SEXUAL reproduction in Drosophila requires the to form sperm at a low frequency, indicating that the

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1 Copyright 2002 by the Genetics Society of America Sex Determination Signals Control ovo-b Transcription in Drosophila melanogaster Germ Cells Justen Andrews 1 and Brian Oliver 2 Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland Manuscript received May 26, 2001 Accepted for publication November 9, 2001 ABSTRACT Nonautonomous inductive signals from the soma and autonomous signals due to a 2X karyotype determine the sex of Drosophila melanogaster germ cells. These two signals have partially overlapping influences on downstream sex determination genes. The upstream OVO-B transcription factor is required for the viability of 2X germ cells, regardless of sexual identity, and for female germline sexual identity. The influence of inductive and autonomous signals on ovo expression has been controversial. We show that ovo-b is strongly expressed in the 2X germ cells in either a male or a female soma. This indicates that a 2X karyotype controls ovo-b expression in the absence of inductive signals from the female soma. However, we also show that female inductive signals positively regulate ovo-b transcription in the 1X germ cells that do not require ovo-b function. Genetic analysis clearly indicates that inductive signals from the soma are not required for ovo-b function in 2X germ cells. Thus, while somatic inductive signals and chromosome karyotype have overlapping regulatory influences, a 2X karyotype is a critical germline autonomous determinant of ovo-b function in the germline. SEXUAL reproduction in Drosophila requires the to form sperm at a low frequency, indicating that the coordinated development of both the soma and male somatic environment can transform germ cells germline (reviewed by Cline and Meyer 1996). The from female (egg) to male (sperm). Likewise, germline primary determinant of sexual identity in Drosophila, phenotypes observed in 2X flies genetically transformed the X chromosome karyotype, was discovered nearly a from females into males in the soma indicate that a century ago. Diploid flies with two X chromosomes (2X) male soma can transform 2X germ cells from female are female, while flies with a single X chromosome (1X) to male (Seidel 1963; Marsh and Wieschaus 1978; are male. The Y chromosome is required for male fertil- Schüpbach 1982; Cline 1984; Nöthiger et al. 1989; ity, but plays no known role in sex determination per se. Oliver et al. 1993). However, the sex of the germ cells Somatic sex determination is relatively well understood, does not strictly follow inductive somatic instruction. but germline sex determination has been more enigfrom For example, some 2X germ cells in flies transformed matic. Both the gametes and the gonads that they debers female to male develop recognizable egg cham- velop in are highly sexually dimorphic. Unsurprisingly, in addition to spermatocytes (Brown and King the communication between sex-specific gametes and so- 1961; Seidel 1963; Nöthiger et al. 1989; Oliver et al. matic gonads is complex. Unfortunately, this complextion 1993). Another example of incomplete sex transforma- ity, especially the overlapping influences of cell-autonoexpress is the finding that 2X flies transformed into males mous and noncell-autonomous signals, complicates our both male- and female-specific products in the understanding of how germline sex is encoded in the germline, instead of uniquely male-specific products genome. High fidelity female gametogenesis requires a (Oliver et al. 1993; Horabin et al. 1995). Thus, female 2X germline karyotype and an ovarian environment. germline sexual identity and oogenesis requires an ovar- Experiments showing that the sex of the soma can ian environment. override the inherent sexual karyotype of the germline Male germ cells (1X) are more resistant to transforma- provide strong evidence for the somatic contribution tion from male to female identity, suggesting that an to germline sex determination (Steinmann-Zwicky et ovarian environment alone is insufficient for induction al. 1989). Karyotypically female germ cells (2X) develop of female germline sexual identity. 1X germ cells trans- poorly when transplanted into a male soma, but are able planted into females with no endogenous germline form ovarian tumors instead of eggs (Steinmann-Zwicky et al. 1989; Steinmann-Zwicky 1994). The ovarian tumor 1 cells associated with defective sex determination resem- Present address: Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN ble arrested primary spermatocytes (Dobzhansky and 2 Corresponding author: LCDB, NIDDK, NIH, 50 South Dr., Bethesda, Bridges 1928; Dobzhansky 1931; Schüpbach 1985; MD oliver@helix.nih.gov Oliver et al. 1988, 1993; Steinmann-Zwicky et al. 1989; Genetics 160: (February 2002)

2 538 J. Andrews and B. Oliver Pauli et al. 1993; Steinmann-Zwicky 1994). These data suggest that 1X germ cells attempt to develop according to the 1X male karyotype in an inappropriate ovarian environment. Again, this is not strict. Very limited female germline development and gene expression occur in 1X germ cells in a phenotypically female soma (Waterbury et al. 2000; Janzer and Steinmann-Zwicky 2001). While the precise sexual state of a germ cell is difficult to determine, it is quite clear that proper female germline sex determination requires both 2X germ cells and an ovary. A network of genes act in germ cells downstream of the autonomous 2X signals, or inductive signals, or both (reviewed by Cline and Meyer 1996). The ovo locus occupies an upstream position in this germline sex-determination hierarchy and acts to regulate ovarian tumor (otu) and 2X germ cell viability. The ovo locus encodes a set of C 2 H 2 zinc-finger proteins that bind to three sites in the otu promoter region (Lü et al. 1998; Lee and Garfinkel 2000; Lü and Oliver 2001) required for otu transcription (Lü and Oliver 2001). Molecular and genetic data suggest that ovo ultimately acts to regulate germline Sex-lethal (Sxl) expression via otu (Oliver et al. 1993; Pauli et al. 1993). The ovo locus is complex, encoding mrnas required for germline (ovo) and somatic (shavenbaby) functions during Drosophila development (Garfinkel et al. 1994; Mével-Ninio et al. 1995; Andrews et al. 2000). The locus is transcribed in somatic cells from ill-defined upstream Figure 1. Organization and function of the ovo locus. (A) Molecular genetic map of the ovo/shavenbaby locus. The top promoters and is transcribed in the germline from ei- shows the genomic region surrounding 0, a SalI site in the locus. ther of two closely linked downstream promoters, ovo-a Just under the scale, the major promoters (P svb, an ill-defined and ovo-b (Figure 1). Alternative promoter use in the somatic promoter, and P gl, the two tightly linked promoters germline gives rise to two major classes of OVO proteins active in germ cells), noncoding and coding exons (open and with common C-terminal DNA-binding domains and solid bars, respectively), introns (thin bent lines), and the region encoding the zinc finger DNA-binding domain are shown. Undifferent N-terminal domains (Figure 1B). The ovo-a der the transcript map is a summary of the genetic data that transcript has an initiating AUG codon in exon 1A that separate the somatic and germline ovo functions. Deletion of is in frame with the long open reading frame in exon 2. P svb by Deficiency (bracket) leaves wild-type ovo function in The ovo-b transcript has an initiating AUG codon well the germline. Transgenes include the indicated wild-type DNA into exon 2. The choice between germline promoters rescue germline ovo phenotypes, but not shavenbaby pheno- types. (B) An expanded view of the germline promoter region. and the resulting protein isoform classes, OVO-A and An AUG in the first exon of ovo-a mrna appends a repression OVO-B, is critical for female germline development. domain onto the OVO-A transcription factor, resulting in negative activity in the germline. The repression domain is not The shorter OVO-B isoform provides necessary and sufencoded due to the absence of an AUG in the first exon of ficient locus activity for female fertility (Andrews et al. 2000). Expression of the antagonistic OVO-A isoform ovo-b mrna. (C) A model of ovo function where ovo responds to the number of X chromosomes in the germline. (D) A results in dominant female sterility (Mével-Ninio et al. model of ovo function where ovo responds to a female somatic 1996; Andrews et al. 1998, 2000). This antagonism is environment. (C and D) OVO binding sites are found at ovo evident at the transcriptional level (Figure 1, C and and otu promoters. The OVO-A protein directly or indirectly D). OVO-B activates the otu promoter, while OVO-A downregulates the ovo and otu promoters, while the OVO-B negatively regulates the otu promoter, as well as both protein directly upregulates the otu promoter. germline ovo promoters (Lü et al. 1998; Andrews et al. 2000; Lü and Oliver 2001). We know little about how ovo-b and ovo-a promoters study concluded that the 2X karyotype controls overall are regulated. However, the effect of somatic signals ovo expression because 2X flies transformed to males and X chromosome karyotype on the expression of ovo expressed high levels of reporter activity, while 1X flies reporter genes ( jointly reporting the ovo-b and ovo-a promoters) has been previously reported (Figure 1, C and D; Oliver et al. 1994; Waterbury et al. 2000). One transformed into females showed feeble expression (Oliver et al. 1994). In contrast, a later study reexamined the same reporters and concluded that the somatic

3 Regulation of ovo-b 539 sexual identity controls overall ovo expression because labeling. RT and PCR primers are listed according to 5 posi- 1X flies transformed into females expressed high levels tion on the genomic map of Mével-Ninio et al. (1995), the direction (P, plus; M, minus), and length (in nucleotides). A of reporter activity (Waterbury et al. 2000). These re- total of g of total RNA was reverse transcribed with ports are largely contradictory. A shortcoming of both 16 ng/ l ofovo-specific primer 1846/M/24, 1 mm dntps, 8 studies is that neither distinguished between ovo-b and mm DTT, 0.8 units/ l RNase inhibitor (Boehringer Mann- ovo-a promoter activity. For these reasons, it is unclear heim, Indianapolis), 50 mm Tris 8.3, 75 mm KCl, 2 mm MgCl 2, if a 2X germline karyotype (Figure 1C) or a female sosearch Laboratories) in a total volume of 25 l. Other RT and 10 units/ l M-MLV reverse transcriptase (Bethesda Re- matic sexual identity (Figure 1D) is most important for primers tested were 1538/M/24 and 1541/M/17. Standard upregulation of the ovo-b promoter. PCR conditions used 2.5 l of RT reaction, 8 ng/ l of either Here we determine how the ovo-b promoter responds primer 446/P/20 (ovo-a specific) or primer 858/P/20 (ovo-b to 2X and somatic signals. We show that endogenous specific), 8 ng/ l primer 1480/M/22, 2.5 Ci/33 nm [ 32 P]dCTP, ovo-b mrna is readily detectable in the female germline, 1X MasterAmp J (Epicentre Technologies, Madison, WI), and 0.02 units/ l Taq polymerase (Bethesda Research Laborabut is quite difficult to detect in the male germline. We tories) in a total volume of 50 l. The cycle profile was 5 min looked at endogenous ovo-b transcripts and ovo-b re- at 94, 25 times (1 min each at 94, 50, and 72 ), and 10 min porter expression in mutants that transform the sex of at 72. For touchdown PCR we used 2.5 l of RT reaction, 8 the germline or soma to determine which cues are criti- ng/ l of either 446/P/20 (ovo-a specific) or 858/P/20 (ovo- cal for the differential expression of ovo-b. We find that B specific), 8 ng/ l 1480/M/22, 2.5 Ci/33 nm [ 32 P]dCTP, 1 MasterAmp G (Epicentre Technologies), and 0.02 units/ 2X germ cells express high levels of ovo-b regardless of l Taq polymerase (Bethesda Research Laboratories) in a total the sexual identity of either the surrounding soma or volume of 50 l. The cycle profile was 5 min at 94, 3 times the germ cells themselves. By analysis of double mutants, (30 sec each at 94, 64, and 72 ), 3 times (30 sec each at 94, we also show that ovo is upstream or independent from 62, and 72 ), 3 times (30 sec each at 94, 60, and 72 ), 3 somatic sex determination signals, while otu, a direct times (30 sec each at 94, 58, and 72 ), 3 times (30 sec each at 94, 56, and 72 ), 15 times (1 min each at 94, 55, and OVO target gene, is downstream of somatic sex deter- 72 ), 5 times (1 min each at 94, 50, and 72 ), and 10 min mination signals. These data indicate that ovo-b is func- at 72. Other PCR primers used were 455/P/17, 914/P/17, tionally controlled by the germ cell sex chromosome 1127/M/20, 1286/M/24, and 1457/M/17. Amplicons were karyotype. However, we also show that somatic sex deter- isolated and sequenced using fluorescent dye terminators (ABI- mination signals do promote significant ovo-b expres- PRISM, drhodamine Terminator cycle sequencing, and an ABI-377 sequencer; Perkin-Elmer, Norwalk, CT). Following sion in 1X germ cells. Thus, while we conclude that the initial verification of amplicon sequence, bands were idenovo-b is regulated primarily by a 2X karyotype and is tified by mobility against a known amplicon and by restriction required only in 2X germ cells, it is also regulated by a digestion. We amplified internal controls in negative samples, female somatic environment. indicating that the absence of product in those samples was not due to failed reactions. MATERIALS AND METHODS RESULTS Flies and histochemistry: We used standard Drosophila techovo-b mrna is expressed at high levels in the wildniques throughout. Flies were grown at Most alleles and transgenes have been previously described and can be type female germline and is barely detectable in the found, with references, at FlyBase (http: /flybase.bio.indiana.edu). male germline: Multiple alleles of ovo result in female Relevant ovo and sex determination alleles and FlyBase accessterility, while none result in male sterility (Oliver et sions are as follows: ovo D1rv22 (FBal ), ovo D1rv23 (FBal ), ovo D2rvBT2 (a spontaneous revertant of ovo D2 obtained from al. 1987). The female-specific requirement is for OVO-B the Daniel Pauli laboratory), ovo D1r (FBql ), Df(1)JC70 (Andrews et al. 2000). However, ovo must be expressed (FBab ), snf 1 (FBal ), otu 1 (FBal ), otu 17 in the male germline too, as otu promoter activity is (FBal ), Sxl fs1 (FBal ), Sxl fs3 (FBal ), Sxl 7BO greatly reduced in males hemizygous for amorphic ovo (FBal ), tra 1 (FBal ), tra-2 B (FBal ), alleles (Hager and Cline 1997; Lü et al. 1998). The Df(2R)TRIX (FBab ), Df(3)dsx M R15 (FBab ), dsx Swe (FBal or FBal ), tra hs.pm (FBal or expression of ovo in the male germline is likely to be FBal ), ovo::lacz 1.1 (FBal ), ovo::lacz Dap (FBal- low, as previous work has failed to detect ovo mrna in ), and ovo::lacz Dbp (FBal ). Genotypes are listed males by Northern blotting (Bae et al. 1994; Garfinkel in the text and figure legends. Gonads were dissected and et al. 1994). We were also unable to detect ovo mrna examined live, under phase contrast and Nomarski optics, in testis by RNAse protection assays (J. Andrews, unor fixed and stained with X-Gal to detect LACZ expression (Andrews et al. 2000). Experimental samples and positive and published results). Expression in the male germline has negative controls were coprocessed in the same tubes. been detected indirectly, using ovo reporter genes (Oli- Reverse transcriptase-pcr: Total RNA was extracted from ver et al. 1994; Mével-Ninio et al. 1995). Nothing is known tissues using TRIZOL (Bethesda Research Laboratories, Gai- about ovo-a vs. ovo-b expression in the male germline. thersburg, MD). To increase the sensitivity of reverse tran- Low abundance in the male germline and problematic scriptase (RT)-PCR, [ 32 P]dCTP was incorporated in the PCR step (Oliver et al. 1993). Touchdown PCR, which increases locus structure make this determination difficult. The sensitivity by negatively ramping the annealing temperatures ovo-a and ovo-b transcripts are nearly the same size, (Don et al. 1991), was also deployed in conjunction with radio- limiting the value of Northern blot analysis using com-

4 540 J. Andrews and B. Oliver Figure 2. RT-PCR analysis of ovo transcription. (A) Diagram showing the location of the RT-PCR primers (arrows) used to detect the indicated transcripts from the ovo-a and ovo-b promoters. The RT primers are located farther downstream. (B) Radiolabeled touchdown RT-PCR results showing that at least some ovo transcript was present in gonads. Detection of ovo-b in ovaries was facile and robust, but detection of ovo-a in females and detection of either transcript in testis were difficult and sporadic. (C) Radiolabeled standard RT- PCR showing the clear detection of ovo-b transcript from the ovary and in testis only when the ovo copy number is increased to five. The ovo copy number was reduced using ovo D1rv22 and increased with P[w ovo D1 ] transgenes. N, number scored. and provide reassuring molecular evidence for ovo expression in the testis, in support of ovo-dependent expression of otu::lacz in the male germline (Lü et al. 1998). While the touchdown RT-PCR data suggest that both forms of ovo mrna are expressed in the gonads, this is not a useful assay for determining which primary sex determination signals are responsible for the high level expression in the female germline. Using standard PCR conditions, we detected ovo-b transcripts in RNA isolated from whole adult females and from ovaries, but not from any adult male tissues or female carcasses (Figure 2C). We failed to detect ovo-a transcripts in any tissue using standard PCR conditions (not shown). Multiple RT-PCR conditions and ovo-b primer pairs gave similar results (see materials and methods). ovo-b mrna was readily detected using as little as 80 ng of total RNA from females, but not when up to 10 g of testis or 20 g of adult male RNA was used. In terms of wild- type gonads, standard RT-PCR provides an essentially plus/minus assay for the nearly female-specific expres- sion of ovo-b. The ovo locus is X-linked; therefore, wild-type females have two doses of ovo, while males have one. But differ- ential expression of ovo-b is not trivially due to this inherent dose difference between males and females. If we failed to detect ovo-b expression in males because there are fewer copies, then we should be able to detect ovo-b expression in males with two copies of ovo (Figure 2C). However, we failed to detect ovo-b expression in testis samples from males with up to three copies of ovo. We did detect ovo-b expression in samples from males with five copies of ovo. However, this is not likely to be a reflection of a simple increase in ovo gene number and a linear increase in ovo-b transcript levels, as increased ovo gene dose results in autoregulation of ovo reporters in trans (Lü et al. 1998). Because we failed to detect ovo-b from testis bearing up to three copies of ovo, any detection of ovo-b in 2X flies transformed from female into male is unlikely to be due to a simple ovo dose artifact. Reporter genes provide a convenient second assay for the expression of ovo isoforms and are especially useful for determining which cells express the individual forms. Additionally, the reporter genes are autosomal, elimi- nating the complicating issue of assessing the influence of X chromosome dose on an X-linked gene. The ex- pression patterns of ovo-a-specific and ovo-b-specific reporter genes reflect the higher levels of endogenous ovo transcripts in females vs. males (Figure 3; Andrews et al. 2000). The ovo::lacz ap reporter shows high ovo-b activity in the female germline and little to no activity in the male germline (Figure 3B). In contrast, the mon probes. Further, the short A/T-rich 1A and 1B exons are poor hybridization probes and thus cannot be used to distinguish the isoforms. Therefore, previously deployed methods were probably insufficiently sensitive to detect the limited quantity of ovo mrna in male germ cells and determination of the isoform expressed is unknown. Our interest in this study is the regulation of the ovo-b isoform in the female germline. However, an understanding of ovo expression in 2X female vs. 1X male germ cells is critical for understanding how inductive or autonomous signals regulate ovo. We examined the expression of ovo-a and ovo-b mrnas in males and females using a sensitive RT-PCR assay, using [ 32 P]dCTP in the reaction and intron spanning oligonucleotide primers (Figure 2A). While we did not experience difficulties amplifying ovo-b mrna from ovaries by RT-PCR, amplification of ovo-a mrna from ovaries was not robust. Unsurprisingly, amplification of either ovo-b or ovo-a from testis required extensive exploration of reaction conditions. The best detection of ovo isoforms in gonads was obtained using Touchdown RT-PCR (Figure 2B), an optimization method that uses gradually decreasing annealing temperatures during cycling (Don et al. 1991; materials and methods). We were ultimately able to detect ovo-b and ovo-a amplicons in both testis and ovary ovo::lacz bp reporter shows feeble ovo-a activity in the preparations. Neither class of ovo transcripts was detected female germline and little to no activity in the male in gonadless carcasses. These data are consistent with germline (Figure 3C). Thus, the reporter genes faithfully replicate the expression of the endogenous the germline specificity of the ovo-a and ovo-b promoters ovo

5 Regulation of ovo-b 541 Figure 3. The expression of ovo reporter genes in gonads. (A) Reporter of both promoters. (B) ovo-b reporter. (C) ovo-a reporter. Shown are schematics of the reporter constructs (left), genotype with respect to the reporter (left of the images), and X-gal-stained ovarioles (bundles of these functional units exist in the ovary) on the left and apical regions of the testes on the right (the apex contains the stem cells and young primary spermatocytes, the cell types expressing low levels of ovo mrna). transcripts, providing another assay for determining in the germline is assessed (Granadino et al. 1993; which sex determination signals control ovo-b expression. Steinmann-Zwicky 1993; Hager and Cline 1997). In ovo-b expression in 2X females transformed into males: the soma, the effects of multiple X chromosome count- The ovo-b isoform is required for female germline devel- ing elements contribute to the initiation of female sexopment, while the ovo-a isoform plays no known zygotic ual development (Cline and Meyer 1996). As there role in the female germline (Andrews et al. 2000). To are multiple X-linked genes in the nascent germline determine if ovo-b expression in females is positively sex-determination hierarchy, we asked if they have addiregulated by inductive female signals, or the number tive or synergistic effects on the expression of ovo-b. of X chromosomes, or is a simple consequence of fe- In addition to the single mutants (Figure 5, C E), we male germline differentiation, we assayed for ovo-b ex- examined females homozygous for mutations in pairs pression in mutations that transform sexual identity. of X-linked genes (otu 1 Sxl fs1 or snf 1 otu 1 ) and females If the somatic sexual identity is a critical determinant heterozygous for multiple X-linked germline sex deterof ovo-b expression, then transformation of somatic sex- mination genes [snf otu Sxl 7BO /snf 1 otu 1 Sxl, Df(1)JC70 ual identity from female to male should result in a marked decrease in germline ovo-b expression. The somatic sex of 2X flies was transformed from female to male by the absence of transformer-2 (tra-2) orbythe expression of only the male-specific form of doublesex (dsx) from the dominant allele, dsx swe. Use of the dominant allele is critical, as the absence of all dsx activity results in intersexual flies, not males. In our experiments, microscopic examination showed that 10% of the sex-transformed 2X flies had testes containing mature primary spermatocytes, spermatids, or sperm. Thus, these 2X male individuals allowed us to test for the dependence of ovo-b expression on both female somatic and germline sexual differentiation. High level expression of ovo-b was noted in the 2X male germ cells, indicating that female sexual identity (somatic or germline) Figure 4. The expression of the ovo-b reporter in pheno- is not an obligate requirement for high level ovo-b extransformed into somatic males. Expression is highest in 2X typic males. (A) Wild-type testis. (B and C) Testes from females pression (Figure 4). As a corollary, these data indicate germ cells. The genotypes with respect to the reporter (left) that a 2X karyotype is important for ovo-b expression. and with respect to sex determination genes (bottom) are We do not know how the number of X chromosomes shown. The X chromosome status is shown at the top.

6 542 J. Andrews and B. Oliver Figure 5. The expression of the ovo-b reporter in phenotypic females. (A) Wild-type female ovariole. (B) Ovary from a 1X fly transformed from a somatic male into a somatic female. (C E) Ovarian tumors from females mutant for germline sex determination genes. X-gal staining is strongest in 2X gonads, but note that there is detectable expression in some of the tumorous chambers of 1X, tra hs.pm females. (ovo snf - ) otu Sxl /ovo snf otu 1 Sxl fs1,ordf(1)jc70 We clearly detected ovo-b amplicons in 1X males trans- (ovo snf - ) otu /ovo snf 1 otu 1 ]. In no case did we observe formed into females (Figure 6, lane 3), but not in wildan overt reduction in ovo-b reporter activity (data not type 1X males (Figure 6, lane 4). These data indicate shown). These data indicate that these X-linked loci do that a female soma does have a positive influence on not provide overt 2X signals contributing to ovo-b expres- the expression of ovo-b mrna. We also confirmed that sion. endogenous ovo-b transcripts are readily detected in 2X Detectable ovo::lacz ap expression in 1X males trans- males (Figure 6, lanes 1 and 2), indicating that a 2X formed into females: Our experiments on 2X females karyotype is sufficient for high level ovo-b expression in transformed into males indicate that female somatic the absence of a female soma. Detectable expression of sexual identity is not required for high level expression ovo-b in 2X flies transformed into males is remarkable, of ovo-b. We next determined if ovo-b expression is posi- given how few germ cells are present in 2X flies transtively regulated by a female soma in the absence of a formed from female to male. Wild-type males with many 2X karyotype. 1X flies were somatically transformed more germ cells fail to express detectable ovo-b even from male to female using the gain-of-function tra hs.pm when there are up to three copies of ovo (Figure 1C). allele. In the female somatic environment of these flies, Finally, ovo-b expression was readily detected in 2X flies the 1X germ cells resemble arrested primary spermato- mutant for otu, snf, orsxl (Figure 6, lanes 6 8). Briefly, cytes, although some female germline differentiation these RT-PCR data are fully consistent with the reporter may also occur (Steinmann-Zwicky et al. 1989; Oliver results and strongly indicate that a germline 2X karyoet al. 1994; Waterbury et al. 2000; Janzer and Stein- type plays a prominent role in high level ovo-b expresmann-zwicky 2001). We compared the expression of sion, but that somatic signaling also positively regulates ovo-b in 1X females to that in 2X females with wild-type ovo-b expression. or tumorous ovaries (Figure 5) or to males (Figure 4). Somatic signal input rests between ovo and otu: We The comparison to 2X tumors is especially apt, because 2X and 1X ovarian tumors show similar phenotypes. The 1X germ cells within the ovaries of flies bearing the transgene did not express ovo-b at the levels seen in 2X flies (Figure 5), indicating that a female soma alone does not support high level ovo-b transcription. However, 1X males transformed into somatic females did express germline ovo-b at a level significantly higher than that in 1X males (Figure 4). The low to intermediate expression of ovo-b in 1X females indicates that a female soma has a positive influence on ovo-b expression. Endogenous levels of ovo-b in sex-transformed flies: We used RT-PCR to directly corroborate the reporter data. To investigate the role of sex chromosome karyotype and somatic sex in regulating ovo-b expression, we performed standard RT-PCRs from sex-transformed flies. Because a female somatic sex had a weak effect Figure 6. RT-PCR on sex-transformed flies. Genotypes, karyotypes with respect to the X, and germline and somatic on ovo-b reporter expression, we were especially interphenotypes of the flies harvested for the reactions are indicated. ested in determining if endogenous ovo-b transcripts The location of the ovo-b product and an anonymous amplicon could be detected in 1X female flies. that serves as a useful positive control are also indicated.

7 Regulation of ovo-b 543 Figure 8. Model of ovo and otu regulation by primary karyotypic and somatic sex determination signals. Shaded somatic signals indicate that while there is molecular evidence for activation of ovo-b via somatic induction, there is no genetic evidence for biological relevance. It is not known if the two somatic signals are the same. Zwicky 1996). Variability, and the possibility of overlapping regulation, makes it difficult to rule out the proposition that a female soma functionally regulates ovo-b. Given the importance of this point, we extensively examined the phenotype of 2X flies transformed from female Figure 7. ovo function does not depend on somatic signals. Paired samples were from 2X brothers trans-heterozygous tive purposes, we examined flies with the same sex- to male, in the presence or absence of ovo. For comparafor tra-2 B and the tra-2 Df(2)TRIX chromosomes. ovo flies transforming genotype in the presence or absence of were trans-heterozygous for ovo D1rv22, ovo D1rv23,orovo D3rvBT2. otu flies were trans-heterozygous for otu the downstream genes otu and snf. There is debate on 1, otu 14,orotu 17. snf flies were homozygous for snf 1. The ovo, otu, and snf siblings whether some or all of the somatic sex determination were heterozygous for the mutant alleles and FM7. N, number scored. About 10% of control 2X females transformed into males due to the absence of tra-2 had a well-developed germline with advanced primary spermatocytes, sper- matids, or sperm. The gonads of the remainder showed mostly debris. In contrast, 2X males that also lacked ovo did not show evidence of germ cells (Figure 7). This highly statistically significant absence of germ cells is due to the 2X karyotype and not due to any requirement for ovo in spermatogenesis, as 1X males hemizygous for the same alleles of ovo are fertile. These data unambiguously indicate that ovo genetic activity is not fully depen- dent on somatic sex determination signals transmitted via the tra-2 locus. In contrast to the results with ovo, the double mutant data clearly indicate that otu and snf genetic activities are under the control of somatic signals as has been previously suggested (Nöthiger et al. 1989; Oliver et al. 1993; Horabin et al. 1995; Nagoshi et al. 1995; Waterbury et al. 2000). The 2X flies transformed from somatic females to males and lacking either otu or snf showed the same frequency of germline development (including spermatogenesis) as 2X females mu- tant for only tra-2. Thus, there is clear somatic signal input downstream from ovo and upstream of otu (Figure 8). were interested in determining whether the contribution of a female soma to ovo-b expression was important for ovo function in the female germline. The characteristic ovo loss-of-function phenotype is female germline death (Oliver et al. 1987, 1994; Staab and Steinmann- Zwicky 1996). Several studies have shown that ovo is not required for 1X germ cell viability in males transformed into females by gain-of-function tra alleles (Oliver et al. 1994; Nagoshi et al. 1995; Waterbury et al. 2000). Thus, any effect of the female soma environment on ovo function must be assayed in 2X flies. If somatic signals are obligatory for proper ovo function, then two related predictions are (1) that transformation of a female soma to a male identity should result in a germline phenotype like that seen in an ovo loss-of- function mutation and (2) that a double mutant for a somatic sex determination gene and ovo should show the same phenotype as flies mutant for only the somatic sex determination gene. Mutations in both ovo and somatic sex transformations result in the loss of germ cells, although this loss is much less extreme in 2X females transformed into males (Nöthiger et al. 1989) than in 2X females without ovo function (Oliver et al. 1987, 1993, 1994). While this argues against a female soma being an obligate regulator of ovo, both phenotypes have been shown to be highly variable (Oliver et al. 1987, 1993, 1994; Nöthiger et al. 1989; Rodesch et al. 1995; Staab and Steinmann- signals pass through the tra and dsx loci, so we restricted our double mutant experiments to tra-2, which has been implicated consistently (Nöthiger et al. 1989; Oliver et al. 1993; Horabin et al. 1995; Waterbury et al. 2000). DISCUSSION Transcription patterns frequently correlate with tissue requirements. The ovo locus is required for female

8 544 J. Andrews and B. Oliver germline viability, sex determination, and differentia- idea that the 2X karyotypic signals are the most important tion, but has no known role in the male germline (Oliver for ovo function. et al. 1987). More specifically, ovo is required only We thank Virginia Boulais for maintaining Drosophila stocks and in germ cells with a 2X karyotype, as 2X males require The Bloomington Stock Center and Daniel Pauli for providing stocks. ovo for germline viability (Oliver et al. 1994; Nagoshi We also thank Jurrien Dean, Alan Kimmel, Jining Lü, and Michael et al. 1995; this study), while 1X females do not (Oliver Parisi for comments on the manuscript. et al. 1994; Nagoshi et al. 1995; Waterbury et al. 2000). Certainly, the requirement for ovo in 2X germ cells might be expected to be reflected in higher ovo expression LITERATURE CITED in 2X germ cells, but this does not have to be the Andrews, J., I. Levenson and B. Oliver, 1998 New AUG initiation case. For example, both males and females express some codons in a long 5 UTR create four dominant negative alleles of the Drosophila C2H2 zinc-finger gene ovo. Dev. Genes Evol. sex determination genes, but achieve sex-specific func- 207: tion by pairing with sex-specific cofactors (reviewed by Andrews, J., D. Garcia-Estefania, I. Delon, J. Lü,M.Mével-Ninio Cline and Meyer 1996). Initial work suggested that ovo et al., 2000 OVO transcription factors function antagonistically in the Drosophila female germline. Development 127: was female-specifically expressed (Bae et al. 1994; Garfin- Bae, E., K. R. Cook, P. K. Geyer and R. N. Nagoshi, 1994 Molecular kel et al. 1994). However, later reporter and genetic stud- characterization of ovarian tumors in Drosophila. Mech. Dev. 47: ies clearly indicated that ovo is also expressed and can Brown, E. H., and R. C. King, 1961 Studies on the expression function in the male germline (Oliver et al. 1994; Mével- of the transformer gene of Drosophila melanogaster. Genetics 46: Ninio et al. 1995; Hager and Cline 1997; Lü et al. 1998) Our RT-PCR and reporter data indicate that ovo transcripproduct Cline, T. W., 1984 Autoregulatory functioning of a Drosophila gene that establishes and maintains the sexually determined tion is highly differential, but not female specific. state. Genetics 107: The existence of ovo isoforms with opposite activities Cline, T. W., and B. J. Meyer, 1996 Vive la difference: males vs (Andrews et al. 2000) raised the possibility that produc- females in flies vs worms. Annu. Rev. Genet. 30: Dobzhansky, T., 1931 Interactions between female and male parts tion of a particular isoform and not the overall expresin gynandromorphs of Drosophila simulans. Wilhelm Roux s Arch. sion of the locus could be sex specific (Andrews et al. 123: ). We provide evidence that ovo-a and ovo-b isoof Dobzhansky, T., and C. B. Bridges, 1928 The reproductive system triploid intersexes in Drosophila melanogaster. Am. Nat. 62: forms are expressed in the female and male germline, albeit at markedly lower levels (failure to detect ovo-b Don, R. H., P. T. Cox, B. J. Wainwright, K. Baker and J. S. Mattick, expression in males by standard RT-PCR is quite stringene 1991 Touchdown PCR to circumvent spurious priming during amplification. Nucleic Acids Res. 19: gent). A simple model where, for example, ovo-b acts Garfinkel, M. D., J. Wang, Y. Liang and A. P. Mahowald, 1994 in the female germline while ovo-a acts in the male Multiple products from the shavenbaby-ovo gene region of Drosophila melanogaster: relationship to genetic complexity. Mol. Cell. germline has not emerged. For our purposes here, the striking expression differences between males and fe- Biol. 14: Granadino, B., P. Santamaria and L. Sanchez, 1993 Sex determimales as assayed by RT-PCR and reporter gene expres- nation in the germ line of Drosophila melanogaster: activation sion are excellent assays to tease out the regulatory in- of the gene Sex-lethal. Development 118: puts to the ovo-b promoter. Hager, J. H., and T. W. Cline, 1997 Induction of female Sex-lethal RNA splicing in male germ cells: implications for Drosophila Two studies have examined the regulation of overall germline sex determination. Development 124: ovo transcription by primary sex determination signals Horabin, J. I., D. Bopp, J. Waterbury and P. Schedl, 1995 Selection and maintenance of sexual identity in the Drosophila germ- (Oliver et al. 1994; Waterbury et al. 2000). Both relied line. Genetics 141: on reporters (Oliver et al. 1994) that were constructed Janzer, B., and M. Steinmann-Zwicky, 2001 Cell-autonomous and before the ovo-a and ovo-b promoters were mapped somatic signals control sex-specific gene expression in XY germ (Mével-Ninio et al. 1995). The authors reached oppo- cells of Drosophila. Mech. Dev. 100: Lee, S., and M. D. Garfinkel, 2000 Characterization of Drosophila site conclusions using the same reporters. The first study OVO protein DNA binding specificity using random DNA oligosuggested that high overall ovo transcription is depen- mer selection suggests zinc finger degeneration. Nucleic Acids dent on a 2X karyotype because of high expression in Res. 28: Lü, J., and B. Oliver, 2001 Drosophila OVO regulates ovarian tumor 2X flies transformed into males and low expression in transcription by binding unusually near the transcription start 1X flies transformed into females (Oliver et al. 1994). site. Development 128: The second study suggested that high overall ovo expreszinc-finger Lü, J., J. Andrews, D. Pauli and B. Oliver, 1998 Drosophila OVO protein regulates ovo and ovarian tumor target promot- sion is controlled by the somatic signals because of high ers. Dev. Genes Evol. 208: expression in 1X flies transformed into females (Water- Marsh, J. L., and E. Wieschaus, 1978 Is sex determination in germ bury et al. 2000). Oliver et al. (1994) discounted weaker line and soma controlled by separate genetic mechanisms? Nature ovo reporter expression in 1X females as background 272: Mével-Ninio, M., R. Terracol, C. Salles, A. Vincent and F. Payre, and Waterbury et al. (2000) did not compare ovo ex ovo, a Drosophila gene required for ovarian development, pression in 1X females to 2X male expression. Neither is specifically expressed in the germline and shares most of its study looked at transcription directly. This study indipatterning. Mech. Dev. 49: coding sequences with shavenbaby, a gene involved in embryo cates that both a 2X karyotype and a female soma con- Mével-Ninio, M., E. Fouilloux, I. Guenal and A. Vincent, 1996 tribute to high ovo-b expression. Epistasis supports the The three dominant female-sterile mutations of the Drosophila

9 Regulation of ovo-b 545 ovo gene are point mutations that create new translation-initiator Schüpbach, T., 1982 Autosomal mutations that interfere with sex AUG codons. Development 122: determination in somatic cells of Drosophila have no direct effect Nagoshi, R. N., J. S. Patton, E. Bae and P. K. Geyer, 1995 The on the germline. Dev. Biol. 89: somatic sex determines the requirement for ovarian tumor gene Schüpbach, T., 1985 Normal female germ cell differentiation re- activity in the proliferation of the Drosophila germline. Develop- quires the female X chromosome to autosome ratio and expression ment 121: of Sex-lethal in Drosophila melanogaster. Genetics 109: Nöthiger, R., M. Jonglez, M. Leuthold, P. Meier-Gerschwiler Seidel, S., 1963 Experimentelle untersuchungen über die grundelagen and T. Weber, 1989 Sex determination in the germ line of der sterilität von transformer (tra) männchen bei Drosophila Drosophila depends on genetic signals and inductive somatic melanogaster. Z. Vererblehre 94: factors. Development 107: Staab, S., and M. Steinmann-Zwicky, 1996 Female germ cells of Oliver, B., N. Perrimon and A. P. Mahowald, 1987 The ovo locus Drosophila require zygotic ovo and otu product for survival in is required for sex-specific germ line maintenance in Drosophila. larvae and pupae respectively. Mech. Dev. 54: Genes Dev. 1: Steinmann-Zwicky, M., 1993 Sex determination in Drosophila: sis-b, Oliver, B., N. Perrimon and A. P. Mahowald, 1988 Genetic evidence a major numerator element of the X:A ratio in the soma, does that the sans fille locus is involved in Drosophila sex determi- not contribute to the X:A ratio in the germ line. Development nation. Genetics 120: : Oliver, B., Y. J. Kim and B. S. Baker, 1993 Sex-lethal, master and Steinmann-Zwicky, M., 1994 Sex determination of the Drosophila slave: a hierarchy of germ-line sex determination in Drosophila. germ line: tra and dsx control somatic inductive signals. Develop- Development 119: ment 120: Oliver, B., J. Singer, V. Laget, G. Pennetta and D. Pauli, 1994 Steinmann-Zwicky, M., H. Schmid and R. Nöthiger, 1989 Cell- Function of Drosophila ovo in germ-line sex determination de- autonomous and inductive signals can determine the sex of the pends on X-chromosome number. Development 120: germ line of Drosophila by regulating the gene Sxll. Cell 57: Pauli, D., B. Oliver and A. P. Mahowald, 1993 The role of the ovarian tumor locus in Drosophila melanogaster germ line sex deter- Waterbury, J. A., J. I. Horabin, D. Bopp and P. Schedl, 2000 Sex mination. Development 119: determination in the Drosophila germline is dictated by the sex- Rodesch, C., P. K. Geyer, J. S. Patton, E. Bae and R. N. Nagoshi, ual identity of the surrounding soma. Genetics 155: Developmental analysis of the ovarian tumor gene during Drosophila oogenesis. Genetics 141: Communicating editor: R. S. Hawley