Developmental Biology 294 (2006) 406 – 417 www.elsevier.com/locate/ydbio
Deadlock, a novel protein of Drosophila, is required for germline maintenance, fusome morphogenesis and axial patterning in oogenesis and associates with centrosomes in the early embryo Kristina Wehr, Andrew Swan, Trudi Schüpbach ⁎ Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA Received for publication 5 August 2005, revised 26 January 2006, accepted 3 March 2006 Available online 17 April 2006
Abstract The deadlock gene is required for a number of key developmental events in Drosophila oogenesis. Females homozygous for mutations in the deadlock gene lay few eggs and those exhibit severe patterning defects along both the anterior–posterior and dorsal–ventral axis. In this study, we analyzed eggs and ovaries from deadlock mutants and determined that deadlock is required for germline maintenance, stability of mitotic spindles, localization of patterning determinants, oocyte growth and fusome biogenesis in males and females. Deadlock encodes a novel protein which colocalizes with the oocyte nucleus at midstages of oogenesis and with the centrosomes of early embryos. Our genetic and immunohistological experiments point to a role for Deadlock in microtubule function during oogenesis. © 2006 Elsevier Inc. All rights reserved. Keywords: Drosophila; Oogenesis; Fusome; Germline stem cell; Microtubules; Spermatogenesis
Introduction Analyses of genes required for female fertility in Drosophila have provided insights into the diverse cellular processes required for stem cell maintenance and egg development. While some of these genes encode proteins with very specific functions, many others appear to function at several stages during oogenesis giving rise to complex phenotypes (Huynh and St Johnston, 2004). We report here that the deadlock gene is required for a wide range of processes including germline maintenance, mitosis, patterning and growth. All of these processes require an organized and dynamic microtubule network and deadlock appears to be required for microtubule functions. Oogenesis begins in a specialized region called the germarium found at the anterior tip of the ovariole. Between 2 and 3 germline stem cells (GSCs) reside there in close contact with somatic cells whose role it is to regulate their division and to maintain their fate as stem cells (Song et al., 2002; Spradling et al., 2001; Szakmary ⁎ Corresponding author. Fax: +1 609 258 1547. E-mail address: [email protected]
(T. Schüpbach). 0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.03.002
et al., 2005; Wang and Lin, 2004). GSCs undergo an asymmetric cell division to yield a new stem cell and a daughter cystoblast. These cystoblasts subsequently undergo 4 rounds of mitosis with incomplete cytokinesis to give rise to a cyst of 16 cells. Cyst cells stay connected and share cytoplasm via intercellular bridges termed ring canals. One of the cells becomes the oocyte while the other 15 differentiate into nurse cells. Each GSC harbors a spectrin-rich membranous structure called a spectrosome positioned at the anterior cortex of the cell (de Cuevas and Spradling, 1998; Deng and Lin, 1997; Lin et al., 1994). It has been suggested that the spectrosome is required to anchor stem cells in their niche in conjunction with adherens junctions (Deng and Lin, 1997; Song et al., 2002). Departure from the niche allows germline cells to differentiate into cystoblasts as they move further away from stem cell maintenance signals (Cox et al., 2000; King and Lin, 1999; Szakmary et al., 2005; Xie and Spradling, 1998, 2000). Proper orientation of GSCs divisions is therefore important for maintenance of GSC fate and cystoblast production. This spatially directed division involves the association of the spectrosome with a single pole of the mitotic spindle. In the
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first asymmetric division, the daughter cystoblast inherits roughly one third of the spectrosome, which, thereafter is called a fusome. The fusome anchors one mitotic spindle pole in each subsequent division, giving rise to a stereotypical cluster of cells. The fusome remains associated with the progenitor cells of each division until cytokinetic arrest at which point a ‘plug’ of fusomal material forms in each new ring canal. The plug fuses with the original fusome to form a branched structure that extends into each cell of the cyst (de Cuevas and Spradling, 1998; Deng and Lin, 1997; Lin et al., 1994; McGrail and Hays, 1997). Two classes of mutants have been described that disrupt fusome morphology. Mutations in genes encoding structural components of the fusome such as hu-li tai shao (hts) and αspectrin (αspc) cause severe disruptions in cystocyte divisions and mutant egg chambers usually degenerate before completing oogenesis (de Cuevas et al., 1996; Lin et al., 1994; Snapp et al., 2004; Yue and Spradling, 1992). The heavy chain of the microtubule motor, Dynein (Dhc64C), and its regulator, Drosophila Lissencephany1 (DLis1), are required for fusome biogenesis as are the non-motor microtubule-associated proteins, Abnormal Spindle (Asp) and Orbit/Mast. Mutations in these microtubule-associated proteins disrupt fusome growth and branching suggesting that fusome assembly is a microtubule-dependent process (Liu et al., 1999; Mathe et al., 2003; McGrail and Hays, 1997; Riparbelli et al., 2004; Swan et al., 1999). In addition to defects in fusome biogenesis, these mutants exhibit aberrations in oocyte growth, differentiation and axial patterning as well. In this study, we analyzed the defects that underlie early arrest and axial patterning phenotypes of del mutants. We found that ovarian phenotypes in del flies are strikingly similar to aberrations associated with defects in microtubule-associated proteins and microtubule motors. In addition, we found that del mutants are very sensitive to normally well-tolerated modifications of microtubule-associated proteins. These results strongly implicate Del in playing a role in the organization of, or transport along, microtubules. Materials and methods Drosophila strains and transgenes Deadlock alleles used in this study, delHN and delWK, were from our lab collection, but had been induced on different chromosomes (Schupbach and Wieschaus, 1991). The CG9252 P-element insertion line, P[SUPor-P] CG9252KG10262, the Glu1 allele, as well as the deadlock Deficiency, Df(2L) DS6, were provided by the Bloomington Stock Center. Flies carrying the UASGFP-tub transgene, were provided by Allan Spradling, and the NOD-lacZ transgenic line was provided by Ira Clark (Clark et al., 1997). Mutations in Dhc64C were provided by Tom Hays, UAS-HA-del was injected into yw flies. The transgene was made by fusing 2 copies of a hemagglutinin tag (HA) in frame at the 5′ end of full-length del cDNA derived from the SD07269 EST clone (ResGen) and cloned into the pUASp vector (Rorth, 1998). Expression was driven by the nos-Gal4-VP16 (Van Doren et al., 1998), or mat-alpha4-GALVP16 (Hacker and Perrimon, 1998).
Histological staining techniques For in situ hybridizations, ovaries were dissected in PBS and fixed for 20 min in 4% paraformaldehyde in PBS +0.1% Tween20, 10% dimethyl
sulfoxide and 3vol of heptane, as well as without heptane. Subsequent steps were performed as previously described, except that proteinase K treatment was varied in length between 1 min. and 5 min. (Queenan et al., 1997; Tautz and Pfeifle, 1989). Testes were dissected in Testes Buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA, 1 mM PMSF), and the procedure of White-Cooper et al. (1998) was followed. All ovaries for immunostaining, with the exception of BamC staining, were dissected in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBST (PBS + 0.3% Triton X-100) plus three volumes of heptane at room temperature for 20 min. Ovaries were next blocked and permeabilized in 3% BSA in PBS plus Triton X-100 (1% Triton X-100 for Grk and Orb staining, 0.3% Triton X-100 for all other antibody staining except BamC staining) for 1 h at room temperature. Ovaries were incubated in primary antibody overnight at 4°C and in secondary antibody for 1 h at room temperature. BamC staining was performed as described above using Tween 20 in place of Triton X-100. Monoclonal Grk antibody ID12 was used at 1:10 dilution (Queenan et al., 1999), rabbit anti-Osk (gift from Paul MacDonald) was diluted 1:500 and monoclonal Orb antibodies 4H8 and 6H4 (gift from Paul Schedl) were each diluted 1:60 and mixed. Rabbit anti-Vas (gift from Paul Lasko) was diluted 1:2000, rabbit anti-Caspase-3 was diluted 1:100 (Cell Signaling Technology) and rat anti-BamC (gift from Dennis McKearin) was used at 1:1000. Monoclonal anti-αSpc 3A9 was diluted 1:100 (Developmental Studies Hybridoma Bank), rat anti-αTub YL1/2 (Cappel) was used at 1:100 and rabbit anti-Asp (gift from David Glover) was used at 1:100. We also used a rabbit anti-Prod antibody (provided by Tibor Torok) at 1:500, and a rat anti-HA antibody (Roche) at 1:500. All secondary antibodies, 568 goat α-rat, 568 goat α-mouse (Molecular Probes), Cy3 donkey α-rat (Jackson Immunoresearch), 488 goat α-rabbit (Molecular Probes), were diluted 1:1000 in PBST (0.3% Triton X-100). Oregon green and Alexa Fluor 546 phalloidin were used at 1:1000 and Hoechst was used at 1 μg/ml (Molecular Probes). A polyclonal antibody against Del was generated by injecting rabbits with a peptide corresponding to amino acids 262–280 (NH2-CLKKKTEKVHNKIMDKPKN-COOH) of the CH9252 gene (Bio Synthesis Inc.). The third bleed from rabbit #65 was used at a 1:200 dilution to immunostain ovaries and embryos. Testes were dissected in Testes Buffer (183 mM KCl, 47 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA, 1 mM PMSF). Fixing and staining of testes was performed in the same manner as described for ovaries. Immunostaining of embryos for Tubulin and DNA was performed as described in Swan et al. (2005). For Del immunostaining, embryos were fixed 10 min in 3.7% formaldehyde in PBST and blocked in PBST, 0.3% Triton X-100, 1% BSA. Mouse anti Histones (Chemicon) and mouse anti γ-Tubulin GTU-88 (Sigma) antibodies were both used at 1/500.
Assay for mitotic divisions Ovaries were dissected in Grace's medium at room temperature and incubated in Grace's medium containing 10 μM BrdU for 1 h and 45 min. After 2 washes in Grace's medium, ovaries were fixed for 20 min in 3.7% formaldehyde in PBST (PBS plus 0.1% Triton). Ovaries were washed 3 times for 5 min and acid-treated for 30 min in 2N HCl followed by neutralization in 100 mM borax solution for 2 min. Ovaries were next washed 3 times for 10 min and blocked in 3% BSA for 1 h. BrdU incorporation was detected by immunocytochemistry using a rat anti-BrdU antibody diluted 1:40 in PBST overnight at 4°C followed by a 1-h incubation in Cy3 anti-rat at room temperature (Jackson Immunoresearch).
Results Deadlock mutations cause mitotic defects in germline divisions Deadlock was identified in an EMS screen for genes required for female fertility in Drosophila (Schupbach and Wieschaus, 1991). This screen yielded four alleles of deadlock (del), delHN, delWK, delPS and delWH, all of which lay eggs with axial patterning defects. Females homozygous for the two strongest
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alleles, delHN and delWK, as well as these alleles over a Deficiency, lay only a few eggs on the first days of adult life and cease to lay eggs altogether thereafter. We analyzed delHN/delWK (hereafter referred to as del) ovaries to determine the cause of this phenotype. Inspection of del ovaries revealed that ovarioles contained fewer than normal developing egg chambers (Figs. 1A, B). In addition, Gurken protein accumulation was reduced (Fig. 1B). Egg chambers were often smaller than normal and they sometimes lacked an oocyte (Fig. 1C). Localization of the oocyte to the posterior pole is also affected in del ovaries. The oocyte was mispositioned in 8% (N = 129) of mutant egg chambers such that it occupied a central or anterior position instead of the normal posterior location (Fig. 1D). Staining with Hoechst DNA dye allowed us to count the number of germline cells in each egg chamber and to determine their states of differentiation. We found that while most older egg chambers in young (1–3 day old) del mutants contained normal numbers of germline cells, younger egg chambers often contained fewer than 16 cells. Of total egg chambers scored (N = 61), 36% had too few germline cells (Figs. 1C, E). Many of those likely resulted from a defect in encapsulation as the missing complement of cells could be found in a neighboring egg
chamber (Fig. 1C). In 3% of egg chambers (N = 61), oocytes in young females harbored fewer than 4 ring canals, which suggests that a mitotic defect exists in addition to a defect in encapsulation (data not shown). Cystocyte mitoses are regulated by a structure called the fusome which is composed of small membranous vesicles that are surrounded by components of the submembranous cytoskeleton (de Cuevas and Spradling, 1998). At each cystoblast division, the fusome grows and becomes more highly branched resulting in a structure that reaches into each cell of the cyst via ring canals (Lin et al., 1994). To determine if the reduction in numbers of cells per egg chamber in del ovaries arose from defects in fusome structure, we stained ovaries from young del females using an antibody to α-Spectrin (αSpc), a key component of the fusome (de Cuevas and Spradling, 1998; Lin et al., 1994). We found that mutant germaria contained at most only one highly branched fusome instead of 3–4 observed in wild type (Figs. 2A–D). Instead, large spectrosome-like and barbell-shaped structures accumulated in del germaria suggestive of an arrest in cystocyte divisions (Figs. 2C, D). These cells do not appear to represent stem cells dislodged from their niche, since their fusome-like structures are larger than stem cell spectrosomes. In addition, we detected expression of Bam in the
Fig. 1. Defects in egg chamber production in delHN/delWK mutants. All egg chambers were stained with phalloidin to highlight the actin cytoskeleton (green) and Hoechst dye to label DNA (blue). (A) Wild type egg chambers. (B–E) DelHN/delWK mutant egg chambers. (A, B) In contrast to wild type, DelHN/delWK mutant ovarioles contain few egg chambers and a reduction in Grk protein accumulation (red) in midoogenesis in delHN/delWK oocytes (arrow) is observed. (C–E) Egg chambers were stained with Orb antibodies to identify the oocyte (red). We observed packaging (C), oocyte positioning (D), and cystocyte division (E), defects in delHN/delWK ovaries.
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Fig. 2. Mitotic division and fusome defects in delHN/delWK ovaries and testes. Spherical and barbell-shaped fusomes accumulate in the place of highly branched structures in germaria and testes. (A, B) Wild type germaria. (C, D) DelHN/delWK mutant germaria. (A–D) Germaria were stained with αSpc antibodies to label fusomes (red). (A, C) The actin cytoskeleton was labeled with phalloidin (green). (B, D) BamC antibodies were used to label mitotically active cysts (green). A cystoblast containing a sphere of fusomal material in late region 1 is still able to express BamC (arrow in D) in delHN/delWK mutant. (E, F) Testes were stained with αSpc antibodies to label fusomes (red), phalloidin to highlight the actin cytoskeleton (green) and Hoechst to label DNA (blue). (E) Wild type testes. (F) DelHN/delWK mutant testes, note the absence of highly branched fusomes.
cytoplasm of some of these cells indicating that they have differentiated into a cystoblast-like state (Fig. 2D). Cytoplasmic Bam (BamC) staining is excluded from GSCs and is found only in the subset of cystoblasts and cysts that are mitotically active (McKearin and Ohlstein, 1995; Szakmary et al., 2005; Fig. 2B). A fraction of cystoblasts and cysts in region 1 accumulate BamC in del mutants as is the case in wild type ovaries. Interestingly, BamC staining was observed not only in cystoblasts at the anterior of the germarium but also in cystoblasts which had progressed into the posterior end of region 1 alongside 4- and 8- cell cysts, indicating a delay in their development (Fig. 2D). In Drosophila males, four incomplete gonial mitotic divisions give rise to 16-cell cysts similar to those observed in females. Male gonialblasts contain a spectrosome and mitotic divisions of developing cysts are marked by a growing fusome which can be visualized with αSpc antibodies (reviewed in Fuller, 1993; Gilboa and Lehmann, 2004). Like del females, del males are sterile and their germline cysts have reduced numbers of highly branched fusomes (Figs. 2E, F). This observation supports the idea that Del is required for the formation of 16-cell cysts and that it affects a shared process, such as fusome formation, in male and female gamete development.
Germline stem cell loss and apoptosis in the del ovaries In both young and old wild type females, all ovarioles contain apparent GSCs, cystoblasts, and developing egg chambers (Fig. 3A and data not shown). The finding that del ovaries contain few developing egg chambers suggested a requirement for Del in maintenance of the germline. To ascertain whether germline stem cells (GSCs) are lost in del mutants, we stained wild type and del ovaries with an antibody against Vasa (Vas), a DEAD box helicase expressed at high levels in all germline cells (Hay et al., 1988; Lasko and Ashburner, 1988) (Figs. 3A, B). Young (1–3 days old) del females have small ovaries with few egg chambers per ovariole, yet 1–2 Vasa-positive cells, presumably corresponding to GSCs, can still be found in region 1 of most germaria. Inspection of old (23 days) del ovarioles revealed a dramatic loss of Vas staining cells indicative of germline cell loss. Most ovarioles from old del females contained no Vasa-positive cells, and few or no developing egg chambers (Fig. 3B). A small number of del ovarioles in each ovary maintained GSCs and those sometimes also contained clusters of germline cells with branched fusomes. In order to determine if the remaining germline cells were still mitotically active, we incubated ovaries from old del females with the nucleotide analog, BrdU.
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Fig. 3. Loss of germline cells in delHN/delWK ovaries. (A, B) Immunostaining of Vas (blue) and αSpc (red) labeled the germline and fusomes, respectively. Phalloidin (green) was used to highlight the actin cytoskeleton. (A) Wild type ovariole. (B) Ovariole from 23-day old delHN/delWK female. Most ovarioles in old delHN/delWK females are void of germline cells indicative of germline stem cell loss. (C, D) Immunostaining of BrdU incorporation (green) labeled mitotically active cells and DNA was stained with Hoechst dye (red) in wild type (C) and delHN/delWK germaria (D). (E, F) Immunostaining of activated Caspase-3 (green), actin (red) and DNA (blue). (E) wild type germarium without Caspase-3 labeling. (F) Young delHN/delWK females have an elevated number of cysts undergoing apoptosis, which further contributes to the observed reduction in developing egg chambers.
Immunostaining of BrdU in cells adjacent to the terminal filament and later in germarial region 1 was observed indicating that mitosis was ongoing in both GSCs and cystoblasts (Figs. 3C, D and data not shown). We conclude that few GSC are maintained in older del females but that these are capable of giving rise to mitotically active cystoblasts. Most del ovarioles contain few or no egg chambers though cell division appears still to be ongoing. We asked whether the cystoblasts and cystocytes produced in del germaria were lost through increased cell death. Staining ovaries with an activated Caspase-3 antibody revealed that 54% (N = 46) of del germaria in young females contained several germ cells undergoing apoptosis (Figs. 3E, F). By contrast, only 2% (N = 50) of del heterozygous germaria contained cells that stained with the activated Caspase-3 antibody. Therefore, loss of stem cells and increased programmed cell death, both contribute to the net loss of differentiating egg chambers in del mutants. Del is required for AP and DV patterning of oocyte and eggshell Del mutants lay few eggs and the eggshells exhibit aberrations indicative of patterning defects along both the anterior–posterior (A/P) and dorsal–ventral (D/V) axes (Figs. 4A–D). In addition, the eggs are often small and collapsed due
to an open chorion at the anterior end (Fig. 4E). Immediately apparent is a variable ventralization of eggshells indicated by a loss of dorsal appendage material. Most del eggs exhibit ventralization phenotypes ranging from a reduction of, to a total absence of appendages (Figs. 4B–D). Eggshell defects along the A/P axis were apparent as well. A respiratory structure called the aeropyle is malformed in the vast majority of all del eggs (Figs. 4F–Fʺ). These axial patterning phenotypes resemble those of mutants in the gurken-Epidermal growth factor receptor (grk-Egfr) pathway (reviewed in Ray and Schupbach, 1996). Strong mutations in this pathway produce eggshells with a duplication of anterior cell fates at the posterior thus replacing the aeropyle with a duplicated micropyle. While the aeropyle is not formed properly in del mutants, it is never replaced by a fully formed micropyle either. This is suggestive of reduced Grk signaling at the posterior end of the developing oocyte, which, can result in such intermediary morphologies (K.W. and T. S., unpublished observations). The deadlock eggshell phenotypes resemble those of the spindle class of mutations. These mutations also exhibit karyosome defects (Ghabrial et al., 1998; Gonzalez-Reyes et al., 1997) which are also observed in deadlock mutants. In early vitellogenic stages of wild type oocytes, the DNA condenses into a compact sphere called a karyosome visible by staining with Hoechst dye (Fig. 4G). This structure persists throughout most of oogenesis during which time the oocyte is
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Fig. 4. Defects in patterning of the eggshell are observed in delHN/delWK mutants. (A) Wild type eggshell. (B–Fʺ) DelHN/delWK eggshells. (A–E) Anterior pole is at left and dorsal side is facing up. Eggshells are at the same scale. (B–D) Variable degrees of dorsal appendage loss indicative of D–V patterning defect. (E) Egg with open chorion at anterior end. (F) Wild type aeropyle at posterior pole. (F′, F′′) Aberrant aeropyle structures at posterior poles of delHN/delWK eggshells reflect a defect in A–P patterning. (G–I) Hoechst dye was used to stain DNA (white) and immunostaining with Orb antibody identified the oocyte cytoplasm (red). (G) Wild type oocyte nucleus with spherical karyosome. (H, I) Nuclear abnormalities were observed in delHN/delWK oocytes. DNA sometimes appears in blobs in the interior of the nucleus (H) and occasionally, polyploid (I).
arrested in prophase I (Mahowald and Kambysellis, 1980; Spradling, 1993). The karyosome does not form properly in over 50% of del mutant oocytes. Instead, DNA staining reveals threadlike structures or blobs of staining (Fig. 4H). In less than 5% of oocytes, chromosomes exhibit polyploidy indicative of a partial loss of oocyte fate (Fig. 4I). A subset of spindle class genes is required for repair of double-stand breaks (DSBs) during meiosis. Suppression of the karyosome defect in these mutants is achieved through disruptions the DSB checkpoint kinase gene, mei-41 (Ghabrial and Schupbach, 1999). However, we found that mutations in mei-41 do not suppress the karyosome phenotype in del mutants suggesting that it is not required for meiotic DSB repair. To determine if the ventralized eggshell phenotype reflects a defect in Grk signaling, we analyzed Grk protein accumulation and localization in del ovaries. We found that, in early oogenesis, Grk protein is diffuse throughout the oocyte rather than concentrated at the posterior pole (Figs. 5A, C). At mid-
oogenesis Grk protein was localized properly at the anterior between the oocyte nucleus and the cortex though protein levels were dramatically reduced (Fig. 1B). Like Grk, oo18 RNAbinding protein (Orb) is normally concentrated at the posterior pole of the oocyte in early egg chambers (Fig. 5B) (Chang et al., 1999). In del mutants, we instead observed the most intense Orb staining at lateral positions (Fig. 5D). We also analyzed the expression pattern of Oskar (Osk) protein, which normally achieves tight localization at the posterior pole of the oocyte beginning at stage 9, in a microtubule-dependent manner. We observed a mild Osk mislocalization phenotype in all del oocytes. Instead of being tightly concentrated at the cortex, slightly looser sub-cortical staining was seen suggestive of an improperly organized microtubule network (Figs. 5E, F). Alternatively, Osk protein may not be anchored properly at the posterior cortex due to disruptions in the cortical cytoskeleton or associated proteins. Taken together, these results suggest that there is a more general effect of mutations
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Fig. 5. Defect in localization of Grk, Orb and Osk proteins to posterior pole and growth defect as well as karyosome defect in delHN/delWK oocytes. (A–F) DNA was stained with Hoechst (blue) and the actin cytoskeleton was labeled with phalloidin (green). (A–D) and (E–F) Egg chambers are at same scale, respectively. (A, B, E) Wild type egg chambers. (C, D, F) DelHN/delWK egg chambers. (A, C) During early vitellogenic stages, Grk (red) normally concentrates at the posterior end of the oocyte. It remains diffuse throughout the oocyte in delHN/delWK mutants at these early stages. (B, D) Orb (red) concentrates in more lateral positions in delHN/delWK oocytes rather than assuming the normal posterior position during early oogenesis (E, F) Osk (red) achieves a tight cortical localization at posterior pole of stage 9 wild type oocytes. Osk concentrates at the posterior pole of stage 9 delHN/delWK oocytes but subcortical staining is apparent as well.
in del on the localization of patterning determinants throughout oogenesis. Del interacts with the microtubule cytoskeleton We analyzed microtubule organization in del ovaries by introducing a GFP-tubulin (GFP-tub) transgene into mutant flies. This transgene was used previously to describe microtubule organization in wild type ovaries (Grieder et al., 2000). Surprisingly, we observed a very strong genetic interaction between GFP-tub and del. Introduction of a single copy of UASGFP-tub expressed under the control of a nos-GAL4-VP16 transgene exacerbated ovarian phenotypes of del mutants such that young flies contained only rudimentary ovaries with few developing cysts. In the few cystocytes that could be analyzed, we were able to observe a more pronounced accumulation of cortical GFP-Tub and disorganization of interior microtubules in cells in region 1 of del mutant germaria as compared to del heterozygotes (Figs. 6A, B). Therefore, while the GPF modification is well tolerated in wild type cells, microtubule organization appears weakened and more sensitive to this change in del mutants. We therefore decided to stain ovaries with an α-Tubulin (αTub) antibody to avoid phenotypes generated in the microtubule network by the GFP domain. While most mitotic
spindles and microtubule arrays appeared normal at a gross level, some abnormalities were observed. More pronounced cortical αTub staining was observed in the germ cells in early region 1 of germaria similar to that seen with GFP-Tub (Figs. 6C–F). This was not a side effect of cells undergoing apoptosis since these cells did not co-stain for activated Caspase-3. Aberrations in mitotic spindles, most commonly totally collapsed spindles (Figs. 6E, F), but occasionally wavy spindles (Fig. 6G) were also observed. Figs. 6H–Hʺ shows an enlargement of a collapsed spindle. In region 2a of the germarium a microtubule array is established that extends from a microtubule organizing center (MTOC) in the oocyte into nurse cells (Clark et al., 1997; Theurkauf et al., 1993). The MTOC moves to the posterior cortex of the oocyte between stage 2b and 3 as evidenced by high concentration of αTub (Grieder et al., 2000). While the general organization of microtubule arrays appears normal in del mutants, we sometimes failed to recognize a dense posterior concentration suggesting failure in focusing minus ends of the array. We introduced a NOD-lacZ transgene into del homozygotes to identify more clearly where the minus ends of microtubules were concentrated. Addition of a NODlacZ transgene into del mutants, however, exacerbated ovarian phenotypes as was the case for GFP-Tub. Interestingly, a negative genetic interaction between the NOD-lacZ transgene
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Fig. 6. Abnormal mitotic spindles and elevated cortical accumulation of Tubulin in delHN/delWK germaria. (A–G) All germaria are at same scale. (A) DelHN/CyO or delWK/CyO germarium. (C, E) Wild type germaria. (B, D, F, G) DelHN/delWK mutant germaria. (A, B) GFP-tub transgene was expressed in del heterozygous and del mutant ovaries (green). Actin cytoskeleton was stained with phalloidin (red) and Hoechst was used to stain DNA (blue). (C, D) Germaria stained with αTub antibodies (red). There is a more pronounced cortical accumulation of αTub in the mutant. (E–G) Mitotic spindles were labeled with αTub antibodies (red), Asp localization was visualized with Asp antibody (green) and Hoechst was used to stain DNA (blue). (F) Collapsed mitotic spindle (arrow). (G) Del spindles are sometimes long and wavy. (H) A collapsed spindle showing Asp (H) αTub (H′), DNA (H′′).
and mutations in DLis-1 has been observed previously (AS and Beat Suter, personal communication), lending further support for the idea that Del is involved in a microtubule-dependent process. However, introducing one copy of a mutation in the Dynein heavy chain gene (Dhc64c[6-6]), or introducing one copy of a Glued mutation (Glu) in a del mutant background, did not significantly enhance the deadlock phenotype (data not shown). The abnormal spindle gene (Asp) encodes a microtubuleassociated protein that is required to properly organize microtubules during mitosis and meiosis and to ensure proper differentiation of the oocyte. (Casal et al., 1990; do Carmo Avides and Glover, 1999; Riparbelli et al., 2002, 2004; Saunders et al., 1997). Mutations in asp in the female germline give rise to abnormal mitotic spindles, cortical accumulation of αTub and abundant enlarged spectrosome-like structures (Riparbelli et al., 2004). Given the similarity in germline
phenotypes of del and asp mutants, we asked if Asp localization was disrupted in the del ovaries. Immunostaining reveals normal localization of Asp on mitotic spindles in del mutants both at the spindle poles and on the central spindle. We even observed Asp protein in the center of collapsed spindles of del mutants suggesting mitotic defects in del do not arise from a failure to recruit Asp to microtubules (Fig. 6H). Del encodes a novel protein Del had previously been mapped to an 70 kb interval in cytogenetic region 38F-39A containing 15 genes (Ghabrial, 1999). We found that a P-element insertion P[SUPor-P] CG9252KG10262 in one gene in this region failed to complement mutant alleles. The insertion site of this P-element in exon 2 of CG9252 was confirmed by PCR analyses using primers specific to 5′ and 3′termini of the P-element coupled with primers
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specific to 5′ and 3′ insertion flank regions, respectively. Nonsense mutations were identified in delHN and delWK allele. A single base pair change from A to T changes the 45th residue from an arginine to a stop codon in delHN mutants and the 125th residue, a cysteine, is changed to a stop codon in delWK mutants. The finding that two alleles of del have premature termination codons in the Celera predicted gene, CG9252, strongly suggests that del is CG9252. CG9252 encodes a predicted protein of 981 amino acids (a.a.) which is relatively hydrophilic. Database searches reveal no homology with a gene of known biochemical function. However, there are two regions (a.a. 75–225 and a.a. 300–500), which share some homologies with a very diverse set of proteins in different organisms. We performed in situ hybridizations on ovaries and testes using an anti-sense probe for CG9252. We were not able to detect staining above background in early stages of oogenesis or spermatogenesis suggesting that it is expressed at very low levels at these stages. CG9252 mRNA did accumulate to detectable levels in the oocyte cytoplasm after stage 7 of oogenesis (data not shown). To prove that del is CG9252, we introduced an Nterminally HA-tagged CG9252 fusion protein (HA-Del) in combination with the nos-Gal4-VP16 driver. One copy of this transgene restored egg chamber production in del females resulting in ovaries of wild type size. Ovarioles in rescued females contained normal numbers of developing egg chambers that lacked defects in encapsulation and oocyte positioning. Inspection of DNA by staining with Hoechst revealed that karyosome formation was restored as well. This demonstrates that ubiquitous expression of CG9252 in the germline rescues del mutant phenotypes. Despite this rescue, females expressing HA-Del laid eggs that do not develop very far. Most eggs exhibited a mild ventralization with two closely spaced appendages or one single appendage indicating a partial rescue of later phenotypes. When a maternal alphatubulin-Gal4 driver was used, oogenesis was also restored, but the females laid few eggs which did not develop very far (see below). Nos-Gal4-VP16-driven expression of HA-Del was also sufficient to render del males fertile. HA-Del protein is therefore able to fully restore some aspects of Del function in both females and males. Taken together, our molecular analysis of three del alleles, in addition to transgenic rescue experiments, indicate that CG9252 is del. Deadlock is required for the resumption of embryonic mitosis and co-localizes with centrosomes in the embryo Since the HA-del transgene rescued the oogenesis phenotypes of the strong del mutations, we were able to investigate the embryonic phenotype of the arrested eggs. Staining eggs from delHN/delWK females that expressed HA-del under the control of the nos-Gal4-VP16 driver, revealed that the majority of the embryos were arrested in the metaphase of the first mitotic division (Figs. 7A–B). This shows that deadlock is not only required for the division of the germline in the germarial stages, but also for the resumption of mitosis in the early embryo.
Fig. 7. Deadlock is required for embryonic divisions and co-localizes with centrosomes in the early embryo and in structures associated with the oocyte nucleus in oogenesis. (A, B) Embryos derived from delHN/delWK ; HA-del/nosGal4-VP16 oocytes, arrested in metaphase of the first zygotic division. The spindle microtubules are stained with an antibody against αTub (red), DNA in green. (A) The polar body is visible in the upper left, the zygotic spindle on the right. (B) Zygotic spindle at higher magnification. (C–E) Del protein mostly colocalizes with centrosomes in interphase. (C) Centrosomes visualized with antibody against γTub. (D) Del protein. (E) Merge of the two pictures with γTub in red, Del in green. Higher accumulation of Del protein is seen in a pattern that is largely congruent, but somewhat more spread out than the centrosome pattern. (F) Early embryonic division in a wild type embryo. αTub in red, DNA in blue. Del protein (green) accumulates to high levels at the centrosomes during nuclear division. (G) HA-Del protein, visualized by an anti-HA antibody accumulates in dot like structures associated with the oocyte nucleus. DNA (blue) indicates the location of the nurse cells on the left, the follicle cells on the right, and the oocyte nucleus in the center. Del (red) is indicated with an arrow.
We raised an antibody to the Del protein. Unfortunately, none of the standard fixation conditions allowed us to reliably detect the Del protein in the ovary. However, using the antibody on early embryos, we found that the Del protein co-localizes with the centrosomes (Figs. 7C–F). This colocalization was detectable in interphase (Figs. 7C–E) but was most apparent during metaphase of the nuclear divisions (Fig. 7F). We also used an antibody to the HA-tag to assess the localization of the HA-Del protein in the ovary, expressed under the control of maternal alpha-tubulin-Gal4. While it was not possible to draw any firm conclusions regarding protein localization from these experiments, given that the protein is presumably overexpressed at all stages of oogenesis under these conditions, we did observe a “dot-like” accumulation of HADel in association with the oocyte nucleus (Fig. 7G). We observed similar nuclear dot-like staining in oocytes in mid-
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oogenesis using the anti-Del antibody and these dots were not visible in ovaries from del mutant females (data not shown). A co-staining with an anti-Prod antibody, which labels centromeric heterochromatin (Wakefield et al., 2000), did not show colocalization (data not shown). This accumulation in discrete dots in association with the oocyte nucleus is reminiscent of the recently published appearance of the centrioles and microtubule organizing center in the oocyte at this stage (Januschke et al., 2006). Discussion In this study, we characterized the del gene and found that it is required for a number of processes at different stages throughout oogenesis. At the very earliest stage, Del plays a role in maintenance of the germline stem cell population. Next, Del is required during cystocyte divisions for fusome formation and mitotic spindle integrity. Del oocytes also exhibit defects in nuclear morphology in that their DNA fails to condense normally and sometimes becomes partially polyploid. Axial patterning defects are apparent in del mutants throughout oogenesis as is evidenced by mislocalization of cytoplasmic determinants and, finally, oocytes often fail to grow at a normal rate causing eggs to be small or collapsed. In the early embryo, Del is required for the resumption of mitosis. Many of these seemingly pleiotropic effects likely find their cause in a common requirement for Del in microtubule-dependent processes. Interestingly, we observed that Del protein colocalizes with centrosomes in the early embryo which strongly supports a microtubule related function. We were not able to detect the intracellular localization of the Del protein in the germarium using either an antibody against Del or the HA-tagged protein expressed under the control of nos-Gal4-VP16. Since there are clear phenotypic effects on the early stages of oogenesis in the del mutants, which can be rescued by the HA-del transgene, the protein must be expressed at these early stages. Possibly the protein only accumulates to very low levels, or alternatively, the protein may not be stable at these early stages under our ovarian fixation conditions. We did observe an accumulation in one or two dot-like structures associated with the oocyte nucleus at midstages of oogenesis, which are reminiscent of the location of the remnants of centrosomes (Januschke et al., 2006), but the significance of these structures is presently unknown. Interestingly, mutations in several genes encoding microtubule-associated proteins give rise to similar ovarian phenotypes in Drosophila. Orbit/Mast, the Drosophila homolog of the human CLASP proteins, is a member of a family of plus-end microtubule tracking proteins. Analysis of orbit6, an allele that affects only fertility, revealed several roles for Orbit/Mast during oogenesis. Like del mutants, orbit6 flies exhibit GSC loss, abnormal fusome growth and reduced cystocyte divisions. When cystocytes do divide, their mitotic spindles are either diminutive or collapsed. Orbit/Mast plays additional roles during oogenesis since oocyte determination and ring canal formation are disrupted in orbit6 as well (Mathe et al., 2003).
Mutant analysis from other systems showed that Orbit/Mast is required for chromosome congression and spindle stability during mitosis. Specifically, Orbit/Mast is required at kinetochores for microtubule attachment and growth and also for maintenance of spindle bipolarity (Inoue et al., 2000; Lemos et al., 2000; Maiato et al., 2002, 2005). Depletion of Orbit/Mast causes spindles to collapse resulting in monopolar spindles with DNA buried within the aster (Maiato et al., 2002). Del shares only a subset of phenotypes with orbit6, again suggesting that Del may play a more specialized role during oogenesis. Another microtubule-associated protein whose mutant phenotypes closely resemble those of del is Asp. Asp is required during oogenesis for GSC maintenance, fusome biogenesis, cystocyte divisions, oocyte determination and mitotic spindle integrity. Interestingly, peripheral accumulation of microtubules was observed in asp germaria as we observed in del germaria as well (Riparbelli et al., 2004). Mitotic spindles in asp mutants are very often longer and wavier than normal spindles (Riparbelli et al., 2004). This is also seen in del though the predominant spindle phenotype we observed in del germaria was a total spindle collapse. Asp localizes primarily to microtubules at spindle poles but is also found at minus-end of microtubules on the central spindle during telophase (Riparbelli et al., 2002; Saunders et al., 1997). At the spindle poles, it is thought to maintain the integrity of microtubule organizing centers and to bundle microtubules. Similarly, it is thought to stabilize the central spindle by cross-linking microtubule minus-ends during telophase (Riparbelli et al., 2002; Wakefield et al., 2001). The central spindle is thought to play an important role in directing furrow ingression during cytokinesis (Gatti et al., 2000). Upon cytokinetic arrest during cystocyte divisions, fusomal material aggregates in ring canals and moves along remnants of the central spindle to join the fusome of the progenitor cell. It is possible that central spindle defects in del and asp mutants disrupt fusome coalescence or that aggregation of new fusomal material is prevented by failures in cytokinesis. Given that Del co-localizes with centrosomes, it is possible that Del cooperates directly with Asp, although we found that Asp protein can still localize to microtubules in del mutants. Fusomes in asp, orbit/mast and del mutants, take on very similar shapes. Instead of becoming highly branched, fusomes exhibit blobby, less branched appearances. Spherical fusomes that accumulate in these mutants appear enlarged compared to spectrosomes indicating that aggregation of new fusomal material is occurring but that branch morphogenesis is disrupted. Fusomes also often appear thin or fragmented arising either from premature fusome dissociation or inhibition of fusome coalescence. It is not known what drives fusome morphogenesis, but the established functions of genes required for this process show that microtubules and associated factors are involved. Given the similarity in phenotypes, it appears that Del functions in this microtubule-dependent process. If it is also associated with centrosomes during these early germline divisions, it may stabilize an interaction between centrosomes and fusome-microtubules. Interestingly in the early embryo, Del is required for the resumption of mitosis, and blocks in mitosis
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have also been reported for the centrosome fall off (cfo) mutation in the embryo, where centrosomes are detached from the spindles (Wakefield et al., 2000). As expected for general regulators of microtubule dynamics, asp and orbit/mast genes are required for viability of the fly in addition to their specialized roles during oogenesis. Interestingly, del is required only for gametogenesis in both males and females. This either points toward redundancy in its function in somatic tissues or it indicates that it performs a highly specialized function in the germline. Acknowledgments We thank Allan Spradling, Tom Hays and Ira Clark for fly strains and Paul Schedl, Paul MacDonald, Paul Lasko, Dennis McKearin, Tibor Torok and David Glover for antibodies. We are grateful to Gail Barcelo for help with sequencing and in situ hybridizations, and to Joe Goodhouse for assistance with confocal microscopy. Thanks to members of the Wieschaus and Schüpbach labs for helpful suggestions, in particular Sharon Chen for stimulating discussions and critical reading of the manuscript. This work was supported by the Howard Hughes Medical Institute and the Public Health Service Grant PO1 CA41086. References Casal, J., Gonzalez, C., Wandosell, F., Avila, J., Ripoll, P., 1990. Abnormal meiotic spindles cause a cascade of defects during spermatogenesis in asp males of Drosophila. Development 108, 251–260. Chang, J.S., Tan, L., Schedl, P., 1999. The Drosophila CPEB homolog, orb, is required for oskar protein expression in oocytes. Dev. Biol. 215, 91–106. Clark, I.E., Jan, L.Y., Jan, Y.N., 1997. Reciprocal localization of Nod and kinesin fusion proteins indicates microtubule polarity in the Drosophila oocyte, epithelium, neuron and muscle. Development 124, 461–470. Cox, D.N., Chao, A., Lin, H., 2000. Piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells. Development 127, 503–514. de Cuevas, M., Spradling, A.C., 1998. Morphogenesis of the Drosophila fusome and its implications for oocyte specification. Development 125, 2781–2789. de Cuevas, M., Lee, J.K., Spradling, A.C., 1996. Alpha-spectrin is required for germline cell division and differentiation in the Drosophila ovary. Development 122, 3959–3968. Deng, W., Lin, H., 1997. Spectrosomes and fusomes anchor mitotic spindles during asymmetric germ cell divisions and facilitate the formation of a polarized microtubule array for oocyte specification in Drosophila. Dev. Biol. 189, 79–94. do Carmo Avides, M., Glover, D.M., 1999. Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal microtubule organizing centers. Science 283, 1733–1735. Fuller, M.T., 1993. Spermatogenesis. In: Martinez-Arias, A. (Ed.), The Development of Drosophila melanogaster, vol. I. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 71–148. Gatti, M., Giansanti, M.G., Bonaccorsi, S., 2000. Relationships between the central spindle and the contractile ring during cytokinesis in animal cells. Microsc. Res. Tech. 49, 202–208. Ghabrial, A. (1999). okra and spindle-B encode DNA repair proteins and affect meiosis and pattern formation during Drosophila oogenesis. PhD thesis, Princeton University, Princeton, NJ. Ghabrial, A., Schupbach, T., 1999. Activation of a meiotic checkpoint regulates translation of Gurken during Drosophila oogenesis. Nat. Cell Biol. 1, 354–357.
Ghabrial, A., Ray, R.P., Schupbach, T., 1998. okra and spindle-B encode components of the RAD52 DNA repair pathway and affect meiosis and patterning in Drosophila oogenesis. Genes Dev. 12, 2711–2723. Gilboa, L., Lehmann, R., 2004. How different is Venus from Mars? The genetics of germ-line stem cells in Drosophila females and males. Development 131, 4895–4905. Gonzalez-Reyes, A., Elliott, H., St Johnston, D., 1997. Oocyte determination and the origin of polarity in Drosophila: the role of the spindle genes. Development 124, 4927–4937. Grieder, N.C., de Cuevas, M., Spradling, A.C., 2000. The fusome organizes the microtubule network during oocyte differentiation in Drosophila. Development 127, 4253–4264. Hacker, U., Perrimon, N., 1998. DRhoGEF2 encodes a member of the Dbl family of oncogenes and controls cell shape changes during gastrulation in Drosophila. Genes Dev. 12, 274–284. Hay, B., Jan, L.Y., Jan, Y.N., 1988. A protein component of Drosophila polar granules is encoded by vasa and has extensive sequence similarity to ATPdependent helicases. Cell 55, 577–587. Huynh, J.R., St Johnston, D., 2004. The origin of asymmetry: early polarisation of the Drosophila germline cyst and oocyte. Curr. Biol. 14, R438–R449. Inoue, Y.H., do Carmo Avides, M., Shiraki, M., Deak, P., Yamaguchi, M., Nishimoto, Y., Matsukage, A., Glover, D.M., 2000. Orbit, a novel microtubule-associated protein essential for mitosis in Drosophila melanogaster. J. Cell Biol. 149, 153–166. Januschke, J., Gervais, L., Gillet, L., Keryer, G., Bornens, M., Guichet, A., 2006. The centrosome-nucleus complex and microtubule organization in the Drosophila oocyte. Development 133, 129–139. King, F.J., Lin, H., 1999. Somatic signaling mediated by fs(1)Yb is essential for germline stem cell maintenance during Drosophila oogenesis. Development 126, 1833–1844. Lasko, P.F., Ashburner, M., 1988. The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor-4A. Nature 335, 611–617. Lemos, C.L., Sampaio, P., Maiato, H., Costa, M., Omel'yanchuk, L.V., Liberal, V., Sunkel, C.E., 2000. Mast, a conserved microtubule-associated protein required for bipolar mitotic spindle organization. EMBO J. 19, 3668–3682. Lin, H., Yue, L., Spradling, A.C., 1994. The Drosophila fusome, a germlinespecific organelle, contains membrane skeletal proteins and functions in cyst formation. Development 120, 947–956. Liu, Z., Xie, T., Steward, R., 1999. Lis1, the Drosophila homolog of a human lissencephaly disease gene, is required for germline cell division and oocyte differentiation. Development 126, 4477–4488. Mahowald, A.P., Kambysellis, M.P., 1980. Oogenesis. In: Ashburner, M., Wright, T.R.F. (Eds.), The Genetics and Biology of Drosophila, vol. 2. Academic Press, London, pp. 141–224. Maiato, H., Sampaio, P., Lemos, C.L., Findlay, J., Carmena, M., Earnshaw, W. C., Sunkel, C.E., 2002. MAST/Orbit has a role in microtubule-kinetochore attachment and is essential for chromosome alignment and maintenance of spindle bipolarity. J. Cell Biol. 157, 749–760. Maiato, H., Khodjakov, A., Rieder, C.L., 2005. Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres. Nat. Cell Biol. 7, 42–47. Mathe, E., Inoue, Y.H., Palframan, W., Brown, G., Glover, D.M., 2003. Orbit/ Mast, the CLASP orthologue of Drosophila, is required for asymmetric stem cell and cystocyte divisions and development of the polarised microtubule network that interconnects oocyte and nurse cells during oogenesis. Development 130, 901–915. McGrail, M., Hays, T.S., 1997. The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila. Development 124, 2409–2419. McKearin, D., Ohlstein, B., 1995. A role for the Drosophila bag-of-marbles protein in the differentiation of cystoblasts from germline stem cells. Development 121, 2937–2947. Queenan, A.M., Ghabrial, A., Schupbach, T., 1997. Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase dorsalizes both the eggshell and the embryo. Development 124, 3871–3880. Queenan, A.M., Barcelo, G., VanBuskirk, C., Schüpbach, T., 1999. The transmembrane region of Gurken is necessary for transport to the oocyte membrane in Drosophila. Mech. Dev. 89, 35–42.
K. Wehr et al. / Developmental Biology 294 (2006) 406–417 Ray, R.P., Schupbach, T., 1996. Intercellular signaling and the polarization of body axes during Drosophila oogenesis. Genes Dev. 10, 1711–1723. Riparbelli, M.G., Callaini, G., Glover, D.M., Avides Mdo, C., 2002. A requirement for the abnormal spindle protein to organise microtubules of the central spindle for cytokinesis in Drosophila. J. Cell Sci. 115, 913–922. Riparbelli, M.G., Massarelli, C., Robbins, L.G., Callaini, G., 2004. The abnormal spindle protein is required for germ cell mitosis and oocyte differentiation during Drosophila oogenesis. Exp. Cell Res. 298, 96–106. Rorth, P., 1998. Gal4 in the Drosophila female germline. Mech. Dev. 78, 113–118. Saunders, R.D., Avides, M.C., Howard, T., Gonzalez, C., Glover, D.M., 1997. The Drosophila gene abnormal spindle encodes a novel microtubuleassociated protein that associates with the polar regions of the mitotic spindle. J. Cell Biol. 137, 881–890. Schupbach, T., Wieschaus, E., 1991. Female sterile mutations on the second chromosome of Drosophila melanogaster: II. Mutations blocking oogenesis or altering egg morphology. Genetics 129, 1119–1136. Snapp, E.L., Iida, T., Frescas, D., Lippincott-Schwartz, J., Lilly, M.A., 2004. The fusome mediates intercellular endoplasmic reticulum connectivity in Drosophila ovarian cysts. Mol. Biol. Cell 15, 4512–4521. Song, X., Zhu, C.H., Doan, C., Xie, T., 2002. Germline stem cells anchored by adherens junctions in the Drosophila ovary niches. Science 296, 1855–1857. Spradling, A.C., 1993. Developmental genetics of oogenesis. In: MartinezArias, M.B.A.A. (Ed.), The Development and Genetics of Drosophila. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 1–70. Spradling, A., Drummond-Barbosa, D., Kai, T., 2001. Stem cells find their niche. Nature 414, 98–104. Swan, A., Nguyen, T., Suter, B., 1999. Drosophila Lissencephaly-1 functions with Bic-D and dynein in oocyte determination and nuclear positioning. Nat. Cell Biol. 1, 444–449. Swan, A., Barcelo, G., Schüpbach, T., 2005. Drosophila Cks30A interacts with Cdk1 to target Cyclin A for destruction in the female germline. Development 132, 3669–3678.
Szakmary, A., Cox, D.N., Wang, Z., Lin, H., 2005. Regulatory relationship among piwi, pumilio, and bag-of-marbles in Drosophila germline stem cell self-renewal and differentiation. Curr. Biol. 15, 171–178. Tautz, D., Pfeifle, C., 1989. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81–85. Theurkauf, W.E., Alberts, B.M., Jan, Y.N., Jongens, T.A., 1993. A central role for microtubules in the differentiation of Drosophila oocytes. Development 118, 1169–1180. Van Doren, M., Williamson, A.L., Lehmann, R., 1998. Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr. Biol. 8, 243–246. Wakefield, J.G., Huang, J., Raff, J.W., 2000. Centrosomes have a role in regulating the destruction of cyclin B in early Drosophila embryos. Curr. Biol. 10, 1367–1370. Wakefield, J.G., Bonaccorsi, S., Gatti, M., 2001. The drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis. J. Cell Biol. 153, 637–648. Wang, Z., Lin, H., 2004. Nanos maintains germline stem cell self-renewal by preventing differentiation. Science 303, 2016–2019. White-Cooper, H., Schafer, M.A., Alphey, L.S., Fuller, M.T., 1998. Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila. Development 125, 125–134. Xie, T., Spradling, A.C., 1998. Decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary. Cell 94, 251–260. Xie, T., Spradling, A.C., 2000. A niche maintaining germ line stem cells in the Drosophila ovary. Science 290, 328–330. Yue, L., Spradling, A.C., 1992. Hu-li tai shao, a gene required for ring canal formation during Drosophila oogenesis, encodes a homolog of adducin. Genes Dev. 6, 2443–2454.