5033
Development 124, 5033-5048 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV5164
Induction of female Sex-lethal RNA splicing in male germ cells: implications
for Drosophila germline sex determination
Jeffrey H. Hager and Thomas W. Cline*
Department of Molecular and Cell Biology, Division of Genetics, University of California, Berkeley, 401 Barker Hall, Berkeley, CA
94720-3204, USA
*Author for correspondence (e-mail: [email protected])
SUMMARY
With a focus on Sex-lethal (Sxl), the master regulator of
Drosophila somatic sex determination, we compare the sex
determination mechanism that operates in the germline
with that in the soma. In both cell types, Sxl is functional
in females (2X2A) and nonfunctional in males (1X2A).
Somatic cell sex is determined initially by a dose effect of
X:A numerator genes on Sxl transcription. Once initiated, the active state of Sxl is maintained by a positive
autoregulatory feedback loop in which SXL protein insures
its continued synthesis by binding to Sxl pre-mRNA and
thereby imposing the productive (female) splicing mode.
The gene splicing-necessary factor (snf), which encodes a
component of U1 and U2 snRNPs, participates in this RNA
splicing control. Here we show that an increase in the dose
of snf+ can trigger the female Sxl RNA splicing mode in
male germ cells and can feminize triploid intersex (2X3A)
germ cells. These snf+ dose effects are as dramatic as those
of X:A numerator genes on Sxl in the soma and qualify snf
as a numerator element of the X:A signal for Sxl in the
germline. We also show that female-specific regulation of
INTRODUCTION
Two cells could hardly be more different than Drosophila eggs
and sperm. This dramatic example of sexual dimorphism arises
through an intimate collaboration of somatic and germline cells
whose sexual identity must match (reviewed by Mahowald and
Wei, 1994; Steinmann-Zwicky, 1994b). Although the
molecular mechanisms by which these two interacting cell
types acquire their appropriate sexual identity are remarkably
different, both cell types require the key regulatory gene Sexlethal for female development (reviewed in Cline and Meyer,
1996). Sxl functions as a switch gene in the soma, but relatively
little is understood about Sxl regulation and function in the
germline. In this paper we explore the role of Sxl in germ cell
development and investigate the nature of the genetic signals
that regulate its functional state in this cell type.
In the soma, Sxl gene products are both necessary and sufficient to dictate the female pathway of sexual differentiation
and X-chromosome dosage compensation. In the absence of
SXL proteins, male somatic development ensues. The sex-
Sxl in the germline involves a positive autoregulatory
feedback loop on RNA splicing, as it does in the soma.
Neither a phenotypically female gonadal soma nor a female
dose of X chromosomes in the germline is essential for the
operation of this feedback loop, although a female X-chromosome dose in the germline may facilitate it. Engagement
of the Sxl splicing feedback loop in somatic cells invariably
imposes female development. In contrast, engagement of
the Sxl feedback loop in male germ cells does not invariably disrupt spermatogenesis; nevertheless, it is premature
to conclude that Sxl is not a switch gene in germ cells for
at least some sex-specific aspects of their differentiation.
Ironically, the testis may be an excellent organ in which to
study the interactions among regulatory genes such as Sxl,
snf, ovo and otu which control female-specific processes in
the ovary.
Key words: Drosophila melanogaster, Sex-lethal, splicing-necessary
factor, germ cell, sex determination, RNA splicing, triploid
intersexes
specific activity state of Sxl in the soma is determined by the
zygotic dose of a set of X:A numerator genes which encode
transcription factors (reviewed in Cline and Meyer, 1996). The
double dose of numerator genes present in chromosomal
females (2X2A) turns on a ‘sexual pathway establishment’
promoter, SxlPe, less than 2 hours after fertilization, but the
single dose of numerator genes in chromosomal males (1X2A)
is insufficient to activate SxlPe. Less than 1 hour later SxlPe
shuts off and a ‘sexual pathway maintenance’ promoter, SxlPm,
comes on in both sexes. In contrast to transcripts from SxlPe,
transcripts from SxlPm are only processed into mRNA that
encodes full-length SXL protein if active SXL protein is already
present to direct RNA splicing. In the absence of such protein,
SxlPm-derived mRNA includes an exon that prematurely terminates translation allowing only inactive product to be
generated. The transient expression of SxlPe, which occurs only
in females, results in a pulse of SXL protein that triggers the
productive splicing of SxlPm transcripts. That productive RNA
splicing mode is self-maintained thereafter by the SXL protein
that arises as a consequence and that interacts directly with Sxl
5034 J. H. Hager and T. W. Cline
pre-mRNA. Because SxlPe is silent in males, males never
engage this positive feedback loop for RNA splicing and only
produce Sxl mRNAs that include the translation-terminating
exon.
SXL proteins execute their female-specific somatic functions
in a cell autonomous fashion by controlling switch genes that
are more functionally specialized. One of these called transformer (tra) controls all aspects of somatic sexual differentiation and is activated by Sxl (McKeown et al., 1988). Another
called male-specific lethal #2 (msl2) controls many aspects of
dosage compensation and is repressed by Sxl (Bashaw and
Baker, 1997; Kelley et al., 1997; Zhou et al., 1995).
Although the functioning of Sxl in the germline is as femalespecific as it is in the soma – Sxl is essential for oogenesis but
dispensable for spermatogenesis – neither of the known somatic
targets for Sxl is required in the germ line (Bachiller and Sánchez,
1986; Marsh and Wieschaus, 1978; Schüpbach, 1985). The fact
that the growth of 2X2A somatic cells but not germ cells is
impaired by the loss of Sxl+ is another key difference between
these two cell types (Cline, 1984; Schüpbach, 1985). The specific
nature of the requirement for Sxl in the germline is unclear. A
role for Sxl in germline sex determination is indicated by the fact
that 2X2A germ cells deficient for Sxl+ activities display an
unambiguously male morphology and express a variety of malespecific molecular markers (Staab et al., 1996; SteinmannZwicky et al., 1989; Wei et al., 1994); however, recent studies
with other molecular markers of germline sex have suggested that
loss of Sxl in the germline may not masculinize as completely as
loss in the soma (Bae et al., 1994; Horabin et al., 1995).
Answering the question of whether Sxl is a switch gene for
sex determination in the germline as it is in the soma is made
difficult by the fact that a sexual mismatch between germ cells
and somatic cells of the gonad blocks differentiation of germ
cells at early stages when few unambiguous molecular markers
of sexual phenotype are available. Other complications arise
from the partially cell-nonautonomous nature of germline sex
determination, from the pleiotropy of many of the genes
involved (which interferes with the use of null alleles), and
from critical gaps in the information available for genes such
as orb that have been used as molecular markers of germline
sexual phenotype.
In the germline, as in the soma, full-length SXL protein is
found only in female cells due to sex-specific alternative
splicing of the translation-terminating exon in SxlPm transcripts
(Bopp et al., 1993; Oliver et al., 1993). Despite this similarity
at the molecular level, it is not known how the female-specific
RNA splicing mode is initiated in germ cells, nor whether that
splicing mode is maintained by positive autoregulation. The
‘establishment’ promoter, SxlPe, which is central to Sxl activation in the soma, seems not to be active in germ cells (Keyes
et al., 1992), and two genes that are essential for SxlPe somatic
activation, daughterless and sisterlessB, are not required in the
female germline (Cronmiller and Cline, 1987; SteinmannZwicky, 1993). The control of Sxl transcript splicing in the
germline is not a strictly cell-autonomous process as it is in the
soma. Proper female SxlPm transcript splicing in the germline
depends not only on a female dose of X chromosomes in the
germ cells themselves, but also on a female-specific expression
mode of regulatory genes downstream of Sxl in the somatic
cells of the gonad (Nöthiger et al., 1989; Oliver et al., 1993;
Steinmann-Zwicky, 1994a; Steinmann-Zwicky et al., 1989).
By investigating the effect of expression of female-specific
SXL proteins in the male germline, we hoped to answer a variety
of important questions regarding the nature and regulation of
Sxl functioning in this cell type. Does Sxl qualify as a switch
gene in the germline as it does in the soma, with its femalespecific protein products able to impose female development on
chromosomally male germ cells? Does Sxl autoregulation occur
in the germline, with SXL female proteins able to impose the
female-specific splicing mode on SxlPm germline transcripts? If
so, is such autoregulation independent of germline X-chromosome dose and/or the sex of surrounding somatic cells and
hence not the control point for either of these sex signal inputs?
Here we describe the consequences of germline-specific
expression of a female-specific SXL protein isoform shown previously in male somatic cells to be able to engage the femalespecific SxlPm transcript splicing feedback loop, impose female
differentiation and disrupt dosage compensation. In the course
of these studies, a remarkable germline-specific dose effect on
Sxl transcript splicing was discovered for splicing-necessary
factor (snf, a.k.a. sans fille). This gene encodes an integral
component of U1 and U2 snRNPs that had already been implicated in somatic Sxl RNA splicing control and seemed likely
to be involved in germline Sxl regulation as well, based on the
phenotypes of antimorphic mutant alleles (Salz and Flickinger,
1996). In a control for one of the autoregulation studies, we
discovered a molecular interaction between two other genes
previously implicated in Sxl germline regulation, ovo and
ovarian tumor (otu). In a control for studies of snf+ dose effects
in 2X3A animals, we extended previous characterization of the
feminizing effects of gain-of-function Sxl alleles to reveal that
2X3A adults only survive with such mutations under conditions where the alleles are not fully expressed and oogenesis
is not entirely normal.
MATERIALS AND METHODS
Drosophila culture, genetics and nomenclature
Flies were raised at 25°C in uncrowded conditions on a standard
cornmeal, yeast, sucrose and molasses medium. wjt is a hypomorphic
white allele induced in our laboratory and used because it allows one
to score cm in the presence or absence of the w+mC transgene marker.
All other mutations and chromosomes are described in Lindsley and
Zimm (1992) or are referenced in the text. For the gene first known
as fs(1)1621 (Gans et al., 1975), but subsequently renamed sans-fille
(Oliver et al., 1988), liz (Steinmann-Zwicky, 1988) and splicingnecessary-factor (Flickinger and Salz, 1994), we have chosen to use
the last name, since it was conferred by those who isolated additional
alleles of the locus, defined its null phenotype, showed the original
allele to be a gain-of-function change, and preserved the most
commonly used and euphoneous abbreviation, snf.
The experiment revealing an effect of ovo on the otu promoter
compared testes of males that were non-recombinant for the ovospanning interval between y and cv from the following cross:
ovoD1rS1cv ct v/y cm ct sn × +/Y; P(ry+ otu::lacZ)1J ry/ry. Males were
fixed and stained separately for β-galactosidase in a preliminary
experiment, then subsequently fixed and stained together to confirm
the difference.
In situations where increased Sxl+ dose was required for induction
of anti-SXL staining, the small tandem duplication designated
Dp(1;1)jnR1-A was far more effective than either of two much larger
duplications. For example, in combination with Dp(4F), 25 of 25
testes showed autoregulatory cysts with this tandem duplication while
Female SXL protein in male germ cells 5035
only 1 of 25 and 4 of 31 testes did so with Dp(1;Y)y+ct+ and
Dp(1;3)sn13a1, respectively. Early in the study, we established that the
SXL-inducing effect of this X chromosome was linked to the tandem
duplication of Sxl in region 6F, since exchanges between cv (5B) and
cm (6E5-6) (two tested), or between ct (7B3) and t (8C) (two tested)
gave recombinant chromosomes whose sensitivity to Dp(4F) was
comparable to those of the parental chromosome. However, as
discussed in the text, something quite close to the tandem duplication
may contribute to sensitivity since an exchange just centromere
proximal to ct was subsequently found to reduce the sensitivity of the
Dp(Sxl+) chromosome to Sxl autoregulation. Nevertheless, two lines
of evidence argue that both copies of Sxl+ on this chromosome are
necessary for the interaction with Dp(4F). First, a very similar
rearrangement, Dp(1;1)jnR1-B, was generated from the same parental
chromosome in the same way and at the same time as Dp(1;1)jnR1A and is nearly as large; however, it lacks the extra copy of Sxl+
(Maine et al., 1985; Nicklas and Cline, 1983) and fails to synergize
with Dp(4F) in the testis. Second, a mutant derivative of Dp(1;1)jnR1A that has only a partial loss-of-function mutation in just one of the
tandem Sxl+ alleles (Cline, unpublished) exhibits a much reduced
level of interaction with Dp(4F). Testes with this mutant derivative
displayed autoregulatory cysts in only 5 of 76 cases examined, and
the staining observed was far less extensive than that for the parental
chromosome.
Transgene construction
A 578 bp NotI-Asp718 fragment of the otu gene (Comer et al., 1992)
was ligated upstream of the Sxl 1.8 kb cF1 cDNA (Bell et al., 1988)
and subsequently cloned into the Casper P-element vector (Pirrotta,
1988). Transgenic lines were generated using standard procedures
(Spradling, 1986) with w1118 hosts and the pTURBO transposase
source (D. C. Rio, personal communication).
Whole-mount immunofluorescence
The procedure was modified from Bopp et al. (1993). Testes were
dissected and collected in chilled (4°C) Drosophila Ringer’s, then
fixed within 1 hour. Fixation was in 4% formaldehyde (E. M. grade
Polysciences Inc.) in 1× PBS at room temperature for 20 minutes with
agitation. Fixed testes were rinsed in 1× PBS with 0.1% Triton X-100
and 0.05% Tween 80 (PBSTT) then transferred to 0.5 ml Eppendorf
tubes and incubated in PBSTT with 0.2% BSA (PBSTTB) for 1.5 hour
with multiple changes. A 30 minutes incubation in PBSTTB with 5%
Normal Goat Serum (PBSTTBN, block) was followed by incubation
with agitation overnight at 4°C with a 1:2000 dilution of mouse antiSXL polyclonal ascites fluid raised against a 183-residue fragment of
Sxl containing both RRM domains (Bernstein et al., 1995). Testes
were washed 3× 5 minutes then 3× 30 minutes in PBSTT, blocked in
PBSTTBN for 30 minutes, then incubated for 1.5-2 hours at room
temperature with a 1:500 dilution (in PBSTTBN) of Goat anti-mouse,
CY3-conjugated antibody (Jackson Immunoresearch). Testes were
again washed 3× 5 minutes and 3× 30 minutes, then DAPI was added
at 50 µg/ml in PBSTT and incubated for 10 minutes, followed by a
final 10 minute wash in PBSTT. Testes were mounted in 70%
glycerol/PBS and viewed through a Zeiss Axiophot.
Western blotting
Extracts were prepared as described in Bopp et al. (1991), but without
8M urea, then electrophoresed on a 4-15% gradient-acrylamide gel
using standard conditions. Proteins were transferred to nitrocellulose
by semi-dry blotting (BioRad) at 15 V for 30 minutes. The filter was
stained with Ponceau-S to verify transfer, then blocked in Trisbuffered saline with 0.05% Tween 20 (TBST) and 5% non-fat dry milk
for 30 minutes, followed by incubation with mouse anti-SXL ascites
fluid at 1:1000 for 2 hours at room temperature. The filter was washed
3× 10 minutes in TBST then incubated 1 hour in a 1:5000 dilution (in
TBST) of goat anti-mouse HRP-conjugated antibody (Jackson
Immunoresearch). Following incubation, the filter was washed 3× 10
minutes in TBST and antigen/antibody complexes were visualized by
chemiluminescence (E. C. L., Amersham).
RESULTS
Ectopic SXL protein triggers the female-specific Sxl
RNA splicing feedback loop in male germ cells
without disrupting spermatogenesis
In male somatic cells, a pulse of the SXL protein isoform
encoded by Sxl cDNA cF1 is sufficient to initiate the femalespecific mode of pre-mRNA splicing for transcripts from the
endogenous Sxl+ allele, and that splicing mode is self-sustaining thereafter due to feedback from the female proteins
generated (Bell et al., 1991). Would the same SXL isoform
trigger the female feedback loop in male germ cells and, if so,
what would the consequences be for spermatogenesis? In view
of the toxicity of SXL protein to male somatic tissues and the
complicated regulatory relationship that exists between
somatic and germline Sxl expression, we sought to simplify the
analysis by employing a transgene that would only express SXL
protein in germ cells, and do so regardless of sex prior to the
point at which the cells embark on terminal differentiation. The
transgene used for this purpose is diagrammed in Fig. 1C. Its
design was based on the finding by Comer et al. (1992) that a
small 5′ fragment of the ovarian tumor (otu) gene would drive
expression of β-galactosidase specifically in germline stem
cells of either sex (Fig. 1A,B).
Ten independent P(otu::Sxl+cF1) germline transformants
were recovered. None had any adverse effect on male viability
or fertility. Only three exhibited levels of SXL protein in the
male germline that could be assayed in situ by indirect
immunofluorescence with anti-SXL polyclonal antibody. These
three plus two non-staining lines were assayed for Sxl+
germline functional activity by complementation tests. None
complemented any of the three germline-specific, femalesterile, point-mutant alleles, Sxlf4, Sxlf5 and Sxlf18, but all five
clearly produced some active Sxl product since they suppressed
the germline-autonomous, female-sterility of snf1621, an antimorphic allele that interferes with Sxl female splicing in both
the germline and the soma (Albrecht and Salz, 1993; Bopp et
al., 1993; Oliver et al., 1993; Steinmann-Zwicky, 1988). There
is no contradiction between these two sets of results, since all
three female-sterile Sxl alleles encode only defective gene
products (Lersch et al., 1994). The single SXL isoform
generated by these transgenes is unlikely to be able to substitute for all the missing isoforms from the three mutant
Sxl alleles, but may be able to trigger the production of the full
spectrum of isoforms from a Sxl+ allele whose productive premRNA splicing would otherwise have been blocked by the
mutation in snf. Suppression of snf1621 is likely to be an exceptionally sensitive assay for Sxl germline activity, since simply
increasing the dose of Sxl+ can partially suppress snf1621
female sterility (data not shown).
Both the level and spatial distribution of SXL protein induced
in testes by transgene P(otu::Sxl+cF1)A depends on the dose of
endogenous Sxl+ alleles (Fig. 2). From this fact, and from the
mosaic nature of the SXL protein staining pattern in these SXLpositive testes, one can conclude that this transgene can engage
the Sxl female-specific RNA splicing feedback loop even in the
absence of female somatic signals and the female dose of X
5036 J. H. Hager and T. W. Cline
Fig. 1. A 578 bp fragment of the otu gene upstream of the otu
translation start site drives heterologous gene expression in both the
male and female germline. (A) Adult testis carrying P(ry+
otu::lacZ)1J (Comer et al., 1992). Stem cells are located at the apical
tip (api). They undergo an asymetrical division (asy) to produce a
spermatogonial cell which will terminally differentiate. The
spermatogonial cells proceed through four incomplete mitotic
divisions (mit) to generate a 16-cell spermatocyte cyst. The
spermatocytes of each cyst then embark on a growth phase (gro),
increasing 25-fold in cell volume. Staining for β-galactosidase
activity generated from expression of the otu promoter used in this
study is limited to the stem cells and early spermatogonial divisions
at the apical tip. (B) Adult ovary carrying P(ry+ otu::lacZ)1J. Stem
cells are located at the apical tip (api). They undergo an asymetrical
division (asy) to produce the oogonial cell that will terminally
differentiate. It proceeds through four incomplete mitotic divisions to
form the 16-cell egg chamber from which the oocyte is ultimately
derived. β-galactosidase generated from expression of the otu
promoter used in this study is present at all stages of oogenesis.
(C) The germline transformation construct used to generate SXL
protein only in germ cells in both sexes. The 578 bp otu gene
fragment present in P(ry+ otu::lacZ)1J was fused to the female Sxl
cDNA, cF1.
chromosomes that would normally be required for femalespecific germline Sxl pre-mRNA splicing.
In the absence of an otu::Sxl+cF1 transgene, the background
anti-SXL signal in testes is very low, particularly at the apical
tip (Fig. 2A). The testis shown has twice the normal dose of
wild-type Sxl+ alleles, but identical non-specific staining was
observed in males that lacked all Sxl genomic sequence
(SxlfP7bo/Y, not shown). The typical signal generated by
P(otu::Sxl+cF1)A in the absence of an endogenous Sxl+ allele
is shown in Fig. 2B. For the 42 testes examined, staining was
unambiguously above background only at or very near the
Fig. 2. The amount and distribution of SXL protein in the germline
of P(otu::Sxl+cF1) transgenic males depends on the dose of
endogenous Sxl+ alleles. Whole mounts of testes from 5- to 7-dayold adult males stained for SXL protein by anti-SXL indirect
immunofluorescence (A-E) or for DNA by DAPI (F,G). (A) Two
copies of Sxl+. No anti-SXL staining above background. (B) No Sxl
allele and two copies of P(otu::Sxl+cF1). Very faint anti-SXL staining
above background in apical tip only. (C). One copy of Sxl+ and two
copies of P(otu::Sxl+cF1). Stronger anti-SXL staining but still limited
to apical tip. (D,E) Two copies of Sxl+ and two copies of
P(otu::Sxl+cF1). SXL-positive autoregulatory cysts are apparent far
beyond the domain of transgene expression. (F) DAPI of B.
(G) DAPI of D. Note persistence of DAPI-positive nuclei beyond the
apical tip, in contrast to (F). Full genotypes of males:
(A) Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ v/Y ; (B) y w cm SxlfP7boct6/Y;
P(w+mC otu::SxlcF1)A/P(w+mC otu::SxlcF1)A ; (C) w1118/Y; P(w+mC
otu::SxlcF1)A/P(w+mC otu::SxlcF1)A; (D,E) Dp(1;1)jnR1-A, y wjt cm
Sxl+Sxl+ v/Y; P(w+mC otu::SxlcF1)A/P(w+mC otu::SxlcF1)A.
apical tip. This is the domain of expression expected for the
transgene in males, based on the extent of β-galactosidase
staining observed for the otu::lacZ transgene (Fig. 1A) on
whose design the otu::Sxl+cF1 transgene was based. When an
endogenous Sxl+ allele was added to this genotype (Fig. 2C),
anti-SXL staining induced by the transgene was markedly more
intense at the apical tip and, in many cases, began to extend
beyond the expected domain of otu-driven transcription to
include some young dividing spermatogonial cysts. The
example shown was the most extreme case among 30 testes.
With the addition of an extra copy of Sxl+ (Fig. 2D,E), there
was a dramatic expansion of the domain of staining to include
Female SXL protein in male germ cells 5037
much older cysts that were well into their growth phase. The
staining took on a strikingly mosaic character: heavily staining
cysts and groups of cysts were intermingled with cysts that did
not stain at all. This phenotype indicates that, in the presence
of a female dose of Sxl+ (two copies), expression of the
transgene in male stem cells triggered the Sxl splicing feedback
loop for endogenous Sxl+ alleles in some but not all of the
clonally developing cysts. Such cysts displaying anti-SXL
staining far beyond the normal (male) domain of otu expression
are referred to as ‘autoregulatory cysts’ hereafter.
The conclusion that the extensive staining in Fig. 2D,E
reflects transgene-triggered engagement of the female Sxl
splicing feedback loop rests on the assumption that the domain
of expression of the transgene itself in males is not expanded
as a consequence of the female SXL proteins that are generated.
Feminization of the germline would be expected to expand this
domain, since otu::lacZ is expressed in all stages of oogenesis
(Fig. 1B). No such expansion was observed for the genotype
in Fig. 2D,E among a total of 43 testes also carrying
P(otu::lacZ) and stained for β-galactosidase, despite the fact
that all 50 testes from their genetically identical brothers
showed autoregulatory cysts when stained instead for SXL
protein (data not shown).
There was also no indication of feminization by morphological and functional criteria. Terminally differentiating cysts
of female germ cells become polyploid and display a nucleolar
and DAPI-staining morphology that clearly distinguishes them
from differentiating male germ cells at comparable stages
(reviewed in Fuller, 1993; Spradling, 1993). No such female
character was seen in any of the many SXL-expressing testes
examined (Fig. 2G). Moreover, there was no significant differ-
ence in fecundity between two groups of ten sibling males,
both of which carried two doses of Sxl+ but only one of which
carried the transgene that triggers the Sxl feedback loop (data
not shown). The only hint of an effect of SXL female proteins
on spermatogenesis was an abnormal persistence of DAPI
(DNA) staining as growing cysts moved away from the tip of
the testes (Fig. 2G) that correlated with the presence of SXL
protein (Fig. 2D). Not all testes staining for SXL displayed this
phenotype, and the DAPI staining did eventually disappear as
the cysts grew and moved farther down the testis. This delay
in the loss of DAPI staining suggests that the ectopic female
SXL protein present in these autoregulatory cysts may
sometimes retard the rate of differentiation of spermatocytes,
but does not abort the process.
The proportion of testes displaying autoregulatory cysts was
somewhat variable from experiment to experiment and
decreased somewhat with age. In one series, it was 95%
(38/40) less than 1 day after eclosion, but only 52% (19/36) 57 days later. Percentages approaching 100 were generally
observed only among males that were not highly inbred, and
only with a particular (v-marked) tandem Sxl+ duplication
chromosome (see below). For the genotype illustrated in Fig.
2, the proportion of autoregulatory cysts was no higher among
males carrying only a single copy of the Sxl transgene than
among those with two copies.
Although autoregulatory cysts were generated by only one
of the three original transgene lines that displayed anti-SXL
staining, the differences among lines appear simply to reflect
non-specific insertion-site position effects on otu promoter
expression level. The other two lines clearly induced female
splicing of transcripts from endogenous Sxl+ alleles in testes,
Fig. 3. Induction of SXL protein in the male germline by Dp(4F),snf+ as a function of Sxl+ gene dose. Three views of the same four whole
mounts of testes from 5- to 7-day-old adult males. Top row: anti-SXL indirect immunofluorescence. Middle row: DAPI fluorescence (DNA).
Bottom row: DIC light microscopy. (A,F,J) Two copies of Sxl+ and one copy of chromosome region 4F. No anti-SXL staining above
background. (B,G,K) No Sxl allele and two copies of region 4F. No anti-SXL staining above background. (C,H,L) One Sxl+ allele and two
copies of region 4F. Faint anti-SXL staining at apical tip, but no autoregulatory cysts apparent. (E,I,M) Two copies of Sxl+ and two copies of
region 4F. Brightly staining autoregulatory cysts. Note persistence of DAPI staining, particularly in abnormal spermatogonial cysts
(arrowheads). Full genotypes of males: (A) Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ v/Y; (B) y w cm SxlfP7boct6/Y; Dp(4F),w+/+, where Dp(4F) =
Dp(1;2)w+64b13 & Df (1)3F-4A; (C) w1118/Y; Dp(4F),w+/+ ; (E) Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ v/Y ; Dp(4F),w+/+.
5038 J. H. Hager and T. W. Cline
since the level of SXL protein that they generated increased
with increasing Sxl+ copy number (data not shown). However,
since staining was confined to the apical tip, the level of SXL
protein generated by autoregulation must have been below the
threshold needed to maintain female splicing of transcripts
from endogenous alleles once the transgenes had shut off.
Transgenes that had failed to generate detectable SXL protein
could induce autoregulatory cysts when hopped to new chromosomal sites (data not shown).
SXL protein is localized to the nucleus in most non-gonadal
cells, but it is predominantly cytoplasmic in wild-type female
germline stem cells (Bopp et al., 1993). However, as the terminally differentiating progeny (cystoblasts) of these female stem
cells divide to form 16-cell cysts (16 cystocytes) in a process
analogous to the division of spermatogonia to form 16-cell spermatocyte cysts, SXL protein shifts to the nucleus. Ectopic SXL
protein in the male germline behaves, at least superficially, in a
similar way. In the male stem cells and young dividing spermatogonia, the protein is predominantly cytoplasmic. It then
assumes a nuclear as well as cytoplasmic location in the
growing 16-cell spermatocyte cysts (data not shown).
Somatic expression of P(otu::Sxl+cF1) occurs but is
irrelevant
Although the otu promoter driving expression of Sxl+cF1 in
these transgenes had been thought to direct β-galactosidase
expression exclusively in the germline (Comer et al., 1992), we
found that all three otu::Sxl+cF1 transgene lines (one original
and two remobilizations) whose expression was high enough
to generate autoregulatory cysts in the male germline also
displayed anti-SXL nuclear staining in at least three different
somatic cell types of the testes: sheath cells, terminal epithelial cells and some cells of the seminal vesicle. Staining was
not obvious in the tissues of normal males because of the sparse
distribution of nuclei, but it was unmistakable in the collapsed
gonadal tissues of males whose germ cells had been genetically ablated (data not shown). The lack of effect on male
viability, morphology and fertility indicates that the level of
female SXL protein produced in the soma by these transgenes
either is too low or arises too late to interfere with development; nevertheless, in order to conclude that expression of the
transgenes in the germline itself was what triggered Sxl
germline autoregulation, we had to exclude the possibility that
this low-level somatic expression was involved. Since the
influence of somatic Sxl activity on the activity of Sxl in the
germline is known to be mediated by the switch gene transformer (tra) and its constitutive partner transformer2 (tra2)
(Horabin et al., 1995; Nöthiger et al., 1989; Oliver et al., 1993;
Steinmann-Zwicky, 1994a), autoregulatory cyst induction in
males should be blocked by elimination of tra or tra2 if
somatic expression of P(otu::Sxl+cF1)A were a causative factor.
Note that neither tra nor tra2 is required in the germline itself
for Sxl regulation (Marsh and Wieschaus, 1978; Schüpbach,
1982).
Data in Table 1 show that somatic expression of
P(otu::Sxl+cF1)A is not a factor in generating autoregulatory
germline cysts. Neither the frequency of testes displaying
autoregulatory cysts induced by P(otu::Sxl+cF1)A, nor the extent
of anti-SXL protein staining in SXL-positive testes (data not
shown) were influenced by the loss of either tra+ or tra2+
(compare class 1 with 2, and class 3 with 4). The correlation
between somatic expression of the transgenes and their ability to
induce autoregulatory cysts seems likely to simply reflect
insertion of the transgenes into chromosomal locations that allow
an unusually high level of otu promoter activity. This hypothesis
is supported by the observation that transgenes effective at triggering the Sxl feedback loop also displayed atypically high level
of expression of the white+mC transgene marker (data not shown).
There are strong genetic background effects on
germline Sxl autoregulation
Throughout this study, we observed that different duplications
of Sxl+, and even differently marked chromosomes carrying
Sxl+ duplications, had consistently different sensitivities to
P(otu::Sxl+cF1)A (see MATERIALS AND METHODS). These
differences suggest that there are significant effects of genetic
background on Sxl autoregulation and hence that the most
reliable comparisons in experiments are between the most
closely related genotypes. One dramatic illustration of genetic
background effects is illustrated by the difference in autoregulation between the controls for the tra and tra2 experiments in
Table 1. To allow scoring the w+mC transgene marker on a bw
background for the tra2 series, v on the Dp(1;1)Sxl+Sxl+ X
chromosome was replaced by v+ (and sn3) by a recombination
event approximately 1 cM centromere proximal to the tandem
duplication. The frequency of autoregulatory cysts generated by
the sn3-marked X chromosome in the presence of two copies of
P(otu::Sxl+cF1)A (class 4) was far below that for the v-marked
X with just a single copy of P(otu::Sxl+cF1)A (class 2).
A different sensitivity of these two X chromosomes to
P(otu::Sxl+cF1)A was also observed in an experiment primarily
designed to determine whether increasing the dose of Sxl+
from two to three would increase the amount of male germline
Sxl autoregulation triggered by the transgene and perhaps
thereby disrupt spermatogenesis. Such 3×(Sxl+) males did show
intense and high-frequency anti-SXL staining, but only when the
X chromosome marked with v rather than sn3 was employed
(compare classes 5 and 6 in Table 1). Even with the v chromosome, less than 100% of the testes scored positive for
autoregulation and there was still no general disruption of
spermatogenesis. For the less sensitive sn3 X chromosome,
the extra dose of Sxl+ did appear to raise the level of Sxl autoregulation relative to that for 2×(Sxl+) males (Table 1, compare
classes 4 and 5).
Effects of mutations in snf, ovo and otu on Sxl
autoregulation in males
Female-sterile mutant alleles of the genes snf, ovo and ovarian
tumor (otu) have been shown to disrupt the female-specific
germline splicing of Sxl pre-mRNA, but it is not yet known
how direct this effect is, nor whether the effect is on female
splicing initiation or maintenance (Bae et al., 1994; Bopp et
al., 1993; Horabin et al., 1995; Oliver et al., 1993). Data in
Table 1 show that mutations in snf (class 8 versus 7) and ovo
(class 9 versus 7) greatly reduce the ability of P(otu::Sxl+cF1)A
to induce female Sxl+ RNA splicing in the male germline. The
block by snf1621 of P(otu::Sxl+cF1)A-induced autoregulation in
males is the inverse of what had been observed in females
where P(otu::Sxl+cF1)A effectively suppressed the block to
autoregulation by snf1621.
The response of the otu− chromosome (class 11) was down
relative to the v-marked X control (class 7), but not down
Female SXL protein in male germ cells 5039
Table 1. Effects of changes in sex determination genes on transgene-induced Sxl feedback loop engagement in the male
germline
Male
progeny
class
1
2
3
4
5
6
7
8
9
10
11
Testes with
“autoregulatory cysts”*
Cross
Key X/Y
genotype
(all Dp(1;1)Sxl+Sxl+)
P(otu::Sxl+cF1)
transgene
dose
%
(number/total)
A
A
B
C
D
E
F
G
H
I
J
v/Y; Df(tra)/tra1
v/Y; Df(tra)/+
sn3/Y; tra2B/tra2B
sn3/Y; tra2+/tra2+
sn3/Yy+ct+,Sxl+
v/Yy+ct+,Sxl+
ovo+snf+otu+v/Y
snf1621v/Y
ovoD1rS1v/Y
otu17/Y
otu17/Y
1
1
2
2
2
2
2
2
2
1
2
82
87
22
20
92
52
88
<3
2
5
27
(27/33)
(20/23)
(11/51)
(10/50)
(33/36)
(22/42)
(37/42)
(0/31)
(3/183)
(1/20)
(12/45)
*Anti-Sxl immunostaining beyond the normal domain of otu-promoter driven expression (see Fig. 1A). All males were 5-7 days old when dissected.
Full genotype of crosses (females × males). Marker phenotypes of the males examined are listed only for crosses in which more than one class of male was
produced:
A. Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ v/w1118; P{w+mC otu:SxlcF1}A/+; Df(3L)st-J7, tra−Ki roe pp/+ × w1118/Y; tra1/TM3, Ser. Ki was used as a closely linked
(3.6 cM) marker for Df(tra), while cm marked Dp(Sxl+) (<0.2 cM). Hence progeny class 1 was y wjt&w+mC cm v Ki while class 2 was y wjt&w+mC cm v Ser.
Because the desired non-recombinant y wjt cm v males (duplicated for Sxl+) with w+mC (the transgene marker) were indistinguishable from the undesired
recombinant y wjt cm+ v+ males without w+mC, the “total” number of testes was reduced by 17% to adjust for these undesired recombinants that carried neither
Dp(Sxl+) nor P(otu::Sxl+cF1).
B. y w f:=/Y; CyO/P{w+mC otu:SxlcF1}A tra2B bw × Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ sn3/Y; CyO/P{w+mC otu:SxlcF1}A tra2B bw. Examine Cy+ males.
C. y w f:=/Y; CyO/P{w+mC otu:SxlcF1}A × Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ sn3/Y; CyO/P{w+mC otu:SxlcF1}A. Examine Cy+ males. The animals for cross C
were closely related to those from cross B.
D. y w f:=/y+ct+Y,Sxl+; P{w+mC otu:SxlcF1}A/+ × Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ v/y+ct+Y,Sxl+; P{w+mC otu:SxlcF1}A/+. Examine w+mCw+mC males.
E. Same females as D × Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ sn3/Y; P{w+mC otu:Sxl+cF1}A/+. Examine w+mCw+mC males.
F. Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ v/Binsinscy; P{w+mC otu:Sxl+cF1}A /P{w+mC otu:Sxl+cF1}A
× Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ v/Y; P{w+mC otu:Sxl+cF1}A /P{w+mC otu:Sxl+cF1}A.
G. Dp(1;1)jnR1-A, y wjt snf1621 cm Sxl+Sxl+ v/Binsinscy; P{w+mC otu:Sxl+cF1}A /P{w+mC otu:Sxl+CF1}A × same males as D.
H. Dp(1;1)jnR1-A, y wjt ovoD1rS1cv cm Sxl+Sxl+ v/Binsinscy; P{w+mC otu:Sxl+cF1}A /P{w+mC otu:Sxl+cF1}A × Dp(1;1)jnR1-A, y wjt snf1621 cm Sxl+Sxl+ v/Y;
P{w+mC otu:SxlcF1}A/P{w+mC otu:SxlcF1}A.
I. Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ otu17/Binsinscy; P{w+mC otu:Sxl+cF1}A /P{w+mC otu:Sxl+cF1}A × Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ v/Y.
J. Same females as I × Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ otu17/Y; P{w+mC otu:Sxl+cF1}A /P{w+mC otu:Sxl+cF1}A.
relative to the sn3-marked X in the tra2 experiment (class 4).
Because construction of the Dp(Sxl+)otu− chromosome entailed
replacement of the same chromosomal region that lowered sensitivity of the Dp(Sxl+) chromosome in the tra2 series, it is not
possible to conclude that the otu genotype has any significant
effect on Sxl germline autoregulation. With this less sensitive
otu− chromosome, a dose effect of P(otu::Sxl+cF1)A on autoregulation was seen for the first time (compare classes 10 and
11).
Although the effect of ovo− on autoregulation reported in
Table 1 seems dramatic, it is likely to be due to a reduction in
the expression of the P(otu::Sxl+cF1)A transgene, rather than a
reduction in autoregulatory effectiveness of its SXL product,
since we found that the level of β-galactosidase activity
generated by the P(otu::lacZ) transgene (Comer et al., 1992)
is distinctly lower in testes that are ovo− versus ovo+ (data not
shown). A similar comparison of otu+ and otu− testes revealed
no effect of otu genotype on otu promoter expression in males
(data not shown).
A duplication of snf+ triggers female-specific
splicing of Sxl in the male germline
It had been noted long ago that in situations where a failure to
fully autoregulate limits expression of a mutant Sxl allele, increasing the dose of the X-chromosome region in which snf and ovo
reside can enhance Sxl autoregulation (footnote a of Fig. 17 in
Cline, 1988). It seemed worthwhile therefore to explore the possibility that a male-viable duplication of this region,
Dp(1;2)4FRDup (Salz, 1992) – referred to hereafter as Dp(4F) –
might enhance the effect of P(otu::Sxl+cF1)A on Sxl+, perhaps
raising the level of female SXL proteins to a level that would
interfere with spermatogenesis. Surprisingly, Dp(4F) did not
display any obvious synergism with the transgene, but it triggered
female Sxl+ splicing even in the absence of P(otu::Sxl+cF1)A.
This effect of Dp(4F) was eventually traced to snf.
This transgene-independent effect of Dp(4F) on Sxl in testes
is shown in Fig. 3. The pattern of anti-SXL staining induced by
Dp(4F) was generally mosaic in the presence of a female dose
(2×) of Sxl+ alleles (Fig. 3E), like that induced by
P(otu::Sxl+cF1)A. 90% of testes had autoregulatory cysts
(28/31) in this series, but the penetrance reached 100% in other
experiments (Table 2, classes 1 and 3). In contrast, with a male
dose (1×) of Sxl+ alleles (Fig. 3C), staining was weak and
limited to the apical tip and no autoregulatory cysts were
observed (41 testes examined). As expected, in the absence of
an endogenous Sxl+ allele, testes with Dp(4F) did not stain
above background (Fig. 3B, 20 testes examined), and there was
also no staining above background in males duplicated for Sxl+
alone (Fig. 3A). The non-staining control males in Fig. 3A,B
were sibs of the brightly staining male in Fig. 3E.
5040 J. H. Hager and T. W. Cline
Table 2. Effects of changes in sex determination genes on Dp(4F)-induced Sxl feedback loop engagement in the male
germline
Male
progeny
Class
Testes with
“autoregulatory cysts”*
Cross
Key X/Y genotype
%
(number/total)
1
A
Dp(Sxl+) v/Y; Dp(4F)/+
2x Sxl+ 2x ovo+ 2x snf+ 1x otu+
100
25/25
2
B
snf- Dp(Sxl+) v/Y; Dp(4F)/+
2x Sxl+ 2x ovo+ 1x snf+ 1x otu+
0
0/87
3
C
ovo- Dp(Sxl+) v/Y; Dp(4F)/+
2x Sxl+ 1x ovo+ 2x snf+ 1x otu+
100
15/15
4
D
Dp(Sxl+) otu-/Y; Dp(4F)/+
2x Sxl+ 2x ovo+ 2x snf+ 0x otu+
2
1/41
5
E
Dp(Sxl+) sn3/Y; Dp(4F) tra2B/+
58
25/43
6
E
Dp(Sxl+) sn3/Y; Dp(4F) tra2B/tra2B
84
48/57
*See Table 1.
Full genotype of crosses (females × males).
Dp(1;2)w+64b13 & Df (1)3F-4A is abbreviated as Dp(4F).
A. Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ v/Binsinscy, y w sn B × y w Df(1)ovoD1rG6/Y; Dp(4F),w+/+. w+ cm non-balancer males were examined.
B. Dp(1;1)jnR1-A, y w snf210 cm Sxl+Sxl+v/Binsinscy, y w sn B × males as in A. w+ cm non-balancer males were examined.
C. Dp(1;1)jnR1-A, y w ovoD1rS1cv cm Sxl+Sxl+v/Binsinscy, y w sn B × males as in A. w+ cv cm non-balancer males were examined.
D. Dp(1;1)jnR1-A, y wjtcm Sxl+Sxl+ otu17/Binsinscy, y w sn B × males as in A. w+ cm non-balancer males were examined.
E. y w f:=/Y; Dp(4F), w+ tra2B bw/CyO × Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ sn3/Y; cn tra2B bw/+. w+ bw+ (tra2/+) and w+ bw (tra2/tra2) males were examined.
As was the case with P(otu::Sxl+cF1)A, induction of female
SXL protein by Dp(4F) was without effect on either male
viability or fertility (data not shown). While Dp(4F) did reduce
male viability to 67% relative to Dp(4F) sisters, and male
fecundity to 14% relative to brothers without Dp(4F), these
effects of this complex chromosome aberration were no more
severe for males with the double-dose of Sxl+ required for
high-level activation of female Sxl expression than for their
brothers that carried no Sxl+ allele (data not shown), hence they
were not caused by inappropriate expression of Sxl.
Notwithstanding the lack of any substantial effect on
fertility, there was a significant morphological effect on spermatogenesis when SXL proteins were induced by Dp(4F) rather
than P(otu::Sxl+cF1)A. Although spermatocyte cysts exiting the
apical region of the testes were unambiguously male in morphology, they did not always proceed normally through the
spermatocyte growth phase. As they moved down the testis,
some failed to grow to the extent of neighboring cysts which
had not activated Sxl and they continued to stain with DAPI.
DAPI staining became particularly intense as the cysts became
morphologically abnormal (Fig. 3I,M). These abnormal cells
were more loosely organized than those of surrounding normal
cysts, and there were more than sixteen cells per cyst as the
cells began to degenerate. Since only cysts displaying anti-SXL
staining exhibited these abnormalities, it was not surprising
that no such abnormalities were seen in genotypes lacking
either Dp(4F) or Dp(Sxl+). At no point did the abnormal cells
display any recognizable female-like morphological character.
Lack of feminization was also evidenced by the fact that the
male-specific enhancer trap #590 (Gönczy et al., 1992)
continued to express normally in Dp(4F) Dp(Sxl+) testes (data
not shown). This enhancer reporter had been shown previously
to be repressed by Sxl+ in XX gonads (Wei et al., 1994).
One of the many genes besides snf whose dose is increased
by Dp(4F) is ovo, another apparent regulator of Sxl in the
germline. Data in Table 2 show that the increased dose of snf
but not ovo is necessary for Dp(4F) germline induction of antiSXL staining. Dp(4F) did not induce SXL spermatocyte cyst
staining in testes of males whose X chromosome carried the
null allele snf210 (Flickinger and Salz, 1994)(class 2), but cyst
staining was undiminished when the X chromosome carried the
null allele ovoD1rS1 instead (class 3).
Subsequent to our thorough analysis of Dp(4F), a genomic
transgene became available that contained only snf+ and the
two immediately flanking genes deadhead and DmMo15, both
unrelated to sex determination (B. Suter, personal communication). With this transgene we found that the increased dose
of snf+ is not only necessary for the effect of Dp(4F), but
actually sufficient to induce the female mode of Sxl RNA
splicing in the male germline. Moreover, with this transgene it
was possible to show that by increasing the dose of snf+ sufficiently, one can trigger autoregulatory cysts even in testes that
have only a single copy of Sxl+.
Data in Table 3 show that a single copy of P(snf+) was as
effective as Dp(4F) in inducing autoregulatory cysts. The most
appropriate comparison is between class 1 of Table 3 and class
5 of Table 2, since the same (less responsive) Dp(Sxl+) chromosome was used in these two crosses. Both the penetrance
and expressivity of anti-SXL staining were nearly identical in
these two male genotypes. Comparison of classes 1 and 2 in
Table 3 shows that increasing the dose of snf+ still further
continued to increase Sxl testes staining, although the level
reached in this experiment with two extra copies of snf+ was
not significantly greater than what had been achieved previously by Dp(4F) alone in genotypes that included a more
responsive Dp(Sxl+) X chromosome. Data in classes 3 and 4
of Table 3 show for the first time that increased dose of snf+
can trigger autoregulatory cysts even in males who have no
duplication of Sxl+. Nevertheless, the anti-SXL staining in
males with four extra copies of snf+ and a single Sxl+ allele
Female SXL protein in male germ cells 5041
Table 3. Increased dose of a snf+ transgene mimics effects of Dp(4F) in the male germline
Male
progeny
Class
Testes with
“autoregulatory cysts”*
Cross
Key X/Y genotype
Dp(Sxl+)
sn3/Y; P(snf+)/+
2xSxl+ 2xsnf+
%
(number/total)
61
30/49
1
A
2
A
Dp(Sxl+) sn3/Y; P(snf+)/P(snf+)
2xSxl+ 3xsnf+
96
102/106
3
B
+/Y; P(snf+)/P(snf+)
1xSxl+ 3xsnf+
2
1/60
4
C
+/Y; P(snf+)P(snf+)/P(snf+)P(snf+)
1xSxl+ 5xsnf+
18
11/62
5
D
+/Y; Dp(4F)/P(snf+)P(snf+)
1xSxl+ 4xsnf+
87
33/38
*See Table 1.
Full genotype of crosses (females × males).
A. y w f:=/Y; P{w+mC snf+}108/+ × Dp(1;1)jnR1-A, y wjt cm Sxl+Sxl+ sn3/Y; P{w+mC snf+}108/+ . Examine w+mC/+ (class 1) and w+mC/w+mC (class 2) males.
B. y w f:=/Y; P{w+mC snf+}108/P{w+mC snf+}108 × w1118/Y; P{w+mC snf+}108/P{w+mC snf+}108.
C. y w f:=/Y; P{w+mC snf+}19 P{w+mC snf+}108/P{w+mC snf+}19 P{w+mC snf+}108 × w1118/Y; P{w+mC snf+}19 P{w+mC snf+}108/P{w+mC snf+}19 P{w+mC
snf+}108 .
D. Same females as C × w1118/Y; Dp(1;2)w+64b13 & Df (1)3F,w+/+ . Examine w+ males.
was still considerably lower than that in males with only one
extra copy of snf+ but a female (2×) dose of Sxl+, showing that
the synergism between dose effects at these two loci is striking.
Induction of SXL staining by snf+ transgenes produced sporadic
morphological abnormalities and delays in spermatocyte maturation (data not shown) that were indistinguishable from those
induced by Dp(4F). The fact that these morphological abnormalities were only observed for genotypes with two copies of
Sxl+ suggests that they are a consequence of female SXL
protein in the male germline at levels above the minimum
required to trigger the RNA splicing feedback loop.
To help eliminate the possibility that signaling from the
gonadal soma might be involved in the induction by Dp(4F) of
SXL germline protein in males, the effect of mutations in tra2
on induction was examined. The rationale was the same as that
presented earlier in connection with autoregulation triggered
by P(otu::Sxl+cF1)A. As the data for progeny classes 5 and 6
in Table 2 show, loss of tra2 did not reduce the effectiveness
of Dp(4F); hence, the soma seems unlikely to be involved in
this induction.
Elimination of a functional otu locus drastically reduced
Dp(4F) induction of SXL staining (Table 2, class 4). The otu−
chromosome was more than 25× less responsive to Dp(4F)
than even the relatively unresponsive sn3 otu+ chromosome
(class 5) discussed earlier. This observation strengthens the
argument that the reduction in this case is truly due to otu,
rather than to some undefined aspects of genetic background
in the vicinity of Dp(Sxl+). Additional evidence in favor of this
interpretation is the fact that in every experiment with an otu+
chromosome, induction of germline SXL protein by Dp(4F)
was stronger than induction by P(otu::Sxl+cF1)A, but for the
otu− X chromosome the converse was true. Thus otu appears
to be important, though not essential, for induction of female
Sxl RNA splicing in the male germline by Dp(4F). Note that
while the sn3 otu+ chromosome (cross 5) was less sensitive to
Dp(4F) than the otu+ v X chromosome (crosses 1 and 3), the
difference was less dramatic than it had been with induction by
P(otu::Sxl+cF1)A, perhaps because Dp(4F) is a somewhat
stronger inducer of germline female SXL expression.
Sxl-positive male germ cells make female SXL
protein isoforms
Because anti-SXL staining induced in the adult testes by Dp(4F)
was strictly limited to the germ cells, a direct test could be made
on the assumption that staining reflects the presence of normal
female SXL protein isoforms in the male gonads, and hence
must arise from a switch to the female mode of Sxl RNA
splicing. Fig. 4 shows a western blot of protein extracts stained
with anti-SXL antibody. Wild-type control females are represented in lane 8. They display the characteristic 36 and 38 kDa
female doublet of major SXL isoforms. The difference between
Fig. 4. Western blot of crude extracts showing that normal female
SXL protein isoforms are induced by Dp(4F), snf+ specifically in
testes of adult males that carry two copies of Sxl+. Blot also shows
that male-specific SXL protein isoforms are not present in testes.
(lanes 1,2,3) Extract from 20 testes. (lane 4) Extract from 0.5 male
with testes removed. (lane 5) Extract from 0.5 male including testes.
(lanes 6,7) Extract from one male. (lane 8) Extract from 0.25 female.
Oregon-R is a wild-type strain. Genotypes: (lanes 1,5) same as Fig.
3A; (lanes 2,6) same as Fig. 3B; (lanes 3,4) same as Fig. 3E.
5042 J. H. Hager and T. W. Cline
these two isoforms arises as a consequence of non-sex-specific
alternative splicing of Sxl transcripts. Wild-type control males
(lane 7) lack these proteins and, instead, show two corresponding fainter bands at 33 and 35 kDa, respectively. These male
proteins were noted earlier by Bopp et al. (1991), who speculated that they are non-functional translational reinitiation
products. Lane 3 shows a testes extract from males with
autoregulatory cysts induced by Dp(4F) in a Dp(Sxl+) background. The characteristic female isoform doublet is apparent.
Controls in lanes 1 and 2 show that the induction of these female
proteins in males requires both Dp(4F) and Sxl+. Lane 4 shows
that the ectopic female SXL proteins are found only in the testis
itself, since the doublet is not seen in extracts of adult males
whose testes have been removed. Incidentally, comparison of
lanes 3 and 4 suggests that the wild-type male-specific SXL
proteins are only found in non-gonadal tissues. Comparison of
lanes 4 and 5 shows that the level of these minor male-specific
protein species was not affected by Dp(4F). Comparison of
lanes 5 and 7 show that, as expected, more of these malespecific proteins are generated as the dose of Sxl+ is increased.
Somatic feminization does not feminize SXL-positive
male germ cells
Might the absence of female development in SXL-positive male
gonads be due simply to the lack of a female soma? It had been
shown that a P(hsp70::tra+F) transgene that constitutively
generates female-specific TRA+ protein feminizes an XY soma
enough to support oogenesis for transplanted XX germ cells,
although XY germ cells continue to develop male in such a
somatic environment (Steinmann-Zwicky, 1994a). Not determined, however, was whether the inability of P(hsp70::tra+F)
to feminize XY germ cells might stem from a lack of female
SXL protein in those chromosomally male cells. Discovering
that Dp(4F) would reliably induce female SXL protein in XY
germ cells even when the somatic phenotype was male, we
were in a position to learn whether SXL-positive XY germ cells
would respond to a feminized soma to follow the female
pathway of differentiation.
Among the 21 control Dp(Sxl+)/Y; P(hsp70::tra+F) /+
pseudo-ovaries containing germ cells but lacking Dp(4F), there
was no hint of female development. Curiously, a few isolated
cells did stain positive for SXL protein in 71% of the cases (data
not shown), suggesting that female signals from the soma may
have a weak but significant effect on Sxl transcript splicing
even for XY germ cells, at least when those cells carry an extra
copy of Sxl+. When Dp(4F) was added to this genotype, the
number of SXL-positive cells increased and the level of staining
was much higher for 53% of the 45 pseudo-ovaries examined;
nevertheless, only two ovaries showed any trace of female
development: a few isolated nurse-like cells scattered among a
large number of small, spermatocyte-like cells. Although a
larger scale experiment would be required to determine the significance of these two exceptional gonads, it is clear that most
SXL-positive XY germ cells follow an abortive male developmental pathway even in a phenotypically female soma.
Increased dose of snf+ and Sxl+ feminizes 2X3A
germ cells
The interactions described so far between Dp(Sxl+) and
Dp(snf+) in the male germline are analogous to the interactions
in somatic cells between Dp(Sxl+) and duplications of X:A
numerator elements, genes that constitute the primary sex
determination signal in the soma (Cline, 1988). In both cases,
simultaneously increasing the dose of Sxl and a class of
uniquely dose-sensitive genes that regulate Sxl causes males to
express Sxl gene products that are appropriate only for females.
Although snf has been shown not to be a somatic numerator
element (Cline, 1988), the genetic behavior described here
suggests that snf might be a numerator element of the cellautonomous X-chromosome dose signal(s) for germline sex
determination that has been inferred to exist. Evidence for such
a role would be strengthened if a snf dose effect on germ cell
sexual fate could be demonstrated, not just an effect on Sxl
transcript splicing. We anticipated that triploid intersexes
(2X3A) might reveal such a dose effect.
In the context of a phenotypically female soma, the
ambiguous X:A balance (0.67) in 2X3A germ cells causes
some of them to follow the female pathway and differentiate
eggs, while others – even in the same gonad – choose an
abortive male pathway and form tumors of spermatocyte-like
cells (Schüpbach, 1985). It has been reported that, in such a
situation, nearly all germ cells can be induced to form eggs by
Sxl mutations that partially bypass normal sex-specific RNA
splicing controls (Nöthiger et al., 1989). This implies that,
regardless of whether Sxl can behave as a sexual switch gene
in the germline of XY diploid animals, it is surely acting so in
2X3A germ cells. Hence increasing the dose of snf+ in such a
2X3A situation should not just bias Sxl transcript splicing
towards the female mode, it should also drive germ cells
towards a female sexual fate. Increasing the dose of Sxl+ itself
should have a similar effect.
A test of these predictions is presented in Table 4. For this
test, we feminized the soma surrounding 2X3A germ cells
using the same P(hsp70::tra+F) transgene (McKeown et al.,
1988) discussed in the previous section. Incorporation of yolk
into growing germline cysts was an unambiguous phenotypic
criterion for female differentiation. Data are included on the
relative viability of the classes of flies compared and on the
number of germ cells actually differentiating into cysts. Only
with such information on viability can one distinguish between
a phenotypic effect caused by a change in the frequency with
which germ cells chose the male pathway over the female
versus an effect that arises as a consequence of differential
survival of germ cells that have chosen one pathway over the
other.
Data in this table establish that an important point shown
previously for somatic sex determination (Cline, 1983b, 1988)
also applies to the germ line: undefined differences in genetic
background can have a profound effect on sexual phenotype of
2X3A animals (compare the +/+; +/+/+ controls from crosses
A, C and E). Hence to help randomize genetic background, fly
stocks were maintained and crosses were carried out allowing
free recombination. In addition, most significance was attached
to comparisons between classes of siblings.
For every pair of 2X3A sibling genotypes compared in three
separate crosses in Table 4 (A, B and C), the flies with the
higher dose of snf+ displayed the greater proportion of yolky
germline cysts and eggs. The similarity between experimentals
and controls from crosses A and B with respect to organism
viability and germ cell cyst number allows one to conclude that
the effect of snf dose is indeed on the choice of sexual fate and
not on female germ cell survival.
Female SXL protein in male germ cells 5043
Table 4. Sxl+ and snf+ dose effects on the germline sexual phenotype of somatically feminized triploid intersexes (2X3A)
Ovarian phenotype*
P(hsp::tra+) XXAAA pseudofemales recovered
Cross to
generate
Relevant
genotype
Viability
relative to
control
siblings¶
Germ cell growth
Germ cell sex
Number
Eclosed
Animals
scored for
ovary
phenotype†
% ovaries
missing or
without
germ cells
“cysts” per
ovary‡,§
±s.e.m.
(range)
% ovaries
with yolk‡
(relative
to control)
yolky cysts
per ovary‡
±s.e.m.
(range)
A
+/+;
Dp(4F)/+/+
108 %
40
24
15%
52 ± 6
(1-115)
58%
(3.1x)
7±1
(1-20)
A
+/+;
+/+/+
reference
for above
37
24
13%
54±5
(1-140)
19%
7±2
(1-21)
B
Dp(Sxl+)/+
Dp(4F)/+/+
76 %
19
16
16%
61±8
(3-126)
67%
(3.0x)
14±4
(2-40)
B
Dp(Sxl+)/+;
+/+/+
reference
for above
25
23
20%
51±6
(2-143)
22%
3±1
(1-7)
C
+/+;
P(snf+)/+/+
135 %
31
22
14%
32±4
(1-101)
26%
(8.2x)
4±1
(1-12)
C
+/+;
+/+/+
reference
for above
23
32
9%
14±1
(1-71)
3%
1
D
Dp(Sxl+)/+;
+/+/+
32 %
73
28
5%
58±4
(1-119)
53%
5±1
(1-18)
D
SxlfP/+;
+/+/+
reference
for above
255
28
13%
17±3
(1-20)
0%
na
E
Dp(Sxl+)/+
+/+/+
49 %
72
32
9%
49 ± 3
(1-106)
43%
(2.3x)
5±0.6
(1-11)
E
+/+
+/+/+
reference
for above
147
32
19%
31±5
(1-112)
19%
4±1
(1-10)
F
SxlM1/+;
+/+/+
17 % @22o
0 % @25o
21
0
8
0%
75±4
(53-101)
88%
9±1
(5-15)
F
SxlfP/+;
+/+/+
reference
for above
127 @22o
0
115 @25o
0
*Animals were aged 3-6 days before scoring to allow vitellogenesis.
†Where this number is less than the number eclosed for the experimental class, the difference reflects deaths during the aging period for adults prior to
germline scoring.
‡Among ovaries with one or more germ cells, and including mature eggs.
§An entire germarium, whether arrested or not, is counted as “one cyst,” as in an entire ovary if it contains only disorganized germ cells.
¶Includes all adults that eclosed, regardless of whether they subsequently died prematurely.
Crosses:
Dp(4F) = Dp(1;2)w+64b13 & Df (1)3F 4A; Dp(Sxl) = Dp(1;1)jnR1-A,Sxl+l(1)7AaaSxl+l(1)7Aac
y w/Y; C(2L)RM, dp; C(2R)RM, px; C(3L)RM, h2 rs2; C(3R) males to the following females (number):
A: (1,200) w1118; Dp(4F),w+/+; ±P{hsp70:traF} (50%)/+
B: (900) w cm Dp(Sxl+); Dp(4F),w+/+; ±P{hsp70:traF} (50%)/+
C: (700) w1118; P{w+mC snf+}108/ +; ±P{hsp70:traF} (50%)/+ .
Based on analysis of the genetalia of animals not carrying P{hsp70:traF}, the zygotic genotype with respect to the snf+ transgene had no effect on the sexual
phenotype of XXAAA individuals.
D: (650) w cm Dp(Sxl+)/w cm SxlM1,fPc-w+mC; ±P{hsp70:traF} (50%)/+
SxlM1,fPd-w+mC is an allele whose activity has been destroyed by the insertion of a w+-tagged P element (Cline, unpublished). A lower fraction of eclosing w
than w+ individuals were scored for ovarian phenotype here and in cross E, but all w pseudofemale 2X3A animals were paired for scoring with w+ sibs that
eclosed at the same time to avoid any bias due to maternal or zygotic age.
E: (1000) w cm DpSxl+/y w cm P[w~]; ±P{hsp70:traF} (50%)/+.
P[w~] is a wmini-tagged P element approximately 0.1 cM centromere distal to Sxl that was generated as a local hop of P(lacW) from SxlM1,fPc-w+mC. It was
subsequently found to be even more closely linked to a truncated duplication of SxlM1 that was generated during the mobilization event. The presence of this
partial duplication should not interfere here with the use of this P element as a benign marker for Sxl+, since the duplication lacks SxlPm, fails to suppress snf1621
female sterility and does not complement female-sterile Sxl alleles.
F: (250) y w cm SxlM1/y w SxlM1,fPd-w+mC ct6
“±P{hsp70:traF} (50%)/+” refers to the fact that half these females carried P{hsp70:traF}, since they were derived from mothers confirmed to be heterozygous
for P{hsp70:traF} by the fact that they generated pseudofemale X/YBs progeny. P{hsp70:traF} is unlinked to Dp(4F). To randomize genetic backgrounds, females
for all crosses were taken only from stocks in which the relevant genetic elements had been maintained in an unbalanced condition for several generations.
5044 J. H. Hager and T. W. Cline
Crosses D and E in Table 4 show that the dose of Sxl+ also
influences the sexual phenotype of germ cells although, in this
case, the analysis is complicated somewhat by significant
effects on both organismal and germ cell viability. Cross D
allows a comparison of animals with three functional copies of
Sxl to those with one, while in cross E the comparison is
between animals with three versus two copies. Germ cells with
only a single copy of Sxl+ (cross D, viability reference) are
expected to be most biased towards the male pathway and that
is indeed what was observed. Even taking into account the fact
that these gonads had only 39% as many cysts as those of
brothers with three copies of Sxl+ and had a somewhat greater
proportion of missing or empty ovaries, one would have
expected to see 14 ovaries with yolky cysts rather than zero if
the degree of feminization had been the same for the two
genotypes.
Where there was a significant difference in germ cell proliferation (measured as ‘cysts’ per ovary) between experimental and control animals in Table 4, the animals with the more
feminized germline showed the greater amount of proliferation
as measured by cyst frequency. This contrasts with the somatic
dose effect of Sxl seen previously (Cline, 1983a, 1984, 1988)
and again in Table 4: 2X3A individuals with more copies of
Sxl+ are less viable, presumably as a consequence of effects on
dosage compensation.
Not all gonads had recognizable germ cells and the number
of germ cells attempting to differentiate (cysts) varied greatly
from gonad to gonad, both within and among individuals. In
no single gonad did all differentiating germ cells attempt to
follow a female pathway. Arrested germaria with ‘quiescent’
germ cells were frequently observed. Invariably some gonads
were either missing, detached and/or not organized into recognizable ovaries, although nothing resembling a testis was
ever observed. None of these abnormalities appeared to
correlate with the dose of snf+. Even in the most feminized
genotypes, germ cells reaching late stages of female differentiation were generally abnormal. Egg chambers were often disorganized and had inappropriate numbers and positions of
nurse cells and oocyte nuclei. The amount and distribution of
yolk was frequently abnormal. Egg chambers displaying a
‘dumpless’ phenotype (abnormal persistence of nurse cells due
to failure to transfer cytoplasm to the oocyte) were common.
These abnormalities in female germ cell differentiation
seemed more likely to be due to problems with gonadal dosage
compensation than to inadequacies in the level of tra+ activity
provided by P(hsp70::tra+F) in cells whose Sxl+ alleles were
expressed in the male mode. The sexual phenotype of all
internal and external somatic dimorphic characters appeared to
be fully female in 2X3A animals carrying P(hsp70::tra+F), in
contrast to the salt-and-pepper mosaic sexual phenotype of
their sibs that did not carry the traF transgene (mosaicism
reflecting cell-to-cell differences in the Sxl RNA splicing
mode). Nevertheless, because the inference that the effects of
snf+ dose on the male/female decision made by 2X3A germ
cells rests on the assumption that the female somatic signal
generated by P(hsp70::tra+F) alone is fully effective, we felt it
was important to explore further the basis for the observed
defects in oogenesis by comparing the gonads of 2X3A individuals feminized by P(hsp70::tra+F) to those feminized by
the constitutively feminizing allele, SxlM1. Since a single Sxl+
allele expressed in the female mode is sufficient to direct fully
female differentiation for all diploid cells, one would expect
the two Sxl alleles expressed in the female mode in 2X3A cells
feminized by SxlM1 to be more than adequate for directing
normal female development. In contrast, one could not be so
sure that expression from a single P(hsp70::tra+F) transgene
would be sufficient to direct fully normal female differentiation
of the gonadal soma (including proper female signaling to
germ cells) for 2X3A somatic cells that did not express their
Sxl alleles in the female mode.
The first study of SxlM1/Sxl+; AAA animals reported that this
genotype was lethal (Cline, 1983b); however, Nöthiger et al.
(1989) recovered viable adults and reported that essentially all
their germ cells chose a female pathway and differentiated
normally. Using an experimental design that included Sxl−/+;
AAA siblings as an internal viability reference for SxlM1/+;
AAA individuals, we traced this apparent discrepancy
regarding viability to differences in culture temperature, but
also discovered that SxlM 2X;3A adults may only survive under
conditions where not all cells follow the female pathway during
development. We observed that feminization of triploid intersexes by SxlM1 is accompanied by defects in oogenesis very
much like those that we observed with P(hsp70::tra+F). The
fact that gonads feminized by SxlM alleles generate gametes as
defective as gonads feminized by P(hsp70::tra+F) supports the
argument that inadequacies in the balance of X-linked and
autosomal gene products, rather than inadequacies in tra+dependent somatic signals, are responsible for abnormal
female germ cell differentiation in feminized 2X3A animals,
and hence that the Sxl and snf gene dose effects that we observe
reflect their germline rather than somatic functioning.
The effects of SxlM1 are shown in Table 4, cross F. No
SxlM1/+ 2X3A animals survived at 25°C, and their viability was
no more than 17% even at the 22°C used by Nöthiger et al.
(1989). Viability was even higher (47%) at 18°C but, at this
temperature, SxlM1 did not invariably feminize the external
cuticle (data not shown). We confirmed that nearly all germ
cells follow a female developmental pathway in viable SxlM1/+;
AAA animals at 22°C; however, we found that the vast
majority of cysts in advanced stages were abnormal, much as
they were in the Sxl+/Sxl+ 2X;3A pseudofemales of crosses AC. Fully wild-type mature eggs were seen in only three of the
eight animals examined and, even for those three, most latestage oocytes were either dumpless or flaccid. In one animal,
no cysts had even begun vitellogenesis after 3 days. The
average number of eggs and vitellogenic cysts per 2X;3A ovary
was actually higher for the Dp(Sxl+); Dp(4F) animals of cross
B than for the SxlM1/+ individuals of cross F. None of the 2X3A
pseudofemales in Table 4 had nearly as many vitellogenic cysts
and eggs as true triploid females (3X3A) aged for a comparable time (26±2 yolky cysts per ovary, range 18-40, n=12).
DISCUSSION
Sxl autoregulation occurs in the germline and is
disrupted by snf1621
Sex-specific regulation of Sxl in the germline, as in the soma,
occurs at the level of alternative pre-mRNA splicing of the
translation-terminating, male-specific Sxl exon #3 (Bopp et al.,
1993; Oliver et al., 1993). With the results presented here, it is
Female SXL protein in male germ cells 5045
clear that SXL protein itself participates in controlling this alternative splicing in the germline. Hence the autoregulatory
mechanism by which somatic cells maintain their sexual
identity long after the primary sex determination signal has disappeared also operates in the germline, at least with respect to
the activity state of Sxl. Germline Sxl autoregulation was
demonstrated by the ability of a P(otu::Sxl+cF1) transgene that
generates a single female SXL protein isoform in germ cells to
suppress the germline-autonomous female sterility of the antimorphic mutant allele snf1621, despite the fact that the
transgene by itself cannot provide all essential Sxl germline
functions. The inability of the transgene to support oogenesis
on its own is shown by its failure to complement recessive Sxl
alleles that are specifically defective in germline functions. The
female sterility of snf1621 was known to be due to the absence
of female Sxl RNA splice forms and (hence) SXL protein in the
adult germline (Bopp et al., 1993; Oliver et al., 1993;
Steinmann-Zwicky, 1988). For this snf allele to be suppressed
by P(otu::Sxl+cF1), the transgene product must induce the
female splicing mode for transcripts from endogenous Sxl+
alleles that would otherwise be spliced in the male mode.
Sxl germline autoregulation was also demonstrated by the
observation that P(otu::Sxl+cF1) can elicit female products
from endogenous Sxl+ alleles in males. From this fact, one can
deduce that neither a female signal input from the surrounding
gonadal soma, nor a female dose of X chromosomes in the
germline are essential for Sxl germline autoregulation.
However, the fact that autoregulation in the male is not as
robust as it is in the female even if the gonadal soma is
feminized indicates that a female dose of X chromosomes in
the germline must facilitate autoregulation. Neither the penetrance nor the expressivity of transgene-initiated engagement
of the Sxl splicing feedback loop in males is as high as it is in
females. The limiting factor does not appear to be simply the
level of transgene expression, since in genetic backgrounds
where a single copy of the transgene is effective at triggering
feedback in at least some cells in a majority of testes, adding
another copy had little effect. The conclusion that autoregulation in males is less robust than in females is also consistent
with the fact that snf1621 blocks P(otu::Sxl+cF1)-induced
germline autoregulation in males, yet P(otu::Sxl+cF1) suppresses the snf1621 block to female Sxl splicing in females.
However, other explanations are possible for this lack of reciprocity, including subtle differences between the sexes in
expression of the transgene’s otu promoter (see below).
It is shown here for the first time that SXL protein can induce
female Sxl pre-mRNA splicing without necessarily engaging
the Sxl feedback loop. Thus there must be a concentration
threshold for SXL protein below which the female mode of
RNA splicing is not self-maintaining. This was apparent in
male germ cells that carried P(otu::Sxl+cF1) and only a single
copy of Sxl+. Autoregulation clearly occurred in this genotype,
since the level of anti-SXL staining within the normal domain
of transgene expression was greater than it would have been in
the absence of an endogenous Sxl+ allele. Nevertheless, the fact
that anti-SXL staining did not extend beyond the domain of
transgene expression showed that the Sxl-positive feedback
loop had not become stably engaged.
Is Sxl a feminizing switch gene in the germline?
Chromosomally male somatic cells induced to engage the Sxl
autoregulatory feedback loop become developmentally
feminized and their dosage compensation is disrupted (Bell et
al., 1991; Cline, 1984). In contrast, it is shown here that male
germ cells can engage this same feedback loop without any
adverse effect on their development. This would seem to imply
that Sxl does not function as a switch gene for any sex-specific
aspect of development in the germline. However, the conservative interpretation of this negative result is only that the
autoregulatory splicing feedback loop for Sxl in the germline
can be maintained in males by a level of SXL protein that is
generally not sufficient to feminize cells to an extent that would
interfere with male development.
It seems premature to reject the possibility that Sxl is a
switch gene for at least some aspects of germline sexual development based on experiments with transgenes that are not fully
effective at bypassing the normal signals for Sxl regulation in
this cell type, particularly when there are adverse developmental effects of SXL female proteins in a significant fraction
of SXL-positive male germ cells. If autoregulation is inefficient
in male germ cells because they lack certain feminizing signals,
a germ cell that engages the autoregulatory splicing loop may
not necessarily ramp up to the full female Sxl RNA splicing
mode; instead, an equilibrium may be reached at which a significant fraction of Sxl transcripts continue to be processed in
the male mode. In such a situation, it would not be surprising
if the levels of SXL protein varied significantly among different
autoregulating cysts, with only those cysts at the higher end of
the distribution aborting spermatogenesis.
The possibility that germ cells might be able to engage the
Sxl autoregulatory feedback loop without ramping up to full
female splicing seems not to have been considered by Horabin
et al. (1995) who concluded that SXL protein in the germline
must not have the kind of global effect on sexual phenotype
that it has in the soma. They argued that Sxl was not a germline
sex switch in part because some XX germ cells of tra2ts2/tra2−
animals that stained positive for female SXL protein may nevertheless have expressed the orb gene in its male-specific
mode. Both Horabin et al. (1995) and Bae et al. (1994) also
argued against a switch-gene role for Sxl in the germline based
on the converse observation: orb expressed in its female mode
in germ cells that may not have had SXL protein. Unfortunately
the genotype used to eliminate SXL protein in these cases is
likely to have allowed the protein to have been made at preadult stages (see Salz and Flickinger, 1996) and does not invariably block female expression of Sxl even in adults (Bae et al.,
1994; Gollin and King, 1981; our unpublished results).
Moreover, the conclusions these workers drew regarding the
role of Sxl in germline sexual development rested on assumptions regarding the functional significance of orb sex-specific
expression and the source and timing of signals controlling orb
that remain to be validated.
If Sxl has no role within the germline as a sex switch for any
substantial fraction of sexually dimorphic regulatory genes, it
is curious that it can have such a profound effect on the gonads
of 2X3A animals. Even more peculiar would be the fact that
Sxl is regulated in a sex-specific fashion in the germline, and
by a mechanism very different from that in the soma, so long
before there is any overt sexual differentiation (Horabin et al.,
1995; Poirié et al., 1995). And if, as has been suggested
(Horabin et al., 1995), germ cells have no ‘sexual memory’ –
no heritable commitment to any aspect of sexual fate prior to
5046 J. H. Hager and T. W. Cline
their terminal differentiation – it seems strange indeed that Sxl
autoregulation, the key to stable sexual pathway commitment
in the soma, is one of the few aspects of Sxl regulation common
to both soma and germline. Regardless of whether Sxl does or
does not behave as a switch gene in the germline, our results
show clearly that one cannot infer from wild-type male morphology alone that a developing germ cell is not splicing a significant fraction of Sxl transcripts in the female mode.
Using males to study female-specific processes
Our study of the effects of ovo and otu illustrate the ironic fact
that the male germline may be a good place in which to study
the effects of certain types of female-sterile mutations on the
female-specific process of germline Sxl autoregulation. Often
the most reliable inferences in genetic studies are made from
the study of null alleles, but in the case of these two femalesterile loci, mutant female germline stem cells lacking these
genes die prior to the adult stage, thereby interfering with
analysis (Nagoshi et al., 1995; Rodesch et al., 1995; Staab and
Steinmann-Zwicky, 1995). Even if leaky alleles are used to
avoid this complication, germ cell physiology is grossly
disrupted, complicating inferences from phenotype (see
Horabin et al., 1995). But because the development of male
germ cells is unaffected by the loss of either ovo or otu, we
could show that null mutations in these two genes can block
the ability of various transgenes to induce Sxl female splicing
in the germline. Thus despite the fact that the essential
germline functions of ovo and otu are female specific (Geyer
et al., 1993; Oliver et al., 1987), and notwithstanding the
evidence that ovo expression is controlled by the dose of X
chromosomes (Oliver et al., 1994), our study shows that ovo
and otu are expressed in the male germline at a level that can
affect female-specific gene functions. The influence of ovo on
otu promoter activity that we discovered is consistent with phenotypic synergism that has been documented between mutant
alleles at these two loci (Pauli et al., 1993).
We were surprised to find similarities between males and
females in the movement of female SXL protein from the
cytoplasm to the nucleus as germ cells differentiate. Such shifts
in location were presumed to reflect important changes in this
protein’s functioning during oogenesis (Bopp et al., 1993), yet
similar shifts occur in a situation where the protein seems
unlikely to be functioning. The situation may be analogous to
that observed for the male-specific (presumably non-functional) forms of SXL protein in D. virilis: although these male
proteins do not trigger Sxl female splicing, their apparent association with nascent Sxl transcripts on salivary gland chromosomes seems indistinguishable from that for female-specific
forms (Bopp et al., 1996). The movement of SXL protein is yet
another of the many similarities that exist between young differentiating germ cells of both sexes and is one more ‘female’
process that can be studied in the context of a normal male
gonad.
The splicing factor gene snf is part of an Xchromosome dose signal that helps determine germ
cell sex
An effort to increase the sensitivity of Sxl to P(otu::Sxl+cF1)
led to the discovery that the female splicing mode of Sxl can
be induced in male germ cells simply by increasing the dose
of wild-type snf alleles. Study of snf1621 had previously impli-
cated this gene in both somatic and germline Sxl regulation;
however, when snf was discovered to encode a U1/U2 integral
snRNP protein and when snf1621 was shown to be an antimorph
rather than a null allele (Salz and Flickinger, 1996), there
seemed little likelihood of functional specificity for the actions
of this ‘generic splicing factor’ (Horabin et al., 1995) in
Drosophila. Our discovery of such a striking and specific effect
of increased dose of snf+ suggests that this gene’s product may
be pleiotropic and have a direct and highly specific role in the
splicing regulation of Sxl that is separate from its role as a
spliceosomal subunit. Pleiotropy of this sort has already been
indicated for a human homolog of snf (Gunderson et al., 1994).
Our demonstration of feminizing effects by increased Sxl
and snf dose on 2X3A germ cells argues that the synergistic
dose effects observed between these two genes in the gonads
of diploid males reflects their participation in a germline Xchromosome dose signal. This signal acts in conjunction with
somatic signals and perhaps with other germline X-chromosome dose signals as well to direct germ cell sexual fate. The
fact that the eggs and late-stage oocytes generated by 2X3A
cells were so often abnormal even when the cells carried SxlM1
suggests that there may be germline X-chromosome dose
effects on aspects of oogenesis that do not involve Sxl and snf,
but it might equally well suggest that normal oogenesis is
impossible if the somatic and/or germ cells of the gonad are
not properly dosage compensated.
Sxl+ and snf+ certainly cannot be the only components of the
germline-autonomous sex-determination signal for gametogenesis, since females carrying a male dose of Sxl and snf
produce normal eggs, at least at 25oC, while bona fide haploX germ cells surrounded by a female soma do not. Moreover,
males with a female dose of these two genes produce female
SXL proteins but remain fertile, not the result expected for bona
fide XX germ cells since XX germ cells expressing Sxl in its
female mode (due to SxlM1) in the context of a male soma have
been shown to embark on an abortive female developmental
sequence (Steinmann-Zwicky et al., 1989). At this time, it is
not possible to say whether the missing feminizing X-linked
elements in these fertile, duplication-bearing males regulate
other sex-determination genes in the germline that work in
pathways separate from Sxl, or whether instead the missing
genes act to increase the level of SXL protein in the germline
or the activity of its product above a threshold needed for Sxl
to behave as a sexual switch.
The basis for the snf germline dose effect
How might the increased dose of snf+ trigger female splicing
of Sxl transcripts in the male germline and why does it not do
so in the soma? From work with low-level gain-of-function Sxl
alleles in the soma (Cline, 1988 and unpublished), it seems
most likely that SNF acts to boost the autoregulatory effectiveness of very low levels of female SXL protein, rather than acting
directly on its own to influence Sxl transcript splicing. If
instead SNF were able to influence Sxl transcript splicing on its
own in both cell types, the protein would have to be much more
effective in the germline than in the soma to account for the
remarkable germline specificity of the dose effect described
here. In the alternative ‘facilitation’ model, germline specificity
might reflect Sxl splicing control being less tightly regulated in
germ cells than in the soma so that there would be a significant level of female SXL protein in the male germline with
Female SXL protein in male germ cells 5047
which SNF could act. Such protein may have escaped detection
so far, either because of limitations in the sensitivity of the
immunological tools used to detect it, and/or because it peaks
during a brief period when males have not yet been carefully
assayed. There is already evidence that levels of female SXL
protein sufficient to support female development can escape
detection by anti-SXL antibodies (Parkhurst et al., 1993).
There are reasons to believe that the sex-specific control of
Sxl splicing might not need to be as stringent in the germline
as it is in the soma. First, we show here that spermatogenesis
is relatively resistant to perturbation by female SXL proteins.
Second, since somatic as well as germline signals are a factor
in Sxl splicing control, the concentration threshold at which
SXL protein would trigger the female splicing feedback loop in
males might be significantly higher for germ cells than for the
soma. Finally, the gonad is uniquely able to compensate for
cells that might accidentally acquire an inappropriate sexual
identity and be lost, since the cells of this organ retain their
ability to divide mitotically and generate differentiated
products throughout adult life.
Initiation of female Sxl transcript splicing in wildtype female germ cells
How is the female Sxl splicing mode normally triggered in
germ cells? It has been proposed that sex-determination signals
might act at the transcriptional level on a germline-specific Sxl
promoter analogous to but separate from Sxlpe, the target of sex
signals in the soma (Bopp et al., 1993; Salz et al., 1989). The
demonstration here that Sxl autoregulation occurs in the
germline is important for the viability of this model. By this
scenario, snf+ might contribute to the germline-autonomous
part of the X-chromosome dose effect by increasing the
autoregulatory effectiveness of a pulse of female SXL proteins
generated from such a germline promoter. Like any X-chromosome dose signal element, snf would have to contribute to
the signal prior to becoming dosage compensated itself (note,
however, that dosage compensation has not yet been shown to
occur in the germline of Drosophila).
An alternative model is based on the idea discussed above
of leaky sex-specific Sxl splicing control in the germline, such
that a low but non-zero level of female mRNA is generated
from germline expression of SxlPm even in the absence of preexisting female SXL protein from some other source. In this
connection, it may be relevant that an anti-SXL signal was
apparent in many of the pseudo-ovaries of Dp(Sxl+)/Y;
P(hsp70::tra+F) /+ pseudofemales, the control class in our
study of the effect of somatic environment on the sexual development of SXL-positive male germ cells. Through germline
dose effects of a variety of X-linked genes including snf and
Sxl, that basal level of female Sxl RNA splicing for germ cells
in a female somatic environment might suffice to engage the
Sxl feedback loop in XX but not X(Y) germ cells. The initial
effect of any one of these X-linked genes might not have to be
very dramatic in order for the Sxl feedback loop to be triggered
eventually; moreover, while some of the effects might be
directly on splicing, others might be on such variables as the
SxlPm expression level. Effects of the magnitude of those that
we observed for ovo on the otu promoter might suffice.
Searching for germline gene targets of Sxl
One motivation for these ectopic expression studies was our
desire to develop a genetic handle on the downstream targets
of Sxl in the germline. We anticipated that ectopic SXL
expression in the germline might sterilize males by specifically
interfering with spermatogenesis. If so, mutations in downstream targets might be identified as suppressors of such
sterility. The fertility of the males described here is unfortunate in this connection, but our study nevertheless provides an
important starting point for continued genetic efforts in this
direction, particularly since some disruption of spermatogenesis by SXL female proteins was observed. In addition, the
genotypes described here should allow one to explore more
effectively than before both the process of Sxl germline
autoregulation and the factors that govern SXL protein localization and turnover in these cells. We continue to believe that
the comparison between somatic and germline control of Sxl
function has enormous potential to contribute to our understanding of the differences that exist between a cell type that
is effectively immortal and a cell type that is altruistic, whose
cooperation is essential to the success of the metazoan lifestyle.
We thank A. R. Comer, S. DiNardo, H. K. Salz, and B. Suter for
fly stocks and plasmids, and J. L. Dines, L. Megna, B. J. Meyer and
L. Sefton for helpful comments on the manuscript. This work was
supported by grant GM-23468 from the U. S. National Institutes of
Health to T.W.C.
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