Characterization of defects in adult germline development and oogenesis of sterile and rescued...

JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 288:205–218 (2000)

© 2000 WILEY-LISS, INC.

Characterization of Defects in Adult GermlineDevelopment and Oogenesis of Sterile and RescuedFemale Hybrids in Crosses Between Drosophilasimulans and Drosophila melanogaster

HOPE HOLLOCHER,1* KRISTIN AGOPIAN,2 JULIE WATERBURY,2

RACHEL W. O’NEILL,1 AND ANDREW W. DAVIS3

1Department of Ecology and Evolutionary Biology, Princeton University,Princeton, New Jersey 08544

2Department of Molecular Biology, Princeton University, Princeton, NewJersey 08544

3Department of Biological Sciences, Webster University, St. Louis,Missouri 63119

ABSTRACT Crosses between Drosophila melanogaster and D. simulans normally result in prog-eny that are either inviable or sterile. Recent discovery of strains that rescue these inviability andsterility phenotypes has made it possible to study the developmental basis of reproductive isolationbetween these two species in greater detail. By producing both rescued and unrescued hybrids andexamining the protein product staining patterns of genes known to be involved in early germlinedevelopment and gametogenesis, we have found that in crosses between D. simulans and D.melanogaster, hybrid female sterility results from the improper control of primordial germline pro-liferation, germline stem cell maintenance, and cystoblast formation and differentiation duringearly oogenesis. Rescued hybrid females are fertile, yet they generally have lower amounts of adultgermline from the outset and show a premature degeneration of adult germline cells with age. Inaddition, older rescued hybrid females also exhibit mutant egg phenotypes associated with defectsin dorso-ventral patterning which may result from the improper partitioning of cytoplasmic factorsduring early oogenesis that could stem from the early defect. Although a variety of germline andoogenic defects are described for the hybrid females, all of them can potentially result from thesame underlying primary defect. Hybrid males from these same crosses, on the other hand, haveno detectable germline in adult reproductive tissues, even when hybrid sterility rescue strains areused, indicating that male sterility and female sterility stem from distinctly different developmen-tal defects. J. Exp. Zool. (Mol. Dev. Evol.) 288:205–218, 2000. © 2000 Wiley-Liss, Inc.

A remarkable and often repeated pattern whencrossing different species is the tendency for in-terspecific hybrids to be completely sterile. Inter-specific sterility very often comes about throughthe degeneration of the gonads (Kerkis, ’33;Dobzhansky, ’36; Coyne, ’92). The profound dis-turbances found in the gonads of hybrids contrastssharply with the normality of the soma. Althoughdegeneration of the gonads in interspecifc hybridshas long been well known, the molecular and de-velopmental genetic causes of this phenomenonare not well understood.

Over the last decade, much progress has beenmade in understanding the evolution of hybrid ste-rility in Drosophila, especially with respect tobroad genetic patterns (reviewed in Laurie, ’97;Orr, ’97; Hollocher, ’98; Wu and Hollocher, ’98).

Studies have shown that the X chromosome andautosomes contribute almost equally to hybrid ste-rility, yet genetic factors causing hybrid male ste-rility are up to 20 times more numerous thanfactors causing female sterility in crosses betweenD. simulans and its two sister species D. mauri-tiana and D. sechellia (Hollocher and Wu, ’96; Trueet al., ’96; reviewed in Laurie, ’97, Orr, ’97 andHollocher, ’98). These results contradict several

Grant sponsor: National Science Foundation; Grant numbers: NSFDEB-9728890 and NSF DEB-9816979; Grant sponsor: Alfred P. SloanFoundation Molecular Evolution Young Investigators Awards; Grantnumber: 98-4-1 ME; Grant sponsor: Princeton University.

*Correspondence to: Hope Hollocher, Department of Ecology andEvolutionary Biology, Guyot Hall/Washington Road, Princeton Uni-versity, Princeton, NJ 08544. E-mail: [email protected]

Received 6 November 1999; Accepted 24 April 2000

206 H. HOLLOCHER ET AL.

earlier models that had been proposed to accountfor the evolution of hybrid sterility in general whicheither predicted that X chromosome factors shouldgreatly outnumber autosomal factors (Charles-worth et al., ’87; Coyne and Orr, ’89) or that ex-pression of recessive genes in the heterogameticsex could explain differences in the prevalence ofhybrid male and female sterility (Muller, ’40; Orr,’93; Turelli and Orr, ’95). Instead, it appears thatdivergence between species generally affects malereproductive function before it affects female re-productive function, whether heterogamety is in-volved or not, and that male and female hybridsterility are quite distinct developmentally, geneti-cally and evolutionarily (reviewed in Wu and Davis,’93, Hollocher, ’98; for an extension of this modelbeyond Drosophila see Presgraves and Orr, ’98).

The ultimate goal of these types of geneticanalyses is the molecular identification of thegenes involved in species differentiation andanalysis of their function. Much can be learnedabout the evolution of reproductive isolation bydetermining whether the genes that cause hybridsterility are the same genes required for normalgametic function within species or are some otherclass of genes. However intriguing this questionis, few Drosophila studies thus far (or studies inany taxonomic group for that matter) have beenable to analyze the genes causing reproductive iso-lation at the molecular level (for two notable ex-ceptions see Wittbrodt et al., ’89 for platyfish andTing et al., ’98 for Drosophila). The tools neces-sary to perform detailed molecular genetics arenot well developed in the Drosophila simulans sis-ter species that have typically been used in stud-ies of this sort, making progress in this directiondifficult. It is natural to gravitate toward D.melanogaster for studies of genetic mechanismsof speciation because this species possesses all themolecular genetic tools that one would ever need.Although D. melanogaster has been studied insome detail in the past with respect to reproduc-tive isolation (Sturtevant, ’20; Kerkis, ’33; Mullerand Pontecorvo, ’40a; Muller and Pontecorvo, ’42;Pontecorvo, ’43a,b; Orr, ’92), it has turned out tobe technically very difficult to use. Crosses be-tween D. melanogaster and its closest relative, D.simulans, normally result in offspring that are ei-ther inviable or completely sterile—making ge-netic analyses of their differences practicallyimpossible. Muller and Pontecorvo (’40a,b, ’42) by-passed these genetic barriers by crossing triploidD. melanogaster females to irradiated D. simulansmales to produce progeny with mixed D. simulans

and D. melanogaster genomes. Muller and Ponte-corvo’s results (’40a,b, ’42) as well as later analy-ses of the “pseudo-backcross” flies they produced(Pontecorvo, ’43a,b; Orr, ’92) are difficult to inter-pret because too few lines were generated in theoriginal experiments to replicate the differentmixed-species effects. In addition, there remainsthe nagging possibility that the sterility effectsattributed to D. simulans genetic material did notresult from hybridization, but were actually newmutations generated by the X-rays used to makeaneuploid sperm in the D. simulans males.

The use of D. melanogaster/D. simulans hybridsto study the genetics of reproductive isolation hascontinued to allure researchers, in spite of the ap-parent obstacles involved (Watanabe, ’79; Hutterand Ashburner, ’87; Hutter et al., ’90; Sawamuraet al., ’93a,c; Orr et al., ’97). To date, hybridiza-tion between D. melanogaster and D. simulans hasbeen used primarily to investigate the genetic ba-sis of hybrid inviability. In crosses between D.melanogaster females and D. simulans males,male hybrids die as third instar larvae or pupae.In the reciprocal cross, male hybrids survive whilefemale hybrids die during embryogenesis. Theembryonic lethality in this direction of the hybrid-ization is temperature sensitive: hybrid femalescan often escape inviability at 18°C but rarely sur-vive in crosses at 25°C (Sawamura et al., ’93a;Orr, ’96). Surveys of laboratory and wild typestocks of these species led to the identification ofseveral important mutant strains which have theability to rescue either hybrid male or hybrid fe-male lethality and the molecular genetic analysisof the genetic factors responsible for the inviabil-ity rescue effects of these lines is currently un-derway (reviewed in Hutter, ’97; Hollocher, ’98).

It was long thought that all hybrids betweenD. melanogaster and D. simulans, whether nor-mally viable or rescued from lethality, were ster-ile, having only atrophied gonads with no obvioussign of a functional germline. Recently, however,a D. simulans strain, C167.4, that rescues femalesterility in crosses between D. simulans femalesand D. melanogaster males was described (Daviset al., ’96). Davis et al. (’96) showed that the ste-rility rescue in this cross is primarily due to oneor more recessive maternal effect genes segregat-ing in the C167.4 strain.

This paper describes the first steps towards us-ing the hybrid inviability and sterility rescuestrains to study the developmental basis of repro-ductive isolation. By staining the adult reproduc-tive tissues of rescued and unrescued hybrids

GERMLINE DEFECTS IN DROSOPHILA HYBRIDS 207

using germline specific markers, we have deter-mined that the primary defect associated withhybrid female sterility in crosses between D.melanogaster and D. simulans most likely resultsfrom improper control of primordial germline pro-liferation, germline stem cell maintenance, andcystoblast formation and differentiation duringearly oogenesis. Unrescued hybrid females gener-ally contain very little germline (most likely indi-cating a severe defect in primordial germlineproliferation during larval development) whichdoes not undergo proper mitotic cell division anddifferentiation at the onset of oogenesis. Rescuedhybrid females, although fertile, generally havelower amounts of adult germline from the onsetas compared to wild type (also indicating a defectin primordial germline proliferation during lar-val development) and show a premature degen-eration of adult germline cells with age (indicatingimproper germline maintenance). In addition tothe phenotypes described above, older rescued hy-brid females also exhibit mutant egg phenotypesassociated with defects in dorsoventral pattern-ing which most likely result from the improperpartitioning of cytoplasmic factors during earlyoogenesis. Hybrid males from these crosses haveno detectable germline in adult reproductive tis-sues, whether the C167.4 rescue strain is used ornot. These germline and gametogenic defects arediscussed in light of what is known about the evo-lution of hybrid inviability and sterility in crossesbetween D. melanogaster and D. simulans.

MATERIALS AND METHODSFly stocks

The D. simulans hybrid sterility rescue strainC167.4, D. simulans nonrescue strain Tsimbazaza,and D. melanogaster female embryonic and malelarval inviability rescue strains In(1)AB and In(1)ABf-M1/C(1)M4,y are all described in Davis et al. (’96).Because inviability rescue is temperature sensitive,all flies were raised at more permissive tempera-tures between 19°C–21°C. D. melanogaster strainswere raised on standard corn-meal media. D.simulans strains were raised on instant Drosophilamedia (Carolina Biological Supply).

CrossesAll crosses were done in multiples (three to five

vials at a time) using standard corn-meal mediaat 19°C–21°C. In order to overcome the substan-tial pre-mating isolation, each vial contained 60–80 males and 20–30 females. Males were collected

at <24 hours old and aged for four to seven daysbefore being crossed. Females were collected as vir-gins, <8 hours old, and crossed immediately to agedmales. Crosses were transferred to fresh vials ev-ery three to four days. F1 hybrid progeny were col-lected daily from each cross and aged (three to fivedays) separated by sex before dissection to assessfertility and perform antibody staining. F1 femaleswere recorded as fertile if they had more than 15eggs and partially fertile if they had more thanone egg but less than 15 eggs. Females were re-corded as sterile if they contained no eggs andshowed atrophied ovaries. Two sets of dissectionsfor staining male adult germline were performedon flies one and three days post-eclosion, respec-tively. Males were recorded as fertile if their tes-tes were healthy-looking and contained motilesperm and recorded as sterile if their testes wereatrophied and contained no motile sperm.

Tissue preparationGonads were dissected in 1× phosphate buffered

saline (PBS) and fixed in 4% paraformaldehyde/1× PBS for 14–20 minutes at room temperature(Lantz et. al., ’94). Fixed samples were rinsed 3×for 3–5 minutes with PBT (0.3% Triton X-100 inPBS). Samples were stored at 4°C for 0–3 daysbefore antibody staining. Preliminary blockingwas done for 1–2 hours rocking at room tempera-ture with 5% Normal Goat Serum (NGS) in PBST(0.1% Triton X-100/0.05% Tween in PBS).

ImmunocytochemistryExtensive molecular genetic analyses of germ-

line development and oogenesis have revealed sev-eral gene products required for the establishmentand maintenance of adult germline stem cells andproper oogenesis (reviewed in Wei and Mahowald,’94; Lasko, ’94; Williamson and Lehmann, ’96; Lin,’98). For several of these genes, protein productsare expressed early and continuously throughoutgermline development making them extremelyuseful as germline markers. In order to assay forthe presence of germline cells in sterile male andfemale hybrids, we used immunofluorescence tostain for the protein products of two germlinemarker genes: orb and vasa, which show germline,but not sex-specific expression (Lantz et al., ’92;Breitwieser et al., ’96). Antibodies were kindly sup-plied to us by Paul Schedl and Paul Lasko.

Orb stainingPrimary Orb antibody staining was done as de-

scribed in Lantz et al. (’94) except that Orb anti-


bodies 4H8 and 5B2 were used. The gonads wereincubated overnight at 4°C with the monoclonal su-pernatants diluted at 1:20 in 5% NGS in PBST.Each round of staining for Orb and all of the fol-lowing antigens was followed by a 3× 10 minuteswash with PBST. Secondary Orb staining with arhodamine-conjugated antibody at 1:500 in NGSwas done either overnight at 4°C, or for 5–6 hoursat room temperature. RNase was included in thesecondary stain cocktail. Nuclei staining was per-formed by incubating for 10 minutes during the fi-nal washes with Yo-Pro (Molecular Probes) at 1:1000in PBST at room temperature (Lantz et al., ’94).

Vasa stainingPrimary Vasa antibody staining was done with

rabbit anti-Vasa sera diluted at 1:500 in 5% NGSand incubated overnight at 4°C. Secondary anti-body staining was performed using a biotinylatedanti-rabbit antibody at a 1:1000 dilution in NGSand incubated either overnight at 4°C or for 5–6hours at room temperature. Finally, the sampleswere incubated with FITC-Streptavidin diluted at1:100 for 4–5 hours at room temperature. Occa-sionally, the secondary stain was done with aTexas Red-conjugated anti-rabbit antibody at a1:100 dilution. Times and temperatures were thesame as for rhodamine described above. Texas Reddid not involve a follow-up stain.

Mounting and viewingOnce the gonads were stained and washed 3×

10 minutes in PBST, they were mounted in Aqua-polymount (Polysciences, Inc.). Samples wereplaced on clean slides and arranged under a mi-croscope using low levels of light. Excess liquidwas drawn off and a coverslip coated with Aqua-polymount was placed over the gonads. Slideswere stored in the dark at 4°C. After solidifyingfor a day, the slides were ready to be examinedby laser scanning confocal microscopy (Krypton-Argon Laser, Bio-Rad MRC 600). A minimum of20 females from each type of cross was stainedand examined.

Egg and early germline quantitationIn addition to the dissections and staining de-

scribed above, a time-series study of female game-togenesis was performed using flies aged 1, 3, 5, 6,7, 9, and 12 days. All ovaries for the time-courseexperiment were fixed, stained with immunofluo-rescence and mounted as described above. In addi-tion, the amount of germ tissue present wasquantitated using light microscopy (Olympus BX40,

Ph3 filter, 10× objective). The filter (Ph3) and theobjective (Ph1) were out-of -phase, creating a dark-field appearance. Any egg chamber that appearedto be older than stage 12 was counted as an egg.Any ovariole with at least one egg chamber thatappeared to be younger than stage 8 was recordedas having early germline tissue.

RESULTSCrosses

Crosses between D. simulans females andIn(1)AB males are inhibited by a strong prematingbarrier (Table 1). Few progeny were obtained inthese crosses despite employing the cross condi-tions designed to encourage mating (see Materi-als and Methods). The premating barrier wasstronger for C167.4 than for Tsimbazaza. Crossesbetween D. simulans females and In(1)AB-f malesperformed much better (Table 2). The use of dif-ferent inviability rescue strains (In(1)AB f-M1/C(1)M4,y or In(1)AB) did not significantly affectthe sterility/fertility of hybrids (compare Tables 1and 2). The relatively high viability ratios of prog-eny from these crosses indicate that rescue offemale embryonic inviability by In(1)AB andIn(1)AB-f was fairly successful. Female hybridsfrom the sterility rescue cross D. simulans C167.4× In(1)AB-f and In(1)AB showed fertility levels

TABLE 1. Progeny numbers, inviability ratios, andfertility proportions resulting from D. simulans

females ´ In(1)AB males1

C167.4 × Tsimbazaza ×In(1)AB2 In(1)AB3

Female Male Female Male

Progeny counts 57 56 271 312Viability ratio 1.02 1 0.87 1Proportion

Fertile % (N) 0.81 (31) 0.0 (14) 0 (51) 0 (15)Partial % (N) 0.19 (31) — 0.06 (51) —

Total proportionproducing at 1.00 (31) 0.0 (14) 0.06 (51) 0 (15)least onegamete

Other defectsAbnormal 0.0 (31) 0.0 (52)

abdomen1(N) indicates sample size. Females were classified as being fertile ifdissection revealed them to be carrying at least 15 eggs. Partiallyfertile females carried at least 1 egg but less than 15 eggs. Sterilehybrids either showed small atrophied ovaries or testes lacking anymature gametes when dissected.2Progeny counts from seven crosses each transferred every 3 to 4days. Because of a severe premating barrier, four of these crossesyielded no progeny.3Progeny counts from five crosses each transferred every 3 to 4 days.


(96% and 100% of F1 females produced at leastone egg for In(1)AB-f and In(1)AB, respectively)comparable to those found by Davis et al. (’96).Female hybrids from the non-rescue cross ofTsimbazaza × In(1)AB-f or In(1)AB showed muchlower levels of egg production also on par withthe earlier results by Davis et al. (’96). Femalehybrid sterility in this direction has often beenfound to be leaky but low, with only 6% to 27% ofthe hybrids in this case producing very few eggs(approx. two eggs per ovary). In both sets ofcrosses, F1 hybrid male progeny were invariablysterile displaying tiny atrophied testes contain-ing no sperm. In addition, hybrid males resultingfrom crosses with In(1)AB-f often showed an ab-normal abdomen phenotype discussed in more de-tail below. Overall, the adult gonadal phenotypesof hybrids from the D. simulans by D. melano-gaster crosses were as expected.

The results for the reciprocal cross—D. melano-gaster females × D. simulans males—are shownin Table 3. Again, there was a pre-mating barrierassociated with crosses in this direction withTsimbazaza males being less discriminated againstthan the C167.4 males. In this direction, the res-cue of male inviability by In(1)AB is incomplete.In crosses with Tsimbazaza, the sex ratio was veryskewed toward the production of females. In ad-dition, hybrid males from both crosses that actu-ally lived to adulthood often displayed a variety

of somatic defects. These defects included abnor-mal abdomens, notched wings, knobby wings andstumpy legs. The frequency of occurrence of thesedefective adult phenotypes varied with the paren-tal D. simulans strain. All hybrid progeny (bothmales and females) produced by In(1)AB motherswere completely sterile except for hybrid femalesfrom crosses involving C167.4 males (Table 3).Forty-one percent of these females showed someegg production (albeit at very low levels—approx.one egg per ovary). In addition, these eggs werenot healthy-looking (small, ventralized, or other-wise abnormal) and were unlikely to be capableof producing a viable embryo.

Assessment of gametogenesis in rescued andunrescued hybrid females

In wild type Drosophila, oogenesis begins in eachovariole with the regenerative mitotic division ofthe totipotent germline stem cell (reviewed in King,’70; Spradling, ’93). One of the two progeny of thisstem cell remains totipotent and acts as the stemcell in the next round of division. The other daugh-ter cell, called the cystoblast, undergoes four mitoticdivisions accompanied by incomplete cytokinesis toproduce a cluster of 16 cystocytes interconnectedby cytoplasmic bridges. Each cell in this 16-cell cystis connected to the others by one, two, three, or

TABLE 2. Progeny numbers, inviability ratios, andfertility proportions resulting from D. simulans

females ´ In(1)AB-f males1

C167.4 × Tsimbazaza ×In(1)AB-f 2 In(1)AB-f 3



Fertile % (N) 0.56 (82) 0 (134) 0.04 (84) 0 (142)Partial % (N) 0.40 (82) — 0.23 (84) —

Total proportionproducing at 0.96 (82) 0 (134) 0.27 (84) 0 (142)least onegamete


abdomen1(N) indicates sample size. Females were classified as being fertile ifdissection revealed them to be carrying at least 15 eggs. Partiallyfertile females carried at least 1 egg but less than 15 eggs. Sterilehybrids either showed small atrophied ovaries or testes lacking anymature gametes when dissected.2Progeny counts from 17 crosses each transferred every 3 to 4 days.3Progeny counts from 19 crosses each transferred every 3 to 4 days.

TABLE 3. Progeny numbers, inviability ratios, and fertilityproportions resulting from

In(1)AB females ´ D. simulans males1

In(1)AB ×In(1)AB × C167.42 Tsimbazaza3



Fertile % (N) 0.02 (61) 0.00 (102) 0.00 (48) 0.00 (11)Partial % (N) 0.39 (61) — 0.00 (48) —

Total proportionproducing at 0.41 (61) 0.00 (102) 0.00 (48) 0.00 (11)least onegamete


abdomenNotched wings 0.51 (61) 0.41 (167)Knobby wings 0.26 (61) 0.00 (167)Stumpy legs 0.00 (61) 0.02 (167)

1(N) indicates sample size. Females were classified as being fertile ifdissection revealed them to be carrying at least 15 eggs. Partiallyfertile females carried at least 1 egg but less than 15 eggs. Sterilehybrids either showed small atrophied ovaries or testes lacking anymature gametes when dissected.2Progeny counts from nine crosses each transferred every 3 to 4 days.3Progeny counts from three crosses each transferred every 3 to 4 days.


four ring canals that form at the site of the incom-plete cell division. One of the cells with four ringcanals of this 16-cell cluster becomes the oocyteand moves to a posterior position in the egg cham-ber. The other 15 cells become the polyploid nursecells that will function (along with the somatic fol-licle cells which surround the cluster) to providematernal mRNAs to the oocyte. As the 16-cell clus-ter develops the oocyte grows in size and the nursecells eventually degenerate. Meiosis begins earlyin oogenesis in regions 2–3 of the germarium withthe pairing and synapsis of homologous chromo-somes following the formation of the 16-cell cyst,but is not completed until after fertilization.

Orb is required early in oogenesis for the for-mation of the 16-cell cluster and also for dorso-ventral and antero-posterior axis patterning. AnRNA-binding protein, Orb helps direct the properlocalization of the Bicaudal-D, K10, oskar, andgurken maternal mRNAs (Lantz et al. ’92; Lantzet al. ’94; Christerson and McKearin, ’94). In fe-males, Orb protein begins to accumulate in regiontwo of the germarium and becomes preferentiallylocalized to the developing oocyte (see Fig. 1A). Orbprotein is found at these stages in a graded distri-bution throughout the 16-cell cluster with highestlevels in the oocyte and lowest levels in the mostanterior nurse cells. When the oocyte begins to ex-pand in size at stage 7, Orb protein becomes local-ized posteriorly in the oocyte (Lantz et al., ’94).During stages 8–10, Orb protein distributes alongthe entire oocyte cortex (in contrast to the local-ization of orb RNA to the anterior and dorsal andventral cortical regions). After stage 11, Orb levelsare low and even throughout the oocyte.

We used Orb staining to assess the condition ofgermline cells and oogenesis in unrescued adulthybrid females from crosses between D. simulansTsimbazaza and the D. melanogaster lines. Themajority of these unrescued hybrids do not showany positive staining for Orb. This is especiallytrue for hybrid females derived from In(1)AB fe-males crossed to Tsimbazaza males. For the re-ciprocal cross (Tsimbazaza females crossed toeither In(1)AB or In(1)AB-f males), however, sev-eral hybrid females stain for Orb expression in atleast one cell (Fig. 1B,C). The presence of cellspositive for expression of Orb protein in these hy-brids is taken as an indication that it is possiblefor unrescued hybrids to produce some germlinetissue even though the majority of the time theirovaries appear completely atrophied upon dissec-tion (see Tables 1–3). Although germline cells aredetected in these hybrid females, often they ex-

hibit an unusual phenotype, which we refer to asthe ‘‘doughnut’’ phenotype (shown in Fig. 1D andE). In the unrescued hybrids, the germline cellstend to be unusually large and stain positive forcytoplasmic Orb protein. A layer of smaller cellslikely to be follicle cells surrounds each of theselarge cells. Inside the “doughnut,” a large clumpof DNA is visible upon nuclear staining. The DNAclump looks like an aggregation of several largenuclei (most likely the polyploid nuclei normallyfound in the nurse cells) or could represent thenucleus of a single nurse cell with an unusuallyhigh ploidy.

We then used Orb staining to determine whetherthe apparent hybrid sterility rescue by the D.simulans C167.4 strain was complete. Rescued hy-brids were produced from the cross of D. simulansC167.4 females to In(1)AB-f or In(1)AB males. Al-though all female hybrids typically produce eggsin this cross (see Tables 1 and 2), Orb stainingrevealed a range of rescue phenotypes in hybrid

Fig. 1. Unrescued and rescued F1 hybrid females show-ing a continuum of phenotypes from practically no germlineto complete sterility rescue. (A) Distal end of a wild type (D.simulans C167.4) two- to three-day-old adult ovary stainedwith an anti-Orb antibody. Orb protein is present in thegermarium and developing cystoblasts, eventually becominglocalized in the developing oocyte. (B) Atrophied ovary of afour-day-old adult hybrid resulting from Tsimbazaza D.simulans females × In(1)AB-f D. melanogaster males stainedwith an anti-Orb antibody. One large cell at the very distaltip of the ovary stains positive for Orb protein as indicatedby the arrow. (C) The same ovary as in (B) showing thenuclear staining pattern. The single cell staining positivelyfor Orb protein shows an enlarged nucleus as indicated bythe arrow. (D) Representation of the “doughnut” phenotypein an ovary of a four-day-old hybrid resulting from TsimbazazaD. simulans females × In(1)AB-f D. melanogaster males andstained with an anti-Orb antibody. Differentiation of the nursecells and the oocyte has not occurred and Orb staining isgenerally cytoplasmic. (E) The same ovary as in (D) showingthe nuclear staining pattern. The nuclei tend to be clumpedtogether and are enlarged indicating unusual ploidy. (F) Ovaryof a three-day-old rescued hybrid female from C167.4 D.simulans females × In(1)AB-f D. melanogaster males andstained with an anti-Orb antibody. Oogenesis and Orb stain-ing patterns appear normal. (G) Ovary of a one-day-old res-cued hybrid female from the same cross as (F) and stainedwith an anti-Orb antibody. For the most part, oogenesis ap-pears normal; however, Orb protein is being mislocalized orsome developing oocytes fail to localize to the posterior of theegg chamber as indicated by the arrow. (H) At the low ex-treme for hybrid sterility rescue, an ovary from a four-day-old hybrid female from the same cross as (F) showing verylittle developing germline at all. (I) An ovary from an olderhybrid female (seven to nine days old) from the same crossas (F) showing the “doughnut” morphology. (J) Nuclear stain-ing pattern of the same ovary shown in (I).


females aged 1–4 days (Fig. 1F–H). At the mostcomplete rescue end of the spectrum, hybrid fe-males match wild type staining patterns (Fig. 1F).Orb protein is localized properly to the oocyte inearly stages and properly within the oocyte in themiddle stages of ovarian development. Less com-plete forms of rescue show oogenesis proceedingnormally for most cysts, yet the Orb staining in-dicates that at times either the protein is beingmislocalized or some developing oocytes fail to lo-calize to the posterior of the egg chamber (Fig.1G). Hybrid females having very few early stageegg clusters (Fig. 1H) typify the most incompleteform of rescue seen in these same crosses. In thesecases, the developing egg chambers that can beseen are often not differentiated and Orb local-ization does not occur. In addition to the range ofphenotypes discussed above, the “doughnut” pheno-type, as described earlier in the nonrescue crosses,appears often in older (7–12 days) rescued femalehybrids which otherwise would exhibit normal oo-genesis at earlier adult ages (Fig. 1I,J). These olderhybrid females typically contained mature but de-fective eggs (see the later section describing eggphenotypes for more details).

The level of gametogenic rescue of hybrid fe-males from the reciprocal cross (In(1)AB females× C167.4 males) lies somewhere between the se-verity of oogenic phenotypes seen for progeny ofTsimbazaza females × In(1)AB or In(1)AB-f andthose seen for progeny of C167.4 females crossedto In(1)AB or In(1)AB-f males.

Assessment of spermatogenesisin hybrid males

We also examined the condition of adult germ-line stem cells and extent of spermatogenesis inhybrid males and addressed whether brothers ofthe rescued fertile hybrid females showed any im-provement in the condition of these traits as com-pared to brothers of unrescued hybrid females.Just by looking at the dissected testes, it appearedthat these males showed no improvement in termsof fertility over other hybrid males (e.g., the tes-tes were equally small and degenerate). However,it was possible that they could have increasedgene expression that would be evident only ingene-specific assays. Therefore, wild type and hy-brid male adults were stained for Vasa protein.

Wild type Vasa staining in male testes is shownin Figure 3. Vasa stains very brightly at the tipsof the testes where stem cells in the earliest stagesof spermatogenesis are localized and decreases inintensity further along the testes where laterstages are localized. In the examination of a mini-mum of 25 testes from three-day-old hybrid malesfrom each of the crosses described in Tables 1through 3, no staining above background was everdetected (see Fig. 2). The entire experiment wasrepeated using males aged only one day and theresults were identical.

Time course quantitation offemale hybrid fertility

The appearance of the “doughnut” phenotype inolder rescued hybrid females suggests a loss of

Fig. 2. Phenotypes of adult male testes stained with anti-Vasa antibody. (A) Wild type (D. simulans C167.4) adult tes-tes showing strong staining of Vasa protein near the distaltips. (B) Higher magnification of (A). (C) Testes of a three-day-old hybrid male from the rescue cross of C167.4 D.simulans females × In(1)AB-f D. melanogaster males and

stained with anti-Vasa antibody. No detectable levels of Vasastaining above background can be seen. Testes of this male,as indicated by the arrows, are completely atrophied and con-tain no germline. The other parts of the male reproductivesystem indicated in this slide are the male accessory glands(AG) and the vas deferens (VD).


fertility as these hybrids aged. In order to investi-gate this possibility further, we performed a timecourse analysis of the average number of eggs andaverage number of ovarioles containing germlinecells in hybrid females aged between 1 and 12 days(Tables 4 and 5). Orb was used as the marker genefor all the following time course studies. All speci-mens were examined and characterized under aphase contrast microscope as described in the Ma-terials and Methods. Any egg chamber older thanstage 12 was recorded as an egg, and any ovariolecontaining at least one egg cluster younger thanstage 8 was counted as containing germline.

The C167.4 D. simulans control shows the typi-cal trend for average number of eggs and averagenumber of ovarioles containing germline cells perovary over 1 to 12 days (Table 4). For this control,the number of eggs steadily increased over timewith the number of ovarioles containing germlinecells remaining relatively constant and only taper-ing off slightly by 12 days of age. For crosses be-tween C167.4 females and In(1)AB or In(1)AB-fmales, a significant decrease in the quality (andquantity) of eggs and ovarioles containing germ-line cells was observed as rescued females aged.The number of ovarioles starts off lower than wildtype and drops off to an average of less than 1.5ovarioles per ovary by day 12 as compared to theaverage of 7.75 seen in the C167.4 control. Notsurprisingly given the ovariole counts, the aver-age number of eggs per ovary for hybrids fromthis cross starts at a relatively normal level, butdrops significantly by day 12. The ovaries from12-day-old hybrid females are in very poor condi-tion and typically show several aberrant early-stageegg clusters, some displaying the “doughnut” phe-notype and others with abnormal Orb localizationas described earlier (see Fig. 1). Because ovarieslike this are rarely seen in young rescued hybrids,it is likely that as rescued hybrids age, they lose a

good amount of the germline they contain initially.It is important to note that variability in the num-bers of eggs and the number of ovarioles contain-ing germline cells is high in these hybrids. Ingeneral, however, younger hybrids tended to havemore eggs and healthier germline cells than olderhybrids.

Progeny of the cross of Tsimbazaza females andIn(1)AB or In(1)AB-f males showed very low eggproduction with a slight peak between days 6 and9 (of about one egg per ovary). The gradual loss ofgermline in these unrescued hybrids was similarto that seen above. At day 3, these unrescued hy-brids tend to have some early oogenic cysts, but byday 12, none has any early germ tissue left. Prog-eny of the reciprocal crosses (In(1)AB females × D.simulans Tsimbazaza males) were always com-pletely sterile and there were no signs of adultgermline cells or oogenesis for flies of any age.

F1 hybrids from the final cross (In(1)AB females× C167.4 males) showed low levels of egg produc-tion (yet slightly better than Tsimbazaza × In(1)ABor In(1)AB-f). Early stage egg clusters often showedaberrant Orb localization and the “doughnut” phe-notype. Time course dissections of females aged3, 6, 9, and 12 days performed on these hybridfemales showed the same general trends as seenin the reciprocal cross, but with much reduced eggproduction and germline cells to begin with. Fromthis analysis, it is clear that C167.4 males, al-though unable to rescue the sterility of their hy-brid daughters, can advance the stage to whichoogenesis proceeds in their daughters.

Egg phenotypesEggs made by unrescued and older rescued hy-

brid females sometimes show a ventralized pheno-type. Ventralization occurs when the specificationof ventral regions of the chorion becomes extendedcausing the dorsal cells and structures to be either

TABLE 4. Results of the time course experiment examining changes in average number of eggs and ovarioles in the hybridoffspring for the control (C167.4 D. simulans) and hybrids from crosses using the C167.4 D. simulans strain

D. simulans (C167.4) C167.4 × In(1)AB-f C167.4 × In(1)AB In(1)AB × C167.4Average no. Average no. Average no. Average no. Average no. Average no. Average no. Average no.

of eggs of ovarioles of eggs of ovarioles of eggs of ovarioles of eggs of ovariolesAge per ovary per ovary per ovary per ovary per ovary per ovary per ovary per ovary

1 0 (8) 8.6 (8) — — 0.2 (5) 5.6 (5) — —3 3.8 (9) 8.1 (9) 7.1 (12) 4.8 (12) 7.0 (7) 7.6 (7) 2.3 (8) 1.1 (8)5 — — — — 6.3 (8) 3.5 (8) — —6 9.6 (22) 10.6 (22) 14.6 (14) 5.1 (14) 11.5 (13) 3.5 (13) 7.1 (8) 1.1 (8)7 — — — — 16.1 (9) 4.2 (9) — —9 — — 18.4 (17) 2.9 (17) 15.9 (8) 3.1 (8) 5.9 (7) 0.4 (7)

12 19.25 (8) 7.75 (8) 8.1 (11) 0.2 (11) 7.2 (6) 1.2 (6) 2.0 (3) 0 (3)


lost or converted to a more ventral fate. Typically,mutations that affect the dorso-ventral patterningof eggs such as those occurring in gurken or tor-pedo cause changes in the appearance of the dor-sal appendages of the egg which become fused orlost altogether (Schüpbach, ’87).

Ventralized eggs have been found in all threetypes of female hybrids capable of egg productionand show a range of phenotypes depending on thecross (see Fig. 3). The eggs showing the most se-vere levels of ventralization also show defectswhich commonly appear as secondary phenotypesassociated with ventralization mutants: short eggsand chorion abnormalities (Schüpbach, ’87; Schüp-bach and Wieschaus, ’91).

DISCUSSIONNone of the hybrid males resulting from any of

the crosses in this study showed any signs of adultgermline stem cells or spermatogenesis. As dis-cussed in the introduction, it has been clearly es-tablished that in Drosophila, male reproductivefunction of hybrids is often severely affected earlyon in species divergence (see Hollocher, ’98). Fromresearch performed in the D. simulans clade, it hasalready been estimated that the number of geneticfactors causing male sterility in crosses betweentwo of these sibling species (D. simulans and D.mauritiana) is well over 100 (Wu and Palopoli, ’94;Wu and Hollocher, ’98). Therefore, the number ofpossible genetic incompatibilities affecting inter-species male fertility that have accumulated be-tween D. melanogaster and D. simulans, whichhave been separated from each other for at leasttwice the amount of time as the D. simulans spe-cies, is probably far too great to be rescued by thestrains used in this study alone. Because femalesterility, on the other hand, has evolved more re-cently (sterile hybrid females are produced incrosses between D. melanogaster and D. simulans,yet all of the hybrid females resulting from crosses

between the species in the D. simulans clade arecompletely fertile), these rescue strains have alarger impact and can directly inform us about theevolution of female hybrid sterility between thesetwo species.

It is clear from this study that the general cat-egorization of female hybrids resulting fromcrosses between D. melanogaster and D. simulansas being either rescued or unrescued is an over-simplification. In reality, these two categories rep-resent two ends of a continuum of germline andoogenic phenotypes that vary in severity depend-ing on which particular set of strains are crossedand in which direction.

The typical agametic phenotype that has oftenbeen used to describe females that result from hy-bridization between these two species can be foundconsistently in crosses between most strains of D.melanogaster females and D. simulans males(Davis et al., ’96; our own unpublished survey re-sults). Rescuing the larval/pupal inviability of thehybrid males in these crosses by using In(1)ABD. melanogaster females does not alter this gen-eral observation. In this study, when we crossedIn(1)AB females to Tsimbazaza, all F1 females stillpossessed small, atrophied ovaries and showed nostaining for the germline marker.

The situation for the reciprocal cross is inter-estingly more complex. In this case, most crossesbetween D. simulans and D. melanogaster areinhibited by a strong premating barrier andtherefore generally have not been performed asoften as crosses using D. melanogaster as the fe-male parent. When the premating barrier in thisdirection can be overcome, the hybrid femalesthat result die during embryogenesis. BecauseF1 females in this direction are genetically iden-tical to F1 females from the reciprocal cross savethe maternal cytoplasmic contribution, the em-bryonic lethality of these hybrid females derivesfrom an incompatibility between maternal com-

TABLE 5. Results of the time course experiment examining changes in average number of eggs and ovariolesin the hybrid offspring from crosses using the Tsimbazaza D. simulans strain

Tsimbazaza × In(1)AB-f Tsimbazaza × In(1)AB In(1)AB × TsimbazazaAverage no. of Average no. of Average no. of Average no. of Average no. of Average no. of

Age eggs per ovary ovarioles per ovary eggs per ovary ovarioles per ovary eggs per ovary ovarioles per ovary1 — — 0 (12) 0.4 (12) — —3 0 (12) 1.8 (12) 0.2 (18) 0.7 (18) 0 (7) 0 (7)5 — — 0 (5) 2.0 (5) — —6 0.7 (12) 0.5 (12) 0.8 (4) 1.0 (4) 0 (10) 0 (10)7 — — 0.3 (8) 0.3 (8) — —9 1.0 (8) 0.4 (8) 0.3 (10) 0.4 (10) 0 (7) 0.4 (7)

12 0.3 (8) 0 (8) 0.2 (13) 0.2 (13) — —


Fig. 3. Unrescued and rescued F1 hybrid females show-ing a continuum of egg phenotypes. (A) Eggs from a 12-day-old ovary of a wild type female (C167.4 D. simulans). Theseeggs show normal chorion development and well-formed dor-sal appendages. (B) Eggs from a three-day-old rescued hy-brid female from C167.4 D. simulans females × In(1)AB-f D.melanogaster males. Eggs appear normal. (C) Eggs from a12-day-old rescued hybrid female from the same cross as (B).Here the eggs show a weak ventralized phenotype with thedorsal appendages fused. (D) Eggs from a different 12-day-

old rescued hybrid female from the same cross as (B). In thiscase, the eggs show a more severe ventralized phenotype withthe dorsal appendages fused, often short, and sometimes miss-ing. (E) Eggs from a three-day-old hybrid female from thereciprocal cross, In(1)AB D. melanogaster females × C167.4D. simulans males. Hybrid females from crosses in this di-rection never yield normal eggs. (F) Eggs from an unrescuedhybrid female from Tsimbazaza D. simulans females ×In(1)AB-f D. melanogaster males. These eggs show a severelyventralized and shortened egg phenotype.


ponents and zygotic genetic factors in the hybridembryos (Hutter and Ashburner, ’87; Hutter etal., ’90; Sawamura et al., ’93a,b,c). The embry-onic lethality of these hybrid females has pre-cluded the easy examination of their germlineand oogenic phenotypes.

Our study shows that the use of strains thatrescue hybrid female embryonic inviability alonein crosses between D. simulans females and D.melanogaster males often reveals that these hy-brid females actually do retain some germlineand oogenic capabilities, albeit highly defective(as was seen in crosses between Tsimbazaza fe-males and In(1)AB or In(1)AB-f males). The useof the C167.4 D. simulans strain rescues thesesame germline and oogenic defects to recover al-most wild type phenotypes (as seen in the re-sults described for crosses between C167.4 D.simulans females and In(1)AB and In(1)AB-f D.melanogaster males). Surprisingly, although therescue effect of C167.4 has a strong maternalcomponent (Davis et al., ’96), this strain can alsorescue the germline and oogenic defects to somedegree in the reciprocal cross of In(1)AB femalescrossed to C167.4 males. These hybrid femalesare not fertile, yet oogenesis does progress fur-ther in these females than in hybrid females re-sulting from crosses not using the C167.4 strainindicating a zygotic (and not merely maternal)effect of C167.4 rescue. Ordering the hybrid fe-males with respect to the severity of theirgermline and oogenic defects starting with theleast defective and going towards the most de-fective, we would rank the crosses as follows:C167.4 females × In(1)AB or In(1)AB-f males >>In(1)AB females × C167.4 males >> Tsimbazazafemales × In(1)AB or In(1)AB-f males >> In(1)AB females × Tsimbazaza males. As good as therescue of C167.4 is in crosses between C167.4females and In(1)AB and In(1)AB-f males, it isonly ephemeral. Although germline cells and oo-genesis are restored almost to wild type levelsin young hybrid females, both traits begin to re-semble the unrescued phenotypes as the flies age.

An interesting question to consider is whetherthe various phenotypes described in this study arecaused by a single factor acting at several differ-ent steps during oogenesis, or represent the accu-mulation of separate factors controlling eachparticular phenotype. Although a variety of de-fects are described for the hybrid females in thisstudy, all of them can potentially stem from asingle, underlying defect. The complete lack or re-duced levels of germline cells in adult female hy-

brid gonads, the arrest of oogenesis during theearly formation of 16-cell cysts (resulting in the“doughnut” phenotype), and the lack of germlinestem cell maintenance in older hybrid females canall result from defects in the control of mitosisand differentiation associated with the formationof the cystoblast from the germline stem cell andthe early stages of oogenesis.

Within this context, a review of the literaturedescribing germline and oogenic mutants in Droso-phila melanogaster has revealed a striking resem-blance between the phenotypes reported here andthose described for mutations of the pumilio (pum)gene (Lin and Spradling, ’97; Forbes and Leh-mann, ’98; Parisi and Lin, ’99). The D. melano-gaster pumilio gene was first shown to be amaternal effect gene required for posterior pat-terning during embryogenesis (Nüsslein-Volhardet al., ’87). More recently, a certain class of muta-tions of this gene (pumovarette) have been shown tobe required zygotically for germline cell divisionand oogenesis (Lin and Spradling, ’97; Forbes andLehmann, ’98) as well as for preoogenic germlineproliferation and ovarian morphogenesis duringlarval and pupal development (Parisi and Lin, ’99).

The pumilio gene codes for multiple proteinisoforms that have different but highly overlappingfunctions in embryogenesis (through the control oftranslation of hunchback mRNA [Murata andWharton, ’95]), germline maintenance (through con-trol of the self-renewing, asymmetric germlinestem cell divisions) and oogenesis (through con-trol of division of cystoblasts and differentiationof germline cysts) (see Parisi and Lin, ’99 and ref-erences therein). Interestingly, null mutations ofpumilio are generally responsible for the mater-nal effect embryonic lethality. This maternal ef-fect can be rescued by introduction of pumiliopartial-loss-of-function mutants that can produceonly a subset of the protein isoforms (Parisi andLin, ’99). Although the maternal function is re-stored in these genetic constructs, more pumilioprotein product of at least one of the proteinisoforms is required to restore the zygotic func-tion of pumilio during germline development andoogenesis. Therefore, it appears that pumilio actsin a dosage-related manner and that zygotic func-tion requires more pum expression than the ma-ternal effect of the gene.

The different expression patterns of pumiliomutant and their resulting phenotypes match verywell the female hybrid inviability and sterilityphenotypes seen in crosses between D. simulansfemales and D. melanogaster males described


here. The resemblance is suggestive that changesin pumilio regulation between species may ac-count for some of the hybrid defects detected inour study. Future experiments are being designedto test that possibility. In any event, whatever theactual underlying genetic basis of female hybridinviability and sterility in these crosses may be,it appears that rescue occurs in a dosage-depen-dent manner. The fact that all of the hybridoogenic phenotypes can possibly be explained bythe action of a single gene known in Drosophilais encouraging, suggesting that determining theunderlying genetic and developmental basis of fe-male hybrid sterility is a tractable problem.

We plan to continue our phenotypic analysis ofmale and female hybrids by shifting our atten-tion to earlier stages in germline development.During larval development, the oogonia prolifer-ate and ultimately serve as the source of stemcells of the germaria. The lower overall levels ofgermline in rescued and unrescued hybrid femalessuggest that reduced proliferation of these cellsduring larval development may be a key factor. Itis also known that male germline proliferation anddifferentiation begin much earlier than in females,with male germ cell differentiation detectable inthe first larval instar whereas female germ celldifferentiation begins the second day of pupation.Because males and females are out of synch, thesame type of germline defect (e.g., reduced germ-line proliferation and improper germline stem cellmaintenance) can lead to complete adult hybridmale sterility but only partial female sterility. Pre-liminary analysis of larval gonadal disks stainedwith Vasa antibody indicates that the germline isoften completely lacking in male disks in crosseswhere female disks are normal in appearance (un-published results). Presence of normal levels ofproliferated germline in female larval disks inmost crosses reinforces our conclusion that defectsin germline stem cell maintenance and differen-tiation during early adult oogenesis are the pri-mary causes of sterility in hybrid females. Thecomplete lack of germline in hybrid male larvalgonadal disks indicates male sterility results fromdefects occurring during embryogenesis, e.g., dur-ing pole cell formation, germband elongation, orgerm cell migration. In addition to using thegermline markers described above, we are alsoexpanding our analysis to include staining of thecytoskeletal structures which will help us describemore precisely the defects we have identified here.

ACKNOWLEDGMENTSWe thank Paul Schedl and Paul Lasko for sup-

plying the antibodies used in this study. We alsothank Paul Schedl, Eric Wieschaus, and TrudiSchüpbach as well as the members of their labo-ratories for technical advice and for several stimu-lating discussions. We are grateful to MelisandeWolf and Laura Jawidzik for their help in the lit-erature search of germline and oogenic mutantsresembling the defects in hybrids. Special thanksgo to Joe Goodhouse in the Department of Mo-lecular Biology Microscope Facility for the Confo-cal Imaging and Gordon Gray for preparing theDrosophila media. The manuscript was much im-proved by the helpful suggestions of two anony-mous reviewers.

LITERATURE CITEDBreitwieser W, Markussen F-H, Horstmann H, Ephrussi A. 1996.

Oskar protein interaction with Vasa represents an essentialstep in polar granule assembly. Genes Dev 10:2179–2188.

Charlesworth B, Coyne JA, Barton NH. 1987. The relativerates of evolution of sex chromosomes and autosomes. AmNat 130:113–146.

Christerson LB, McKearin DM. 1994. Orb is required for an-teroposterior and dorsoventral patterning during Drosophilaoogenesis. Genes Dev 8:614–628.

Coyne JA. 1992. Genetics and speciation. Nature 355:511–515.Coyne JA, Orr HA. 1989. Two rules of speciation. In: Otte D

and Endler J, editors. Speciation and its consequences.Sunderland, MA: Sinauer Press. p 180–207.

Davis AW, Roote J, Morley T, Sawamura K, Herrmann S,Ashburner M. 1996. Rescue of hybrid sterility in crosses be-tween D. melanogaster and D. simulans. Nature 380:157–159.

Dobzhansky T. 1936. Studies on hybrid sterility, II: localiza-tion of sterility factors in Drosophila pseudoobscura hybrids.Genetics 21:113–135.

Forbes A, Lehmann R. 1998. Nanos and pumilio have criti-cal roles in the development and function of Drosophilagermline stem cells. Development 125:679–690.

Hollocher H. 1998. Reproductive isolation in Drosophila: howclose are we to untangling the genetics of speciation? CurrOpin Genet Devel 8:709–714.

Hollocher H, Wu C-I. 1996. The genetics of reproductive isola-tion in the Drosophila simulans clade: X versus autosomaleffects and male versus female effects. Genetics 143:1243–1255.

Hutter P. 1997. Genetics of hybrid inviability in Drosophila.Adv Genet 36:157–185.

Hutter P, Ashburner M. 1987. Genetic rescue of inviable hy-brids between Drosophila melanogaster and its sibling spe-cies. Nature 327:331–333.

Hutter P, Roote J, Ashburner M. 1990. A genetic basis for theinviability of hybrids between sibling species of Drosophila.Genetics 124:909–920.

Kerkis J. 1933. Development of gonads in hybrids betweenDrosophila melanogaster and Drosophila simulans. J ExpZool 66:477–509.

King RC. 1970. Ovarian development in Drosophila melano-gaster. New York: Academic Press.

Lantz V, Ambrosio L, Schedl P. 1992. The Drosophila orb geneis predicted to encode sex-specific germline RNA-binding


proteins and has localized transcripts in ovaries and earlyembryos. Development 115:75–88.

Lantz V, Chang JS, Horabin JI, Bopp D, Schedl P. 1994. TheDrosophila orb RNA-binding protein is required for the for-mation of the egg chamber and establishment of polarity.Genes Dev 8:598–613.

Lasko PF. 1994. Molecular genetics of Drosophila oogenesis.Austin, TX: R.G. Landes Co.

Laurie CC. 1997. The weaker sex is heterogametic: 75 yearsof Haldane’s rule. Genetics 147:937–951.

Lin H. 1998. The self-renewing mechanism of stem cells inthe germline. Curr Opin Cell Biol 10:687–693.

Lin H, Spradling AC. 1997. Germline stem cell division andegg chamber development in transplanted Drosophilagermaria. Dev Biol 159:140–152.

Muller HJ. 1940. Bearing of the Drosophila work on system-atics. In: Huxley JS, editor. The new systematics. Oxford:Clarendon Press. p 185–268.

Muller HJ, Pontecorvo G. 1940a. The artificial mixing of in-compatible germ plasms in Drosophila. Science 92:418.

Muller HJ, Pontecorvo G. 1940b. Recombinants betweenDrosophila species, the F1 hybrids of which are sterile. Na-ture 146:199–200.

Muller HJ, Pontecorvo G. 1942. Recessive genes causing in-terspecific sterility and other disharmonies between Droso-phila melanogaster and D. simulans. Genetics 27:157.

Murata Y, Wharton RP. 1995. Binding of pumilio to maternalhunchback mRNA is required for posterior patterning inDrosophila embryos. Cell 80:747–756.

Nüsslein-Volhard C, Frohnhofer HG, Lehmann R. 1987. De-termination of anterior polarity in Drosophila. Science238:1675–1681.

Orr HA. 1992. Mapping and characterization of a “specia-tion” gene in Drosophila. Genet Res 59:73–80.

Orr HA. 1993. A mathematical model of Haldane’s rule. Evo-lution 47:1606–1611.

Orr HA. 1996. The unexpected recovery of hybrids in a Droso-phila species cross: a genetic analysis. Genet Res 67:11–18.

Orr HA. 1997. Haldane’s rule. Ann Rev Ecol Syst 28:195–218.Orr HA, Madden LD, Coyne JA, Goodwin R, Hawley RS. 1997.

The developmental genetics of hybrid inviability: a mitoticdefect in Drosophila hybrids. Genetics 145:1031–1040.

Parisi M, Lin H. 1999. The Drosophila pumilio gene encodestwo functional protein isoforms that play multiple roles ingermline development, gonadogenesis, oogenesis and em-bryogenesis. Genetics. 153:235–250.

Pontecorvo G. 1943a. Hybrid sterility in artificially producedrecombinants between Drosophila melanogaster and Droso-phila simulans. Proc Roy Soc Edinb B 61:385–397.

Pontecorvo G. 1943b. Viability interactions between chromo-somes of Drosophila melanogaster and Drosophila simulans.J Genet 45:51–66.

Presgraves DC, Orr HA. 1998. Haldane’s rule in taxa lackinga hemizygous X. Science 282:952–954.

Sawamura K, Taira T, Watanabe TK. 1993a. Hybrid lethalsystems in the Drosophila melanogaster species complex, I:the maternal hybrid rescue (mhr) gene of Drosophilasimulans. Genetics 133:299–305.

Sawamura K, Watanabe TK, Yamamoto MT. 1993b. Hybridlethal systems in the Drosophila melanogaster species com-plex. Genetica 88:175–185.

Sawamura K, Yamamoto M-T, Watanabe TK. 1993c. Hybridlethal systems in the Drosophila melanogaster species com-plex. II. The Zygotic hybrid rescue (Zhr) gene of D.melanogaster. Genetics 133:307–313.

Schüpbach T. 1987. Germ line and soma cooperate duringoogenesis to establish the dorsoventral pattern of egg shelland embryo in Drosophila melanogaster. Cell 49:699–707.

Schüpbach T, Wieschaus E. 1991. Female sterile mutationson the second chromosome of Drosophila melanogaster, II:mutations blocking oogenesis or altering egg morphology.Genetics, 129:1119–1136.

Spradling AC. 1993. Developmental genetics of oogenesis. In:Bate M, Martinez-Arias A, editors. Drosophila development.Cold Spring Harbor, NY: Cold Spring Harbor LaboratoryPress. p 1–70.

Sturtevant AH. 1920. Genetic studies with Drosophilasimulans, I: introduction: hybrids with Drosophila melano-gaster. Genetics 5:488–500.

Ting C-T, Tsaur S-C, Wu M-L, Wu C-I. 1998. A rapidly evolv-ing homeobox at the site of a hybrid sterility gene. Science282:1501–1504.

True JR, Weir BS, Laurie CC. 1996. A genome-wide surveyof hybrid incompatibility factors by the introgression ofmarked segments of Drosophila mauritiana chromosomesinto Drosophila simulans. Genetics 142:819–837.

Turelli M, Orr HA. 1995. The dominance theory of Haldane’srule. Genetics 140:389–402.

Watanabe TK. 1979. A gene that rescues the lethal hybridsbetween Drosophila melanogaster and D. simulans. Jpn JGenet 54:325–331.

Wei G, Mahowald AP. 1994. The germline: familiar and newlyuncovered properties. Ann Rev Genet 28:309–324.

Williamson A, Lehmann R. 1996. Germ cell development inDrosophila. Ann Rev Cell Dev Biol 12:365–391.

Wittbrodt J, Adam D, Malitschek B, Maueler W, Raulf F, Tell-ing A, Robertson SM, Schartl M. 1989. Novel putative re-ceptor tyrosine kinase encoded by the melanoma-inducingTu locus in Xiphophorus. Nature 341:415–421.

Wu C-I, Davis AW. 1993. Evolution of postmating reproduc-tive isolation: the composite nature of Haldane’s rule andits genetic basis. Am Nat 142:187–212.

Wu C-I, Palopoli MF. 1994. Genetics of postmating reproduc-tive isolation in animals. Ann Rev Genet 28:283–308.

Wu C-I, Hollocher H. 1998. Subtle is nature: the genetics ofspecies differentiation and speciation. In: Howard D,Berlocher S, editors. Endless forms: species and speciation.Oxford: Oxford University Press. p 339–351.

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