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Home , Sexual differentiation

Copyright 6 1995 by the Genetics Society of America

Genetic Characterization of small ovaries, a Required in the Soma for the Development of the Drosophila Ovary and the Germline

Sigrid Wayne, Kristen Liggett, Janette Pettus and RodN. Nagoshi

Department of Biological Sciences, The University of Iowa, Iowa City, Iowa 52242 Manuscript received September 1, 1994 Accepted for publication November 29, 1994

ABSTRACT The small ova? gene (sou) is required for the development of the Drosophila ovary. Six EMS-induced recessive alleles have been identified. Hypomorphic alleles are female sterile and have no effect on male , whereas more severe mutations result in lethality. The female-sterile alleles produce a range of mutant that affect the differentiation of both somatic and germlinetissues. These mutations generally produce small ovaries that contain few cysts and disorganized ovarioles, and in the most extreme case no ovarian tissue is present. The mutant egg cysts that develop have aberrant morphology, including abnormal numbersof nurse cells and patches of necrotic cells. We demonstrate that sou gene expression is not required in the germline for the development of functional egg cysts. This indicates that the sou function is somatic dependent. We present evidence using loss-of-function and constitutive forms of the somatic regulatory that sou activity is essential for the developmentof the somatic ovary regardless of the chromosomal sex of the fly. In addition, the genetic mapping of the sov locus is presented, including the characterization of two lethal sou alleles and complementation mapping with existing rearrangements.

HE developing Drosophila egg is a mosaic of so- The germline is in physical proximity to the somatic T matic and germline cells whose coordinate differ- beginning at early stages in embryogenesis. The entiation is essential for normal . An active pole cells migrate during germ band extension to the interaction between the germline and soma controls mesodermal precursors of the gonad. These somatic the deposition of yolk protein into theegg, the produc- cells can differentiate into either ovary or testes de- tion of the chorion egg “shell” ( MAHOWALD and KA” pending on the action of the somatic sex regulatory BBELLIS 1980) and dorsal-ventral patterning in the egg genes. in the become visible chamber and embryo (WIESCHAUS1979; SCH~PBACH during the early larval stages both in terms of gonad 1987; STEVENSet al. 1990). In addition, the prolifera- size and morphology. This sexual differentia- tion and differentiation of both male and female germ tion is dependent on sex-specific interactions between cells are influencedby the sexual identity of the somatic the soma and germline that occur during this period gonad. Polecell transplantation studies demonstrate ( STEINMANN-ZWICKY1994). that functional are only produced when X/X Despite this close interaction between the soma and germ cells develop in ovaries and X/Y germ cells in germline, the sexual differentiation of these tissues is testes (VANDEUSEN 1976; MARSH and WIESCHAUS1978; regulated by two distinct sets of genes. Somatic sex de- SCHUPBACH 1982; STEINMANN-ZWICKYet al. 1989) . termination depends on the interpretation of the XA Therefore, it appears that thegenotype of the germline ratio by the Sex-lethal ( Sxl) gene (reviewed in PARK- is not sufficient to support or oogen- HURST and MENEELY1994). A female XA ratio of 1:l esis in somatic tissue of the inappropriate sex. In fact, ( X/X) activates the SxlRNA splicing activity that causes sex-specific somatic signals can induce germ cells to the transformer ( tra) gene to produce a female-specific undergo a differentiation pathway that is contrary to product. The tra function acts with the product of an- what would be expected from their XA ratio. When X/ other unlinked gene, transformer-2 ( tra-2), to control Xgerm cellswere transplantedinto testes,they at- the sex-specific expression of the functionally dimor- tempted to undergo what appeared by morphological phic (dsx) gene. Mutations inany of these criteria to be the initial stages of spermatogenic differ- genes can alter the somatic sexual differentiation of the entiation ( STEINMANN-ZWICKYet al. 1989). Apparently fly. For example, loss-of-function mutations in tra cause the male soma can “impose” its sexual identity on the X/Xflies to develop as males with fullydeveloped male X/X germline. somatic structures (STURTEVANT1945; BROWN and KING 1961). However, these sexually altered flies are Cmesfmnding author: Rod N. Nagoshi, Department of Biological sterile with only rudimentary germline development, Sciences, University of Iowa, Iowa City, IA 52242. indicating that the germline is not similarlysexually

Genetics 139: 1309-1320 (March, 1995) 1310 S. Wayne et al. transformed (BROWN andKING 1961; N~THIGERet al. soma, as controlled by the somatic sex determination 1989). Furthermore, pole cell transplantation experi- genes, as well ason thegenes that control thedifferenti- ments demonstrate that X/Xgerm cells mutant for tra, ation of the follicle cells. How the female germline is tra-2 or dsx can develop normally if placed in a female affected by these somatic factors is not well understood. somatic environment (MARSH and WIESCHAUS1978; In this manuscript we examined the soma-germline SCHUPBACH1985). These results suggest the existence interaction by characterizing a gene,small ovaries (sou) , of a separate genetic pathway to regulate sexual differ- that is required both for the formation of the somatic ention of the germline. Several genes have been impli- ovary and for the developmentof the female germline. cated in this process, including otu, ouo, Sxl and sans We demonstrate that the expression of the sou gene is jille (snf), based on morphological and molecular ob- required in the soma for both the developmentof the servations that suggest mutations in these genes can somatic ovary and the normal differentiation of the cause X/Xgerm cells to take on some male characteris- female germline. This ovary-specific sou function is de- tics. However, this presumed sexual transformation is pendent on regulation by the somatic sex determina- incomplete and subject to other interpretations ( BAE tion genes. We suggest that sou servesto mediate at et al. 1994). least a subset of the interactions that occurbetween the Later in development, the differentiation of the egg somatic and germline tissues. cysts becomes dependent oninteractions that occurbe- tween the germline and the somatically derived follicle MATERIALSAND METHODS cells. By the late larval and pupal stages, the female Fly strains: The sou”’ alleles were isolate in an EMS muta- germline has begun the process of differentiating into genicscreen designed to identify sex-specific sterility egg chambers. Initially, germline stem cells divideasym- (MOHLER 1977).Descriptions of other mutations and bal- metrically to produce a daughter stem cell and a cys- ancer used in this study are found in LINDSLEY toblast. The cystoblast undergoes four mitotic divisions, and ZIMM ( 1992). Flies were raised on a standard cornmeal, molasses, yeast, agar media containing propionic acid as a each characterized by incomplete cytokinesis, to form mold inhibitor and supplementedwith live yeast. a 16-cellsyncytium. One of these cells becomes the EMS mutagenesis used to isolate lethal sov alleles: , whereas the other 15 differentiate into nurse and SOU"^^'*^ were isolated in an F2 screen for EMSinduced cells that provide much of the material for the matura- X-linked lethal and sterile mutations. W””/Y males were fed tion of the oocyte. As the 1Gcell syncytium develops, it 25 mM EMS in 5% sucrose for 24 hr using standard protocols (ASHBURNER 1989). Themutagenized (w’”’*/Y) males were becomes surrounded by follicle cells and together these mated enmasse to FMO/ClB and thew1118x/FM0 and form the egg chamber. W”’’*/ClBfemale progeny were individually mated to FMO/ Follicle cells havespecialized behaviors and functions Y males. The progeny from each pair mating was examined that are essential for the development of the egg. For for the presence of an X-linked lethal by the absence of B+ example, the delamination of follicle cells at the ante- (W””*/Y) males. Pair matings withviable mutagenized X chromosomes were tested for the presence of female-sterile rior end of the cyst and their subsequentdifferentiation lesions. In these cases the mutagenized chromosomes were into stalk cells act to separate individual egg chambers made homozygous and the resulting females were tested for as they leave the germaria (KING 1970). Polar follicle fertility. From 1821 mutagenized chromosomes we obtained cells thatbecome localized atthe anterior-posterior 756 (41.5% ) X-linked lethals and 66 ( 3.6% ) X-linked female ends of the egg chamber will eventually give rise to steriles. We tested each mutation against sou2 for femalefertil- ity. Two of the lethal lines were shown to be allelic to sou. dorsal appendages and may also be required for ante- Morphological analyses of gonads: The morphology of the rior-posterior microtubule organization ( RUOHOLAet mutant gonads were examined by either Feulgen or DAF’I al. 1991; CLARKet al. 1994) . Border follicle cells migrate staining, both of which specifically label nuclei. Fly cultures into the lumen of the developing egg chamber and act were kept under uncrowded conditions at 25”. Female flies to separate the growing oocyte from the nurse cells. of the appropriate genotypes were aged 2-3 days after eclo- sion at 25”. The ovaries were dissected in phosphate-buffered These follicle cells are also required for the develop- saline (PBS; 130 mM NaCl, 7 mM NapHP04.2H20,3 mM ment of the micropyle ( MONTELLet al. 1992). Muta- NaHpP04.2H20) and thenstained by Feulgen reaction using tions that disrupt the differentiation of specific follicle a modification of the procedure described in GALICHERand cells can have dramatic effects on the morphology of KOZLOFF ( 1971 ) . Ovaries were hand dissected and fixed in the ovary and the viability of the female germline. For Carnoy’s solution ( 1:4 acetic acid:ethanol) for 2-3 min. After fixation, the ovaries were incubated in 5 N HCI for 3-4 min. example, the neurogenicNotch and Delta genes are also This was followed by incubation in Feulgen reagent until the required in the ovaries for the establishment of follicle nuclei were appropriately stained. Staining was stopped by a cell fate and oocyte polarity. Mutations in these genes 5-min incubationin dilute sulfuric acid. The ovaries were result in fused and disorganized egg chambers and are dehydrated by a series of washes in lo%, 30%, SO%, 70%, 90%, 100% ethanol. The stained ovaries were cleared in xy- often associated with necrotic germ cells ( RUOHOLAet lene and mountedin permount. Specimens can be visualized al. 1991). under visible light or fluorescence using a green excitation These results indicate that the development of the filter. female germline is dependent on thesexual state of the For DAPI staining, adult gonadswere dissected in PBS and Som atic-Dependent Ovarian Gene 131 AGene Somatic-Dependent Ovarian 1 then incubated in 50% fixative:50% heptane in a covered and cells withpycnotic nuclei that appear necrotic(Fig- depression slide with agitation froma rotary shaker for3 min. ure 1C). Themutant ovaries are often associated with The tissue was rinsed three times in in PBS + 0.1% Triton and incubated in DAPI solution (0.5 pg/ml in 180 mM Tris- unencysted polyploid cells located in theoviduct. These HCl, pH 7.5) for 1 hr to fluorescently stain nuclei. The prepa- are morphologically similar to nurse cells, perhaps re- ration was washed for 20 min five times with PBS. The tissue sulting from aberrant cyst formation or the degenera- was mounted in 50% glycerolin PBS. The stock solutions tion of follicle cells. In the most severely affected ova- used for this procedure were as follows: solution B, 1.4 g/l ries, the oviducts (ovd) end in small nubs (nb) that NanHPOl, 0.1 g/ 1 KHpPOl, take to pH 7 with NaOH, and solution C, 6.75 g/l NaCl, 6.63 g/1 KCl, 0.66 g/1 are absent germcells and somatic ovarian tissue (Figure MgS04.7H20,0.54 g/l MgC12.6H20, 0.33 g/l CaC12.2H20 1D). These could result from necrosis, reduced prolif- in 3.7% formaldehyde. The fixative was made up by mixing eration of the ovarian tissue or from a failure of the nine parts solution C with 10 parts solution B. DAPI-stained oviduct to join with the female gonad during develop specimens were observedunder fluorescence usinga UV exci- ment. Inmost cases, we believe that the nubphenotype tation filter. Germline clonal analysis: Germline clones were produced occurs when the ovary lobe fails to develop, because we by thewellestablished dominant female-sterile procedure usually cannot find a detached gonadelsewhere in the ( PEWMON and GANS 1983). The dominant female-sterile abdomen. Occasionally, however, a failureof the gonad allele, ovoD’ (or Fs( I)K1237), blocks oogenesis whenpresent and oviduct to attach must occur as free-floating clus- in one copy in the germline stem cells. The ro eny from ters of egg chambers are sometimes found. the mating of y cv sov- vf/FM6 females to ouoE v‘/Y males were irradiated with 1000 rads from a 13’Cs gamma source at In addition to these affects on the somatic structure 44-52 hr postoviposition to induce mitotic recombination in of the ovary, sou mutations also affect the viability, prolif- the germline. Clones induced by this method often occupy eration anddifferentiation of the germline. sou mutant several ovarioles ( WIESCHAUSand SZBAD 1979). Irradiated ovaries often contain egg cysts that contain fewer than females of the genotypey ovo+ cv soo- vf/ovo”’ sou+ vZ4were the normal 15 nurse cells. These could result from cell tested for fertility by matings with y cv v f/Y males. Clones resultingfrom recombination events proximal to f (and death as the cysts often contain condensed, irregularly therefore sou and ovo as well) must be ova+ sou-, producing shapednuclei that are associated with degenerating of the genotypey ovo+ cv sou- vf. These proximal clones nurse cells (KING 1970).However, some hyponumerary were identifiedby the productionof progeny that were yellow, cysts appear to be undergoing advanced stages of oo- crossveinless, vermillion and forked when crossedto y c1) uf/ genesis without evidence of necrosis (Figure 1E). This Y males. Confirmation that the recombinant chromosomes were sou- came by complementation testing against sou2. suggests that the sou mutations can affect the number of mitotic divisions that individual cystoblasts undergo, RESULTS without affecting their capacity to differentiate in a fe- male-specific manner.Hypernumerary cysts are also sou mutations affect both somatic ovary and germline found that can contain more than the normal 15 nurse development: A normally developing ovary consists of cells (Figure 1F). Totest whether these cysts resulted egg cysts organized in lineararrays called ovarioles. Egg from thefusion of egg chambersor increased germ cell chambers of different developmental stages are found proliferation, we examined the number of ring canals in each ovariole (ovl) , with the least mature cysts lo- that form in the hypernumerarycysts. In normal oogen- cated apically andthe mature yolky (y) ar- esis the cystoblast undergoes fourmitotic rounds associ- rangednear the oviduct (Figure 1A). sou mutations ated with incomplete cytokinesis to produce a 16 cysto- affect both the somatic and germline components of cyte syncytium.The cytoplasmic bridges connecting the the ovary, affecting the organization of the gonad as cystocytes are associated with a ring canal that can be well asthe differentiationand viability ofthe germ cells. visualized using phalloidin, an actin-specificfluorescent Three EMS-induced female-sterile sou alleles had been stain. Therefore, the number of ring canals indicates previously identified ( MOHLERand CARROLL1984) and the number of cell divisions that each cystocyte has were used in the characterization of the sou mutant undergone, with a maximum of four divisions in normal . Because the sov mutations result in a range oogenesis. In sou mutant cysts, we found no germ cells of ovarian phenotypes, we could not unambiguously that contained more than four ring canals, and most determine the relative severity of the different alleles, had only one (data not shown) . We therefore believe although mutant combinationswith sou3 generally pro- that most hypernumerary cysts occur by the fusion of duce the most severely affected ovaries (Table 1). one or more egg chambers containingcysts undergoing The most common mutant phenotype obtained is the normal number of mitotic divisions. However, we the disruption of ovariole structure thatresult in ovaries cannot preclude the possibility ofsome aberrant prolif- ( OV) containing a haphazard arrangement of the cysts eration, particularly if it occurs with complete cytokine- (Figure 1B; Table 1) . In many cases, ovarioles do not sis and no ring canal formation. appear to form at all, producing large sacs filled with sou mutations can also result in abnormal germ cell irregularly shaped cysts containing a mix of yolk glob differentiation. Cysts often form that arephenotypically ules, cells with large nuclei that resemble nurse cells similar to the ovarian tumor egg chambers (tc) that 1312 S. Wayne et al.

A B

D

E F

FIGURE 1.-The morphologies of wild-type and sov mutant ovaries. A wild-type ovary (A) is compared with the range of mutant ovarian phenotypes that result from the genotype sou4 l(I)EA42/y m sou2 (B-F) . The mutant phenotypes are similar to those obtained from other sov allele combinations. Ovaries are stained with the nucleic acid-specific Feulgen reagent and photographed under bright field optics. Nuclei stain dark. B, C and E were photographed under twofold higher magnification than A, D and F. (A) Wild-type ovary with ovarioles and yolky egg cysts. (B) Mutant ovary with some ovariole structure and aberrant egg chambers. (C)Disorganized ovary with no ovarioles. (D) Oviduct with one small lobe containing a tumorous egg cyst and a "nub." (E) Mutant cyst containing eight nurse cells (normally have 15 nurse cells). (F) Mutant cyst containing 230 nurse cells. ec, egg chamber; H, hypernumerary cysts; h, hyponumerary nurse cell cysts; nb, nub; nc, nurse cell; ovd, oviduct; ovl, ovariole; tc, tumorous cyst; y, yolky egg chamber. A Somatic-Dependent Ovarian Gene 1313

TABLE 1 TABLE 2 Comparison of sou mutant ovarian phenotypes Effect of sou mutations on male sterility

w ym(s0b) Ovary lobes Disorganized -x- with ovary,with no No ovary lobes W Y Genotype ovarioles" ovariolesb (nubs) % fertile Average no. -+ Genotype males progeny/male" + 1.00 (20/20) 0.00 (0/20) 0.00 (0/20) p (20/23) 87 76.8 5 6.7 -soul 0.19 (5/26) 0.54 (14/26) 0.27 (7/26) Y soul ym sou2 100 (32/32) 90.4 5 3.7 - 0.40 (34/84) 0.49 (41/84) 0.11 (9/84) Y sou2 "Values are means -C SE. sod - 0.02 (2/92) 0.28 (26/92) 0.70 (64/92) sod quently contain irregularly shaped pycnotic nurselike cells that may represent abnormal developmentor cell 0.14 (9/66) 0.42 (28/66) 0.44 (29/66) death (Figure 1, B and C). In contrast to the ovary, testis morphology is not af- SOY1 - 0.52 (28/54) 0.29 (16/54) 0.19 (10/54) fected by any combination of the three sou alleles. sou- sou2 males are fertile and contain mature testes that have sou3 wild-type pigmentation and coiling. No morphological - 0.53(50/94) 0.38 (36/94) 0.09 (8/94) sou1 aberrations in spermatogenesis can be discerned. To examine more subtle effects on spermatogenesis, males sod - 0.12 (27/223) 0.38 (84/223) 0.50 (112,023) mutant for sou2 were individually crossed to sou+ fe- so4 males to test for fertility and fecundity. We found no -EA42 0.03(2/68) 0.48 (33/58) 0.48 (33/68) increase in male sterility nor was the numberof progeny sob produced by each male deleteriously affected (Table 2). Therefore, it appears the female-sterile alleles of s0s"'50 0.00 (0/126)0.13 (16/126) 0.87 (110/126) sou do not have a male function. sob Germline clonal analysis demonstrates that sov is so- 0.00 (0/111) 0.02 (2/111) 0.98 (109/111) matic-line dependent: Mutations in the sou gene affect sou2 the development of both somatic and germline ovarian so,yWI-l 50 cells. This could reflect a requirement in both tissues 0.00 (0/52) 0.00 (0/52) 1.00 (52/52) EA42 for sou expression. Alternatively, it is possible that sou so#I-185 is expressed in only one tissue but is required in a cell 0.00 (0/20) 0.15 (3/20) 0.85 (17/20) nonautonomous manner in the other. To examine this EA42 question, we created mosaic females in which the so- Parental cross: sozf/Balancer (FM6 or FMO) X sorY/Y. Val- matic tissue carried a wild-type sou allele and the germ- ues in parentheses are number of labes per total. line was homozygous mutant. If the mosaic animals re- a Ovary has ovarioles but defective egg chambers. 'Ovary is disorganized,contains germ cells butno dis- main sterile, this would indicate that the requirement cernable ovarioles. for sou activity in female germ cells is cell autonomous. "No somatic or germline gonadal tissue present, oviducts Alternatively, the production of progeny would mean end in nubs. that sou expression in the somatic tissue is sufficient to Because l(l)EA42 is a recessive lethal, homozygous and support oogenesis even in the absence of germline sou hemizygous 1(1)EA42 flies were obtained in combination with the duplication Dp(l,3))snf3"'.Parental cross: l(l)EA42/FM6 X function. I(l)EA42/Y;. Dp(l,3)snf~"'/TM6.Full genotypes: sod = y cv sod Germline clones were generated by the dominant v f; sod = y cv sou2; sou' = y cv sod v f; EA42 = l(l)EA42; female-sterile technique in first instar larvae ( PERRIMON so#I.150 = w1118s07/wL150. so#12185 = w1118so#L185. and GANS1983) . Flies heterozygous for sou- and the dominant female sterile mutation, ovool, were irradi- develop from some female-sterile mutations, including ated to induce mitotic crossing over. In the absence of Sex-lethaland ovarian tumor (KING 1979; KING and RILEY recombination, the adult females are sterile due to the 1982; PERRIMONet al. 1986; SALZ et al. 1987) (Figure presence of ouo''. Mitotic crossing over proximal to 1D). The tumorous cysts are filled with hundreds to both the dominant female-sterile mutation and sou will thousands of small germ cells that fail to undergo fe- produce recombinant germline cells that are homozy- male differentiation. Themutant egg cysts also fre- gous for sou- and wild-type for the ouo gene. If the 1314 S. Wayne et al.

TABLE 3 and WIESCHAUS1978; SCH~PBACH1982), these genes Germline clonal analysis are similar to sou in that mutations in themcause necro- sis and aberrant germ cell morphologies that are the Fertile mitotic indirect effect of disrupted female somatic develop Genotype of No. crossovers ment ( MCKEOWNet ul. 1988; STEINMANN-ZWICKYet ul. irradiated irradiated resulting in ovo+ female" females 1989; STEINMANN-ZWICKY1994). These observations germ cellsb could be explained by a mechanism where the somatic OVB' sou+ sex regulatory genes determine thestate of sou activity, I. 406 12b (sou- OVO+) ovo+ sou' which in turn is required for the formation of the so-

OVB' sou+ matic ovary and the supportof X/Xgerm cell develop 11. 142 8b (sou- ovo+) ovo+ sod ment. Alternatively, sou may repond to the the XA ratio by ov8' sov+ 111. 142 8 (sou+ ova+) a pathway independent of that controlled by tru, tru-2 ovo+ sou+ and dsx. At least one such pathway is known to exist OVB' sou+ for dosage compensation in males (BAKERand BELOTE Iv. 554 16" (sou- ova+) ovo+ so3 1983; LUCCHESIand MANNING 1987). Themale-specific lethal genes (msls) control the hypertranscription of OVB' sou+ V. 39 1 20d (SOU+ OVO+) the male Xchromosome but appear to have no role in ovo+ sou+ females (BELOTE and LUCCHESI1980; KURODAet ul. First instar larvae were irradiated from the crossesof I, y 1991). The functions of these genes are not affected m sou' v f / FMO X OV~'d4/K 11, y m sod v f;/FMO X ov8' by mutations in tru, tru-2 or dsx, indicating a separate d4/y;111, cv vf;/yCV f x 0~8'd4/r; IV, CV sovz,/~~~x ov8' v24/Y; V, W'"~,/FMOX ov8' d4/Y. mechanism for their sex-specific activity.The same may bProgeny derived from crossovers were f-, indicating a also be true for the regulation of sou. crossover event proximal to ov8' and sou resulting in ovo+ One direct way to examine how sex-specific sou activ- sou-/ovo+ sou- germ cells. ity is regulated is by determining whether the loss of 'All progeny carried the sod allele and were v+, indicating sou activity can alter the gonad mutantphenotypes asso- they were derived from ovo+ sou-/ovo+ sou- germ cells. dAll progeny carried the w'"~allele, indicating they were ciated with tru, tru-2 or dsx mutations. Loss of function derived from a w1'18 ovo+/w''~80vo+germ cell. mutations in tru or tru-2 and certain allele combinations of dsx result in chromosomally female (X/X) flies de- veloping somaticallyas males (FUJIHARAet al. 1978; expression of sou is not required in the germline, then BAKER and RIDGE 1980). These X/X "pseudomales" these cells should be able to produce functional eggs. produce malelike somatic testes (pseudotestes)that Alternatively, a cell autonomous germline requirement contain a degenerating and mophologically aberrant for sou activity would preclude the induction of fertile germ cell population (Figure 2A) (BROWNand KING sou- clones. As shown in Table 3, fertile clones were 1961; BELOTEand BAKER1982). If sou is regulated by induced with the sou', sou2 and sou3 alleles, indicating the somatic sex regulatory genes to promote ovarian that sou function is not requiredin the female germline development, then X/Xflies transformed to a male for fertility despite the severe effects of sou mutations identity by mutations in tru, tru-2 or dsx will not express on germ cell morphology. Therefore, the somatic ex- the sou ovarian activity even if the sou+ allele is present. pression of sou, at least from the time of clonal induc- Therefore, the presenceof sou mutations inthese pseu- tion (first instar larvae) , is sufficient to allow female domales should have no effect on thepseudotestis phe- germline development. notype. Alternatively, if sou reponds to the the XA ratio The requirement for sou activity in ovarian develop by a pathway independent of that controlled by tru, ment is controlled by thesomatic sex regulatory tru-2 or dsx, then the pseudotestis will still require sou genes: The sex specificity of sou function for both the function because of its X/Xgenotype. In this case, we development of the somatic ovary and the completion would expect that sou mutations will disrupt the devel- of oogenesis indicates that sou is ultimately responding opment of the X/Xpseudotestis in a manneranalogous to the XAratio, the initial signal of sex determination. to sou mutant X/X ovaries. This can occur in one of two ways. Most aspects of the Our results indicate that the sou product is required sexually dimorphic somatic differentiation of the gonad in somatic cells undergoing ovarian development re- are regulated by the activities of the trunsformer (tru), gardless of their XA ratio. Typically, sou+ pseudotestes transforme-2 ( tru-2) and doubksex (dsx) genes (BAKER are short with most of the germ cells located apically and RIDGE 1980; BELOTEand BAKER1982; WIESCHAUS and in an undifferentiated and degenerating state (Fig- and NOTHIGER1982). Although clonal analysis has ure 2A). sou- pseudotestes have a range of gonadal demonstrated that tru, tru-2 and dsx activity are not re- phenotypes that are indistinguishable from those seen quired in the female germline for oogenesis (MARSH in sou+ pseudomales (compare Figure 2A with B) . This A Somatic-Dependent A Ovarian Gene 1315

n Y

FIGURE2.-The effect of sou mutations on the gonads of X/X pseudomales (A) Pseudotestis from X/X flies transformed into a somatic male by a mutation in the tm-2 5ene. Germ cells (gc) proliferate into the adult stage but are largely undifferentiated. These flies have two copies of sou+. (B) sou-/sou2, tm-2- pseudotestes are indistinguishable from sov+/sou+ pseudotestes. (C) sou2/ sou+ pseudotestis from sibling of fly shown in B. Flies of this genotvpe typically give rise to large testes with multiple clusters of germ cells distributed throughout the lumen of the gonad. (D) An extreme mutant phenotype often seen in sou2/sov2, pseudotestes are gonads that are largely devoid of germ cells. Even in this extreme case, the somatic gonad still develops. (E) X/Y testis from a tudur- mother. The tudm mutation acts maternally to block germ cell development in both male and female progeny. Even in the absence of germ cells, the somatic gonad can still develop. The nuclei of the preparations were stained by Feulgen reaction. t, testis; gc, germ cells. is true even with the most severely affected pseudotes- 25”, X/Y flies carrying one copy of this construct devel- tes. In the absenceof sovactivity, many ofthe pseudotes- oped somatically as females. The ovaries of these X/Y tes contain few germ cells, resulting in gonads that ap pseudofemales generally produce egg cysts that contain pear as elongated collapsed tubes (Figure 2D). We hundreds of small undifferentiated cells ( MCKEOWNd compared the morphology of these pseudotestes with nl. 1988) (Figure 3A). This phenotype is very similar those resulting from males derived from tudm- moth- to the “ovarian tumor” cysts resulting from mutations ers. Mutations in ludm cause females to giverise to in the olu gene. When X/Y pseudofemales were made progeny that completely lack germ cells ( BOSWELLand sou-, the resulting gonads were severely deformed in a MAHOWALD1985). X/Y testes that are somatically nor- manner similar to sou- X/X ovaries (Figure SB) . They mal but lack a germline are virtually indistinguishable lacked ovarioles and often the oviducts ended in from the severe sou- X/X pseudotestes (Figure 2E). “nubs” (see Figure 1D). These observations demon- These results indicate that X/X mesodermal cells that strate that the sex-specific requirement for sou in go- give rise to the somatic gonad do not require ~071activity nadal development is controlled by the somatic sex reg- when developing as testes. ulatory genes, tm, tm-2 and hx. Support for this conclusion comes from the comple- In addition to the abnormal development of the so- mentary experiment inwhich X/Y flies were trans- matic ovary, sou mutations also cause morphological formed into “pseudofemales” by the ectopic expres- aberrations and necrosis in X/X germ cells. We were sion of the tru gene. Atransgenic fly strain was obtained interested in determining whether X/X germ cells de- that carried a construct inwhich the female-specific veloping in a male soma still required sou function. This tru cDNA was fused to the lzsp83 promoter (from the was tested by examining the morphology of the germ laboratory of Dr. P. SCHEDL,Princeton University). At cells produced in pseudotestes carrying different com- 1316 S. Wayne et al.

FIGURE 3.-The effect of sou mutations on the gonads of X/Ypseudofemales. (A) Pseu- doovaries produced by the expression of the tra gene in an X/Y fly. The tra structural se-

d- quence was fused to the Drosophila hsp83 heat shock promoter. At 25", X/Y flies car- rying a single copy of the hsptru construct are transformed to phenotypic females.The pseudoovaries contain egg cysts filled with hundreds of small, undifferentiated cells. (B) X/Ypseudoovary mutant for sm2.These ovaries are small and rarely contain eggcysts, resembling severesou-, X/Xovaries. The nu- clei of the preparations were stained by Feul- H B genreaction. ov, ovary; tc, tumorous cyst.

binations of wild-typeand SOD- alleles. We classified the 2C). These cells are often found in clusters separate pseudotestes into three phenotypic categories. Group from the less-differentiated germ cells and may repre- A is composed of agametic gonads in which few,if any, sent an abortive attempt at oogenesis. We compared germcells could be detected (Figure 2, D and E). the pseudotestes phenotypeof SOD- /sou- pseudomales Group B consists ofgonads containingvarying numbers with their sibling sou-/sm+ pseudomale gonads. Both of germ cells that appear either undifferentiated and genotypes were derived fromthe same parentsand were degenerating or are arrested during early stages of sper- grownsimultaneously under identical culture condi- matogenesis (Figure 2, A and B) . Group C is repre- tions. Both inturn were compared with sou+ /sou+ pseu- sented by gonads carrying one or more polyploid cells domales obtained in a separate set of crosses. Our re- that are morphologically similar to nurse cells (Figure sults showed no consistent effect of different doses of

TABLE 4 The effect of sov dosage on the pseudotestes phenotype

Group A Group B Group C Genotype (agametic) (male or undifferentiated) (nurselike cells) Total + Ira - .- 0.07 0.89 0.04 82 + ' tra"'

0.05 0.92 0.03 157

sod tra - .- 0.03 0.80 0.17 113 sod ' tra"' + tra-2 - .- 0.01 0.99 0 72 + ' tra-2B sod tra-2 - .- 0.01 0.59 0.40 154 + ' tra-2B sod tra-2 - .- 0.24 0.67 0.09 70 sod ' tra-2B

0 0.78 0.22 54

0 0.80 0.20 50

0.10 0.57 0.33 40

Results are given as ratio (observed pseudotestes:total) Genotypes: sod = y cu sod vf: tra"' = k? $ Ira"' red. + = FM6.tra-2B = cn2 tra-2B bw.&x' = &x' Pp. A Somatic-Dependent Ovarian Gene 1317

Df(7)N73 I5D5-6

I In( 1 IG4elH24iR I 68

b-sov+ FIGURE4.-Location of the sov locus on the cytological map of the Drosophila melanoguster X . A diagram of the handing pattern of the 6CE region is shown with the approximate cytological location l(l)EA42 (LEFEVKE1981; LEFEVKEand WATKINS1986). SeQuences contained in relevant duplications are shown hy a shaded box and regions deleted hv deficiencies are denoted hy an dpen box. sou activity on the viability or differentiation of the X/ by the deletionsD f (I)N73, D f (I)ct", In (I)cm-dfand X germ cells developing in pseudotestes (Table 4). In(I)G4eLH24iR (Figure 4). In addition, two duplica- Although there was substantial variability in the distri- tions were tested for their ability to rescue sou. Neither bution of the phenotypic classes, no convincing pattern a duplication of 3F3-5E8 ( Tp ( 1;2) rb+ 71g) nor a du- emerged in pseudomales resulting from mutations in plication of 6D-7C (Dp(I;?)sn"'") included the sou tm, tm-2 or dm. Therefore, although X/X germ cells gene. This places sou in the 6B-6C region (Figure 4) . require somatic sou activity when developing in ovaries, Our initial studies suggested that the sou female-ster- this is not the case when developing in a somatic testis. ile mutations wereallelic to a lethal mutation that This indicates that themale soma can support theviabil- mapped in the cytological interval between 6D1 and ity of the X/X germline independent of sou function. 6D7. We tested sou alleles against an EMSinduced lethal A curious result was seen in tm-2mutant pseudotestes allele of 1(1)6Dd, l(l)EA42. The lethal mutation was heterozygous for sou' (Figure 2C; Table 4). Forty per- viable in all combinations with the sou alleles but re- cent of thegonads produced by this genotype con- sulted in female sterility and theformation of rudimen- tained clusters of nurselike cells and similar high fre- tary ovaries. This inability to complement sou mutations quencies of this phenotype were consistently attained indicates that the 1( 1)EA42 chromosome is mutant for in multiple experiments with other tru-2allele combina- sou function. However, we found that the I( I)EA42 le- tions (data notshown ) . In contrast, nurselike cells were thality is separable from the sou mutant phenotype. The only rarelyfound in sibling sou- /sou- tru-2mutant pseu- Dp ( I;?) sn 13n1 duplication does not carry the sou gene, domales or in sou+ /sou+ control pseudomales (Table as shown by its inability to suppress the mutant pheno- 4). dsx-derived pseudomales also produced a substan- type of female-sterile sou alleles. However, this duplica- tial proportion of gonads (20-30%) thatcontained tion can rescue the 1(I)EA42 lethality, although the one or more clusters of nurselike cells, although in this surviving females are sterile and display an ovarian phe- case the phenotype was independent of the dosage of notype similar to severe sou mutations (Table 1) . These sou'. The reason for these consistent shifts toward fe- data suggest that the sou and I( l)EA42 mutations may male germline differentiation is not clear but may re- map to separate genes. flect the effects of genetic background or the degree The sou gene is associated with recessive lethality:A of male transformation caused by the alleles used. mutagenic screen was performed that was designed to Thecytological and genetic mapping of the sov isolate both sterile and lethal mutations on the X chro- gene: Three female-sterile alleles of sou, mosome. Some 756 recessivelethals and 66 female-ster- were isolated from a single EMS mutagenicscreen ile EMSinduced mutations were individually tested for ( MOHLER 1977). Recombinationmapping experi- allelismwith sou. The lethals were identified by the ments localized the sou mutations to recombinant map absence of hemizygous males.It was not possible to test position 18.5 on the , placing it in the the homozygous condition to confirm that they are also vicinity of cytological region 6C (MOHLERand CAR- lethal in females. Two of the lethal lines gave rise to ROLL 1984). The sou mutations can be complemented sterile females when made heterozygous with sou' and 1318 S. Wayne et al. are designated s~u~'~'~~andWe believe thatthe tions can result in the formation of "tumorous cysts" lethal phenotype associated with ~ou~~~'~~and so~~'.'~~issimilar to that seen in ovaries mutant for certain alleles due to the disruption of sou function. Recombination of the ovarian tumor gene. These egg chambers contain mapping of the lethality of both souM12150and hundreds of small undifferentiated germ cells that fill place the mutations to within five map units of the sou the entireegg chamber. Thesestudies demonstrate that gene (data not shown) and neither lethal allele are sou is required for the developmentof the somatic orga- rescued by nearby duplications that do notcontain the nization of theovary and can also influence theviability sou locus ( Tp (1;2)rbf 71g and Dp (1;3)sn . In com- and differentiation of the X/X germline. plementation testswith l(l)EA42, both souM12150and Despite the severe effects of sou mutations on oogen- SouMl.IX5 complementthe nonsex-specific lethality of esis,mosaic studies indicate that germ cells that are 1( 1)EA42 but not the female sterility. Females that are made sou- during the embryonic and early larval stages SouM1.150 /l(l)EA42 and so~~~~~~~/l(l)EA42are fully via- can develop intofunctional oocytes. This can result ble, but mostfail to produce ovaries (>95%, Table from either oneof two mechanisms. The first possibility 1 ) . The complementation between 1 (1)EA42 and lethal is that the germline requiressou activity during the em- alleles of sou support the contention that I( l)EA42 is bryonic period. By the time the sou- germline clones closely linked butnot associated with the sou gene. are made during the larval stages, the requirement for Therefore, we tentatively designate the sou mutation on sou expression has passed. Alternatively, the sou gene the 1 (I)EA42chromosome ,sou4. may need to be expressed only in the somatic tissues, When the sou female-sterile alleles were made hetero- which in acell nonautonomous mannerexerts an essen- zygous for souM',150or SOU~I.'~~,the mutant ovarian phe- tial function on germline development.We believe that notype became more severe. In sou2 homozygotes, the latter interpretationis more likely because the mor- >lo% of the females completely lack one orboth ovar- phological examination of sou mutant ovaries indicate ian lobes and -50% of the ovaries had a disorganized that relatively late stages in oogenesis are affected. For structure in which ovarioles were not detected (Table example, aberrations in the number and morphology 1 ) . When sou' was made heterozygous with SOV~'.'~~)or of nurse cells and the increase in nurse cell mortality souMI.IX5 , there was a substantial increase in the fre- suggest that the sou mutations affect nurse cell/ oocyte quency of females absent one or bothovary lobes. The differentiation and viability, processes that occur late increase in the severity of the average mutant pheno- in larval development. This suggests that sou function type is consistent with and s0vM1.'X5eliminating is required well after embryogenesis and past the time sou activity. From these results we propose that the sou of clonal induction. lethal alleles represent null mutations in which souactiv- sou is essential for organismal viability: The deleteri- ityis completely blocked, whereas the viable female- ous effects of the female-sterile sou alleles appear to be sterile alleles are hypomorphic lesions that either spe- completely sex specific. Examination of male fertility, cifically disrupt an ovarian specific sou product or re- fecundity and testis morphology failed to demonstrate duces sou function such that only ovarian development any effect of these alleles on male gonadal develop- is affected. ment. However, we believe that sou is essential for the viability of the fly. In a mutagenic screen for X-linked DISCUSSION lethals and steriles, the only two sou alleles isolated were associated with a recessive lethality. When the female- SOY is a somatic function required for both somatic sterile sou alleles aremade heterozygous with either and germliie ovarian development: The sou gene is es- lethal allele, the females are viable but their ovarian sential for the developmentof the somatic ovary as well mutant phenotype becomes more severe than when ho- as the female germline. Mutations in sou result in a mozygous. We therefore believe that the female-sterile dramatic decrease in the size of the ovary, in the most alleles represent hypomorphic mutations in what is an severe casescausing the completeabsence of the female essential gene for males and probably females as well. gonad in the adultfly. In the intermediate phenotypes, In this regard, sou appears similar to the neurogenic the mutantovaries display varyingdegrees of disorgani- genes Notch and Delta. Null mutations in either of these zation consistent with aberrant somatic development, loci result in embryonic lethality due to disruptions in including the absence of ovarioles, the formation of neurogenesis. Hypomorphic alleles of either Notch or fused egg cysts and misshapen yolky egg chambers. The Delta can cause female sterility associated with defects effects of sou mutations on the morphology and devel- in the differentiation of the somatically derived follicle opment of the germline are equally severe. sou mutant cells. Because the focus of this study is on the role of egg cysts generally contain abnormal numbersof nurse sou on ovarian development, a detailed study of the cells and rarely develop a recognizable oocyte. Many lethal phenotype will be presented elsewhere. cysts carry pycnotic nuclei that likely represent instances The ovarian requirement for son is regulated by the of nurse cell degeneration. Less frequently, sou muta- somatic sex differentiation genes: In flies with two X Som atic-Dependent Ovarian Gene A Somatic-DependentOvarian 1319 chromosomes and two sets of autosomes, the tru gene must reflect some undefined interaction between the is expressed and acts with tru-2 to control theexpression somatic cells and germline that is essential for oogen- of the dsx gene, which in turn regulates the differentia- esis. In the absenceof sufficient sov activity in the soma, tion of female-specific somatic structures. This includes this interaction is disrupted and germline development the development of the female gonad from certain me- is arrested. However, we find that if the X/X soma is sodermal cells that surround the embryonic germline. transformed to a male differentiated state, then X/X In the absence of tra or tra-2 function, or with certain germ cell proliferation can occur and is not afTected by combinations of dsx alleles, these same mesodermal reductions in sou activity. This suggests that the pseu- cells take on a male identity and give rise to somatic dotestes soma can support X/X germ cell viability and testes. However, not all sexually dimorphic processes proliferation by a mechanism different than thatwhich are controlled by the action of these somatic sex regula- occurs in a female differentiated somatic gonad. A simi- tory genes. For example, the twofold difference in X- lar effect occurs in the reciprocal experiment. When linked gene expression between male and female flies, X/Yflies are transformedto a somatic female differenti- that is, dosage compensation, is regulated by a different ated state, thepseudoovaries are capable of supporting genetic pathwayinvolving male-specific lethalgenes substantial proliferation of the X/Y germ cells (Figure (reviewed in LUGCHESIand MANNING 1987) . Similarly, 3A) . In the absence of sou activity however, the pseudo- germline sexual differentiation,the choice between ovaries contain few germ cells (Figure 3B). This indi- spermatogenesis and oogenesis, requires germline-spe- cates that ovarian somatic tissue, even if X/Y, cannot cific genes that act independent of tra, tru-2 and dsx support the proliferation or viability of either X/Y or (reviewed in PARKHURSTand MENEELY1994). Further X/Xgerm cells in the absence of sou activity. complexity is demonstrated by the finding that thesex- In conclusion, sex determination and differentiation specific development of certain male abdominal mus- in Drosophila are initiated by the interpretation of the cles depends on the activity state of tra and tra-2 but XA ratio and is subsequently controlled by a hierarchy not dsx (TAYLOR1992). These observations indicate of regulatory genes thathave progressivelygreater spec- that although eachsex-specific process is ultimately con- ificity in their actions. The Sxl gene is required systemi- trolled by the XA ratio, they depend ondifferent regu- cally for all aspects of sexually dimorphic characteris- latory loci for the regulation of subsequent steps in tics, whereas the tra, tra-2 and dsx genes are limited to sexual differentiation. controlling sexual differentiation in the somatic tissue. We were interested in determining the basis for the The sex-specific instructions imposed by tra, tra-2 and sex-specific requirement for sou activity in gonad devel- dsx must in turn be read by a set of genes that have opment. Using fliessexually transformed because of more defined roles in the differentiation ofspecific mutations in the somatic sex regulatory pathway, we sexually dimorphic tissues. We believe that sou repre- found that both X/YandX/Xmesodermal cells require sents one of these genes. We propose that sou partici- sou activity for the development of ovaries but neither pates in the development of the somatic ovary in re- cell type requires sou for testis development. This indi- sponse to the sexually differentiated state of the cates that the sou requirement in ovarian development gonadal precursors. This functionof sou has apparently can be separated from the XA ratio by mutations in been appropriated for other developmental pathways the somatic sex regulatory genes. Therefore, in the me- as well because sou is also essential for both male and sodermal precursorsto the somatic gonads, the somatic female viability. Both the ovarian and viability functions sex regulatory genes tra, tra-2 and dsx must act before of the sov gene are under investigation. sou to initiate sex-specific gonadal differentiation. The We thank Dr. AIXAALFONSO, Dr. PAM GEYERand members of the sou gene then responds to the sexual state of soma such Nagoshi laboratory for helpful commena and discussion. A special that only cells with a female identity require the ovary- thanks toJEmY BEACHfor timely fly media. This work was supported specific sou function. Thissex- and ovary-specificregula- by National Institutes of Health grant GM-45843 and by a March of tion contrasts with the male, and presumably female, Dimes, Basil O'Connor grant (5-Ek92-1224). lethality associated with theabsence of sou activity. 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