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Gonadal Dysgenesis Reveals Sexual Dimorphism in the Embryonic

Germline of Drosophila

Grace Wei,' Brian Oliver2 and Anthony

P. Mahowald'

Department of Genetics, Case Western Reserue University, Cleveland, Ohio 44106 Manuscript received March 26, 199 1

Accepted for publication May 3 1, 199 1

ABSTRACT

In hybrid dysgenesis, sterility can occur in both males and females. At 27.5", however, we found that P element-induced germline death was restricted to females. This sex-specific gonadal dysgenesis

(GD) is complete by the first larval instar stage. As such, GD at 27.5" reveals the sexually dimorphic character of the embryonic germline. The only other known dimorphic trait of the embryonic germline is the requirement for ovo. ovo is required for germline development in females only and has been implicated in germline sex determination. Dominant mutations of ovo partially suppressed female GD. Although embryonic germ cells are undifferentiated and morphologically indistinguisha- ble between males and females, the functional dimorphism seen in ovo requirement and GD at 27.5"

indicates that sexual identity in Drosophila germ cells is established in embryogenesis.

D

ROSOPHILA melanogaster is a sexually reproduc- ing species in which adults are also sexually dimorphic in both the soma and germline. Somati- cally, external differences in bristle pattern, abdomi- nal pigmentation, and genitalia distinguish males and females. Extensive genetic and molecular studies have uncovered the pathway and mechanism of somatic cell sex determination in Drosophila (reviewed in SLEE and BOWNES 1990; BAKER 1989). In the soma, sex is specified by the ratio of X chromosomes to autosomes (X:A). Thus, a cell which is XY has an X:A ratio of 0.5, and will develop as male; a cell which is X X has an X:A ratio of 1 and will develop as female. Somatic cells assess their X:A ratio and become sexually determined at the beginning of embryogenesis (SANCHEZ and NO-

THIGER 1983). Hence somatic cells are sexually deter-

mined well before the sexual differentiation of tissues, which occurs in larval and pupal stages (WIESCHAUS and NOTHIGER 1982; SANCHEZ and Nothiger 1983). T h e X:A ratio is a primary signal which must be translated into a somatic sexual identity through the sex specific splicing of Sex-lethal (Sxl), transformer ( t r a ) , and doublesex ( d m ) gene products.

Adult germline dimorphism consists of sperm pro- duction in males and egg production in females. How this dimorphism is established is not as fully charac- terized as for the soma, but some determinants are known. In part, the sex of somatic tissues influences the competence of germ cells to differentiate com- pletely (STEINMANN-ZWICKY, SCHMID and NOTHIGER

1989; NOTHIGER et al. 1989; for Drosophila simulans,

I Present address: Department of Molecular Genetics and Cell Biology,

' Present address: Department of Biological Sciences, Stanford University, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637. Stanford, California 94305.

Genetics 1 2 9 203-210 (September, 1991)

DOBZHANSKY 193 1). An example of the somatic influ- ence is the ability of the XY gonad to induce partial sexual transformation of X X germ cells. T h e X to autosome balance has a cell autonomous role in nor- mal gametogenesis. For example, 2X:3A germ cells fail to differentiate in a chromosomally female (X:A = 1) soma (SCHUPBACH 1985). Germline sex is also reg- ulated by a downstream pathway distinct from the somatic sex determination pathway. T h e majority of somatic sex determination genes are not required in germ cells (MARSH and WIESCHAUS 1978; SCHUPBACH

1982). Candidate germline sex determination genes include ovo, sansfille (snf), Sxl, and ovarian tumor (otu) (SCHUPBACH 1985; OLIVER, PERRIMON and MAHOW-

ALD 1988; STEINMANN-ZWICKY 1988; OLIVER, PAULI

and MAHOWALD 1990a,b; reviewed in PAULI and MA-

Studies on the establishment of germ cell sex have relied primarily on detecting differentiation in adult gonads (CLINE 1984; SCHUPBACH 1985; STEINMANN-

ZWICKY, SCHMID and NOTHIGER 1989; NOTHIGER et al.1989; OLIVER, PAULI and MAHOWALD 1990a,b). T h e earliest stages at which germ cell differentiation is detectable are the first larval instar stage in males (GARCIA-BELLIDO 1964) and the second day of pupa- tion in females (KING 1970). First instar testes contain primary spermatocytes, and pupal ovaries contain de- veloping egg chambers. Prior to differentiation, XY and X X gonia1 cells are morphologically very similar. Given the lack of morphological markers for the un- differentiated germline, it is uncertain when germ cells actually become determined as male or female.

Are germ cells sexually determined before game- togenesis occurs? While there is no definitive evidence

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A. Mahowald

for early sex determination in Drosophila germ cells, there are some dimorphic germline traits which occur before gametogenesis is evident. T h e requirement for ouo activity is one such trait. ovo activity is probably essential throughout female germline development

(PERRIMON 1984) and is first required before gastru- lation (OLIVER, PERRIMON and MAHOWALD 1987).

Weak or dominant ovo mutations allow egg chambers to develop, but functional gametes are never formed

(BUSSON et al. 1983; OLIVER, PERRIMON and MAHOW-

ALD 1987). ovo does not appear to be required for male fertility (BUSSON et al. 1983; OLIVER, PERRIMON

and MAHOWALD 1987). Thus, ouo always gives a germ- line phenotype which is sex-specific.

Another dimorphic trait is sensitivity to sterilization from P-M hybrid dysgenesis. Females are generally more sensitive to sterilization with increasing temper- ature. P-"induced sterility, o r gonadal dysgenesis (GD), describes the germline death in hybrid progeny from a cross of M strain females to P strain males. P strains harbor P transposable elements while M strains d o not. GD is one of several germline-dependent traits in hybrid dysgenesis (KIDWELL and NOVY 1979; EN-

GELS and PRESTON 1979; KIDWELL, KIDWELL and

SVED 1977). Hybrid dysgenesis is germline dependent because production of P transposase depends upon a splicing reaction that only occurs in germ cells (LASKI, RIO and RUBIN 1986). We have investigated this dif- ferential sensitivity to GD because it fulfills several key criteria for a trait to use in examining early germline dimorphism. GD is a phenomenon which is germ cell autonomous (NIKI 1986), has a temperature sensitive period beginning in embryogenesis (ENGELS

and PRESTON 1979), and shows sex specificity at 27.5"

(this study).

We found that GD at 27.5" is characterized by germline loss which was restricted to females and was complete by the first instar stage. T h e timing and sex specificity of germline death indicates sexual dimorph- ism has been established in embryogenesis. We have also investigated the possible basis of the sex bias in

GD. One explanation for female sensitivity is the potentially greater number of P elements inherited by daughters of an M X P cross via the paternal P strain

X chromosome. If this explanation is true, then forc- ing sons to inherit extra P elements could result in male GD at 27.5". Alternatively, female GD could reflect cellular properties unique to female germ cell development. If this latter explanation is true, then the incidence of female GD should be reduced by mutations in genes which establish the female char- acter of X X germ cells. We have tested the alternative hypotheses and found female GD was not simply a reflection of P element dose. Rather, female GD is a conditionally dimorphic trait which is influenced by ouo activity.

ycvvfcar

ycvvfcar

21.5OC

ycvvfcar

+

ycvvfcar

7

# scored as y+ = 102 #scoredasy=51

# actual females = 98 # actual males = 46

Accuracy 96% 90%

FIGURE 1.-Method and accuracy of sexing first instar larvae using mouth-hook pigmentation to score yellow (y). As yellow is X - linked, only sons should have the yellow phenotype. Larvae were sorted, then allowed to develop to adulthood to determine the accuracy of scoring. The yellow phenotype was used in determining male ZIS. female first instar larval germ cell numbers for the data in

Table 3.

MATERIALS AND METHODS

The M strains used in this study were Oregon R (Ore-R),

y v f car, and DjTl)ovof4 vz4/FM6. The P strains were )r2 and

7r2 with C(l)DX, y f / Y and either ovoD2 vZ4 or 0voD3 vZ4 (referred to in the text as either ovoDZ 7r4 or ovoD3 7r2, respectively). The Of (l)ovoJ4 vZ4 stock was obtained from M. STEINMANN- ZWICKY (1988); descriptions of mutations in other stocks are found in LINDSLEY and ZIMM (1985, 1990). Virgin females were mated with males at 25" overnight. Flies were then transferred to either 20", 27.5" or 29" f 0.5" and allowed to lay eggs for 24 hr prior to the first embryo collection.

For Ore-R crosses to 7r2 males, the fertility of the progeny was tested by mating single adult flies with either virgin Ore-R females or Ore-R males. Embryos were collected on agar-molasses medium. Embryos were stained for germ cells using anti-vasa antibody (LASKO and ASHBURNER 1990) or embedded in plastic for histological sectioning. Older em- bryos, ie., stage 16/+, were examined for germ cells by serial sections. Subsets of the collected embryos were trans- ferred to standard Drosophila medium and grown to adult- hood at the same temperature at which they were collected. For y v f car crosses to 7r2 males, the first instar larvae

were scored for the yellow "mouth part" phenotype and sorted. The X linkage of this marker allows the sex of the individuals to be scored (see Figure 1). Sexed larvae were split into two groups: one group was fixed immediately in Carnoy's solution (60% ethanol, 30% acetic acid, 10% chlo- roform), embedded in JB4 plastic, sectioned, and stained for histological examination; the second group was allowed

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nads were also fixed (2% glutaraldehyde in PBS), embedded in plastic, and sectioned for histological examination. Larvae classified as lacking germ cells (see Table 3) included those in which a gonad could not be found. In documenting the effect of polar irradiation on gonadal development, ABOIM (1945) noted that loss of the germline leaves first instar larvae with minute gonads comparable in size to embryonic gonads. In examining hybrid third instar larvae, SCHAEFFER, KIDWELL and FAUSTO-STERLING (1 979) also could not locate gonads in most female larvae raised above 25 "

.

Data from control larvae (Table 3, cross 2) strongly suggest that our inability to find gonads in some dysgenic females reflects lack of germ cells. Gonads could be found in all the control larvae, and germ cell numbers were determined.

Antibody staining: Embryos were washed free of yeast with ice-cold 0.02% Triton followed by cold 70% ethanol. Fresh ethano1:bleach (1 : 1) was used to dechorionate at room temperature. After rinsing again with 70% ethanol, embryos were fixed by adding two volumes of 10% formaldehyde/ buffer B (1 00 mM K . HB. P04/50 mM K . Cl/l4 mM

.

Na.

c1/

2 mM Mg.Cl, pH 7.2-7.4) and formaldehyde-saturated heptane to 4 X volume of the aqueous phase. Embryos were shaken moderately during the 15-20-min fixation step. The aqueous phase was then removed and an equal volume of methanol was added to the heptane phase containing the embryos. Embryos were devitellinized by shaking the em- bryos vigorously in the heptane/methanol mix (1 5-20 min) and then in 100% methanol (5-10 min). Embryos were then washed and allowed to equilibrate in PBS, pH 7.4, for 1 hr at 4'. After blocking overnight at 4" in incubation buffer (5% bovine serum albumin (BSA)/O.l% Triton/PBS), anti- vasa antibody (LASKO and ASHBURNER 1990) diluted in fresh incubation buffer was added and embryos were incubated overnight at 4". Embryos were washed of the primary antibody with several changes of PBS at 4" over a 24-hr period. Fluorescein isothiocyanate-conjugated anti-mouse secondary antibody preabsorbed to wild-type embryos was then incubated with the experimental embryos overnight at 4 " . Embryos were then washed again at 4" with several changes of PBS over a 12-16-hr period. Finally, embryos were mounted in ethano1:glycerol (1: 1) and stored at 4". All 4" incubations were performed with continuous agita- tion.

RESULTS

Temperature sensitivity of GD: GD occurs in the hybrid offspring generated by M strain females crossed to P strain males. Hybrid F1 from P strain females crossed to M strain males do not suffer GD (KIDWELL, KIDWELL and SVED 1977). Previous studies have determined that susceptibility to hybrid dysge- nesis depends on maternal genomic and cytoplasmic factors (ENGELS 1979). T h e incidence of GD is also greatly affected by temperature. Below 2 1 O , the levels

of P-"induced sterility are negligible. T h e incidence

of GD sterility consistently increases in both sexes as the temperature is raised from 21 O to 29". Above

24", the incidence of GD is greater among females

than males (KIDWELL and NOVY 1979; reviewed in ENCELS 1989). In temperature trials, we saw an ex- pected background level of sterility from a dysgenic cross at 20°, and greatly increased sterility levels in both sexes when the flies were reared at 29" (Table

1). At 27.5", we found GD-induced sterility to be

essentially female specific (Table 1, crosses 2 and 4). Thus GD is a conditionally dimorphic trait.

Developmental profile of GD: Since GD at 27.5

"

is a sexually dimorphic trait, the time of germ cell death should indicate when germline dimorphism is established during development. To determine the time of germline loss, we scored germ cell number in embryos and sexed larvae. Early embryos were stained with anti-vasa antibody (LASKO and ASHBURNER 1990)

to detect pole cells, the precursors of the germline. As noted by NIKI and CHICUSA (1 986), pole cell for- mation in hybrid dysgenic embryos appears normal. We found 24 .t 1 pole cells at the cellular blastoderm stage (ie., stage 5) in progeny of dysgenic crosses (Oregon-R x T Z ) and 23 .t 1 in progeny of control ( T Z

x Oregon-R) crosses at 27.5". Before stage 16, all embryonic gonads stained positive for germ cells in both dysgenic and control crosses (Table 2). At stage

16, embryos begin to secrete cuticle and have almost completed all the steps in organogenesis and morpho- genesis required to form the first instar larva (stage

16 is equivalent to 13- 16 hr of development at 25 O ,

see CAMPOS-ORTECA and HARTENSTEIN 1985 for stag- ing).

Older, cuticularized embryos (ie., L stage 16) were examined by serial sections for germ cells. Roughly one fifth of stage 16+ embryos from a dysgenic cross were devoid of germ cells at 27.5". Another quarter of their siblings had reduced numbers of germ cells (Table 2). Embryos with reduced numbers of germ cells, also showed necrosis of these remaining germ cells. In short, close to half the progeny of a dysgenic cross at 27.5" have already lost their germline com- pletely or exhibit only a few dying germ cells by the end of embryogenesis. Although embryonic sex was not scored, dysgenic embryos were most likely female given that pole cells are formed only once in devel- opment and adult sterility is female specific.

T o test the notion that the embryonic population showing dysgenesis was indeed female, we scored both sex and dysgenesis in first instar larvae. Figure 1 (see MATERIALS AND METHODS) outlines our method for larval sexing, and our accuracy of sexing. Table 3

shows that most of the hybrid 1st instar population lacking germ cells are female (cross 1). Females from the dysgenic cross which had only 1-5 germ cells often showed necrotic cells in their gonads (Figure 2).

T h e data from sexed larvae indicate that the dysgenic half of the embryonic population was indeed female. T h e embryonic origin of female specific sterility es- tablishes GD as a functional marker of early germline dimorphism.

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G. Wei, B. Oliver and A. P. Mahowald

TABLE 1

Temperature sensitivity and female specificity of GD

Female progeny Male progeny

Cross

Temperature Percent

( " C ) Parental cross. No. scored dysgenicb No. scored dysgenicb

Percent

1 20.0 Ore-R X 7r2 100 4 88 12

2 27.5 Ore-R X 7r2 100 86 87 2

3 27.5 7r2 X Ore-R 144 4 119 1

4 27.5 y m v f c u r x 7 r 2 118 90 66 0

5 27.5 C( I)DX, yf x 7r2 62 77 34 0

6 29.0 Ore-R X r 2 100 98 92 58

a All crosses are shown as maternal genotype X paternal genotype.

Adult flies were scored for fertility and the presence of germ cells in dissected gonads. Flies without any germ cells were scored as dysgenic; flies with germ cells in one or both gonads were scored as nondysgenic.

' Ore-R indicates the Oregon-R strain was used.

TABLE 2

GD becomes apparent at the end of embryogenesis at 27.5"

Percent embryos with given No. germ cells'

No.

Embryos Parental cross" Stageb scored 0 g . c d 1-5 g.cd 5 + g . ~ . ~

A Ore-RfX 7r2 12-15 38 0 0 100

a 2 X Ore-R 12-15 66 0 0 100

B Ore-R X 7r2 16-17 50 22 24 54

a All crosses are shown as maternal genotype X paternal geno-

type.

Embryos were staged according to CAMPOS-ORTEGA and H A R - TENSTEIN (1 985); stage 17 is the last stage of embryogenesis.

'Total germ cell number per embryo was determined by anti- vasa staining in precuticularized embryos (A) or by morphological criteria in serial sections of cuticularized embryos (B).

This class includes embryos in which a gonad could not be found. See MATERIALS AND METHODS section for discussion of detecting gonads.

g.c. stands for "germ cells." The columns indicate number of germ cells per embryo.

/Ore-R indicates the Oregon-R strain was used.

TABLE 3

GD is restricted to females and is complete by 1st instar

Percent larvae with given no. germ cells'

Cross Parental cross' Sexb No. larvae 0 g.c.d 1-5 g.c.d 5+ g.c.d

1 y c v v f cur X 7r2 Female 23 83 17 0

Male 25 20 0 80

2 y m v f cur X Ore-R' Female 55 0 7 93

Male 61 0 0 100

All crosses are shown as maternal genotype X paternal geno- Larvae were sexed using the X-linked yellow marker (see Figure

' Total number of germ cells per 1st instar larva at 27.5" was

,I g.c. stands for "germ cells." The columns indicate the number 'YP;.

1 ) at 24-48 hr (after egg-lay).

scored from serial sections.

o f germ cells per larva.

Ore-R indicates the Oregon-R strain was used.

germ cells. In an M x P cross, progeny with an XX constitution have a greater dose of P elements because they alone inherit the P strain X chromosome. Thus,

females may be more sensitive to dysgenesis because they inherit more P elements than males. Alterna- tively, the sexual bias in GD could be because female development activates processes which promote P-M dysgenesis. Embryonic XX germ cells already differ from XY germ cells in their requirement for ouo func- tion. Sex specific GD may reflect the ability of P elements to exploit female specific properties like ovo requirement. Each of these hypotheses makes a pre- diction which we have tested.

P

element dose and dimorphic GD: In a dysgenic cross, all progeny receive equal numbers of paternal, P-bearing autosomes, but females normally also re- ceive the paternal, P-bearing X chromosome. T h e presumably greater number of P elements in a female could render her more susceptible to sterilization from the potentially higher levels of transposase pres- ent. This hypothesis predicts a reversal in sex specific susceptibility to GD among progeny from an attached- X, M strain mother and a P strain father. T h e at- tached-X construction in cross 5 (Table 1) forces cos- egregation of the maternal X chromosomes. Hence, in cross 5, sons presumably have more P elements than daughters because sons receive the paternal, P strain X chromosome and daughters receive both the M strain X chromosomes. Even when the sex receiving extra P elements was reversed, GD remained female specific at 27.5'. Thus female specific GD at 27.5' is not caused by a greater dose of P elements in females than males.

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FIGURE 2.--Gonadal phenotype of hybrid first instar larvae raised at 27.5'. Males show healthy germ cells (arrows) in first instar testes

(A). First instar females have, at best, a few necrotic germ cells (arrowhead in B) or empty ovaries (arrowhead indicates where germ cells should be in C). Cells not indicated with arrows are somatic cells which form the gonadal tissue. The 10 pm scale bar in A indicates size for

B and C as well.

such as Sxl have not been shown to be required as early as embryogenesis. In short, we only know of mutations at ovo which can block female germline development at the time of female specific GD. The hypothesis predicts ovo mutations might diminish or eliminate female GD.

Most loss of function ovo alleles produce ovaries totally lacking germ cells, whereas dominant alleles cause incomplete or abnormal oogenesis (BUSSON et

al. 1983; OLIVER, PERRIMON and MAHOWALD 1987).

Given that both GD and o m - genotypes result in early germline death, we could not test for any GD-rescuing ability with loss of function ovo genotypes. Since the strongest dominant ovo mutation, ovoD', also causes germ cell death (PERRIMON 1984; OLIVER, PERRIMON and MAHOWALD 1987), we tested the weaker ovoD2 and ovoD3 mutations on female specific GD at 27.5".

Both ovoD2 and ovoD3 allow germ cell survival to vitellogenic stages of oogenesis. The majority of egg chambers in ovoD2, however, arrest before vitelloge- nesis, while ovoD3 ovaries usually contain many late stage eggs (BUSSON et al. 1983; OLIVER, PERRIMON and MAHOWALD 1987). Instead of having empty ova- ries, P-M hybrid females with ovoDz or 0voD3 had ova- ries containing chambers closely resembling the usual ouoD2 or 0 ~ 0 ' ' ~ phenotypes respectively (data not

shown). Quantitatively, the effect of ovoDZ and ouoDJ was to dramatically reduce the incidence of female GD at 27.5" (Table 4).

ovoD2and ovoD3 are antimorphic mutations. Dupli- cation of the wild-type gene in the presence of either dominant mutation restores fertility. Either ovoD2 or

ovoD3 over a deficiency for the locus produces atrophic ovaries similar to ovo- phenotypes (BUSSON et al.

1983). These genetic tests also demonstrate ovoD2 and

ovoD3 do not produce novel activities, i.e., these are not neomorphic mutations. Given the antimorphic nature of the ovoD alleles, we tested whether GD rescue can be achieved by simply reducing the amount of wild-type ovo product. Reduction in the dose of wild- type ovo product was insufficient for GD rescue. P-M hybrid females bearing a deficiency for ouo still suf- fered complete germline death at 27.5" (Table 4,

cross 4). Thus the ability to rescue female GD appears to be a property of the two dominant mutations used. T o confirm that the rescuing ability was specific to o m , we tested the ability of the ovoD3 7r2 stock to induce

GD after the ovd" chromosome had been removed. Without the ovoD3 chromosome, this 7r2 stock induced

the high levels of female GD expected at 27.5" (Table

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Oliver and A. P. Mahowald

TABLE 4

o w D suppresses the female specific GD usually seen at 27.5"

No. Percent Parental cross. scoredb dysgenic'

Cross

1 Ore-Rd X ova"*; r 2 5 8 5 2

2 Ore-R X 0voD3; r 2 5 2 5 0

3 Ore-R X x 2 bcl' 4 2 91

4 0x1) 0 ~ 0 ' ~ vZ4/FM6 x x 2 3 1f 97

' All crosses are shown as maternal genotype x paternal geno-

This column indicates the number of female progeny scored.

' Adult females were dissected and ovaries scored. Females with tYqe.

any egg chambers were scored as nondysgenic. Ore-R indicates the Oregon-R strain was used. Generated by back-crossing ovoD3; x 2 to ~ 2 .

'This number represents DJI)ovo" v Z 4 / r 2 females scored for

gonadal phenotype.

DISCUSSION

Germline dimorphism in Drosophila has been char- acterized mainly from the study of gametogenesis. Gametogenesis is a postembryonic event in both males and females. The lack of markers for the undifferen- tiated germline has blocked inquiry into whether germ cell sex is established prior to the onset of gametogenesis. We investigated GD because of its potential as a functional marker of early germline dimorphism. Females are generally more sensitive to GD. This sensitivity is most pronounced at 27.5",

where complete germline death was virtually re- stricted to females. At 27.5" GD could be traced back to the end of embryogenesis and complete germline

loss was seen in first instar female larvae. As such, the developmental profile of female specific GD indicates germline dimorphism is established in embryogenesis. What is the basis of the sex specificity of GD at

27.5"? Any one of several differences between the

sexes could account for the female bias. First, a female bias could arise from having a germline proliferation rate lower than that of males and also lower than that required to offset the rate of germ cell death in GD. Second, females, in having two X chromosomes, could be more susceptible if there are X-linked factors which promote GD. These factors could be P elements them- selves or endogenous loci. In a dysgenic cross, females may have more P elements because they inherit the paternal P strain X chromosome. Third, susceptibility to GD may be a function of the sexual identity of germ cells. For example, the mechanism of GD may tap into some cellular function which is more active in female development. We have experimentally ad- dressed each of these hypotheses, and find support only for the notion that female GD is influenced by the sexual identity of germ cells.

The generally greater temperature sensitivity of females to GD has been attributed to the difference between male and female germ cell numbers (ENGELS and PRESTON 1979). Both male and female germlines

undergo extensive proliferation in larval stages

(ABOIM 1945), although at different rates. By 3rd instar, males have a larger germ cell population than females. By a hypothesis of stochastic germ cell death, complete germline loss would be less likely in males because of the greater population of larval cells. At the time when we found germline loss to occur in female specific GD (27.5"), however, germline mitosis is only beginning. By the 1st instar stage, only one, or possibly two germline divisions have occurred (SON-

and CAMPOS-ORTEGA 1986). Our results show that germline death is complete in first instar female lar- vae, ie., before the major proliferative phase of the germline. Thus, female specific GD must reflect a sexually dimorphic state present from embryogenesis rather than a difference in larval germ cell numbers between males and females.

We have tested the P element dosage hypothesis of susceptibility by reversing the sex receiving the P strain X chromosome. Redirecting the P strain X chro- mosome into sons rather than daughters did not cause male GD at 27.5". GD at 27.5" remained female specific even when females inherited both X chromo- somes from the maternal M strain. Thus the greater female sensitivity to GD is not due to the inheritance of a potential extra source of P elements via the paternal X chromosome. In fact, comparisons of mo- lecularly characterized or genetically defined P stocks have previously shown there is no simple quantitative relationship between the incidence of P-"induced sterility and increasing numbers of P elements (EN- GELS 1984; RASMUSSON et al. 1990).

XX germ cells could be more susceptible to GD if there is even one endogenous X-linked locus that promotes GD. This hypothesis assumes that such a locus is not dosage compensated in the germline. Dosage compensation refers to the equalization of X- linked gene activities between males, which have one X chromosome per cell, and females, which have two X chromosomes per cell: this equalization is achieved by elevating transcription of the X chromosome in males (LUCCHESI and MANNING 1987). If a GD-pro- moting gene were dosage compensated, there would be no sex difference in GD susceptibility because males and females would have equivalent activity from this locus. Without dosage compensation, such a gene would be expected to induce male GD at 27.5 " if duplicated and reduce female GD if deleted. While ovoD mutations mitigate female GD, ovo does not fit

one criterion for being simply a gene whose dose determines P element activity. Reducing ovo dosage with a deficiency had no effect on the incidence of female GD. This result indicates ovo is not a non- dosage-compensated X-linked locus which simply ful- fills a metabolic requirement for P-M sterility.

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Finally, activation of a female developmental path- way in

XX

germ cells could confer the greater sensitiv- ity to GD. This hypothesis predicts that

XX

germ cells will be more resistant to GD if their female character is reduced. In support of this model, we found that the dominant mutations ovoD2 and ovoD3 partially sup- pressed female specific GD. Although these particular alleles by themselves allow some female germline dif- ferentiation, they have also been shown to interact with the known sex determination genes, Sxl and snf, in a way that suggests ovoD2 and ovoD3 effectively cause a reduction of an activity required for the normal female program in

XX

germ cells (OLIVER, PAULI and

MAHOWALD 1990a,b).

ouo is required for germline development solely in females (OLIVER, PERRIMON and MAHOWALD 1987). The dominant ouo mutations are antimorphic inas- much as they produce activities which antagonize the wildtype ouo activity but do not provide novel func- tions (BUSSON et al. 1983). In short, female GD is mitigated by reducing the activity of a gene required only in females for germline development.

The nature of Drosophila germline sex determina- tion has not been characterized sufficiently to identify which dimorphic quality underlies female specific GD. Any hypothesis on early germ cell dimorphism, how- ever, should be able to account for certain features of GD. It is widely accepted that transposase is required for P-"based sterility (ENCELS 1984; KOCUR, DRIER and SIMMONS 1986). Transposase production, in turn, depends on tissue-specific splicing of the P transcript; only germ cells are capable of producing transcripts encoding functional transposase (LASKI, RIO and RUBIN 1986). Incorporating this understanding of P- "induced sterility, one hypothesis for early dimorph- ism and sex-biased GD is that female germ cells may allow greater efficiency of RNA splicing for transpos- ase production. This efficiency difference may arise from either qualitative or quantitative differences in splicing machinery. Whatever the basis of female biased GD, the relevant female characteristic is influ- enced by, and likely downstream of, ovo activity.

In summary, we have investigated temperature sen- sitive P-"induced sterility with a focus on the nature of sex specific GD. Our results suggest GD at 27.5" uncovers an early sexual dimorphism in the embryonic germline. The dimorphic character responsible for female GD appears to involve ovo, a locus required continually for female germline development. Hence, whatever the cellular properties are that distinguish X X from XY germ cells, these properties are likely to

arise from

XX

germ cells following an ovo-dependent developmental pathway.

We thank B. BAKER for his suggestion of the attached-X experi- ment, and M. D. GARFINKEL, C. SCHONBAUM, D. WRIGHT, our anonymous reviewers and T. SCHUPBACH for their critiques of this

paper. We also thank J. CAULTON who collaborated in some initial experiments which led to these experiments. The anti-vasa antibody used was kindly given to us by P. LASKO and the Dfll)ouo" stock by M. STEINMANN-ZWICKY. This work was supported by a grant from the National Institutes of Health (HD-17608 to A.P.M.). B.O. was supported by a National Institutes of Health predoctoral training grant (T32HD07104). G.W. was supported by a Howard Hughes Medical Institute predoctoral fellowship.

LITERATURE CITED

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Figure

FIGURE 1.-Method using mouth-hook pigmentation
TABLE 1 Temperature sensitivity  and  female  specificity of GD
FIGURE 2.--Gonadal phenotype of hybrid first instar larvae raised at should be in  C)

References

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