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  • Copyright � 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 ‘‘a disintegrin 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.

    AN emerging picture of the regulation of oogenesisin 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.

    1Corresponding author: Biology Department, University of Virginia, P.O. Box 400328, Charlottesville, VA 22904-4328. E-mail: crc2s@virginia.edu

    Genetics 183: 1027–1040 (November 2009)

    http://www.genetics.org/cgi/content/full/genetics.109.107367/DC1 http://www.genetics.org/cgi/content/full/genetics.109.107367/DC1 http://www.genetics.org/cgi/data/genetics.109.107367/DC1/3 http://www.genetics.org/cgi/content/full/genetics.109.107367/DC1 http://www.genetics.org/cgi/content/full/genetics.109.107367/DC1 http://www.genetics.org/cgi/content/full/genetics.109.107367/DC1

  • example, daughterless (da) regulates cell proliferation and apoptosis in the germarium, as well as stalk cell recruitment 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 Janus kinase/signal transducer and activator of transcription ( JAK/STAT) pathway to initiate stalk cell differentiation (McGregor et al. 2002; Torres et al. 2003). Further, the stalk induces the posterior polar cell fate of the younger follicle, leading to upregulation of adhesion molecules, such as DE cadherin [shotgun (shg)] and b-catenin [armadillo (arm)], in the oocyte and posterior somatic cells. Homophilic interactions and cell sorting ulti- mately position the oocyte to the posterior of the egg chamber (Godt and Tepass 1998; Gonzalez-Reyes and St. Johnston 1998). Mutations in many of the genes that control these events disrupt follicle separation, resulting in the packaging of multiple germline cysts within a single somatic epithelium (Ruohola et al. 1991; Cummings and Cronmiller 1994; McGregor et al. 2002). This phenotype is shared by stall (stl) mutants, in which 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 oogenesis have been described in detail, less is known about the extraovarian control of this process. Hormonal input has been shown to affect oocyte production and mat- uration through germarial cell proliferation and cell death, follicle apoptosis, and yolk protein synthesis and uptake: Insulin, juvenile hormone ( JH), and 20-hydroxy- ecdysone (20E) affect egg development in response to the 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 maturation have 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 and ovary transplantation, we identified stall as an additional important extraovarian regulator of follicle morpho- genesis; however, the molecular identity of the stl gene product was then unknown (Willard et al. 2004).

    Here, we extend our analysis of stl ’s control of oogenesis by further defining the stl mutant phenotype, elaborating on the gene’s functional interactions, and identifying its molecular product as a metalloprotease with distinguishing similarities to a disintegrin and me- talloprotease with thrombospondin (ADAMTS) domain proteins. We show that, although stl mutant ovaries fail to maintain the fusome within early germline cysts and contain a moderate number of mislocalized oocytes, stl does not alter oocyte determination. We also address a genetic 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 the study of ovarian follicle formation. These evolutionarily conserved enzymes participate in a wide range of bi- ological processes, and the characterization of Stall as an ADAMTS offers a new approach to considering the roles of these proteins in oogenesis.

    MATERIALS AND METHODS

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

    Genetic and molecular analysis of stl: Bloomington De- ficiency Kits (2003) for chromosomes X, 2, and 3 were crossed to stl a16, and ovaries were dissected from doubly heterozygous adult progeny. Ovaries were 49,6-diamidino-2-phenylindole (DAPI) stained and scored for percentage of ovarioles exhibiting follicle formation defects to identify dominant genetic interactions. To locate the stl transcription unit, meiotic recombination frequencies were determined between stl and l(2)06496 and between stl and l(2)k06908 on the basis of segregation of the P{w1}markers. Both stl a16 and stl pa49 were used 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 opposite cn stl pa49