Abstract
A genetic screen to identify mutations in genes in the 45A region on the right arm of chromosome 2 that are involved in oogenesis in Drosophila was undertaken. Several lethal but no female sterile mutations in the region had previously been identified in screens for P-element insertion or utilizing X rays or EMS as a mutagen. Here we report the identification of EMS-induced mutations in 21 essential loci in the 45D–45F region, including 13 previously unidentified loci. In addition, we isolated three mutant alleles of a newly identified locus required for fertility, sine prole. Mutations in sine prole disrupt spermatogenesis at or before individualization of spermatozoa and cause multiple defects in oogenesis, including inappropriate division of the germline cyst and arrest of oogenesis at stage 4.
THE study of oogenesis in Drosophila melanogaster has contributed to a general understanding of developmental biology in part because many different developmental processes are required for oogenesis, including localization of determinants, cell-cell signaling, cell migration, and cell cycle regulation. Oogenesis occurs in an organized, stepwise manner, beginning with mitotic division to form a germline cyst of 16 interconnected cells. One cell of the germline cyst becomes the oocyte, and the remaining 15 cells, the nurse cells, undergo several rounds of DNA synthesis without intervening mitoses. The polyploid nurse cells provide RNAs and proteins for the developing oocyte. The germline cyst becomes surrounded by a layer of somatically derived follicle cells, forming an egg chamber. During oogenesis, the oocyte becomes larger, and determinants required for establishment of the axes of the embryo are localized within the oocyte. Oogenesis culminates in the formation of a properly organized oocyte, surrounded by a vitelline membrane and an eggshell (reviewed in Spradling 1993).
The isolation of mutations that disrupt oogenesis has been critical for the study of oogenesis (reviewed in Spradling 1993; Ray and Schüpbach 1996). We focused on polytene region 45A on the right arm of chromosome 2 because the physical map of the region was well defined at the time we initiated our study by the Berkeley Drosophila Genome Project (BDGP) and other work (Mohr and Boswell 1999). Several inviable mutations in the 45A region were previously isolated in screens for P-element insertion and by EMS and X-ray mutagenesis (BDGP; Konevet al. 1994; Dockendorffet al. 2000). Female sterile mutations in this region had not been previously reported, although some female sterile mutations map near the region genetically (Schüpbach and Wieschaus 1989, 1991).
Here we describe a screen for EMS-induced lethal and female sterile mutations in the 45A region. Several lethal complementation groups were identified, including 13 previously unidentified lethal loci. In addition, a locus required for male and female fertility, sine prole (Latin for “without offspring”), was isolated and characterized. Mutations in sine prole cause defects in gametogenesis consistent with disruption of germline division control and progression through gametogenesis.
MATERIALS AND METHODS
Screen for inviable and female sterile mutations: cn bw sp males were fed 25 mm ethyl methanesulfonate (EMS) according to the method of Lewis and Bacher (1968) and crossed to nub b nocSco cn bw sp/SM6a, al2 Cy dplvI cn2P sp2 virgin females. Next, individual males with a mutagenized chromosome (* cn bw sp) over SM6a, al2 Cy dplvI cn2P sp2 were crossed to Df(2R)Np3, bw/SM6a, al2 Cy dplvI cn2P sp2 virgin females. For inviable mutations, the F1 progeny of that cross were scored for the absence of brown-eyed flies. For female sterile mutations, F1 brown-eyed females were crossed to wild-type males and scored for fertility. Mutations were recovered by crossing F1 sibling * cn bw sp/SM6a, al2 Cy dplvI cn2P sp2 males to an appropriate strain. Lethal and female sterile mutations were maintained over SM6a, al2 Cy dplvI cn2P sp2.
Complementation analysis involved reciprocal crosses of pairwise mutant combinations. Crosses were performed at 25° and 200–500 progeny were scored for each cross. To identify classes that might develop more slowly, progeny were scored throughout the eclosion period.
Whole mount preparation and staining of testes and ovaries: Testes were dissected in PBSTX (PBS, 0.1% Triton X-100), fixed in 4% formaldehyde (Polyscience, Niles, IL) PBSTX for 10 min, washed in PBSTX, and stained in 0.1 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI) in PBSTX for 10 min, followed by extensive washing in PBSTX. Ovaries were treated identically except that they were fixed for 15–20 min. Testes or ovaries were resuspended in PBS, 70% glycerol plus one drop per 0.5 ml Slowfade Light Component A (Molecular Probes, Sunnyvale, CA) and examined under epifluorescence illumination with a Zeiss Axiophot.
Polytene region 44D1–45F8. Proximal is to the left, distal is to the right. (Top) Drawing of polytene chromosome region 44D1–45F8 (after Bridges 1935). (Bottom) The deletion used in the screen (Df(2R)Np3), deletions used for mapping, and the intervals defined by these deletions. Breakpoint uncertainties are represented as dashes; deleted regions are indicated with solid lines.
For detection of ring canals, ovaries were dissected and fixed as described above, washed in PBSTX, blocked in PBSTX, 1% bovine serum albumin (BSA), and incubated with 1:100 diluted α-phosphotyrosine (Pierce, Rockford, IL) in PBSTX, 1% BSA overnight at 4°. Secondary antibodies conjugated to Alexa 488 (Molecular Probes) were used at a dilution of 1:250. Then ovaries were stained with DAPI, mounted, and examined as described above.
The distribution of Encore protein was examined using α-Encore antisera, kindly provided by T. Schüpbach (Princeton University). Ovaries were prepared as described above and the antisera used at 1:1000.
Whole mount in situ detection of oskar mRNA: Antisense digoxigenin-labeled RNA probes were synthesized as described by the supplier (Roche Molecular Biochemical); ovaries were fixed and stained as described by Klingler and Gergen (1993).
RESULTS
Isolation of lethal mutations in polytene region 45A: To identify genes in the 45A region that are essential for viability, we screened for EMS-induced mutations that cause lethality in trans to Df(2R)Np3 [44D2–E1; 45B8–C1]. Confirmation that the screen was correctly targeting the 44–45 region came from the observation that hemizygous flies with a lightoid phenotype were recovered at a frequency of ~1 in 1000 (lightoid was previously mapped to 44E1–46E9; Lindsley and Zimm 1992). About 4000 chromosomes were screened for lethality and 180 mutations were isolated. The mutations were mapped to intervals along the chromosome by complementation analysis with deletions that overlap with Df(2R)Np3: Df(2R)44CE [44C1–2; 44E1–4], Df(2R)G75 [44F4–5; 44F11], Df(2R) H3E1 [44D1–4; 44F12], Df(2R)Np5 [44F10–11; 45D9–E1], Df(2R)w45-30n [45A6–7; 45E2–3], and Df(2R)w73-2 [45A9–11; 45D5–8] (Figure 1; Konev et al. 1991a,b, 1994; Zhanget al. 1996). Using these deletions, the mutations were mapped to seven intervals along the polytene chromosome map (Figure 1; Table 1).
The mutations were then crossed to one another to determine the number of complementation groups represented. Next, representatives of each complementation group were tested for their ability to complement previously isolated lethal mutations (Table 1). Four P-element insertion mutations were tested: l(2)k16529, l(2)k00116, tsunagi1 [EP(2)0567], and zimp03697 (Rorthet al. 1998; Mohr and Boswell 1999; Spradlinget al. 1999; Mohret al. 2001); all of the available EMS and X-ray-induced mutations in the region were also tested (Bridgeset al. 1936; Li 1936; Nusslein-Volhardet al. 1984; Hooper and Scott 1989; Konevet al. 1994; Bokor and DiNardo 1996; Faulkneret al. 1998; Brummelet al. 1999; Dockendorffet al. 2000). New alleles of l(2)44DEa, lines, filzig, tsunagi, l(2)45Ab, Notoplural, l(2)45Ah, and l(2)45Aj were identified; 13 previously unidentified loci were also identified (Table 1).
We categorize 27 mutations identified in the screen as new alleles of the Notoplural locus (Table 1; Bridgeset al. 1936; Li 1936). The complex pattern of complementation among these alleles, shown in Table 2, suggests that more than one gene is affected in some of the mutant strains that we isolated. Furthermore, the observation that some of these mutations complement Df(2R)w45-30n [45A6–7; 45E2–3] and some do not, placing them in intervals V and VI, suggests two predictions: first, that the Notoplural locus is close to the deletion breakpoint of Df(2R)w45-30n, and second, that a subset of the mutant strains that we place in the Notoplural complementation group affects genes on both sides of the deletion breakpoint. Testing of these predictions and further investigation of this complex group await the availability of molecular and genetic tests for specific disruption of the Notoplural transcript(s).
Lethal mutations identified in the screen with Df(2R)Np3
Characterization of the lethal period: To determine if the mutations cause lethality during embryogenesis or later in development, heterozygous males carrying mutations that map to intervals V or VI [with the exception of l(2)45Aj3 and the Np group mutations] were crossed to Df(2R)Np5/SM6a virgin females, and the resulting embryos were collected and scored for hatching. Of the embryos, 25% should be hemizygous for the mutation, and 75% should be heterozygous or homozygous balancer genotypes that hatch as larvae.
Animals homozygous for most mutations that were tested had a frequency of hatching comparable to wild-type and normal cuticles. Thus, the lethal period of these mutations is during larval or pupal stages (n > 600 for each). This is in agreement with the results of Dockendorff et al. (2000), which indicate that the earliest lethality detected in l(2)45Ah is during larval stages. However, one mutation tested resulted in 24% unhatched embryos (n = 711), suggesting that the lethal period for this mutation is during embryogenesis. We have named the locus criss-cross (cri) and named the mutant allele of the locus that was identified cri1 (Table 1). Examination of cuticles from unhatched cri1/Df(2R)Np5 embryos revealed patterning defects in ~65% of these embryos (n = 27; Figure 2). Thoracic segments and abdominal segments 1–3 are unaffected in cri mutant embryos, but embryos have variable defects in head formation, and fusions of more posterior abdominal segments are commonly observed (Figure 2).
Complementation within the Notoplural group
Isolation of female sterile mutations in polytene region 45A: To identify genes in 45A that are required for female fertility, we screened for EMS-induced mutations that cause female sterility in trans to Df(2R)Np3. For the purposes of this screen, female sterile mutations were defined as mutations that cause hemizygous females to lay no eggs, lay eggs with defective eggshells, or lay eggs that fail to hatch.
Phenotype of criss-cross mutant embryos. (A) Wild type. (B) Hemizygous cri1 mutant embryo. Head defects (double-headed arrow) and fusion of abdominal segments (arrow) are indicated. Fusions of abdominal segments 1 and 2 were not observed.
About 800 chromosomes were screened and 11 recessive female sterile mutations were obtained. Eight of these reduce but do not eliminate hatching and have variable effects on eggshell pattern and thus were difficult to characterize further. However, 3 of the mutations isolated in the screen (sip1, sip2, and sip3; see below) cause hemizygous females to lay no eggs and thus were suitable for further genetic and cytological analysis. The hemizygous sip1, sip2, and sip3 males are also sterile. These mutations map to 45A4–6; 45A9–11 (interval VI) on the basis of complementation with deletions (Figure 1; Table 3). Each of the mutations fails to complement one another for sterility; therefore, they define a single locus (Table 3). We named the locus sine prole (sip). The phenotypes of all trans-heterozygous and hemizygous combinations of the sip mutations isolated are indistinguishable cytologically, and thus the phenotype is referred to in general as the sip mutant phenotype.
Characterization of the sip mutant phenotype: To characterize the defects in spermatogenesis in sip mutant males, testes were fixed and stained with DAPI to visualize DNA. Testes from sip mutant males are equivalent in size to wild type and are cytologically indistinguishable from wild type through the spermatid cyst stage. However, individualized spermatozoa are not detectable in the testes or in the seminal vesicle in sip mutant testes; instead, the spermatids appear to degenerate (data not shown).
Genetic analysis of the sine prole locus
Oogenesis in wild-type and sine prole mutant ovaries. Anterior is to the left and posterior is to the right in A–D′. (A and B) Ovarioles stained with DAPI to visualize DNA. g, germarium; 1, stage 1 egg chamber. (A) Wild-type ovariole. (B) sine prole mutant ovariole. (C) Egg chamber from a sip mutant ovariole stained with DAPI. Arrows indicate the extra layer of follicle cells detected at the posterior of the egg chamber. (D) The same sip mutant egg chamber as in C stained with antiphosphotyrosine to detect ring canals. Note that the oocyte (box) is connected to the surrounding nurse cells by five ring canals, one more than the normal number (enlarged view of oocyte shown in D′).
To characterize the defects in oogenesis in sip mutant females, we examined egg chamber formation, oocyte determination, and RNA and protein import into the oocyte in wild-type and sip mutant ovaries. The number of egg chambers in wild-type and sip mutant ovaries is approximately equivalent; however, ovaries from sip mutant females contain only early stage egg chambers (Figure 3). Egg chambers arrest during previtellogenic stages and with condensed nurse cell chromatin typical of stage 4. Older egg chambers (more posterior in the ovariole) appear larger, as they have more than one layer of follicle cells surrounding part of or the entire egg chamber (Figure 3, B and C).
About 25% of sip mutant egg chambers have more than the normal number of nurse cells. At least two types of events could lead to an abnormal number of nurse cells. First, more than one 15-cell germline cyst could be enclosed in a single egg chamber. Second, the germline could undergo an extra round of division to form an egg chamber with two times the normal number of cells. In the former case, two oocytes would be present, and oocytes would be connected to nurse cells with the normal number of ring canals (i.e., four). In the latter case, 31 nurse cells and a single oocyte would be detected, and the oocyte would be connected to nurse cells with an extra ring canal (i.e., five), indicative of an extra round of division. In sip mutant egg chambers with 31 nurse cells, only one oocyte is detected, and the oocyte is connected to nurse cells by five ring canals (as determined by examining the oocyte ring canals in 23 egg chambers with extra nurse cells from 23 pairs of ovaries), indicating that an extra round of germline division has occurred (Figure 3, D and D′). The observation that an inappropriate division of the germline cyst has occurred in some sip mutant egg chambers and the presence of an extra layer of follicle cells could suggest that sip is involved in cell cycle regulation. However, this involvement is not at the level of control of G1 or mitotic cyclins, as the distribution of cyclins E and B in the germline and follicle cells is unaffected by mutations in sip (data not shown).
To further investigate the germline division defect, we examined the distribution of Encore protein in sip mutant ovaries. The encore gene encodes a protein required for germline division control. Mutations in encore cause an extra round of division of the germline (Hawkinset al. 1996; Van Buskirket al. 2000). In wild-type ovaries, Encore protein is concentrated in the oocyte cytoplasm in early oogenesis (Figure 4A; Van Buskirket al. 2000). In sip mutant ovaries, however, Encore protein is not localized to the oocyte (Figure 4B). Mutations in sip do not cause a general defect in RNA or protein accumulation in the oocyte or in oocyte differentiation, because oskar mRNA and Bicaudal-D protein, which are concentrated in the oocyte in early oogenesis (Ephrussiet al. 1991; Kim-Haet al. 1991; Suter and Steward 1991), are distributed normally in sip mutant egg chambers (Figure 4, C, D, and D′, and data not shown).
The distribution of Encore protein and osk mRNA in wild-type and sip mutant ovaries. (A and B) Egg chambers stained with DAPI (blue) and anti-Encore (red); arrows indicate the position of the oocyte. (A) In wild type, Encore accumulates in the oocyte. (B) In sip mutant egg chambers, Encore is not concentrated in the oocyte. (C) osk mRNA accumulates in the oocyte cytoplasm in wild-type egg chambers. (D) osk mRNA accumulates normally in the oocyte cytoplasm in sip mutant egg chambers; the oocyte nucleus is visible at the posterior of the oocyte (arrow; enlarged view of oocyte shown in D′).
DISCUSSION
In a screen for inviable and female sterile mutations that fail to complement Df(2R)Np3 [44D2–E1; 45B8–C1], we identified new alleles of l(2)44DEa, lines, filzig, l(2)45Ab, tsunagi, Notoplural, l(2)45Ah, and l(2)45Aj. In addition, mutations in 13 previously unidentified lethal loci were isolated. This suggests that the 44D–45A cytogenetic region contains many essential genes, making it of particular interest for study. The largest number of mutations that were identified correspond to the Notoplural locus and have a complex pattern of complementation (Table 2). The Notoplural locus was originally reported in the study of a deletion (Li 1936) and the isolation of recessive lethal, EMS-induced alleles has been reported previously (Dockendorffet al. 2000). A full understanding of the complexity of the Np locus awaits molecular characterization of the Np gene(s). We were particularly interested in identifying genes in 44F–45B that are involved in oogenesis. An estimated 75% of the genes that can be mutated to lethality are also required during oogenesis (Perrimonet al. 1984). Thus, we expect that some of the lethal mutations isolated in the screen will be useful for the study of oogenesis; indeed, this has been shown for tsunagi (Mohret al. 2001). A locus required for fertility but not viability, sip, was also identified in this screen.
Three mutant alleles that define the sip locus were recovered in the screen on the basis of female sterility in trans to Df(2R)Np3. These mutations have indistinguishable effects on gametogenesis. In males, sip mutations disrupt spermatogenesis at or before individualization of spermatozoa. Failure to individualize spermatozoa is a common “editing function” to prevent abnormal spermatids from becoming mature sperm (reviewed in Fuller 1993). Thus, failure to produce viable, individualized sperm in sip mutant testes is likely to reflect an earlier defect not visible at the light microscopic level.
The defects in sip mutant ovaries include arrest in early oogenesis. This defect is reminiscent of defects caused by dominant mutations in ovo and strong loss-of-function alleles of fs(2)cup (Oliveret al. 1987; Keyes and Spradling 1997). In ovoD or fs(2)cup mutant females, egg chambers fail to progress beyond stage 4 and instead arrest before the onset of vitellogenesis and with condensed nurse cell chromatin, similar to what we have observed for sip (Figure 3B). The ovo gene, which encodes a family of transcription factors, and fs(2)cup, which encodes a novel protein, along with stand still and several members of the sex determination pathway, have been implicated in control of expression of ovarian tumor (otu), which is required for proper cyst formation, nurse cell chromatin structure, and progression through oogenesis (Pauliet al. 1993; Keyes and Spradling 1997; Oliver and Pauli 1998; Sahut-Barnola and Pauli 1999; Andrewset al. 2000). The biochemical role(s) of Otu proteins is not revealed by their primary amino acid sequences (Rodeschet al. 1995; Sasset al. 1995).
In addition to the failure of sip mutant egg chambers to mature, ~25% of egg chambers show a defect in germline division control. This phenotype is shared by mutations in encore, in which egg chambers with 31 nurse cells and a single oocyte are detected in ovaries from flies raised at the restrictive temperature for this defect (Hawkinset al. 1996; Van Buskirket al. 2000). The observation that Encore is not localized to the oocyte in sip mutant egg chambers is consistent with a requirement for Encore in germline division control. Furthermore, these data suggest that the failure to accumulate Encore in the oocyte in sip mutant egg chambers is sufficient to explain the germline division defect in sip. However, encore mutations do not affect egg chamber maturation or nurse cell chromatin decondensation; therefore, the absence of detectable Encore in sip mutant oocytes is not sufficient to explain all of the sip mutant defects. Thus, we propose that sip is required for both encore-dependent germline division control and progression through oogenesis.
All mutant stocks generated in this study are available from the authors upon request; representative alleles of all newly isolated lethal and sterile mutations will be made available through the Bloomington Drosophila Stock Center at Indiana University.
Acknowledgments
We thank Drs. Esther Van de Vosse and Tin Tin Su for helpful discussions and Drs. Helena Richardson, Trudi Schüpbach, Ruth Steward, and Tin Tin Su for reagents used in this study. Several fly strains used in this study were kindly provided by the National Drosophila Stock Center, the European Drosophila Stock Center, and the Berkeley Drosophila Genome Project. This work was supported by the National Institutes of Health (NIH) training grant 5T32-GM 07135 to S. E. Mohr and a grant from NIH (GM-57989) to R.E.B.
Footnotes
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Communicating editor: T. C. Kaufman
- Received October 4, 2001.
- Accepted January 21, 2002.
- Copyright © 2002 by the Genetics Society of America
