Ectopic expression of the female transformer gene product leads to female differentiation of chromosomally male drosophila

Cell, Vol. 53, 887-895, June 17, 1988, Copyright © 1988 by Cell Press

Ectopic Expression of the Female transformer Gene Product Leads to Female Differentiation of Chromosomally Male Drosophila Michael McKeown,* John M. Belote,t and Russell T. Boggs* * Molecular Biology and Virology Laboratory The Salk Institute P. O. Box 85800 San D~ego, Califorma 92138 t Department of Biology B~ological Research Laboratories Syracuse University Syracuse, New York 13210

Summary The t r a n s f o r m e r (tra) gene of Drosophila is necessary for all aspects of female somatic sexual differentiation. tra uses a single set of precursor RNAs to produce female- and non-sex-specific RNAs by alternative splicing. Ectopic expression of the femalespecific RNA causes chromosomal males to develop as females, indicative of a linear pathway of regulated genes controlling sex. Genetic and molecular tests with this ectopically expressed gene are consistent with the following order of gene action: X chromosome to autosome ratio -- S e x l e t h a l --, t r a n s f o r m e r --" t r a n s f o r m e r . 2 -~ d o u b l e s e x --" i n t e r s e x - " terminal differentiation. Expression of the female-specific tra RNA in tra mutants is sufficient to lead to female differentiation. Expression of the non-sex-specific tra RNA in tra mutants is not sufficient to lead to female differentiation. The tra female-specific activity is not required for female-specific splicing of the tra precursor RNAs. Introduction Many of the genes controlling sexual differentiation in Drosophila melanogaster have been identified, and their interactions inferred, from genetic studies. The thoroughness of these studies makes it possible to manipulate mutant versions of these genes in a number of ways to test models about their mechanism of action. Since many of these genes are not necessary for viability, it is possible to examine the way in which complete loss of gene function or the inappropriate gain of gene function affects the development of otherwise healthy individuals. The primary determinant of somatic sex in Drosophila is the ratio of the number of X chromosomes to the number of sets of autosomes (the X:A ratio) (see Figure 1 for a diagram of the models consistent with the genetic and molecular data for the genes discussed ~nthis paper; (see Baker and Ridge, 1980; Baker and Belote, 1983; Cline, 1979, 1984, 1985; Nagoshi et al., 1988). Information about this ratio is transmitted, in a process requiring the action of the maternally contributed daughterless gene product and the zygotlcally acting sisterless-a gene, to the Sex lethal (Sxl) gene (Chne, 1976, 1980, 1983, 1984, 1985, 1986;

Maine et al., 1985; Cronmiller and Cline, 1987). Sxl activates, directly or indirectly, the transformer (tra) gene and also controls, either directly or through the action of tra, the transformer-2 (tra-2) locus. The product(s) of the tra-2 locus or of the tra and tra-2 loci together regulate the bifunctional doublesex (dsx) locus. The intersex (ix) gene product acts downstream of or in conjunction with dsx to regulate terminal differentiation. In normal females (X:A = 2:2) Sxl is activated, tra and tra-2 are therefore active, and, as a result, dsx is expressed in its female mode. The female dsx product acts, either in conjunction with or by the activation of the ix product, to repress male differentiation and allow female differentiation. In normal males (X:A = 1:2) Sxl is not activated, tra and tra-2 are not activated, and, as a result, dsx is expressed in its basal, male mode. In its male mode, dsx represses female differentiation and allows male differentiation. The ix gene is not required in males. Germ line sex determination is also controlled by the X:A ratio and Sxl, but appears to be independent of genes that act downstream of Sxl in the somatic sex determination pathway (Cline, 1984, 1985; Maine et al., 1985; Marsh and Wieschaus, 1978; SchLipbach, 1982, 1985). To understand the regulatory interactions within the pathway controlling somatic sexual differentiation, we (Belote et al., 1985; McKeown et al., 1986; 1987) and others (Butler et al., 1986) have isolated the tra gene. We have shown that alternative splicing of tra transcripts gives rise to two different RNAs (Boggs et al., 1987). One of these is present only in females and contains a single long open reading frame, while the other is present in both sexes and contains no long open reading frame. The regulatory switch between these two RNAs is promoter independent. In this paper we examine whether these alternatively spliced RNAs are in fact derived from the same precursor RNAs. We also examine the potential functions of these different RNAs in proper male and female differentiation and use ectopic expression of these RNAs to examine the relationships among the genes of the regulatory hierarchy controlhng sexual differentiation. Results Female and Non-Sex-Specific RNAs Have the Same 5' Ends Our previous results showed that regulation of tra splicing is independent of the tra promoter and suggested that the female-specific and non-sex-specific RNAs have similar 5' ends (Boggs et al., 1987). We have used primer extension and $1 nuclease mapping to verify this (Figure 2). Since the level of tra RNAs is relatively low, we synthesized a comparatively long primer with 32p inserted at C residues throughout its length. The position of this primer is such that it should hybridize to both the female-specific and non-sex-specific RNAs and allow wsualization of primer extension products from both sets of RNAs in a sample of female RNA or from the single set of RNAs in

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Reaulatorv Interactions Genetic Data

Linear Pathway

Branched ~athway

Controlling

Sex

A

Additional Genes Additional Branches

K I "-V

N ! m

V-~.male

and female

.~,~-~#~female ~ Sxl

Sx/

B

TOTAL L~ J TOTAL M 0~0 (3=0

tra

tra tra-2

32p PRIMER

,# tra -2

dsx

Terminal Dlfferentlahon

dsx

622 527

~////////~

Figure 1. Models for Gene Interactions Controlling Sex The models shown give possible regulatory relationships among the different genes examined m this study. Additional genes including daughterless and sisterless-a are involved in the transmtssion of refermarion about the X.A ratio to Sxl (Cline, 1976, 1978, 1984, 1986). The genettc model at left shows a simple model consistent with the genetic observations of Chne (1978, 1979, 1984) and Baker and Rtdge (1980) (for reviews see Baker and Belote, 1983; Cline, 1985). Note that the genettc studtes do not order the interaction of the tra and tra-2 genes and only tentatwely place ix action downstream of dsx. The other three models show possible arrangements of the hierarchy conststent both wtth genetic data and with the molecular data of Nagoshi et al. (1988) The data of Nagosh~et al. (1988) also show that ix does not act before dsx in the regulatory hierarchy. The model at far right indicates the possibility of the extstence of genes that have not yet been tdentified, but which are regulated m a sex-specific manner and do not fall on a linear pathway between Sxl and dsx. In addition to any as-yet-unknown genes, the ix gene could also be a candidate for a gene present on such a branch of the sex determination hterarchy. The hatching strikes out models mconslstent wtth the molecular and genetic data derived from the hs-tra-female construct descnbed in Ftgure 3.

a sample of male RNA (Figure 2A). On the basis of seq u e n c e analyses of cDNA clones, we expect the primer extension products from the non-sex-specific RNA to be between about 400 and 475 nucleotides long, while the primer extension products of the female-specific RNA should be 175 nucleotides shorter. Note that the 5' end of the first intervening sequence lies quite close to the 5' end of the tra RNAs (within as few as 56 nucleotides for some RNAs). Our attempts to use primers 5' to the first intervening sequence have been unsuccessful because of our inability to achieve a high e n o u g h specific activity in labeling of the shorter primers needed in this region. Figure 2B shows the results of primer extension experiments using both female and male RNAs. In the male lanes there are a n u m b e r of bands in the region between 400 and 475 nucleotides. These are not seen if male poly(A)- RNA is used and suggest that the non-sexspecific tra RNA has multiple potential 5' ends. The positions of these bands are consistent with the 5' ends seen in various different cDNA clones (Boggs et al., 1987). The multiple bands seen for the 5' end of the tra RNA are also consistent with the relative broadness of the tra RNA bands seen on RNA blots (McKeown et al., 1987; Boggs et al., 1987). Multiple transcription start sites have been

309

242 238

217 204 190 180

Ftgure 2. Mappmg of the 5' Ends of tra RNAs (A) A schemattc representation of the non-sex-specific and femalespecific tra RNAs, showmg the position of the primer used for primer extension experiments. K and N represent Kpnl and Ncol restriction sttes, respectively. The Ncol stte marks the 3' end of the primer. The Kpnl site marks the 5' end of the first intron in both non-sex-specific and female-specific RNAs. (B) A primer extenston analysis of male and female RNAs Two separate sets of reacttons usmg total RNA from males and females and one set of reactions using poly(A)- RNA from males and females are shown. Lane M contams labeled marker DNAs of the sizes indicated. There is a smgle blank lane between the pely(A)- lanes and the second set of total RNA lanes. The brackets at right mdtcate the positions of two sets of bands found in primer e×tens~onproducts of total RNA but not in primer extension products from poly(A)- RNAs.

observed for other promoters (e.g., Rio and Tjian, 1984; Hahn et al., 1985; Sikorav et al., 1987). The same set of bands ts also seen in the primer extension products derived from female RNA. This shows that the non-sexspecific RNAs seen in females have the same 5' ends as the non-sex-specific RNAs found in males. In addition, there is a set of bands in the primer extension products derived from female RNA that is not seen in the primer extension products derived from male RNA. These bands are approximately 175 nucleotides shorter than the bands derived from the non-sex-specific RNA, consistent with our expectations. As with the non-sex-specific bands, there are multiple bands in the female lane. The pattern of female-specific bands is similar to the pattern observed a m o n g the non-sex-specific bands. To verify that the multiple bands we see are truly representative of 5' ends of RNAs rather than the result of sequences present in both sets of RNAs that are resistant to reverse transcription, we have performed $1 nuclease

Male to Female Sex Changes 889

tides and that these initiation sites are not regulated in a sex-specific manner.

A. h~70 hs

Expression of the Female RNA Induces Female Development

pUCg

B. Wild Type

C. Wtld Type

D, XY hs-tra

Figure 3. Expressron of the tra Female RNA in Males (A) Structure of the hs-tra-female plasmtd used to express tra female RNA in males. This plasm~d was constructed from pUChsneo (Stellar and Pirrotta, 1985) by the addition of a heat shock promoter from pUChspUT-D (S Poole, personal communication) A tra female cDNA begmmng at position +31 of the sequence of Boggs et al (1987) and extending into the poly(A) tall was then cloned downstream of this promoter. The basic vector is the same as was used to express the genomrc tra sequences in Boggs et al (1987) except the tra genomlc sequences have been replaced with a tra female cDNA clone (Boggs et al, 1987). The structure of the plasm=d is such that just over 200 bases of 5' untranslated sequence from the heat shock promoter and linker DNA are transcribed before transcription of the tra sequences. (B-D) A wild-type male (B), a wild-type female (C), and an XY fly carrying a single copy of the hs-tra-female plasmid (D). The latter fly has undergone female somatic differenbatlon rather than the male somat=c differentiation expected from its sex chromosome constttutlon. The Bar eye results from a dominant Bar mutation carried on the Y chromosome This serves as a marker of sex chromosome const=tutton. Nine independent transformed lines carrying hs-tra-female were isolated All of them showed at least partial female differentiation of XY flies. Two of them showed complete female differentiation. Two lines were lost because the single primary transformants recovered from those lines were XY and therefore infertde as a result of sex transformation

mapping on the RNAs derived from males or females (not shown). In these studies we have used a hybrid gene containing the 5' end of the tra gene fused to a non-sexspecific tra cDNA. The probes used for these mapping experiments were synthesized in such a m a n n e r that their 3' ends correspond to the 3' ends of the primers used for the primer extension experiments. The results of these $1 mapping experiments are fully consistent with the primer extension results. If the relative mobilities of the different bands are used to infer the approximate position on the g e n o m e at which the RNAs from which they were derived begin, we see that corresponding female- and non-sex-specific RNAs have similar or (within experimental limits) identical 5' ends. These positions are coincident with, or very nearly coincident with, the 5' ends of cDNA clones e x a m i n e d in Boggs et al. (1987). We conclude that the tra promoter gives rise to transcripts that initiate over a region of about 70 nucleo-

The genetic analyses of Baker and Ridge (1980) and Cline (1979, 1984) suggested that Sx/controls tra and tra-2 and that these two genes control dsx. A molecular genetic analysis (Nagoshl et al., 1988) showed that Sx/+ is necessary for the production of the female-specific RNAs from tra and dsx, and that tra + and tra-2 ÷ are necessary for the production of the female-specific dsx RNA. These results show that Sxl is above tra, tra-2, and dsx in the hierarchy and that dsx is downstream of both tra and tra-2. Additional results have shown that tra-2 mutants do not alter tra expression, indicating that tra-2 does not regulate tra. These genetic and molecular data suggest that the regulatory pathway controlling sexual differentiation has a structure s~milar to one of the three alternatives shown in Figure 1. In the first alternative, tra regulates the activity of tra-2, leading to female express=on of dsx. Any as-yetunidentified genes that are part of the pathway e=ther act at some point in the pathway prior to tra, or they are regulated, directly or indirectly, by tra. In the second model, Sxl regulates the tra and tra-2 activities independently and the tra and tra-2 activities together cause dsx to be expressed m its female mode. In the third model, there are additional genes (for example, the ix gene or other as-yet-unidentified genes) that are regulated in a sex-specific m a n n e r by an element upstream of tra and tra-2 (for example, the X:A ratio or Sxl), but act downstream of or in conjunction with tra and tra-2 We shall refer to the first alternative as the "linear model7 while the second and third alternatives will be called "branched m o d e l s " If the pathway is linear, such that Sxl regulates tra and tra regulates, directly or indirectly, all of the other sexually regulated genes, then ectopic expression of the tra female RNA in males should induce female development. On the other hand, if either of the branched-pathway models is true, then ectopic expression of the tra female RNA in males should be insufficient to induce female development. We have previously isolated cDNA copies of the tra female RNA (Boggs et al., 1987). One of these cDNAs was fused downstream of an hsp70 promoter and its 5' untranslated region, and this construct (hs-tra-female) was introduced into the g e n o m e of wild-type flies by germ line transformation (Rubin and Spradling, 1982; Spradling and Rubin, 1982). As can be seen in Figure 3, XY flies that carry the hs-tra-female construct show at least partial female development, even when raised w~thout heat shocks. Some lines undergo complete female somatic differentiation. Such flies are invariably sterile, however, because their germ lines are not transformed to femaleness. These results show that expression of the tra female RNA in an otherwise male background is sufficient to induce female somatic differentiation. Any other genes that must act in a female-specific m a n n e r were either induced by the expression of tra in these flies, or they were already expressed but the products were inactive in the absence

Cell 890

Table 1. Interactions between Hs-tra-Female and Mutations in the Genes of the Sex Determinatton Hierarchy Gene

Specific Allehc Combinatton

Phenotype

Sxl Sxl Sxl tra-2 tra-2 tra-2 tra-2 dsx dsx dsx D dsx D ix

Sxl M#~.~*"#31SxFn#7,M#7 SxlM#1,frn#3/y Sxffm#7,M#11Y XX, tra-2BItra-2 B XY, tra-2eltra-2 e XX, tra-21tra-2 a XY; tra-21tra-2 B XX, dsxldsx XY, dsxldsx XX; dsxDI + XY; dsxDI + ixhx 2

Femate Female Female Male Male Male Male Intersex Intersex Intersex Intersex Intersex

All fhes carry a single copy of hs-tra-female inserted at 66E on the left arm of the thtrd chromosome Th~s inserted element g~ves rtse to full female development in both XX and XY flies m an otherwtse wild-type background or m a tra- background.

of the female-specific tra product. These results are consistent with a linear model for the regulatory hierarchy controlling sexual differentiatton and are not easily reconciled with models that place tra on one of multiple regulatory branches. If the regulatory pathway is linear, as shown in Figure 1, then the action of hs-tra-female should be capable of overriding mutations in genes for upstream regulatory functions such as Sxl. On the other hand, female differentiation induced by hs-tra-female should require the presence of functional copies of genes responsible for downstream functions such as tra-2, dsx, and ix. One line in which hs-tra-female gives rise to full female development contains the hs-tra-female transposon at position 66E on the left arm of the third chromosome. We have used standard genetic crosses to create fhes that contain this insert and are mutant for other genes of the sex determination hierarchy. The sexual phenotypes of these flies have been examined to determine if the above predictions are supported. The results of these studies are summarized in Table 1. If Sxl acts upstream of tra, then hs-tra-female should overcome the sex-transforming effects of Sxl mutations. Since null alleles of Sxl are normally lethal to females, we have exammed the effects of two Sxl alleles that in heteroallelic combination, lead to full male differentiation but allow the survival of some XX indtviduals (Cline, 1984). Previous experiments have shown that the wild-type tra gene is expressed in its male mode and not in its female mode in such flies (Nagoshi et al., 1988). XX flies of this type that also carry the hs-tra-female construct develop as females, as do their XY; Sxl brothers that carry hs-trafemale. The results are conststent with the linear model shown in Figure 1 and demonstrate that hs-tra-female is capable of inducing female differentiation in the absence of Sxl function. This shows that there are no genes acting downstream of tra that are absolutely necessary for female sexual differentiation and that are induced by the action of Sxl in a tra-independent manner. In the hnear model presented in Figure 1, tra-2 is shown

acting downstream of tra. If this is the case, then female differentiation induced by hs-tra-female should depend on wild-type tra-2 function. Although the action of hs-trafemale rules out the possibility that tra and tra-2 perform independent functions under mdependent control by Sxl, tt is possible that tra and tra-2 are on separate branches of a branched pathway if tra and tra-2 perform related functions such that overexpression of either one will substitute for the action of the other. If this is the case, then the female differentiation induced by hs-tra-female could result from simple overexpression of the tra product in the absence of tra-2 product. If this version of the branched model is true, then the female differentiation induced by hs-tra-female should be independent of mutations that eliminate tra-2 function. As a way of distinguishing between these possibilities, we have produced flies lacking tra-2 function but that carry the hs-tra-female insert. Both XX and XY flies of such constitution develop with male morphology (the tra-2 mutant phenotype) and not as females (the hs-tra-female phenotype), indicating that tra-2 acts downstream of tra and that tra-2 function is necessary for the induction of female differentiation by hs-tra-female. These results are consistent with the linear model and are not consLstent with a branched model in which tra and tra2 have partially interchangeable functions. Both genettc and molecular data suggest that tra exerts its effect on sexual differentiation via control of dsx. The dsx gene has two distinct functions, one for female differentiation and one for male differentiation. In the absence of both activities--the dsx null condition- both XX and XY fhes develop as identical intersexes (Baker and Ridge, 1980). If dsx is downstream of tra, as suggested by genetic (Baker and Ridge, 1980) and molecular (Nagoshi et al., 1988) evidence, and if hs-tra-female does not overcome some normal downstream controls, then the dsx- phenotype should be epistattc to the hs-tra-femaie-induced female differentiation. In genetic tests of this hypothesis, both XX and XY; hs-tra-female dsx flies developed as dsxlike intersexes. This is consistent with the idea that dsx acts downstream of tra and is necessary for the induction of female differentiation by tra. In addition to showing that dsx is necessary for hs-trafemale to induce female differentiation, it is also possible to demonstrate genetically that hs-tra-female causes the wild-type dsx gene to be expressed in tts female mode. Dominant mascuhntzing mutants of dsx such as d s x Mas and dsx D behave both genetically and molecularly as if they express the male dsx function constitutively and do not express the female function (Baker and Ridge, 1980; Baker and Belote, 1983; R. Nagoshi, K. Burtis, and B. Baker, personal communication). XX; dsxDI+ flies develop as intersexes because they express both the female dsx function from the wild-type gene and the male dsx function from the mutant gene. XY; dsxOl+ flies develop as males since they express the male dsx function from both the mutant and wild-type genes. If hs-tra-female acts by causing dsx to be expressed in its female mode rather than its male mode, then expression of hs-tra-female in XY; dsxOl+ fhes should lead to intersexual rather than male development since hs-tra-female will induce the wild-type copy of dsx to be expressed in tts female mode.

Male to Female Sex Changes 891

oJ

~o .,=

A ~

-,~

~

~ o

~B

Figure 4. Expression of dsx RNAs in Flies Carrying HS-tra-Female In wild-type females dsx gives rise to a sex-specific RNA of about 35 kb; ~nwdd-type males there are two sex-speclhc dsx RNAs, of sizes 3 9 and 3.0 kb (Baker et al, 1987, Burtls, Nagoshl, and Baker, personal communication; Nagosh~ et al., 1988). These RNAs have alternative 3' exons, which has enabled us to make probes that are specific for the male or female RNAs from cDNAs derived from these RNAs (A) An RNA blot of poly(A)-contalning RNA from Canton S (wild-type) female or XY; hs-tra-female flies, This blot was probed with a fragment from the 3' end of a dsx female-speclhc cDNA clone. Both lanes show the characteristic dsx female RNA (arrow) Reprobmg of this blot w~th a ribosomal protein RNA-specific probe (rp49; O'Connell and Rosbash, 1984) shows that there ~smore RNA in the Canton S female lane than in the bs-tra-female lane. (B) An RNA blot of poly(A)-contaimng RNA from XY; hs-tra-female or Canton S (wTId-type)male flies. Th~s blot was probed with a fragment from the 3' end of a dsx male-specific cDNA clone. The wdd-type male shows the characteristic dsx male RNAs (arrows) There is no signal from the XY; hs-tra-female lane with this probe The hs-tra-female lane of this blot did show hybnd~zat~onto the dsx female RNA when this blot was reprobed with a dsx female-specific probe (not shown) Reprobmg of the same blot w~th an rp49 probe (O'Connell and Rosbash, 1984) shows that both lanes have essentially equal levels of hybridizable RNA. The blots in (A) and (B) were derived from different parts of the same gel

In tests of this hypothesis, both XX and X Y flies that contain hs-tra-female and are heterozygous for dsx ÷ and dsx D develop as intersexes, indicating an induction of dsx female function in X Y flies (Table 1). These intersexes are indistinguishable from XX; dsxOl+ intersexes. This is a particulary informative test because the p h e n o t y p e of the X Y double mutant is one that ~s not characteristic of either mutant gene alone, but is a predicted result of the genetic model. We have also tested, at the molecular level, if hs-trafemale leads to a shift in dsx expression from its male mode to its female mode. dsx normally expresses either female- or male-specific mRNAs. These have c o m m o n 5' regions but differ in the splicing pattern of their 3' exons and the choice of poly(A) addition sites (Baker et al., 1987; K. Burtis, R. Nagoshi, and B. Baker, personal c o m m u n i c a tion; Nagoshi et al., 1988). We have used sex-specific regions of cDNA clones derived from these RNAs as probes to determine both if the dsx female RNAs are expressed and if the dsx male RNAs are repressed in XY; hs-tra-female flies. As can be seen in Figure 4, XY; hs-trafemale flies express substantial amounts of the dsx fe-

male RNA and very little of the dsx male RNAs. This is consistent with the female sexual differentiation of these flies but is not consistent with their sex c h r o m o s o m e constitution. This is a direct molecular demonstration that expression of the tra female product causes dsx to be expressed in its female mode and not m its male mode. The ix gene is believed to act downstream of or in conjunction with the dsx female function (Baker and Ridge, 1980; Nagoshi et al., 1988). It is necessary for female differentiation but is not required for male differentiation. As described above for tra-2, the p h e n o t y p e of hs-tra-female flies rules out the possibility that tra and ix have independently regulated, n o n o v e r l a p p l n g activities. On the other hand, it is possible to draw models in which ix is on a separate branch of the sex determination hierarchy from tra but in which the two genes have partially overlapping activities. According to this model, overexpression of tra from hs-tra-female results in a substitution of tra female function for ix function and results in female differentiation. If this model is correct, then female differentiation induced by hs-tra-female should be independent of ix function. On the other hand, if ix acts downstream of tra in a linear pathway, then ix-; hs-tra-female flies should develop as intersexes. We have used genetic crosses to produce fhes that are tx-; hs-tra-female. These flies develop as intersexes, consistent with the idea that ix acts downstream of tra to allow female differentiation (Table 1).

Does tra Self-Regulate? Molecular data show that tra function alters (directly or indirectly) the processing of the dsx RNAs. One can imagine that the female product of tra also acts to stab~hze the female differentiation pattern by altering the processing of the tra precursor RNA or that the tra product acts in conjunction with the Sxl product to bring about femalespecific processing of the tra precursor RNAs The genetic data of Sanchez and NSthiger (1982) showing that Sxlsomatic cell clones develop as male even if they are induced after a time at which the female-specific tra RNA is known to be present (Boggs et al., 1987), coupled with the results of this paper showing that the regulatory hierarchy is linear, make it highly unlikely that tra product is capable of independently inducing female-specific sphcing of the tra precursor RNAs. Our preliminary blots with RNA from XY; hs-tra-female flies are consistent with this idea (unpublished). We have also been able to demonstrate that tra + is not necessary for the proper expression of the tra female RNA. As shown in Figure 5, XX flies carrying an ethyl m e t h a n e s u l f o n a t e - i n d u c e d tra mutation, tra v2 (a gift from F. Michael Hoffmann), and as a result develop as males, still express the female-specific tra RNA from the mutant gene. This shows that tra ÷ is not necessary for femalespecific splicing of the tra RNAs.

The Non-Sex-Specific RNA Is Not Necessary for Male or Female Differentiation Our genetic, molecular, and transformation results show that the tra female RNA has a critical function in the induction of female differentiation, The non-sex-specific RNA, on the other hand, has no long open reading frame and

Cell 892

O ~J

,i i ' ' i'~ Figure 5. tra Is Not Necessaryfor Its Own Sphcmg An RNA blot of poly(A)-contamingRNA from wild-typeof tra v2 mutant fhes ts shown• The blot was probed with a rock-translatedtra cDNA probe Reprobmgwith an rp49 (O'Connelland Rosbash. 1984)probe indicates that differences m hybndtzatlon intensity correspond to differences in RNA amount. The tra v2 RNA is actually a mixture of RNA from traV21Df(3L)stl7 and traV21trafhes In both casesthe non-tra v2 chromosomeis knownto be deletedfor the tra gene (seetext, and also McKeownet al, 1987)•In all casesthe sex chromosomesweremarked in such a waythat XX and XY fhescould be dtstmguishedwithout reference to thetr sexual dlfferenttationpatterns•All fhes used to produce the "tra v~' RNA had male morphology.The band resultmgfrom hybridizationto the small antisenseRNA derivedfromthe tra mutantchromosome (seetext)was cropped The female RNA m the "tra v~' lane comlgrateswith the wild-typetra female RNA in other lanesof thts and other blots

appears as if it might be without function, yet it is present in both sexes. We wished to determine if this RNA has any function in males or females. Previous results (Belote et al., 1985; McKeown et al., 1986, 1987; Butler et al., 1986) showed that the original spontaneous tra mutation isolated by Sturtevant (1945) is a deletion that appears to remove most or all of the tra gene. We have isolated DNA containing this deletion and sequenced both of its endpoints. This analysis shows that the tra deletion removes all DNA from positions - 6 9 to +1018 (coordinates relative to the 5' end of the longest cDNA sequenced by Boggs et al. [1987]). This removes all but the 3' 38 nucleotides of tra and leaves none of the potential peptide-coding sequences. DNA blotting using a full-length tra non-sex-specific cDNA as probe reveals no hybridization to DNA from traltra flies under conditions in which there is intense hybridization to tra + DNA in adjacent lanes containing DNA from tra + flies. In addition, XX or XY flies homozygous for this tra allele produce neither the female-specific nor the non-sex-specific tra RNAs. However, it is likely that there is a small fragment of DNA derived from tra + transposed to a nearby location in the t r a - chromosome, since in RNA derived from t r a l + and traltra flies there is an RNA of about 400 bases that is antisense to tra RNA and hybrid=zes to cloned tra cDNA probes. We believe that this RNA is not functional since it is derived from the opposite strand from the tra + RNAs.

We conclude the following: first, the original tra mutation is unable to produce either of the tra + RNAs; and second, this allele appears to be a true null allele for both the non-sex-specific and female-specific RNAs and their potential functions. XY flies homozygous for this allele are male, viable, and fertile, indicating that there is no necessary function of the non-sex-specific tra RNA in males. To test if the non-sex-specific RNA has any necessary function in females, we have constructed flies that are deleted for tra but that carry hs-tra-female. Despite their inability to make the non-sex-specific tra RNA, both XX and XY flies of this type develop as females. This indicates that the non-sex-specific RNA is either without funct=on in female differentiation or that it has an extremely subtle function that is difficult to detect in the laboratory. The only caveat to this interpretation is that the XX flies of this constitutLon that have been examined have been sterile. The cause of this sterility is not obvious, as both internal and external morphology appear female and flies of this genotype are courted and mate as females. Moreover, these females store sperm and the sperm remain motile for several days, as in wild-type females. It is conceivable that the non-sex-specific RNA is necessary for some aspect of fertility, e.g., egg laying behavior. We have tested this possibility by creating flies that are XX; t r a - and carry both the hs-tra-female construct and an hs-tra-non-sex-specific construct (see below). These flies are indistinguishable from XX; t r a - hs-tra-fernale flies in terms of morphology and egg laying behavior; i.e., addition of the tra non-sex-specific RNA does not restore fertility. An alternative we consider more likely is that the level of expression of hs-tra-female in some critical tissue is insufficient for female fertility. This could be the result of using the heterologous hsp70 promoter, or it could be due to genomic position effects in our transformed lines.

The Non-Sex-Specific RNA Is Not a Precursor of the Female-Specific RNA It is possible, based on the structure of the tra RNAs, that the non-sex-specific RNA serves as a precursor for the female-specific RNA. All of the sequences found in the female RNA are found in the non-sex-specific RNA. Secondary splicing of the non-sex-specific RNA could lead to the production of the female RNA. Since the non-sexspecific RNA has lost the standard splice donor site at the 5' side of the first intron, this model suggests that the direct or indirect action of Sx/+ is to allow the junction produced by non-sex-specific processing to be used as a nonstandard sphce donor site. To test this possibility, we have inserted a non-sex-specific cDNA, extending from position +1 of Boggs et al. (1987) to the poly(A) tail, downstream of the same heat shock promoter used to express hs-tra-female. A similar construct using genomic instead of cDNA sequences is known to give tra + activity in females (Boggs et al., 1987). If the non-sex-specific RNA is a precursor of the female-specific RNA, then this hs-tranon-sex-specific construct should be capable of complementing tra +. Using germ line transformation and the appropriate genetic crosses, we have produced XX; hstra-non-sex-specific; t r a - individuals. These flies have full

Male to FemaleSex Changes 893

male morphology and are indistinguishable from their XY or tra- sibs. There appears to be no complementation of tra- by the hs-non-sex-specific construct. The simplest explanation of these results is that the non-sex-specific RNA is not a precursor of the female-specific RNA. Discussion

Usmg genetic means alone it is difficult to determine the order in which a series of positively acting regulatory elements act, since null alleles of any one of the genes in the pathway have similar phenotypes and loss of two sequential functions is no worse than the loss of one. In addition, it is not possible to determine if two positively acting elements are on a single pathway or are parts of a branched pathway in which both branches are necessary for any pathway function. It has been possible to order some of the positively acting elements in Drosophila sex determination. For example, Sxl controls processes other than sex and can be placed above tra and tra-2 on the basis of, first, the additional processes affected by Sxl mutations that are not affected by tra and tra-2 mutations (Baker and BeIote, 1983; Cline, 1984) and second, dominant Sxl mutations that transform male tissue to female tissue and appear to depend on tra ÷ for activity (Cline, 1979). The recent isolation of some of the genes of the sex determination hierarchy has made it possible to use molecular and genetic techniques to study the order in which the genes controlling sex interact. Since these studies can use the expression of particular RNA species as a phenotype, it is possible to determine the order in which two genes with similar mutant phenotypes act. One such set of studies showed that tra is downstream of Sxl but is either upstream of or on a separate branch from tra-2 (Nagoshi et al., 1988). These studies also showed that tra is upstream of dsx. These studies did not determine if tra-2 ts regulated by tra or if the pathway ts branched. Studies of this type can only involve genes previously identified as a result of mutation. As a result, it was not determined if there are additional genes present on other, unidentified branches of the hierarchy. We have used the ectopic expression of a tra female cDNA as a way of approaching the question of branched versus linear pathways. Two factors have made this analysis possible. First, the expression or lack of expression of tra appears to have no effect on the viability of XX or XY flies; thus viability has not been altered by ectopic expression of the tra female RNA. Second, the low levels of tra expression necessary for tra function have allowed us to use the constitutive expression of a heat shock promoter to drive tra female expression. It has not been necessary to induce tra expression via periodic heat shock treatments. This particular kind of analysis is similar to the use of dominant gain-of-function mutants, which appear to express a wild-type product m an inappropriate manner. For example, the hs-tra-female construct is formally analogous to the dominant Sxl allele Sxl M#~, and our genettc analysis is in many ways similar to that of Cline (1978, 1979) using this Sxl allele. These studies with Sxl M#1 pro-

vide the strongest purely genetic evidence that Sxl acts upstream of tra. Our approach has an advantage over the use of spontaneous dominant mutations in that it starts with a single gene product of known structure and a promoter that has been characterized in terms of tissue distribution and expression characteristics. In addition, our approach can be used for any gene and does not require either fortuitous isolation of dominant alleles or specific screens that make the detection of dominant alleles more efficient. Our results suggest that the genes of the sex determination hierarchy that are regulated in a sex-specific manner are arranged in a linear array, at least at the level of the hierarchy containing tra. Our results appear to rule out the existence of genes controlling somatic sexual differentiation that are regulated in a sex-specific manner by either the X:A ratio or Sxl and that are on separate branches of the hierarchy from tra. Specifically, our results show that if tra-2 and ix are regulated in a sex-specific manner, then thts regulation is dependent on the function of the tra female product. Our results cannot rule out the existence of genes that are expressed constitutively but are required only for processes that are sex-specific. For example, there is nothing in our data to show that the tra-2 or ix gene (or any other untdentified genes) are not expressed at all times and in both sexes and that their products are necessary for the specific functions of female sexual differentiation. What our data do imply is that the sexually regulated genes either act between the X:A ratio and tra, as Sxl does, or they are dependent on tra, as dsx is. Genetic data (Sanchez and N6thiger, 1982) coupled with our analysis of the hs-tra-female construct suggest that the tra female product is not sufficient, in the absence of the Sxl female activity, to induce female-type splicing of tra precursor RNAs. One ethyl methanesulfonate-induced tra allele has allowed us to demonstrate that the Sxl female activity does not requtre functional tra product for the production of the tra female-specific RNA. We have not yet addressed the question of whether tra female suppresses its own production, although the results with the tra v2 point mutant suggest that it does net. Specifically, if the tra female product represses the production of the female-specific RNA, then lack of female function, as in tra v2, should lead to a higher level of female RNA at the expense of the non-sex-specific RNA. This does not appear to be the case. The sensitivity of tra transformants to gene position and expressively would also seem to argue against self-repression, since in at least one case (McKeown et al., 1987) a partially complementing tra transformant produced both tra RNAs in about the wildtype ratio, but at reduced levels. Our results suggest that the non-sex-specific RNA from tra is without function. It appears to be dispensable in both males and females and it does not appear to act as a precursor for the female-specific RNA. The reasons for the production of this RNA in females are unclear. One possible explanation is that the tra precursor RNA is an inefficient substrate for action by the female-specific splicing activity, which is dependent on Sxl. If so, some precur-

Cell 894

sors m i g h t be spliced in a n o n - s e x - s p e c i f i c m a n n e r before f e m a l e - s p e c i f i c splicing occurs. T h e fact that e x p r e s s i o n of the tra f e m a l e p r o d u c t leads to f e m a l e d i f f e r e n t i a t i o n

has a n u m b e r

of useful ex-

p e r i m e n t a l c o n s e q u e n c e s . First, it indicates that it might

marked "adverbsement" m accordance w~th 18 U S C. Section 1734 solely to indicate th~s fact Received February 8, 1988, revised March 22, 1988.

References

be possible to use f e m a l e d i f f e r e n t i a t i o n of X Y flies as a f u n c t i o n a l b i o a s s a y for m u t a t i o n s within t h e tra g e n e itself that lead to c o n s t i t u t i v e p r o d u c t i o n of the tra f e m a l e RNA. This test s h o u l d w o r k for either in vivo or in vitro synthesized mutations. S e c o n d , the ability to p r o d u c e X Y flies with s o m a t i c f e m a l e c h a r a c t e r i s t i c s might be used to es-

Baker, B S, and Belote, J. M. (1983). Sex determination and dosage compensation in Drosophila melanogaster. Annu. Rev Genet. 17, 345-397 Baker, B. S., and Ridge, K (1980) Sex and the single cell: on the action of major loci affecting sex determination in Drosophla melanogaster Genetics 94, 383-423

zatKon of the g e r m line.

Baker, B S, Nagoshi, R. N, and Burt~s, K C (1987). Molecular genetic aspects of sex determination in Drosophila. Bioessays 6, 66-77

Experimental Procedures

Belote, J M, McKeown, M , Andrew, D. J., Scott, T. N, Wolfner, M F, and Baker, B. S. (1985). Control of sexual differentiation in Drosophda melanogaster. Cold Spring Harbor Symp Quant B~ol 50, 605-614

tablish a s c r e e n for d o m i n a n t m u t a t i o n s that c a u s e femini-

Standard Techniques All standard molecular biological techniques involved in DNA or RNA preparation, blotting, restriction digestion, plasmid construction, or hybridization were essentially as in Manlatls et al. (1982) or as m McKeown et al (1987) or Boggs et al. (1987). Primer Extension Primer extension was performed essentially as described elsewhere (Jones et al., 1985). An ohgonucleotlde 18 bases long was hybridized to a single-stranded plasmld containing genomic DNA from tra and extended using the Klenow fragment of DNA polymerase I (Bethesda Research Laboratories) and [~-32p]dCTP. The resulting duplex DNA was cut with Ncol, and the labeled, single-stranded 122-mer was purified by gel electrophoresis. This labeled DNA was annealed to 45 p.g of RNA and extended using 330 ~M nonrad~oactive dNTPs and 10 U of AMV reverse transcriptase (L=fe Sciences) for 1 hr at 37°C Total RNA was used since m our hands and others' (K. Jones, personal commumcation), the resolut=on and sens~tw=tywere as great or greater than with poly(A) + RNA The products were size-fractlonated on polyacrylamlde gels containing urea. S1 Nuclease Mapping $1 nuclease mapping was as described in Boggs et al (1987) To increase the length of homology between the probe and the tra RNAs, a special construct was made ~n which the 5' region, including the 5' untranscnbed region, of tra was fused to a non-sex-specific tra cDNA. Probes were then synthesized using the same primer used in the pnmer extension analyses. It was thus poss=ble to compare the patterns of $1 protect=on to the patterns of primer extension products. Drosophila Culture and Transformation Nonselected crosses and most stocks were kept on standard cornmeal-molasses-agar reed,urn at 25°C or at room temperature. Most mutat=ons are listed =n Lmdsley and Grell (1968) or in LIndsley and Zimm (1985, 1986, 1967, and unpublished) The trav2 allele was isolated by F Michael Hoffmann as part of a screen for ethyl methanesulfonate-mduced mutations in chromosome region 73A (personal communication). Transformation was performed as described in Boggs et al. (1987) using a nontransposlng P helper vector (Karess and Rub=n, 1984)

Boggs, R T., Gregor, P., Idnss, S, Belote, J M, and McKeown, M (1987). Regulation of sexual differentiation in D melanogaster via alternative splicing of RNA from the transformer gene Cell 50, 739-747. Butler, B, PLrrotta, V., Irmlnger-Fmger, I, and N6thiger, R. (1986) The sex-determining gene tra of Drosophila: molecular cloning and transformation studies EMBO J. 5, 3607-3613. Cllne, T. W. (1976) A sex-specific, temperature sens~ttve maternal effect of the daughterless mutation of Drosophila melanogaster. Genetics 84, 723-742 Chne, T W (1978). Two closely-linked mutations in Drosophila melanogaster that are lethal to opposite sexes and interact with daughterless Genetics 90, 683-698 Chne, T. W. (1979) A male-specific lethal mutation m Drosophila melanogaster that transforms sex Dev B~ol 72, 266-275. Cline, T W. (1980). Maternal and zygotic sex-specific gene interactions =n DrosopMa melanogaster. Genetics 96, 903-926 Cllne, T W. (1983) The interaction between daughterless and Sexlethal m trlplotds a lethal sex-transforming maternal effect mutation linking sex determination and dosage compensation in Drosophila melanogaster Dev Biol 95, 260-274 Cline, T W (1984) Autoregulatory functioning of a Drosophila gene product that establishes and maintains the sexually determined state Genet=cs 107, 231-277. Chne, T W (1985). Primary events =n the determlnat=on of sex m Drosophila melanogaster In Origin and Evolut=on of Sex, H. O. Halvorson and A Monroy, eds (New York' Alan R Liss), pp 301-327. Chne, T W. (1986). A female-specific lethal lesion in an X-linked posltwe regulator of the Drosophila sex determination gene, Sex-lethal Genetics 113, 641-663. Cronm~ller, C., and Chne, T. W (1987). The Drosophila sex determination gene daughterless has different functions in the germ hne versus the soma. Cell 48, 479-487, Hahn, S, Hoar, E. T., and Guarente, L. (1985). Each of three "TATA elements" specifies a subset of the transcription m~tlatlon sites at the CYC1 promoter of Saccharomyces cereveslae Proc Natl. Acad. Sci USA 82, 8562-8566 Jones, K. A., Yamamoto, K. R., and Tjian, R. (1985) Two distinct transcription factors bind to the HSV thymldme klnase promoter in vitro Cell 42, 559-572.

Acknowledgments

Karess, R E, and Rubln, G M (1984). Analysis of P transposable element funct=ons in Drosoph=la Cell 38, 135-146

We thank Barbara Taylor for examination of the sexual phenotypes of some of the flies in th~s study; Kevln Nash, Anthony Manly, and Karen Kaczman Darnel for technical assistance; and Katherine Jones for advice on primer extension experiments. We also thank Bruce Baker and coworkers for a gift of dsx male and female cDNAs for use as probes. This work was supported by grants from the National Institutes of Health to M. M. and J M B M.M. is a Pew Scholar in the Biomedical Sciences. R. T B. was supported by an NIH postdoctoral training grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby

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Male to Female Sex Changes 895

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