized organelle, the spectro- some/fusome, anchors the GSC mitotic spindle to direct the axis of division and subsequently divides to be inherited by the cystoblast (supporting information, Figure S2). Following four rounds of mitosis with in- complete cytokinesis, the 16 germline cystocyte daugh- ters are connected by elongated fusomes through actin- rich ring canals (Lin et al. 1994; Roper and Brown 2004). Of the 16 cystocytes, 1 retains the most fusome material and differentiates into the oocyte (Lin and Spradling 1995): Its nucleus remains diploid in preparation for meiosis. The remaining 15 cells of each germline cyst become nurse cells: Each nucleus becomes polyploid to produce sufficient nutrients for the oocyte. The germline cyst travels toward the posterior of the germarium where somatic stem cells lie laterally (Nystul and Spradling 2007). Here, a controlled number of somatic progeny encapsulate each germline cyst with an epithelial monolayer (Margolis and Spradling 1995). The germline cyst and its somatic epithelium bud off from the germarium as a discrete follicle and are separated subsequently from the next formed egg chamber by a somatic stalk. This process of follicle individualization requires regulated somatic cell proliferation, cell fate determination, stalk cell recruit- ment, differential adhesion, and cell migration; many of the genes that contribute to these processes have been identified, and functions both inside and outside the ovary have been described (reviewed in Bastock and St. Johnston 2008; Berg 2008; Gruntenko and Rauschenbach 2008). Control of follicle formation within the ovary requires multiple cell signaling and adhesion pathways. For Supporting information is available online at http://www.genetics.org/ cgi/content/full/genetics.109.107367/DC1. 1 Corresponding author: Biology Department, University of Virginia, P.O. Box 400328, Charlottesville, VA 22904-4328. E-mail: [email protected] Genetics 183: 1027–1040 (November 2009)

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Copyright � 2009 by the Genetics Society of AmericaDOI: 10.1534/genetics.109.107367

stall Encodes an ADAMTS Metalloprotease and Interacts Genetically WithDelta in Drosophila Ovarian Follicle Formation

Emily F. Ozdowski,*,† Yvonne M. Mowery* and Claire Cronmiller*,1

*Department of Biology, University of Virginia, Charlottesville, Virginia 22904-4328 and †Institute for Genome Sciences and Policy/Departmentof Biology, Duke University Medical Center, Durham, North Carolina 27710

Manuscript received July 15, 2009Accepted for publication September 8, 2009

ABSTRACT

Ovarian follicle formation in Drosophila melanogaster requires stall (stl) gene function, both within andoutside the ovary, for follicle individualization, stalk cell intercalation, and oocyte localization. We haveidentified the stl transcript as CG3622 and confirmed the presence of three alternatively spliced isoforms,contrary to current genome annotation. Here we show that the gene is expressed in both ovarianand brain tissues, which is consistent with previous evidence of an ovary nonautonomous function. Onthe basis of amino acid sequence, stl encodes a metalloprotease similar to the ‘‘a disintegrin andmetalloprotease with thrombospondin’’ (ADAMTS) family. Although stl mutant ovaries fail to maintainthe branched structure of the fusome and periodically show improperly localized oocytes, stl mutants donot alter oocyte determination. Within the ovary, stl is expressed in pupal basal stalks and in adult somaticcells of the posterior germarium and the follicular poles. Genetically, stl exhibits a strong mutantinteraction with Delta (Dl), and Dl mutant ovaries show altered stl expression patterns. Additionally, apreviously described genetic interactor, daughterless, also modulates stl expression in the somatic ovary andmay do so directly in its capacity as a basic helix-loop-helix (bHLH) transcription factor. We propose acomplex model of long-range extraovarian signaling through secretion or extracellular domain shedding,together with local intraovarian protein modification, to explain the dual sites of Stl metalloproteasefunction in oogenesis.

AN emerging picture of the regulation of oogenesisin Drosophila includes multiple, diverse molecular

and cellular mechanisms that take place in the ovaryitself, as well as a growing number of regulatory pro-cesses that act from outside the ovary to coordinate theexternal/internal environmental conditions with thefounding and development of the oocyte. In the ovarythis process requires molecular communication betweensoma and germline for proper cell fate determination,adhesion, and migration and organization of follicularstructure; it begins in the germarium, at the anterior endof each of the ovary’s 15–20 oocyte assembly linestructures (called ovarioles) (Figure 1A) (for reviews, seeKing 1970; Spradling 1993). Here, 2–3 germline stemcells (GSCs) divide asymmetrically to produce daughtercystoblasts, while maintaining stem cells within themolecular niche. A specialized organelle, the spectro-some/fusome, anchors the GSC mitotic spindle to directthe axis of division and subsequently divides to beinherited by the cystoblast (supporting information,Figure S2). Following four rounds of mitosis with in-complete cytokinesis, the 16 germline cystocyte daugh-

ters are connected by elongated fusomes through actin-rich ring canals (Lin et al. 1994; Roper and Brown

2004). Of the 16 cystocytes, 1 retains the most fusomematerial and differentiates into the oocyte (Lin andSpradling 1995): Its nucleus remains diploid inpreparation for meiosis. The remaining 15 cells of eachgermline cyst become nurse cells: Each nucleus becomespolyploid to produce sufficient nutrients for the oocyte.The germline cyst travels toward the posterior ofthe germarium where somatic stem cells lie laterally(Nystul and Spradling 2007). Here, a controllednumber of somatic progeny encapsulate each germlinecyst with an epithelial monolayer (Margolis andSpradling 1995). The germline cyst and its somaticepithelium bud off from the germarium as a discretefollicle and are separated subsequently from the nextformed egg chamber by a somatic stalk. This process offollicle individualization requires regulated somatic cellproliferation, cell fate determination, stalk cell recruit-ment, differential adhesion, and cell migration; many ofthe genes that contribute to these processes have beenidentified, and functions both inside and outside theovary have been described (reviewed in Bastock andSt. Johnston 2008; Berg 2008; Gruntenko andRauschenbach 2008).

Control of follicle formation within the ovary requiresmultiple cell signaling and adhesion pathways. For

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.109.107367/DC1.

1Corresponding author: Biology Department, University of Virginia, P.O.Box 400328, Charlottesville, VA 22904-4328. E-mail: [email protected]

Genetics 183: 1027–1040 (November 2009)

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example, daughterless (da) regulates cell proliferationand apoptosis in the germarium, as well as stalk cellrecruitment at the budding follicle border (Smith et al.2002). In the newly formed follicle, Notch (N) and Delta(Dl) induce anterior polar cell fate, and the ante-rior polar cells subsequently signal through the Januskinase/signal transducer and activator of transcription( JAK/STAT) pathway to initiate stalk cell differentiation(McGregor et al. 2002; Torres et al. 2003). Further, thestalk induces the posterior polar cell fate of the youngerfollicle, leading to upregulation of adhesion molecules,such as DE cadherin [shotgun (shg)] and b-catenin[armadillo (arm)], in the oocyte and posterior somaticcells. Homophilic interactions and cell sorting ulti-mately position the oocyte to the posterior of the eggchamber (Godt and Tepass 1998; Gonzalez-Reyes andSt. Johnston 1998). Mutations in many of the genesthat control these events disrupt follicle separation,resulting in the packaging of multiple germline cystswithin a single somatic epithelium (Ruohola et al. 1991;Cummings and Cronmiller 1994; McGregor et al.2002). This phenotype is shared by stall (stl) mutants, inwhich multicyst ovarioles lack interfollicular stalk struc-tures as early as pupal ovary development (Bakken 1973;Schupbach and Wieschaus 1991; Tworoger et al. 1999;Smith et al. 2002; Willard et al. 2004).

While many of the ovarian regulators of oogenesishave been described in detail, less is known about theextraovarian control of this process. Hormonal inputhas been shown to affect oocyte production and mat-uration through germarial cell proliferation and celldeath, follicle apoptosis, and yolk protein synthesis anduptake: Insulin, juvenile hormone ( JH), and 20-hydroxy-ecdysone (20E) affect egg development in response tothe nutritional environment of the fly (Soller et al.1999; Drummond-Barbosa and Spradling 2001;Lafever and Drummond-Barbosa 2005). In addition,neural influences on follicle formation and maturationhave been shown to involve the activity of the neuro-transmitters, serotonin and dopamine (Willard et al.2006). Finally, on the basis of mitotic clonal analysis andovary transplantation, we identified stall as an additionalimportant extraovarian regulator of follicle morpho-genesis; however, the molecular identity of the stl geneproduct was then unknown (Willard et al. 2004).

Here, we extend our analysis of stl ’s control ofoogenesis by further defining the stl mutant phenotype,elaborating on the gene’s functional interactions, andidentifying its molecular product as a metalloproteasewith distinguishing similarities to a disintegrin and me-talloprotease with thrombospondin (ADAMTS) domainproteins. We show that, although stl mutant ovaries failto maintain the fusome within early germline cysts andcontain a moderate number of mislocalized oocytes, stldoes not alter oocyte determination. We also address agenetic interaction between stl and Dl in follicle in-dividualization and oocyte polarity. Finally, the identifi-

cation of Stl as a metalloprotease is a critical leap in thestudy of ovarian follicle formation. These evolutionarilyconserved enzymes participate in a wide range of bi-ological processes, and the characterization of Stall as anADAMTS offers a new approach to considering the rolesof these proteins in oogenesis.

MATERIALS AND METHODS

Drosophila stocks: Flies were maintained on molasses–cornmeal–yeast medium at 25�. Stocks used in this study arelisted in Table 1.

Genetic and molecular analysis of stl: Bloomington De-ficiency Kits (2003) for chromosomes X, 2, and 3 were crossedto stl a16, and ovaries were dissected from doubly heterozygousadult progeny. Ovaries were 49,6-diamidino-2-phenylindole(DAPI) stained and scored for percentage of ovariolesexhibiting follicle formation defects to identify dominantgenetic interactions. To locate the stl transcription unit,meiotic recombination frequencies were determined betweenstl and l(2)06496 and between stl and l(2)k06908 on the basisof segregation of the P{w1}markers. Both stl a16 and stl pa49 wereused to calculate the distances in map units. Male recombina-tion was performed with insertions in l(2)rG270, CG3732,CG3875, l(2)k17002, ppa, jbug, blw, asrij, and Nop60B oppositecn stl pa49 bw or cn stl ph57 bw via D2,3 transposase. Deletionswere produced by mobilization between Exelixis insertionpairs, P{WH}f06717 to P{XP}d06151 and P{XP}d02208 toP{WH}f07572, as screened by the loss of w1. Ovaries of thesterile Df/stl a16 females were dissected and DAPI stained; flycarcasses were used for genomic DNA analysis to confirm thegenotype. The specific primer pairs for genomic PCR wereGACGCATGATTATCTTTTACGTGAC and ATGATTCGCAGTGGAAGGCT for the P insertion and TTGCCTTTGTTCTACGCTCTC and GCCCAAGAACACGACGATAA for the flankinggenomic region. To sequence candidate genes within thedeleted region, RNA was isolated from cn bw (the parentalstrain for stl pa49 and stl ph57), stl a16, stl pa49, stl ph57, stl awk26, and stl wu40

females with Trizol (GIBCO-BRL, Gaithersburg, MD), Phase-lock gel (Eppendorf), and poly(A) selection kits (Sigma, St.Louis). cDNA was produced by reverse transcription (Prom-ega, Madison, WI) and amplified by PCR [Invitrogen (Carls-bad, CA) Platinum Taq High Fidelity]. Amplification ofCG3622 cDNA was accomplished with the forward primersGGCCGGTTGTTAATTCTTCA (isoform A), GCCACCAATGCCACAAAT (isoform B), and CAGAGAAGCCGTCATCATCA(isoform C) and the reverse primer GAACTTTCTCGTTCGCACTG (common to all isoforms). Sequencing of both strandsof these PCR products was performed in overlapping frag-ments with the following primers: GGCCGGTTGTTAATTCTTCA (F1A), GATCGTAGTGCAGCGGATCT (R1), GCCACCAATGCCACAAAT (F1B), CTTGACGACGCCTCACTGTA (R1B),CAGAGAAGCCGTCATCATCA (F1C60), CACCGTCGTTTGGCTTTAAT (F1C), GGCCCATTCCCATCTTCT (RTBCR), GTGCTGAAGCGCCTAGAGAT (F2), GAGATCCCGACAGATTTCCA (R2), GAGCAAGATGGAAATCTGTCG (F3), GGTGGAATTATCGCAGGAGA (R3), CGCTTCTCCTGCGATAATTC (F4),CACCTGCCATACACGCAATA (R4), GGTGTGGAGAGCATCCAACT (F5), and GAACTTTCTCGTTCGCACTG (R5). To con-firm the identified mutations, independently isolated genomicDNA was amplified by PCR and sequenced with the aboveprimers as well.

59 rapid amplification of cDNA ends: The Ambion First-Choice RLM–rapid amplification of cDNA ends (RACE) kitwas used on total RNA from white Canton-S adult flies. Theresulting adaptor-ligated cDNA was amplified with kit adaptor

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primers and a gene-specific primer to a common exonTTGACGACGCCTCACTGTAG (3622RACEouter); a nestedprimer to a common exon CTCTTGTCCACCCTCGCTTGGTAGAG (3622 RACEinnerLong); and nested primers to theunique exon for each isoform AATGCCATTGTAGTGCAAGCTCCTCT (RACERCLong), CATCGGTGGCATTTGTGGCATTGGTG (RACERBLong), and GATCGAGGCGTGAAGAATTAACAACC (RACERALong). Products were gel purified(QIAGEN, Valencia, CA) and sequenced to identify the 59-UTR.

Tissue staining and analysis: Ovaries were fixed in 4%paraformaldehyde and stained with DAPI, as described pre-viously (Cummings and Cronmiller 1994). Antibodies fortissue staining included rabbit anti-Vasa 1:1000, rabbit anti-Mio 1:1000, rabbit anti-C(3)G 1:1000, mouse anti-Hts (1B1)1:10, mouse anti-Bic-D (Bicaudal-D 4C2) 1:10, and mouse anti-Orb (Orb4H8) 1:10. [The last three antibodies were obtainedfrom the Developmental Studies Hybridoma Bank (DSHB)maintained by the University of Iowa, Department ofBiological Sciences.] Whole tissue immunostaining was per-formed as previously described (Cronmiller and Cummings

1993). Secondary antibodies were used at a concentrationof 1:300 [FITC-conjugated goat anti-mouse and TRITC-conjugated goat anti-rabbit ( Jackson Immunoresearch Lab-oratories)]. Stained ovaries were visualized on a Zeiss

(Thornwood, NY) Axioskop microscope. Images were cap-tured by an Olympus Magnafire digital camera and falsecolored in Adobe Photoshop.

In situ hybridization: In vitro transcription plasmids wereconstructed by ligation of 699 bp of stl isoform A (of which only�100 bp are unique to A) or 635 bp of isoform B/C intopGEM-T Easy. Riboprobes were digoxigenin labeled via in vitrotranscription and quantified by chemiluminescent dot blot.The hybridization protocol (Wahli and Braissant 1998) wasmodified as follows: Ovaries were dissected in 13 PBS, fixedfor 10 min in 4% paraformaldehyde, treated with 0.1% activediethylpyrocarbonate (DEPC) for 20 min, and hybridized in400 ng/ml riboprobe at 58� for 40 hr. After NBT/BCIPstaining was stopped with TE buffer, ovaries were stored in13 PBS at 4� (without dehydration) and mounted in 50%glycerol. Tissues were analyzed under DIC.

In silico analysis: The Stl protein sequences were analyzed bySignalP 3.0 (Bendtsen et al. 2004) for signal peptide sequenceprediction (http://www.cbs.dtu.dk/services/SignalP/), byTMHMM v2.0 and Phobius for transmembrane domain andsignal peptide prediction (http://www.cbs.dtu.dk/services/TMHMM/ and http://phobius.sbc.su.se), by NetNGlyc 1.0for N-glycosylation consensus site prediction (http://www.cbs.dtu.dk/services/NetNGlyc/), by BLAT and Evoprinter

TABLE 1

Drosophila stocks used in this study

Stock genotype Originally obtained from

Oregon-R (wild-type strain)cn bw/CyO T. Schupbachcn stl pa49 bw/CyO T. Schupbachcn stl ph57 bw/CyO T. Schupbachstl wu40 bw/CyO T. Schupbachc stl awk26 bwD/CyO T. Schupbachw/w; al b pr cn stl a16/CyO R. NagoshiP{XP}d06151 Exelixis, Harvard Medical SchoolPBac{WH}RpS24[f06717] Exelixis, Harvard Medical SchoolP{XP}d02208 Bloomington Stock CenterP{WH}f07572 Bloomington Stock Centery1 w 67c23; P{lacW}l(2)06496 k14618/CyO Bloomington Stock Centery1 w 67c23; P{lacW}chrw k06908/CyO Bloomington Stock CenterP{PZ}l(2)rG270 rG270/CyO; ry 506 Bloomington Stock Centery1 w 67c23; P{EPgy2}CG3732 EY00923 Bloomington Stock Centery1 w 67c23; P{SUPor-P}CG3875 KG07486 Bloomington Stock Centery1 w 67c23; P{lacW}l(2)k17002 k17002/CyO Bloomington Stock Centery1 w67c23; P{w1mC¼lacW}l(2)k00611k00611/CyO Bloomington Stock Centery1 w 67c23; P{SUPor-P}jbug KG00131 Bloomington Stock Centery1 w 67c23; P{lacW}blw k00212/CyO Bloomington Stock Centerw 1118; P{SUPor-P}asrij KG08071/CyO, P{sevRas1.V12}FK1 Bloomington Stock Centery1 w 67c23; P{lacW}Nop60B k05318/CyO Bloomington Stock Centerstat92E1681/TM3 C. DearolfDf(3R)H-B79 Bloomington Stock CenterDl3/In(3R)C, sprd1 e Bloomington Stock CenterDlX/In(3L)P, In(3R)P Bloomington Stock CenterDl9/In(3R)C, sprd1 e1 Bloomington Stock Centerry 506 P{ry1t7.2¼PZ} Dl 05151/TM3, ry RK Sb1 Ser 1 Bloomington Stock CenterDf(3R)Dl-BX12, ss1 e4 ro1/TM6B, Tb1 Bloomington Stock CenterN spl-1 Bloomington Stock Centery1 w* N1/FM7c, P{w1mC¼GAL4-twi.G}108.4, P{UAS-2xEGFP}AX Bloomington Stock CenterDf(1)N-8/FM7c Bloomington Stock CenterSer 1/In(3R)C, sprd1 e 1 l(3)e 1 Bloomington Stock Centerro1 Ser Bd-1 ca1/In(3R)C, sprd1 l(3)a1 Bloomington Stock Center

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for evolutionary conservation sequence analysis among Dro-sophila species (http://genome.ucsc.edu/cgi-bin/hgBlat andhttp://evoprinter.ninds.nih.gov/), and by ClustalW2 to alignADAMTS molecules of multiple species (http://www.ebi.ac.uk/Tools/clustalw2/index.html).

RESULTS

The stl mutant phenotype reflects a complete failureof follicle individualization (Figure 1, A and B) (Bakken

1973; Schupbach and Wieschaus 1991). While theearliest defects appear in the pupal gonad (Willard

et al. 2004), the disruption of ovarian morphology iscomplete even in newly eclosed adults. Subsequently,ongoing deterioration of ovarian structure as femalesage results in the grossly aberrant stl mutant phenotypethat includes widespread cell degeneration (Figure 1C).To verify that these ovarian defects, which are commonto all of the five extant stl mutant alleles, compose thenull phenotype, we generated a deletion chromosome(see materials and methods), examined the pheno-type of hemizygous mutant flies (stl/Df), and found thatit was indistinguishable from that of any mutant homo-zygotes (stl/stl) (Figure 1D). Using these five null alleles,we were able to identify the stl transcript and fill a criticalgap in our understanding of the regulation of oogenesis.

stall encodes an ADAMTS metalloprotease: Multipleapproaches, including meiotic recombination, male re-combination, deficiency mapping, and candidate genesequencing of mutational lesions, were used to identifystl as CG3622 at 59B2 (Figure 2, A and B). The meioticrecombination frequencies between stl a16 and l(2)06496[II-102.0] and between stl a16 and l(2)k06908 [II-104.5]were 1.5% (n¼ 1850) and 3.7% (n¼ 2000), respectively;this mapping placed stl at 100.5–100.8 and was con-firmed with stl pa49. Male recombination with multiple Pinsertions further refined the stl location to an 87-kbgenomic region between blw and asrij. Of the 21 pre-dicted coding regions in this interval, sequencing re-vealed only one gene with mutational lesions in each ofthe five extant stl alleles: This identified stl as CG3622.

Although the genome annotation of CG3622 variedover the course of our work, we confirmed three alter-native splice forms of stl mRNA in wild-type tissues(Figure 2C). By RT–PCR and sequencing analyses, allthree isoforms were found to share five common exonsat their 39 ends (Figure 2, D and E). Isoforms B and Calso shared three middle exons that were excluded fromisoform A, while 59 exons for the three isoforms werefound to be partially overlapping and/or unique. Weused 59 RACE to characterize the 59-UTRs for isoformsB and C, although low expression levels of isoformA apparently prevented precise determination of thistranscript’s 59 end by this method. Sequence analysis ofthese three stl mRNAs would predict proteins with 1136aa (Stl-C), 1091 aa (Stl-B), or 790 aa (Stl-A). Moreover,the predicted amino acid sequences of all three Stl

Figure 1.—The stl null phenotype. (A) Wild-type ovariolestructure includes the separation of follicles by interfollicularstalks. (B) A stla16 ovariole from a young female (1 day poste-closion) lacks any individualized follicles or interfollicularstalks: Multiple germline cysts are enclosed in a single somaticepithelium. (C) A whole ovary from an older stl ph57/stl pa49

female (�10 days old) shows severe cell degeneration. Thisphenotype was observed for all homozygous and heteroallelicstl genotypes. (D) Df(2R)d02208-f07572/stla16 ovarioles showthe same severe phenotype as stl point mutations, confirmingtheir identification as null alleles. The nuclei are stained withDAPI to visualize cellular organization. In this and all subse-quent figures, scale bar is 50 mm, and anterior is positioned atthe top or the left of each panel.

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mRNA isoforms identified the conceptual protein of thegene as a metalloprotease, with many structural featuresin common with the ADAM and ADAMTS family ofthese proteins (Figure 2E).

The Stl protein is most similar to members of theADAMTS proteins. ADAM and ADAMTS proteins arerelated in their enzymatic activity: They are metallopro-teases that bind zinc ions to catalyze proteolytic cleavage of

Figure 2.—stl is CG3622, encoding anADAMTS metalloprotease. (A) Diagram of thetransposable element insertions used in the ge-netic and molecular analysis of stl. P-element in-sertions (short triangles) were used to map stl bysequential male recombination analyses, delimit-ing the stl gene location to an 87-kb region be-tween blw and asrij (arrows). PBac insertions(tall triangles) were used to generate molecularlydefined deficiency chromosomes that confirmedboth the stl gene location and the null pheno-type. Within the 87-kb region, 21 candidategenes were sequenced from five independentstl mutant strains, leading to the identificationof stl as CG3622 (B, red). (C) SemiquantitativeRT–PCR analysis of stl expression in whole adults,dissected ovary tissue, or adult heads shows thepresence of all three predicted isoforms, withthe long isoform C most highly expressed. Theamplified bands shown are 2985, 3445, and3552 bp long for isoforms A, B, and C, respec-tively, and each band includes the coding regionof its isoform. (D) Diagram of the three alter-nately spliced isoforms of CG3622 that were con-firmed by RT–PCR and sequencing. The diagramincludes previously undefined 59-UTR for iso-form C, as determined by 59 RACE. Dark grayrepresents the protein-coding region, while lightgray indicates the untranslated regions. Coloredlines mark the positions of stl mutational lesions,all of which fall within the protein-coding region(triangle, missense; asterisk, nonsense). (E) TheStl amino acid sequence, which identifies theprotein as a member of the ADAMTS family.The protein domains are indicated as follows:prodomain, lavender highlight; reprolysin metal-loprotease domain, green highlight; disintegrin-like domain, yellow highlight; thrombospondin(TS-1) repeat, purple highlight; cysteine-rich do-main, orange highlight; and the C-terminalspacer region, blue highlight. This sequenceshows the most N-terminal region unique toStl-C (-C: 60 amino acids), the unique portionof Stl-B (-B: 15 amino acids), the region commonto B and C (286 amino acids), and the remain-der, which corresponds to the entire Stl-A iso-form (-A: starting at M301/346) and is commonto all three isoforms. The predicted signal se-quence in Stl-C is italicized, the Zn-binding met-alloprotease consensus sequence is boxed with adotted line, potential furin cleavage sites areshown in boldface type (RxK/RR), and possibleN-glycosylation sites are underlined (NxS/T). Fi-nally, the positions of the molecular lesions ofthe stl point mutations are indicated by theboxed amino acids (color coordinated with theline indicators in D): red, stl awk26; yellow, stl wu40;purple, stl a16; black, stl ph57; and green, stl pa49.

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extracellular matrix (ECM) components. However, mul-tiple structural domain differences between these proteinfamilies distinguish their associated substrates, cellularfunctions, and localization. Structural motifs of thecanonical, secreted ADAMTS proteins include a prodo-main, a Zn-binding and catalytic protease domain, adisintegrin-like domain, a central thrombospondin repeat(TSR) domain, a cysteine-rich region, a spacer region, andvariable C-terminal TSRs (Kuno et al. 1997). The mem-brane-associated ADAMs differ from ADAMTSs primarilyby the inclusion of a transmembrane domain and theabsence of recognizable thrombospondin repeats. Thesedifferences appear to distinguish between membrane andextracellular sites where these proteins carry out theirproteolytic functions. Importantly, the Stl protein con-tains a highly conserved Zn-binding and metalloproteaseconsensus sequence (Figure 2E, dotted outline), and thisfunctional domain is most similar to the reprolysindomain of the ADAMTS family (Kuno et al. 1997). Onthe basis of overall sequence homology, the presence of acentral thrombospondin repeat, and the secretion signalsequence in the long C isoform (Figure 2E, italics), theconceptual Stl protein appears most like the ADAMTSproteins (Cal et al. 2001; Nicholson et al. 2005).

As has been found for other ADAM/ADAMTS pro-teins (e.g., ADAM12 and ADAM28: Gilpin et al. 1998;Roberts et al. 1999; Howard et al. 2000), not all isoformsof Stl are likely to be secreted, on the basis of theirN-terminal amino acid sequence differences (Figure 2E).By SignalP analysis, the longest isoform, Stl-C, contains acharacteristic signal sequence (D-score ¼ 0.723). Forcomparison, the closest mammalian ortholog, ADAMTS-16, possesses a strong signal sequence in both human(D-score ¼ 0.733) and mouse (D-score ¼ 0.650) and ispredicted to be secreted (Cal et al. 2002). In contrast,neither Stl-A nor Stl-B Signal P analysis gave evidence ofan unambiguously identifiable signal peptide (isoform A,D-score ¼ 0.301; isoform B, D -score ¼ 0.199) (Nielsen

et al. 1997; Bendtsen et al. 2004); however, further studyof their N-terminal sequences did suggest possible trans-

membrane positioning of these isoforms. The hydropho-bicity plot of Stl isoform A was consistent with a singletransmembrane domain at the N terminus (amino acids13–32), and such a domain within the first 60 amino acidsof a protein could act as a signal peptide (as defined bythe transmembrane domain prediction programs, Pho-bius and TMHMM). The same amino acid sequence ofStl isoform B gave a far less convincing transmembraneprediction, with that sequence located much fartherfrom the N terminus (amino acids 314–333); however,the sequence of Stl-B, like that of other ADAMTS pro-teins, contains a strong furin cleavage site at amino acidposition 266–269 (Figure 2D, text in boldface type), andcleavage at this site would position a weak putativetransmembrane domain much closer to the N terminus(amino acids 43–62). In the absence of any disruptingmutational lesions within the putative signal sequence/transmembrane domains (see below), further experi-ments would be necessary to verify the cellular localiza-tion of Stl.

Detection of Stl functional domains through muta-tional analysis and identification of sequence conser-vation: To discriminate functionally among the threeisoforms of Stl and to search for domains that are criticalto the gene’s roles during oogenesis, we identified themutational lesions of all extant mutant alleles. While themutational lesions for all five stl alleles were found to bedistributed throughout the coding region of the gene(Table 2), the nucleotide changes associated with two ofthese mutations suggest that at least one of the longerprotein isoforms (Stl-B and/or Stl-C) is required foroogenesis. A missense mutation in stlwu40 was found toalter Gly214 of Stl-C (Gly169 of Stl-B), and although thisresidue has not been identified as part of a previouslydefined functional domain, it is conserved in the Stlorthologs from insects, rodents, and humans (FigureS1). Another mutant allele, stl awk26, was found to containtwo lesions, including a nonsense mutation that wouldseverely truncate both Stl-B and Stl-C, leaving only 101or 146 amino acids, respectively. The second lesion in

TABLE 2

Molecular lesions in stl alleles

Allele nameAmino acidalteration

Stl-A isoformresidue no.

Stl-B isoformresidue no.

Stl-C isoformresidue no.

stl wu40 G / S NA 169 214stlawk26 Q / stop NA 101 146

T / I 99 NA (truncated) NA (truncated)stl ph57 Q / stop 520 821 866stl pa49 W / stop 206 507 552stla16 H / Y 145 446 491

D / E 766 1067 1112A / T 782 1083 1128

The locations of protein sequence changes in each of the stl alleles are shown (numbers refer to their iso-form-specific positions). Since the phenotypic severity of each of the five mutant alleles is equivalent to that ofthe deletion (i.e., stl/stl ¼ stl/Df), they can all be classified as null alleles.

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this allele would affect only the Stl-A peptide, and sincethis change represents a conservative missense muta-tion in a nonconserved residue (T99I, Figure S1), it isnot clear whether the lesion would handicap anyfunction associated with Stl-A. The mutational lesionsof the remaining stl alleles were found to delete or alterdefined functional domains that would be shared byall Stl isoforms. Two simple alleles, stl ph57and stl pa49,contained single nonsense mutations to truncate theprotein(s). The more severe of these truncation alleles(stl ph57) would delete the disintegrin-like, sole thrombo-spondin and cysteine-rich domains, as well as thevariable C-terminal spacer region. The less severetruncation (stl pa49) would remove only the C-terminalspacer. The identical null phenotypes that are associ-ated with both of these truncation alleles affirm theimportance of the C-terminal spacer region, whichmediates substrate specificity in other ADAMTS pro-teins (Flannery et al. 2002; Zheng et al. 2003; Majerus

et al. 2005). Finally, stl a16 was found to be the mostpolymorphic strain, and in this mutant we found severalmissense mutations. One of these mutations changedthe highly conserved middle histidine (His145/446/491)that defines the catalytic domain, and two mutationsaltered nonconserved amino acids at the extreme Cterminus (Figure S1). Although the significance of thetwo distal mutations is unknown, mutation of theindispensable histidine illustrates the importance ofZn binding and protease activity for Stl function.

We extended our analysis of stl structure and functionby comparing the genome database sequences of Stlorthologs for eight Drosophila species (melanogaster,sechellia, simulans, yakuba, erecta, ananasssae, pseudoobscura,virilis, and grimshawi). BLAT search and EvoPrinterrevealed four regions of high similarity (Table 3): thehypothetical transmembrane domain of Stl-A, the metal-loprotease consensus, a small portion of the disintegrin-like domain, and a significant region of the C terminus.Although the signal sequence of isoform C is not con-served among all eight Drosophila, it is extremely similarin the six most closely related species. Taken togetherwith the characterization of all extant stl alleles asfunctional nulls and the diversity of the mutationallesions associated with these alleles, the strong sequence

conservation suggests that all of these regions of theprotein are critical to its function. Moreover, the impor-tance of these domains is evident in cell biological andbiochemical studies of mammals as well (reviewed inRocks et al. 2008).

stl mutant effects in early oogenesis: Because thespecific cellular processes that are regulated by stlduring early oogenesis have not been identified, weexamined critical aspects of follicle morphogenesis,namely oocyte determination/localization and somaticcell differentiation/organization, in stl mutants. In theearliest stages of oogenesis, germ cell behavior andoocyte specification depend upon spectrosome/fusomefunction, and although we found altered fusomestructure in stl mutant ovaries, oocyte determinationwas not affected. In the ovaries of newly eclosed stlmutant females, spectrosomes and fusomes were pre-sent and appropriately branched in the majority ofgermaria (Figure S2, A and B); however, in 4-day-oldfemales fusomes were often not detected, and, whenpresent, they appeared unbranched (Figure S2 C). Inolder females, both organelles were completely absent(Figure S2 D). Thus, fusome instability might be aprimary consequence of stl loss-of-function. Conse-quently, if stl function directly regulates fusome main-tenance, we would expect oocyte determination to bedisrupted in stl mutant ovaries, since the fusomemediates this process (Lin and Spradling 1995). Weused oocyte-specific molecular markers to examineoocyte determination in stl mutants and found nosignificant defects: For several different markers, boththe relative distributions and expression levels wereunaltered, even in severely defective mutant ovarioles(Figure S2, E–K). For example, Orb, C(3)G, Bic-D, andMio were all appropriately restricted to a single cell pergermline cyst (Figure S2, G and K, and data not shown).In �20% of stl ovarioles, morphological defects didinclude mispositioned oocytes, such that the oocyte ofthe oldest germline cyst was not found at the posteriorend of the cyst (e.g., Figure S2 K). However, thesemislocalized oocytes still expressed oocyte-specificmarkers and exhibited the characteristic oocyte-specificdiploid nucleus. Thus, with apparently normal oocytedetermination in stl mutants, altered fusome structure

TABLE 3

Conserved sequences in Stl orthologs among eight Drosophila species

Stl-A isoformresidue no.

Stl-B isoformresidue no.

Stl-C isoformresidue no. Description of domain

1–23 287–324 332–369 Putative signal sequence of Stl-A116–166 417–467 462–512 Metalloprotease consensus220–227 521–528 566–573 Disintegrin-like domain599–701 900–1002 945–1047 C terminus

From analysis of homologous genomic sequences among eight Drosophila species, these four regions ofhighest homology potentially identify important Stl functional domains.

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and stability are most likely indirect effects of theseverely abnormal morphology.

Delta is a dominant enhancer of the stall mutantfollicle formation phenotype: Since previous geneticinteraction analyses successfully identified only oneother gene potentially involved in stl-mediated regula-tion of follicle morphogenesis, namely daughterless (da)(Smith and Cronmiller 2001), we screened deficiencychromosomes for dominant enhancement of the stlphenotype (see Table S1 for a listing of the specificdeletions tested). This search yielded three regions ofinterest. The first cytological region (30D; 31F) in-cluded da, confirming the previously described non-complementation of stl by da point mutations andunderscoring the question of the molecular relation-ship between stl and da function (see below). Thesecond region (44F10; 45E1) was found to be geneti-cally complex, and analysis of overlapping deficienciesin this interval failed to distinguish a single interactinglocus (Figure S3). Finally, the third region (91F1–2;92F3–6) identified stat92E and Delta as putative inter-actors, and since both of these genes had previouslybeen connected to da regulation of follicle formation(Cummings and Cronmiller 1994; Smith et al. 2002),we examined their interactions with stl in greater detail.The stat92E–stl interaction was confirmed with a stat92Enull mutation; however, this interaction turned out to

be specific to the stla16 allele. When examined molecu-larly, the stla16 chromosome was found to carry a muta-tion in a nearby, stl-unrelated, putative STAT-bindingsite. It is possible that this binding site regulates expres-sion of another gene that functions during early oogen-esis, thereby accounting for the coincidence of the ovaryinteraction phenotype. The Dl–stl interaction, however,was not merely allele specific for either gene.

To investigate the potential regulatory link between stland the N-Dl signaling pathway, we expanded ourexamination of stl genetic interactions. We foundsignificant phenotypic interactions between stl andmultiple Dl alleles; however, surprisingly, we observedno genetic interaction between stl and Notch (N), whichis generally believed to partner universally with Dl in itsvarious developmental functions. Three strong alleles,DlX, Dl9, and Dl3, displayed oocyte mislocalization andsevere follicle formation defects when doubly heterozy-gous with various recessive alleles of stl (Figure 3, A–C),and all of these interactions were statistically significant(Figure 3D). Interactions with a Dl� deletion [Df(3R)Dl-BX12] were also significant (e.g., Figure 3D), althoughfor both this deletion and the DlX allele, considerablevariation in the frequency of defects among individualfemales suggested complications associated with both ofthese genetic backgrounds. For example, the frequencyof defects in DlX–stl ph57 heterozygotes ranged from 15.4

Figure 3.—Combined reduction of Dl and stlresults in dominant follicle formation defects,but reduction of N and stl has no effect. (A–C)DAPI-stained ovary samples, which show thedominant mutant interaction of stl, Dl doubleheterozygotes. (A) stlpa49/1; Dl9/1 ovarioles dis-play defects in follicle separation, resulting incompound follicles and compound ovariolesnot seen in either heterozygote alone. (B)stlph57/1; Dl9/1 ovarioles show that defects arenot allele specific. (C) A stlph57/1; DlX/1 ovariolethat contains compound follicles also shows amislocalized oocyte; the arrow points to the dip-loid oocyte nucleus that is located centrallyamong the nurse cells. This ovariole also con-tains a partially dispersed border cell cluster (ar-rowhead) that contains a higher than normalnumber of cells, probably contributed by morethan one set of polar cells in this multicyst ovar-iole. (D) The frequency of follicle defects in stlph57

plus Dl, N, or Ser double heterozygotes, expressedas the percentage of ovarioles that containedcompound/morphologically disrupted follicles.Control genotypes were Dl, Df, N, or Ser singleheterozygotes (see Table 1 for complete geno-types). Significant increases over the controlsare in boldface type (a, P > 0.01; b, P , 0.05).The numbers of ovarioles scored are in brackets.

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to 82.4%, and even the DlX heterozygous controlsshowed substantial variation (2.6–48.8%). Nevertheless,positive genetic interactions for all of these Dl–stl alleliccombinations were obvious, and the only Dl allele thatdid not exhibit a dominant interaction with any stl alleletested was the weak recessive mutation, Dl05151 (Figure3D). Neither Ser allele tested demonstrated any en-hancement of stl in the ovary. Surprisingly, we alsoobserved no synergistic phenotypes between stl andNotch (N). While this result could indicate that Nexpression or N signaling is stronger than that of Dlduring oogenesis, making N less likely to show a geneticinteraction when reduced by only one genetic copy, it isalso possible that stl is involved in an N-independentfunction of Dl. Further experiments will be necessary todistinguish between these alternative explanations.

stl is expressed in head and ovarian tissue: Because stlhas been shown to have both ovary autonomous andnonautonomous functions (Willard et al. 2004), welooked at wild-type stl mRNA expression in the ovary, aswell as in nonovarian tissues that could be involved inlong distance regulation of ovarian follicle formation.By RT–PCR, we detected all three alternative spliceforms in adult ovaries and heads; however, the relativeabundance of the three isoforms was different. Wemeasured high levels of stl-C in all tissues, but compar-atively low levels of stl-A and especially low amounts ofstl-B in heads (Figure 2C). To identify where stl wasexpressed in these tissues, we carried out in situhybridization. Unfortunately, expression in the adultbrain, as the primary component of the head sample,was too low or in too few cells to be detected by thismethod, although brain expression was detectable bythe more sensitive real-time RT–PCR (Figure S4). Othercomponents of the nervous/neuroendocrine system inwhich we were unable to detect stl mRNA by in situhybridization included the corpus allatum, the corpuscardiacum, and the ventral nerve cord. In contrast, wedetected expression of stl in both pupal and adultovaries, beginning at �48 hr after puparium formation(APF). At this stage of ovary development, stl wasexpressed in the somatic basal stalk cells of each ovariole(Figure 4A), consistent in timing with the first stl mutantdefects, which develop between 48 and 60 hr APF(Willard et al. 2004). In the more mature ovaries ofpharate adults, stl was confined to follicular poles andwas no longer present in the basal stalk cells (Figure 4B,arrows). Just before eclosion, stl was expressed strongly

Figure 4.—The stl expression pattern is altered in da andDl mutants. (A–E) Detection of stl mRNA in the ovary by insitu hybridization. (A) Wild-type pupal ovaries (48–60 hrAPF) show expression of stl mRNA in the somatic basal stalks.(B–D) In pharate adult ovaries, stl expression is downregu-lated in the basal stalks (B, arrows), increases in region 3 ofthe germarium and at the follicular poles (C, arrowhead),and is not detected in interfollicular stalks (D, arrow). Inadult ovaries (E), stl mRNA remains abundant in region 3of the germarium, and lower levels are also detectable atthe somatic-germ cell border of more mature follicles(arrow). Although no longer restricted to polar cells, mRNAlevels remains slightly elevated at the poles of each follicle.Detection of stl mRNA in stl (F and G), Dl (H), and da(I and J) mutant ovaries shows altered expression. In stl ph57 pu-pal ovaries (F), stl is still strongly expressed in the basal stalks,and in mutant defective adult ovarioles (G), stl is also ex-pressed normally in cells corresponding to region 3 of the ger-marium. In DlX heterozygous ovarioles (H), there is noexpression of stl mRNA in the germarium, ectopic expressionin interfollicular stalks, and high polar region expression thatpersists aberrantly in later follicle stages. In da lyh mutant ova-ries, stl mRNA expression is severely reduced. In pupal ovaries(I), fewer basal stalk cells express stl, and the levels are signif-icantly lower than wild type (arrows); adult ovarioles ( J) alsoshow severely reduced levels of stl expression. (K) Ovarioles

hybridized with a sense riboprobe control demonstrate theabsence of nonspecific staining. (L) A diagram of the up-stream and 59-end structure of the stl gene (length shown,5700 bp) displays the locations (vertical hatch marks) ofE-box sequences, as potential Da-binding target sites. Tran-scribed regions are indicated relative to the E boxes belowthe map: stl-C 59-UTR (red), stl-B 59-UTR (green), and stl-A59-UTR (yellow). The full transcripts are not illustrated.

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in the somatic cells of region 3 of the germarium,restricted to the polar cells of the most mature follicle(Figure 4C), and was conspicuously absent from theinterfollicular stalks (Figure 4D, arrow). The strong ex-pression of stl in region 3 of the germarium persisted inadult ovaries (Figure 4E); these cells play critical rolesduring follicle individualization, stalk formation, andoocyte localization. Finally, a peculiarity of stl mRNAdistribution was the apparent apico-lateral localization atthe plasma membrane of the somatic epithelium (Figure4E, arrow). We were not able to account for this sub-cellular bias, although its absence from negative controls(data not shown) and its loss specifically in da mutantovaries (Figure 4K) suggest that it was not a stainingartifact.

stl expression in the ovary is independent of thegene’s extraovarian function: Because the stl mutantphenotype becomes apparent early during pupal oo-genesis and results principally from a loss of extraovar-ian gene function (Willard et al. 2004), we examinedwhether a primary consequence of that loss was theinstability of stl-expressing cells in the ovary itself. Theexpression pattern of stl in both pupal and adult mutantovaries was indistinguishable from that of wild-typetissues (Figure 4, F and G). In mutant pupal ovaries,stl was still strongly expressed in the basal stalks.Further, in spite of the gross morphological disruptionof the adult mutant ovary, a strong band of stl expres-sion, corresponding to cells found at region 3 of the wild-type germarium, was still present. In the absence ofany interfollicular stalks, this dense population of stl-expressing cells spreads across the width of the defectiveovariole. Thus, the extraovarian and/or early ovarianfunction of stl does not appear to be required for themaintenance of stl-expressing ovarian cells in the adult.

stl expression in the ovary is regulated by Delta anddaughterless: The strong mutant interactions that stlexhibited with Dl and da suggested that these genesmight function in the same regulatory pathway as stl.Since da encodes a known transcription factor, weexamined the possibility that Dl and da function up-stream of stl, at least in the ovary. Indeed, we found that stlexpression was disrupted in both Dl and da mutantovaries. In ovaries from DlX heterozygous females, it wasprimarily the pattern of stl mRNA distribution that wasaltered (Figure 4H). Most ovarioles exhibited the ex-pected stl expression pattern in the germarium and at thepoles of early stage follicles. While the levels of expressionappeared to be normal early, high levels of expression atthe follicular poles persisted even in the later stages,when stl expression was found to decrease normally(compare Figure 4E and 4H). There was also oftenectopic expression in interfollicular stalks. We did notobserve, however, the distinct apico-lateral localization ofthe stl mRNA that characterized wild-type expression inindividualized follicles. And, consistent with our failureto detect any genetic interaction between stl and N, the

stl expression pattern was not significantly changed in Nmutant ovaries (data not shown). In ovaries from dalyh

mutant females, it was primarily the levels of stl mRNAexpression that were altered (Figure 4, I and J): stlexpression was dramatically reduced in both pupal andadult ovaries. Since the Da protein has been identifiedas a helix-loop-helix (HLH) transcription factor, thisresult suggested that Da could be directly involved inthe regulation of stl transcription. This possibility issupported by the presence of multiple HLH targetsequences (E boxes, ‘‘CAnnTG’’) in and around the stltranscription unit (Figure 4L). Direct transcriptionalregulation of stl by Da, particularly at the extraovariansite(s) of stl function, would account for the strongsynergistic mutant interaction between these genes.

DISCUSSION

The molecular identification of the Drosophila Stall(Stl) protein as a metalloprotease reveals a previouslyunrecognized mechanism through which the metaboli-cally demanding process of oogenesis can be modulatedby the physiological condition of the fly. Previouslyknown mechanisms of significant external regulation ofoogenesis include the neurosecretory and hormonalsystems: Responses to nutritional input through insulin-like peptides trigger long-range control of ovarian stemcell division and apoptotic checkpoints, while JH and 20Eregulate yolk protein synthesis and uptake by ovarianfollicle cells (Soller et al. 1999; Drummond-Barbosa

and Spradling 2001; Lafever and Drummond-Barbosa

2005). Further, pharmacological studies have discov-ered nervous system regulation of follicle formationand vitellogenic follicle survival/maturation pointsthat are mediated by the neurotransmitters, dopamineand serotonin, as well as by hormones (JH and 20E)(Willard et al. 2006). Finally, extraovarian regulation offollicle individualization and stalk formation has beenassociated with stl gene function, on the basis of theanalysis of stl mutant somatic clones (Willard et al.2004). The identification of the stl gene product as amember of the ADAMTS family of metalloproteases andthe discovery that the gene is expressed in the braintogether suggest a biochemical model in which Stlenzymatic activity is required to initiate a long-rangeneurosecretory signaling pathway. Moreover, the pre-dicted structural differences of the three Stl isoforms,together with their comparisons to other ADAMTS familymembers, hint at ways in which stl functions both locally(autonomously) and remotely (nonautonomously) toregulate ovarian follicle formation.

Metzincins (metalloproteases that require zinc ionsfor activation), such as ADAMs and ADAMTSs, havebeen associated with multiple cellular roles, with con-siderable variation in both substrate specificity andsubcellular sites of action (reviewed in Seals andCourtneidge 2003; Porter et al. 2005). Generally

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implicated in cell adhesion and migration throughECM remodeling, many of these enzymes cleave ECMcomponents, such as procollagen, aggrecan, versican,and brevican (Abbaszade et al. 1999; Tortorella et al.1999). Non-ECM functions include ectodomain shed-ding of transmembrane cell signaling molecules andthe activation of cell surface molecules. For example,transmembrane ADAM-dependent cleavage can targetgrowth factors, cytokines, and cell surface receptors(Massague and Pandiella 1993; Black et al. 1997;Reddy et al. 2000), and secreted ADAMTS-dependentcleavage of von Willebrand factor is essential for plateletaggregation (Levy et al. 2001; Zheng et al. 2001). Whileboth families affect the local cellular environment,ADAM proteins are more likely to influence long-rangesignaling through release of extracellular cell signalingpeptides.

By structural criteria alone, it is not easy either toclassify Stl as strictly an ADAM vs. ADAMTS or to declareif/how each Stl isoform functions. By BLAST sequenceanalysis, the metalloprotease domain of Stl is most similarto that of human ADAMTS16. Notably, ADAMTS16 isalso highly expressed in ovarian follicles, is induced hor-monally by follicle stimulating hormone, has detectableproteolytic activity, and consists of alternatively splicedisoforms (Gao et al. 2007). However, both ADAMTS16isoforms are predicted to be secreted. The Stl protein isexpected to have proteolytic function, on the basis of aperfect Zn-binding metalloprotease consensus sequenceand a disrupting mutation in that domain in stla16, but itsprimary sequence suggests isoform-specific processing.On the one hand, Stl-B resembles the ADAMs, whichgenerally contain a transmembrane domain that or-ganizes their metalloprotease domains outside the cellfor interactions with other integral membrane proteinsand the ECM. With a predicted transmembrane domainin the middle of the protein, Stl-B is not expected to besecreted, yet some aspects of Stl’s structure suggest theprotein may be processed more like an ADAMTS. Theseenzymes are generally expressed as inactive proproteinforms (zymogens), in which a long (�230-aa) prodomainblocks the zinc-binding catalytic domain; following trans-port to the cell membrane as an inactive protein, furin-dependent prodomain removal results in enzymaticactivation. ADAMTS prodomains are generally longerthan those of ADAMs, and there are typically two furinrecognition sites. Like an ADAMTS, Stl-B contains twofurin target sites in its N terminus, and cleavage at thestronger site would yield a prodomain of 269 amino acids.Alternatively, ADAMTS1 is processed intracellularly andsecreted as an active enzyme (Longpre and Leduc 2004;Koo et al. 2006). In fact, the signal sequence of ADAMTS1is so weak that the signal peptide prediction program,SignalP, fails to predict the protein’s secretion. BecauseSignalP also failed to detect a signal sequence in Stl-B, it ispossible that Stl-B is processed intracellularly and se-creted in a manner similar to ADAMTS1. Finally,

ADAMTS13 has an abnormally short prodomain(�40 aa) and can be secreted and/or proteolytically activeeven in the absence of prodomain cleavage (Majerus et al.2003). Thus, the shorter isoform Stl-A, which has noprodomain N terminal to its metalloprotease domain,could similarly be functional in the absence of anyprocessing. Most definitive of the three isoforms, Stl-Ccontains a clear signal sequence and is overwhelminglypredicted to be secreted as a typical ADAMTS. In the end,there may not be a single mechanism by which Stl reachesits proper subcellular localization(s), and the apparentcomplexity of this issue could relate to the protein’sfunction within vs. outside the ovary. The details of Stlfunction require further in vivo analysis to resolve.

Although Stl appears to be somewhat of a structuralanomaly within the ADAMTS family, a comparison ofknown ADAMTS processing with the essential structuralfeatures of Stl that were identified by mutational lesionssuggests possible mechanisms of regulation. First, sev-eral ADAMTSs undergo processing at the C terminusfor regulated proteolytic activity, and the C-terminalspacer region has been shown to be important forsubstrate binding and the specificity of the enzyme’sbiological activity (Zheng et al. 2003; Majerus et al.2005). ADAMTS4 is cleaved extracellularly, therebygreatly increasing its aggrecanase activity (Flannery

et al. 2002; Gao et al. 2002, 2004), and ADAMTS1 loses aportion of its C terminus, thereby losing affinity forheparin (Rodriguez-Manzaneque et al. 2000). It islikely that the C-terminal spacer region is essential forStl function as well: The least extreme nonsense allele,stl pa49, truncates only the C-terminal spacer region (of allisoforms), and yet the mutant phenotype is as severe as ifthe metalloprotease domain itself were mutated. It isunlikely that the null phenotype of stl nonsense muta-tions results from nonsense-mediated decay of mutanttranscripts, since amplification and sequencing of allstl alleles was performed on easily generated cDNA.However, it is possible that instability of truncated mu-tant protein results in stl loss-of-function; antibody anal-ysis could address this issue. Next, ADAMTS proteins arefrequently substrates for oligosaccharide modification,and these modifications affect membrane targetingand secretion. Generally, the propeptide has sites forN-glycosylation [e.g., ADAMTS9 (Koo et al. 2006)]. Stl-Band Stl-C have two potential N-glycosylation targets(NxS/T) within Stl-B’s putative prodomain, and threeadditional sites within the thrombospondin and cys-richregions are shared by all three Stl isoforms. SinceN-glycosylation can affect protein folding and secretionefficiency [e.g., synaptotagmin 1 and inhibin/activin(Han et al. 2004; Antenos et al. 2007)], it is possible thatthese sites in Stl either facilitate secretion in the absenceof a strong signal peptide (Stl-B) or boost a weak signalsequence (Stl-A). Other ADAMTS oligosaccharide mod-ifications, C-mannosylation and O-fucosylation, typi-cally occur within the TSRs and can be important for

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function (e.g., ADAMTS13) (Gonzalez De Peredo et al.2002; Paakkonen et al. 2006; Ricketts et al. 2007).Capacity for C-mannosylation is noteworthy in thecontext of Stl, because the solitary central TSR of Stl(with no C-terminal TSRs) is one of the major structuraldifferences that marginalize Stl within the ADAMTSfamily and because C-mannosylation occurs only onsecreted proteins. The Stl amino acid sequence containsC-mannosylation sites (WGEW) at the very beginning ofits TSR and in the metalloprotease domain; however,there are no apparent O-fucosylation consensus sites.Such deviation from the ‘‘typical’’ ADAMTS profilefurther confounds the question of Stl’s subcellularlocalization (secreted vs. not). It also could suggest thatStl is an evolutionary intermediate between ADAMs (noTSRs, no C-mannosylation, transmembrane rather thansecreted) and ADAMTSs (multiple TSRs, with prom-inent C-mannosylation, generally secreted).

For now, stall represents a unique form of regulationof oogenesis. But does this gene function in the contextof any known regulatory pathways? On the basis ofdominant genetic interactions during follicle forma-tion, stl, da, and Dl are all clearly functionally connected(Cummings and Cronmiller 1994); however, stl can-not be a simple common component in the previouslydescribed da/Dl oogenesis pathways. First, and mostsignificantly, all of the essential requirements for daand Dl during oogenesis have so far been identified asfunctions within the ovary itself (Lopez-Schier and St.Johnston 2001; Smith et al. 2002). In contrast, the mostcritical stl function originates outside the ovary, with onlya minor role for the gene in the somatic ovary accordingto mitotic clonal analysis (Willard et al. 2004). It ispossible that da and/or Dl could also have roles outsidethe ovary. Both have neurogenic functions earlier inDrosophila development (Alton et al. 1988; Cabrera

1990; Cronmiller and Cummings 1993), so an addi-tional function in the adult nervous system is feasible.However, at least for da, such a nonautonomous role incontrolling oogenesis is improbable, since an ovary-specific mutant allele already displays the null pheno-type for oogenesis (Smith and Cronmiller 2001).Second, a simple linear relationship between stl andda or Dl is unlikely because of plain differences in thecellular processes that are affected by these genes and/or some demonstrated genetic mechanisms for theirfunctions. For example, although mutations in both stland da affect follicle individualization, one reason forthe da effect is disruption of the gene’s normalregulation of cell proliferation within the germarium(Smith et al. 2002); however, BrdU incorporationstudies have shown that stl has no such activity (E. F.Ozdowski and C. Cronmiller, unpublished data).Moreover, all previously described Dl functions involveits receptor Notch, and even da interacts genetically withboth N and Dl (Cummings and Cronmiller 1994); yet,the stl genetic interaction with Dl does not extend to N.

Perhaps this functional connection between stl and Dl inthe ovary is related mechanistically to an N-independentDl function that has been inferred from in vitro cellculture studies, which also demonstrated da involve-ment (Mok et al. 2005).

Finally, elucidating the specific cellular mechanism(s)of stl-mediated regulation of ovarian follicle morphogen-esis will certainly require the identification of the sub-strate(s) of Stl metalloprotease activity. It is theextraovarian metalloprotease activity of Stl that is thecritical one to discover, since it is that activity that leads tolong-range communication with the ovary. While sub-strates of metalloproteases have generally been difficultto identify, genomewide yeast two-hybrid analyses haveidentified two potential physical interactions for the Stlprotein: products of CG12517 and CG31357 (Giot et al.2003). These should certainly be examined for molecularcontributions to the stl regulatory pathway. Otherwise,comprehensive biochemical approaches, together withgenetic strategies, will probably be required to identifyStl’s target(s).

We thank Maho Shibata for technical assistance with anti-Daimmunofluorescence and appreciate the gifts of anti-Mio and anti-C(3)G antibodies from M. Lilly and anti-Vasa from P. Lasko. This workwas supported by grants to C.C. from the Thomas F. Jeffress and KateMiller Jeffress Memorial Trust and the National Institute on DrugAbuse. E.O. was supported by a National Institute of Child Health andHuman Development training grant (HD07528-01). Y.M. was sup-ported by a Harrison Undergraduate Research Award from TheUniversity of Virginia.

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Communicating editor: T. Schupbach

1040 E. F. Ozdowski, Y. M. Mowery and C. Cronmiller

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Supporting Information http://www.genetics.org/cgi/content/full/genetics.109.107367/DC1

stall Encodes an ADAMTS Metalloprotease and Interacts Genetically With Delta in Drosophila Ovarian Follicle Formation

Emily F. Ozdowski, Yvonne M. Mowery and Claire Cronmiller

Copyright © 2009 by the Genetics Society of America DOI: 10.1534/genetics.109.107367

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FIGURE S1.—Amino acid sequence comparison reveals evolutionarily conserved residues and location of mutational

lesions. The closest orthologs of Stl in mammals are ADAMTS-16 and ADAMTS-18, while one of the nearest insect orthologs is Drosophila pseudoobscura 17566. The primary sequences are compared with CG3622 isoforms to show which residues, especially those mutated in stl alleles (asterisks), are highly conserved and likely to be important to protein function.

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FIGURE S2.—Fusome maintenance and oocyte localization are disrupted in stl mutants, but oocyte identity is wild-

type. A-D) Localization of Hts to visualize spectrosomes and fusomes. A) Wild-type germarium shows spherical spectrosomes, normally located in germline stem cells (arrow) that are situated just posterior to the terminal filament. The fusomes (arrowheads) of the germline cyst cells are more elongated and branched. B) In a germarium of a young stlph57 female (1 day post-eclosion), spectrosomes (arrows) and fusomes (arrowhead) both appear normal. C) Four days post-eclosion, spectrosomes are still visible (arrows), but branched fusomes are no longer present. D) A germarium of a 10-day-old stl mutant female, both spectrosomes and fusomes are absent. (The punctate staining densities at the anterior end of this ovariole are located within the terminal filament.) E-G) Analysis of oocyte specification in 4 to 10-day-old stl mutant females by examination of Orb localization. A wild-type ovariole (E, F) shows the normal distribution of Orb protein: in the germline of the germarium and restricted to the oocyte of individualized follicles. G) Despite the absence of individualized follicles, stla16 ovarioles retain the capacity to specify a single oocyte: Orb is localized normally to one cell of the oldest (most posterior) germline cyst and enriched at the posterior of younger cysts. Even when the oocyte is mislocalized laterally (asterisk), Orb remains restricted to a single cell. H-K) Analysis of oocyte specification by examination of C(3)G localization. A wild-type ovariole (H, I) shows the normal distribution of C(3)G: throughout the germline cells of nascent cysts with subsequent restriction to the oocyte. A stl mutant ovariole (J, K) lacks individualized follicles and contains at least one mislocalized oocyte (asterisk), but still shows normal C(3)G localization in the early germline (short arrows), as well as the later stage, mislocalized oocyte (long arrow). Staining: DAPI (E, H, J), anti-Hts (A-D), anti-Orb (F, G), anti-C(3)G (I, K). stl mutant ovarioles shown: stlph57 (B-D, J, K), stla16 (G).

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FIGURE S3.—stl interacts with deficiencies of cytological region 44-45, but a single responsible locus has not been

identified. Deleted regions, as evaluated in the literature by polytene chromosome banding patterns, are indicated by solid lines. Areas of uncertainty are marked by dotted lines. Black lines represent deficiencies that show no interaction with stl: ovarioles appear completely normal with only an occasional compound follicle. Light pink lines represent deficiencies that result in 70-100% defective ovarioles when transheterozygous with stl. The most common defects observed are relatively mild, compound follicles and loss of mid-stage follicles. In contrast, dark pink lines represent deficiencies that interact strongly with stl and result in 100% of ovarioles with severe defects, even in newly eclosed females. Some defects include compound follicles and loss of mid-stage follicles, but most show the complete absence of discrete follicles and the presence of extensive degradation. Combining these results, the blue hatched background demarcates two potential areas of interaction (44F12-45A6 and 45A9-45A11), in which all deletions manifest at least mild defects. The green hatched background shows an area of conflicting evidence, in which a strongly interacting deletion is overlapped completely by deficiencies that show no interaction at all (45C8-45D8). Further screening of new deficiency collections may aid in narrowing down the interacting gene(s) within this region.

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FIGURE S4.—stl is expressed in adult brain tissue. In addition to cDNA pools of whole adult heads, stl amplifies in cDNA from dissected adult brains. Six replicates are shown of isoform A (green) and isoforms B and C (red), while there are five replicates of all three isoforms combined (blue). The second derivative of Sybr Green fluorescence is plotted at each amplification cycle to show the peak of the curve, or the threshold cycle number (Ct): a lower Ct indicates higher expression levels. In this experiment, the stl-A isoform has a Ct of 30.6, whereas the combination of stl-B and stl-C has a Ct of 33.7. cDNA levels were normalized to an rp49 control. Although isoform C levels are high in whole heads, this shows lower relative levels in brain tissue.

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TABLE S1

Deficiency screen for dominant enhancers of stl follicle formation phenotype

Interaction BL# Genotype Breakpoints

X chromosome

- 1329 Df(1)BA1, w/FM7; Dp(1;2)E1, y[+]/+ 1A1; 2A

- 1546 T(1;3)sc[J4] / C(1)DX, y f; Dp(1;f)z9 1B2-14;3A3

- 936 Df(1)64c18, g sd/ Dp(1;2;Y)w[+]/C(1)DX, y w f 2E1-2; 3C2 (Df)

- 935 Df(1)JC19/FM7c 2F6;3C5

- 729 Df(1)N-8/FM7c 3C2-3;3E3-4

- 939 Df(1)dm75e19/FM7c 3C11;3E4;5E Df3C11-3E4

- 940 Df(1)A113/C(1)DX, y w f; Dp(1;2)w[+]64b/+ 3D6-E1; 4F5, 3C2;5A1-2;2F;26D7

- 944 Df(1)JC70/FM7c, sn[+] 4C15-16;5A1-2

- 5705 Basc, Df(1)BA2-8, w[a] B/ bi rb cx 4F5;5A13

- 945 Df(1)C149/ FM6 5A8-9;5C5-6

- 946 Df(1)N73/FM6 5C2;5D5-6

- 5281 Df(1)dx81, w/ Dp(1;Y)dx[+]1/C(1)M5 5C3-10;6C3-12, 5A8-9;6D8;Y

- 3196 Df(1)Sxl-bt, y / Binsinscy 6E2;7A6

- 948 Df(1)ct-J4 , In(1)dl-49, f/ C(1)DX, y w f;

Dp(1;3)sn[13a1]/+

7A2-3;7C1, 6C;7C9-D1;79E

- 3221 Df(1)ct4b1, y/ Binsn 7B2-4;7C3-4

- 949 Df(1)C128/FM 7D1;7D5-6

- 950 Df(1)RA2/FM7c 7D10;8A4-5

- 951 Df(1)KA14/FM7c 7F1-2;8C6

- 3651 Df(1)lz-90b24, y[2] w[a]/ FM7c 8B5-6;8D8-9 or 8D1-2;8E1-2

- 952 Df(1)C52, flw[C52]/ FM6 8E;9C-D

- 954 Df(1)v-L15/FM6 9B1-2;10A1-2

- 3560 Df(1)v-N48, f Dp(1;Y)y[+] v[+] #3/C(1)DX, y, f 9F;10C3-5, 9F3;10E3-4;h1-

h25+1A1;1B2-3

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+/- 957 Df(1)KA7/ C(1)DX, y w f; Dp(1;2)v[+]65b/+ 10A9;10F6-7, 10A1;11A7-8;40-41

- 959 Df(1)HA85/FM7c 10C1-2;11A1-2

- 962 Df(1)N105/FM6 10F7;11D1

- 964 Df(1)JA26/FM7c 11A1;11D-E

- 966 Df(1)N12, ras v/ FM6 11D1-2;11F1-2

- 967 Df(1)C246/FM6 11D-E;12A1-2

- 727 Df(1)g, f B/In(1)AM 12A3-10;12E9

- 998 Df(1)RK2/FM7a 12D2-E1;13A2-5

- 1039 Df(1)RK4/FM7k/Dp(1;Y)y[+], y 12F5-6;13A9-B1, 1A1;1B2;Y

- 3347 Df(1)sd72b/FM7a 13F1;14B1

- 125 Df(1)4b18, y cv v nonA[4b18] f car/ Basc 14B8;14C1

- 3217 Tp(1;2)r[+]75c, sl[3]/ Cy); C(1)M4, y[2] 14B13;15A9;35D-E

- 993 Df(2)r-D1, v f C(1)DX, y w f; Dp(1;4)r[+] /+ 14C2-4;15B2-C1,14A1-2;15A7-

B1;102F

- 4741 Df(1)B25, Sh[14]/FM6 15D3;16A4-6

- 4953 Df(1)BK10, r f / Dp(1;Y)W73/ C(1)DX 16A2;16C7-10, 15C1-D6;16F;Y h1-

h25

- 970 Df(1)N19/FM6 17A1;18A2

- 971 Df(1)JA27/FM7c, sn[+] 18A5;18D

- 972 Df(1)HF396/ FM7c 18E1-2;20

- 977 Df(1)DCB1-35b/FM6/Dp(1;Y)y[+]mal[106],

mal[106]

19F1-2;20E-F, 1A1;1B2+18F;20h;Y

- 3714 Df(1)A209/FM7a 20A;20F

2nd Chromosome

+/- 3638 Df(2L)net-PMF/SM6a 21A1;21B7-8

+/- 6283 Df(2L)BSC4, w[+mC], net cn/ SM5 21B7-C1;21C2-3

- 3548 Df(2L)al/In(2L)Cy, Cy 21B8-C1;21C8-D1

- 3084 Df(2L)ast2/SM1 21D1-2;22B2-3

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E. F. Ozdowski et al. 11 SI

- 3133 Df(2L)dp-79b, dp[DA] cn/ In (2LR)bw[V1], ds[33k]

b bw[V1]

22A2-3;22D5-E1

- 90 Df(2L)C144, dpp[d-ho] ed/ In(2LR)Gla, Gla Bc

Egfr[E1]

23A1-2;23C3-5

+/- 97 Df(2L)JS32, dpp[d-ho]/ SM6a 23C3-5;23D1-2

+/- 3573 In(2LR)DTD16[L]DTD42[R], bw sp/ CyO 23C;23E3-6

- 4954 Df(2L)S2590/CyO P{ry[+t7.2]=sevRas1.V12}FK1 23D2;23E3

- 693 Df(2L)sc19-8/SM6b; Dp(2;1)B19, y ed dp[o2] cl 24C2-8;25C8-9

- 3813 Df(2L)sc19-4/In(2L)Cy[L]t[R] In(2R)Cy, Cy Roi

cn[2] sp[2]; Dp(2;1)B19 y ac sc pn ed dp[o2]cl

25A5;25E5

- 781 Df(2L)cl-h3/SM6b 25D2-4;26B2-5

- 490 In(1)w[m4]; Df(2L)E110/CyO 25F3-26A1;26D3-11

+/- 6338 Df(2L)BSC6; dp[ov1] cn /SM6a 26D3-E1;26F4-7

- 1357 Df(2L)J-H/SM5 27C2-9;28B3-4

- 3077 Df(2L)spd, al dp[ov1]/ CyO 27D-E;28D3

- 4955 Df(2L)XE-2750/CyO,

P{ry[+t7.2]=sevRas1.V12}FK1

28B2;28D3

- 140 Df(1)w67c23 y; Df(2L)Trf-C6R31/CyO 28DE

- 179 In(1)w[m4]; Df(2L)TE29Aa-11/CyO 28E4-7;29B2-C1

- 2892 Df(2L)N22-14/CyO 29C1-2;30C8-9

+/- 556 w[*]; Df(2L)s1402, P{lacW}s1402/CyO 30C1-2;30F

+ 1045 Df(2L)Mdh, cn/ DP(2;2)Mdh3, cn 30D-F;31F

+ 5869 Df(2L)J39 / In(2L)Cy; Dp(2;Y) cb50,

Dp(1;Y)B[S]Yy[+]/C(1)RM

32D1;32F1-3

- 3079 Df(2L)FCK-20, dp[ov1] bw/T(2;3)SM6a-TM6b 32F1-3;33F1-2

- 3344 Df(2L)Prl, Prl nub[Prl]/CyO 33B2-3;34A1-2

- 3138 Df(2L)prd1.7, b Adh[n2] pr cn sca/ CyO P{elav-

lacZ.H}YH2

34B12-C1;35B10-C1

- 3588 Df(2L)b87e25/CyO 35B4-6;35F1-7

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E. F. Ozdowski et al. 12 SI

- 1491 Df(2L)TE35BC-24, b pr pk cn sp/CyO 35D1;36A6-7

- 3180 Df(2L) r10 cn/ CyO 36A8-9;36E1-2

- 420 Df(2L)H20 b pr cn sca/ CyO 36C2-4;37B9-C1

- 3189 Df(2L)TW137, cn bw/ CyO, Dp(2;2)M(2)m[+] 36E4-F1;38A6-7

- 167 Df(2L)TW50 cn /CyO, Dp(2;2)M(2)m[+] 38A6-B1;40A4-B1

- 4959 Df(2L)C'/CyO 40h35;40h38L

+/- 749 In(2R)bw[VDe2L]Cy[R]/In(2LR)Gla, Gla 41A-B;42A2-3 Dp58

- 739 Df(2R)M41A5/SM1 41A

- 1007 Df(2)nap9/In(2LR)Gla, Dp(2;2)BG, Gla 42A1-2;42E6-F1

- 1888 Df(2R)ST1, Adh[n5] pr cn / CyO 42B3-5;43E15-18

+/- 3368 Df(2R)cn9/ Roi sp 42E;44C

+/- 198 w[118]; Df(2R)H3C1/ CyO 43F;44D3-8

- 201 w[118]; Df(2R)H3E1/ CyO 44D1-4;44F12

+ 258 Df(2R)Np3, bw[1] / CyO 44D2-E1; 45B8-C1

++ 6091 Df(2R)Np1, bw[1]/CyO 44F2-4; 45C5-6

+ 3591 w; Df(2R)Np5, In(2LR)w45-32n, cn /CyO 44F10;45D9-E1

++ 5423 Df(2R)Np4, bw[1]/CyO 44F11; 45C1

- 6227 Df(2R)G53/CyO 45A9; 45A6

+ 4966 w;Df(2R)w45-30n cn / CyO 45A6-7;45E2-3

+ 6246 w[1]; Df(2R)w73-1, cn[1]/CyO 45A9-10; 45D5-8

- 6247 w[1]; Df(2R)w73-2, cn[1]/CyO 45A9-11; 45D5-8

++ 68 w[118]; Df(2R)wun-GL/CyO 45C8;45D8

- 6245 w[1]; Df(2R)w45-19g, cn[1]/CyO 45C8-D10; 45D9-E1

- 6917 Df(2R)BSC29, cn[1] bw[1] sp[1]/CyO 45D3-4; 45F2-6

- 1743 w[1118]; Df(2R)B5, px sp/CyO Adh[nB] 46A;46C

- 1702 Df(2R)X1, Mef2[X1]/ CyO Adh[nB] 46C;47A1

- 190 Df(2R)en-A/CyO 47D3;48B2

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E. F. Ozdowski et al. 13 SI

- 1145 Df(2R)en30/SM5; Dp(1;Ybb[-])B[S] 48A3-4;48C6-8

- 4960 Df(2R)CB21/CyO; ry[506] 48E;49A

- 5879 Df(2R)BSC3, w[+mC] unch[k15501] cn bw sp/

SM6a bw[k1]

48E12-F4;49A11-B6

- 754 Df(2R)vg-C/SM5 49A4-13;49E7-F1

- 442 Df(2R)CX1, b pr/SM1 49C1-4;50C23-D2

- 1896 Df(2R)trix/CyO? 51A1-2;51B6

- 1150 w/Dp(1;Y)y[+]; Df(2R)knSA3,

Tp(1;2)TE21F22A/CyO

51B5-11;51D7-E2

- 3518 w[a] N[fa-g]; Df(2R)Jp1/CyO 51D3-8;52F5-9

+/- 3520 w[a] N[fa-g]; Df(2R)Jp8 w[+]/CyO 52F5-9;52F10-53A1

- 5680 Df(2R)robl-c/CyO y[+] 54B17-C4;54C1-4

- 3064 Df(2R)Pcl7B/CyO 54E8-F1;55B9-C1

- 1547 y[*] w[*]/Dp(1;Y)y[+]; Df(2R)P34/CyO 55A;55F

- 757 Df(2R)PC4/ CyO 55E2-4;56C1-11

- 543 Df(2R)017/ SM1 56F5;56F15

- 3467 Df(2R)AA21, c px sp/SM1 56F9-17;57D11-12

- 5247 Df(2R)2-65, b pr cn sca/CyO 57C2;58B2

- 5442 Df(2R)XE3030/CyO, P{sevRas1.V12}FK1 57C2;58C

- 5246 Df(2R)Egfr5, b pr cn sca/CyO P{sevRas1.V12}FK1 57D2-8;58D1

- T. Orr-Weaver Df(2R)X58-6 58A3-4;58E4

- 283 Dp(1;Y)y[+]/y; Df(2R)X58-7 pr cn/CyO bw 58B1-2;58E1-4

+/- T. Orr-Weaver Df(2R)X58-5 58B3;58F8

+/- 100 y[1]; Df(2R)X58-8, pr cn/ SM5 58B3;59A1

- 103 Dp(1;Y)y[+]/y; Df(2R)X58-3 pr cn/CyO bw 58C3-7;58D6-8

- T. Orr-Weaver Df(2R)X58-2 58C7-D2;58F4-5

+/- 282 Dp(1;Y)y[+]/y; Df(2R)X58-12/SM5 58D1;59A

- 5421 Df(2R)02311/CyO 58D2;58E1

- T. Orr-Weaver Df(2R)X58-1 58D6-8;58F3-5

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E. F. Ozdowski et al. 14 SI

- 590 w[*]; Df(2R)59AB/SM1 59A1-3;59B1-2

- 7559 w[1118]; Df(2R)Exel6079, P{XP-U}Exel6079/CyO 59A3;59B1

+/- 3909 w[*]; Df(2R)59AD/SM1 59A1-3;59D1-4

- 7905 w[1118]; Df(2R)Exel7176,

P+Pbac{XP5.WH5}Exel7176

59B4;59C2

- 7313 w[1118]; Df(2R)WI213 cn/CyO,

P{sevRas1.V12}FK1

59B;59D

- 7266 w[1118]; Df(2R)WI327 cn/CyO bw 59B;59D

- 7273 Df(2R)vir130/CyO 59B;59D8-E1

- 7256 y/Dp(1;Y)y[+]; Df(2R)3-70 cn/CyO 59B3;59D5-11

- 7265 Df(2R)Frd-R1 wg[Sp-1] Pin[2]/CyO 59C1;59C4

- 7906 w[1118]; Df(2R)Exel7177,

P+PBac{XP5.RB3}Exel7177/CyO

59C3;59D2

- 7270 w[1118]; Df(2R)WI3117, cn/CyO bw 59C;59C

- 7269 w[1118]; Df(2R)WI3100, cn/ CyO bw 59C;59C

- 7272 Df(2R)vir-12 pr cn Adh[D]/CyO 59D4-8; 59D9-E1

- 1682 Df(2R)or-BR6, cn bw sp/

In(2LR)lt[G16L]bw[V32gR]

59D5-10;60B3-8

- 2355 Df(2R)bw-S46/CyO 59D8-11;60A7

+/- 5225 Df(2R)bw-HB132, Frd[HB132]/SM6a 59D11;59F6-8

- 2604 Df(2R)Px2/SM5 60C5-6;60D9-10

- 2471 Df(2R)M60E/In(2LR) bw[V32g] bw[V32g] 60E2-3;60E11-12

- 3157 Df(2R)ES1, b pr cn wx[wxt] Kr[lf-1]/ SM1 60E6-8;60F1-2

- 4961 Df(2R)Kr10/CyO 60F1;60F5

3rd Chromosome

- 2577 Df(3L)emc-E12/TM6B, Tb 61A; 61D3

- 439 Df(3L)Ar14-8, red / TM2 p[p] Ubx 61C5-8;62A8

- 5411 Df(3L)Aprt-32/TM6 Ubx 62B1;62E3

- 2400 Df(3L)R-G7, ve/ TM6B Tb 62B8-9;62F2-5

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E. F. Ozdowski et al. 15 SI

- 3650 Df(3L)M21, kni[ri] p[p]/ In(3LR) T33[L]f19[R] 62F; 63D

- 3649 Df(3L)HR119/TM6B, Tb 63C2;63F7

- 463 w[1118]; Df(3L)GN34/ TM3 ry su [2] Sb 63E6-9;64A8-9

- 3686 Df(3L)GN24/TM8, l(3)DTS4 Sb 63F4-7;64C13-15

- 3096 Df(3L)ZN47, ry[506]/ TM3 Sb Ser 64C;65C

- 4393 w; Df(3L)XDl98, e /TM6B Tb 65A2;65E1

- 1420 Df(3L)pbl-X1/TM6B Tb 65F3;66B10

- 5877 w; Df(3L)ZP1/TM3 Sb Ser 66A17-20; 66C1-5

- 1541 y w N[spl-1]; Df(3L)66C-G28/TM3, Sb 66B8-9;66C9-10

- 6460 Df(3L)BSC13 rho[ve] e / TM6a? 66B12-C1;66D2-4

- 3024 Df(3L)h-i22 h[i22] Ki roe p[p]/TM3 Ser 66D10-11;66E1-2

- 4500 Df(3L)Scf-R6 th st cu sr e[s] ca/ TM3 Sb 66E1-6;66F1-6

- 1688 Df(3L)Rdl-2, e /TM3, Sb 66F5;66F5

- 2479 Df(3L)29A6, kni[ri] p[p]/TM3, Sb 66F5;67B1

- 997 Df(3L)AC1 roe p[p]/ TM3 Sb 67A2;67D7-13 OR 67A5;67D9-13

- 6471 Df(3L)BSC14 rho[ve] p e /TM3, Ser 67E3-7;68A2-6

- 2611 Df(3L)vin5 ru h gl[2] e[4] ca/TM3 Sb Ser 68A2-3;69A1-3

- 2612 Df(3L)vin7 ru h gl[2] e[4] ca/TM3 Sb Ser 68C8-11;69B4-5

- 5492 W; Df(3L)eyg[C1]/TM3, P{ftz/lacC}SC1, Sb

ry[RK]

69A4-5;69D4-6

+/- 6456 Df(3L)BSC10, rho[ve] e /TM3, Ser 69D4-5;69F5-7

- 6457 Df(3L)BSC12, rho[ve] e/TM3, Ser 69F6-70A1;70A1-2

- 4366 In(3LR)C190[L]Ubx[42TR], Ubx[-]/sti 69F3-4;70C3-4 +89;89

- 3124 Df(3L)fz-GF3b, P{wA[R]}66E/TM6B, Tb 70C1-2;70D4-5

- 3126 Df(3L)fz-M21, th st/TM6 Ubx 70D2-3;71E4-5

- 2992 Df(3L)BK10, ru Ly red cv-c Sb[sbd] sr e / TM3, Sb 71C;71F

- 3640 Df(3L)brm11/TM6c, cu Sb ca 71F1-4;72D1-10

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E. F. Ozdowski et al. 16 SI

- 2993 Df(3L)st-f13, Ki roe p[p]/ TM6B Tb 72C1-D1;73A3-4

- 2998 Df(3L)81k19/TM6B, Tb 73A3;74F

- 6411 Df(3L)BSC8/ TM3, Ser 74D3-75A1;75B2-5

- 2608 Df(3L)W10, ru h TM6B, Tb 75A6-7;75C1-2

- 2990 Df(3L)Cat kni[ri] Sb[sbd] e /TM3 75B8;75F1

- 3617 Df(3L)kto2/TM6B, Tb 76A3;76B2

- 5126 Df(3L)XS-533/TM6B, Tb 76B4;77B

- 2052 Df(3L)rdgC-co2, th st in kni[ri] p[p]/TM6C, cu Sb

Tb ca

77A1;77D1

- 3127 Df(3L)ri-79c/TM3, Sb 77B-C;77F-78A

- 5878 Df(3L)ri-XT1, ru st e ca/TM3, Ser 77E2-4;78A2-4

- 4429 Df(3L)ME107, mwh red e/ TM1, red 77F3;78C8-9

- 3627 Df(3L)31A/Dp(3;3)C126 st cp in kni[ri] p[p] 78A;78E, 78D;79B

- 4430 Df(3L)pPc-2q, ry[506]/ TM2 Ubx 78C5-6;78E3-79A1

- 4506 Df(3L)Ten-m-AL29/TM3, ry[RK] Sb 79C1-3;79E3-8

- 5951 y[1] w; Df(3L)HD1/TM6C, Sb Tb 79D3-E1;79F3-6

- 4370 Df(3L)Delta1AK, ru h ry[506] sr e[s] ca/TM3,

ry[RK] Sb Ser

79F;80A

- 1518 Dr(3R)ME15, mwh red e[4]/ MKRS 81F3-6;82F5-7

- 4787 Df(3R)3-4, ru th st/TM3, Sb 82F3-4;82F10-11

- 5694 w[*]; Df(3R)e1025-14/TM6B, Tb 82F8-10;83A1-3

- 1990 Dr(3R)Tpl10, Tp(3;3)Dfd[rv1], kni[ri] Dfd[rv1] p[p]

Doa[10]/TM3, Sb

83C1-2;84B1-2

- 2393 Df(3R)WIN11, Ki rn[roe] p[p]/TM3, Sb 83E1-2;84A4-5

- 1884 Df(3R)Scr, p[p] e[s]/TM3, Sb 84A1-2;84B1-2

- 1842 Df(3R)Antp17/TM3, Sb 84B1-2;84D11-12 or A6, D14

- 1968 Df(3R)p712, red e /TM3, Sb 84D4-6;85B6

- 1962 Df(3R)p-XT103, ru st e ca/TM3, Sb 85A2;85C1-2

+/- 1931 Df(3R)by10, red e /TM3 Sb 85D8-12;85E7-F1

- 1893 Df)3R)by62, red e /TM1 85D11-14;85F6

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E. F. Ozdowski et al. 17 SI

- 3128 Dr(3R)M-Kx1/TM3, Sb 86C1;87B1-5

- 3003 Df(3R)T-32, kni[ri] cu sr e[s]/MRS Sb 86E2-4;87C6-7

- 3007 Df(3R)ry615/TM3, Sb Ser 87B11-13;87E8-11

- 1534 Tp(3;Y)ry506-85C/MKRS 87D1-2;88E5-6;Y

- 383 Kf(3R)ea, kni[ri] p[p]/TM3, Ser 88E7-13;89A1

- 1467 Dr(3R)P115, e[11]/TM1; Dp(3;1)P115/+ 89B7-8;89E7-8;20

- 4431 Df(3R)DG2/TM2 89E1-F4;91B1-2

- 3071 Df(3R)C4, p/Dp(3;3)P5, Sb 89E3-4;90A1-7

- 3011 Df(3R)Cha7/TM6B, Tb 90F1-4;91F5

+/- 5600 Df(3R)Cha9, Kar[2] ry[5] red[1]/ TM6C Sb[1] 91C7-D1; 92A2

+ 3012 Df(3R)Dl-BX12 ss e[4] ro/TM6B, Tb 91F1-2;92D3-6

+ 4962 Df(3R)H-B79 e /TM2 92B3;92F13

- 3340 Df(3R)e-R1, Ki/TM3, Sb 93B6-7;93D2

- 2425 Df(3R)e-N19/TM2 Ubx 93B;94

- 2586 Df(3R)23D1, ry[506]/ TM3, Sb ry 94A3-4;94D1-4

- 4940 cn; Df(3R)mbc-30/TM3 95A5-7;95C10-11

- 2585 cn; Df(3R)mbc-R1, ry[506]/TM3, Sb ry 95A5-7;95D6-11

- 4432 Df(3R)crb-F89-4, st e /TM3, Ser 95D7-11;95F15

- 2363 Df(3R)crb87-5, st e /TM3 95F7;96A17-18

- 3468 Df(3R)slo[8]/Dp(3;3)Su[8] 96A2-7;96D2-4

- 5601 Df(3R)Espl3/TM6C Sb 96F1;97B1

- 1910 Df(3R)TI-P e ca/ TM3 Ser 97A;98A1-2

- 823 Df(3R)D605/TM3 Sb Ser 97E3;98A5

- 430 w[1118]; Df(3R)3450/TM6B Tb 98E3;99A6-8

- 669 w; Df(3R)Dr-rv1, ry[506]/TM3, ry[RK] Sb Ser 99A1-2;99B6-11

- 3547 Dr(3R)L127/TM6; Dp(3;1)B152 Ubx 99B5-6;99E4-F1

- 3546 Df(3R)B81, P{RP49}F2-80A e/TM3 Sb;

Dp(3;1)67A

99C8;100F5