Download Chase, B. A., and Baker, B. S.

Copyright 6 1995 by the Genetics Society of America
A Genetic Analysis of intersex, a Gene Regulating Sexual
Differentiation in Dvosophila melanogaster Females
Bruce A. Chase * *+ and Bruce S . Baker *
*Department of Biological Sciences, Stanford University, Stanford, California 94305 and tDepartment of Biology,
University of Nebraska, Omaha, Nebraska 68182
Manuscript received July 22, 1994
Accepted for publication December 23, 1994
ABSTRACT
Sex-type in Dros@hila mlanogaster is controlled by a hierarchically acting setof regulatory genes. At
the terminusof this hierarchy lie those regulatory genes responsible for implementing sexual differentiation: genes that control the
activity of target loci whose products give rise to sexually dimorphic phenotypes. The genetic analysisof the intersex (ix) gene presented here demonstrates that
ix is such a
terminally positioned regulatory locus. The ixlocus has been localized
to the cytogenetic interval between
of homozygotes
47E3-6 and 47F11-18. A comparison of the morphological and behavioral phenotypes
and hemizygotes for three point mutations atix indicates that the null phenotypeof ix is to transform
diplo-X animals into intersexes while leaving haplo-X animals unaffected. Analysisof X-ray induced,
mitotic recombination clones lackingix’ function in the abdomenof diplo-Xindividuals indicates that
the ix+ product functionsin a cell-autonomous manner and that it is required at least until the termination of cell division in this tissue. Taken
together with previous analyses, our results indicate that the
ix’ product is required to function with the female-specific product
of doublesex to implement appropriate
female sexual differentiation in diplo-X animals.
I
N Drosophila, extensive genetic and molecular analy-
ses of sex determination have elucidated a set of
genes that act togetherwithin a regulatory hierarchy to
control sex type. This hierarchy can be subdivided into
three levels accordingto the function and mode of
regulation of the genes actingat each level. Atthe apex
of the hierarchy lies the primary determinant of sex
type: the ratio of the number of Xchromosomes to the
number of sets of autosomes (the X:A ratio; BRIDGES
1925). Animals having an XA ratio of 1:2 develop as
males while animals having an X A ratio of 2:2 develop
as females. This initial primary sex-determining signal
is assessedand communicated to a key regulatory gene,
Sex-lethal (Sxl) , through the action of dose-sensitiveX A
numerator and denominator elements including the
products of the sisterless-a (sis-a) , sisterless-b(sis-b), deadpan ( d p n ) and runt (run) loci which act together with
maternally produced gene products, including that of
the daughterless ( d a ) and extra machrochaetae( emc) genes
(reviewed in CLINE1993; CLINE1976,1980,1983,1984,
and
1986, 1988, 1989; MAINE et al. 1985; CRONMILLER
CLINE1987; GERGEN
1987; TORRESand SANCHEZ
1989,
1991, 1992; PARKHUEST et al. 1990; DUFFYand GERGEN
1991; ERICKSON
and CLINE1991; YOUNGER-SHEPERD
et
al. 1992). Indiplo-Xindividuals, this results in theearly
female-specific transcriptional activation of Sxl (KEYES
et al. 1992). Once female-specific function is initiated
Corresponding authw: Bruce A. Chase, Department of Biology, AH
503, 60th and Dodge Sts., Omaha, NE 68182-0040.
E-mail: [email protected]
Genetics 139: 1649-1661 (April, 1995)
at Sxl, thefemaledetermined
state is maintained
througha positive autoregulatory feedback loop in
which Sxl proteins and the products of the sans Jille
( s n f ) andfl( 2 ) d loci direct thefemale-specific splicing
of later transcribed Sxl mRNAs (OLIVERet al. 1988;
BELL et al. 1988, 1991; STEINMANN-ZWICKY
1988; GRANADINO et al. 1990, 1992). This female-specificsplicing
of Sxl primary transcripts marks the beginning of a second levelof control within the regulatory hierarchy
by initiating a cascade of female-specific RNA splicing
events both at Sxl and at regulatory loci lying downstream from Sxl [ transfmrner ( tra) and doublesex ( dsx) ]
that is directed by the products of genes lying within
the regulatory hierarchy itself [ Sxl, tra, and t r a n g i 2 ( tra-2) ] ( BOGGS et al. 1987; MCKEOWNet al. 1988;
NAGOSHI et al. 1988; BURTISand BAKER1989) . The
presence of the RNA-splicing cascade effectively serves
to “amplify” the primary sexdetermination signal to
loci lying at the terminus of the regulatory hierarchy
(BAKER1989). The alternative splicing that occurs in
females results in the production of a female-specific
protein product at dsx, DSXF,while the defaultsplicing
that occurs in males results in the productionof a malespecific proteinproductat
dsx, DSXM (BURTISand
BAKER
1989). Theproducts of the dsxlocus, with those
of additional terminally positioned regulatory loci, direct the implementation of sexual differentiation and
establish a third level of control within the regulatory
hierarchy. These regulatory loci function to repress target loci not required for the acquisition of either the
female (in diplo-X individuals) or the male (in haplo-
1650
B. A. Chase and B. S. Baker
X individuals) sex type (BAKERand RIDGE 1980) ; in
The conclusions from previous studies of ix are subsome cases it appears they may also act in a positive
ject to two important considerations. First, it was not
manner (TAYLOR
1992; TAYLOR
and TRUMAN
1992; Coknown if the two i x alleles available for these studies
SHIGANO and WENSINK
19%; JURSNICH and BURTIS
represented complete loss-of-function alleles. Thus, it
1993; see also HALL.
1994).Extensive investigations into
was possible that the phenotypesobserved were the conthe mechanisms by which the regulatory genes at the
sequences of partial loss-of-function alleles or alleles
last level ofthe hierarchy act to bring about thefemalethat did notreflect the full spectrum of ix function. For
specific expression of the genes encoding theyolk proexample, ix’ and i x 2 , which morphologically transform
teins ( BELOTEet al. 1985; SHEPHERD
et al. 1985; GARABE- diplo-X individuals into phenotypic intersexes while
DIAN et al. 1986) have shown that they function at the
leaving haplo-X individuals morphologically unaflevel of controlling the transcriptional activity of loci
fected, could be partial loss-of-function alleles at a trarequired for terminal sexual differentiation, Furtherlike locus, where full loss-of-function mutations transmore, as the dsx protein products, which share a zincform diplo-X individuals into phenotypic males. Alterfinger related DNA binding domain ( ERDMAN and
BURnatively, they might be female-specific alleles at a dsxTIS 1993), are capable of binding to a sex-specific enlike locus, where null mutations transform both diplohancer of the yolk protein genes (BURTISet al. 1991;
and haplo-X individuals into phenotypic intersexes.
COSHIGANO
and WENSINK
1993), the genes positioned
Second, effects of genetic background, not directly reat the terminus of the regulatory hierarchy appear to
lated to intersex function, but having an impact on sexdirectly exert this transcriptional control.
specific behavior, may have contributed to the effects
That dsx may not be the sole locus functioning at the
seen in the behavioral analyses. For example, the peneterminus of the regulatory hierarchy has been sugtrance and expressivity associated with many mutations
gested by studies on two additional genes, hmaphrodite
having sex-specificphenotypes, including behavior, can
( her) ( PUI.TZet al. 1994;PLJLrZand BAKER,unpublished
be quite sensitive to genetic background ( e.g., see Table
data) andintprsex ( ix) (MORGANet al. 1943; MEEK and
2 in CLINE 1988)
. It would therefore be useful to reasEDMONDSON
1951; MEYER1958; KROEGER 1959; BAKER
sess the behavioral phenotypes of ix individuals that
and RIDGE 1980). Inthis paper, wewill focus on the
are homozygous, hemizygous or heteroallelic for known
ix locus. Genetic analysis of the epistatic interactions
loss-of-function i x alleles. We have undertaken a genetic
between ix and the other loci in the sex-determination
analysisof ix to specifically address these two issues,
regulatory hierarchy, using ix‘ and ix2, the point mutaand to clarify the role ( s ) and position of ix in the sextions, whichhave thus far defined the
determination regulatory hierarchy.
ix locus, sugIn this paper, we describe the results of investigations
gested that the ix+ product functions to repress male
differentiation functions in diplo-Xindividuals and acts
into thefunction and action of ix using extant andnewly
there eithersequentially to, together with, or in parallel
generated ix point mutations and deficiencies. We spewith, DSXF (BAKERand RIDGE 1980). The possibility
cifically address the phenotype associated with complete
loss-of-functionalleles, readdress whether ix’ function
that ix may not only function with dsx to control many
aspects of somatic sex, but also function independently
is required for normal male courtship behavior and, by
of dsx to regulate other aspects of somatic sex has been
analyzing the morphological characteristics of clones of
raised by subsequent behavioral analyses of dsx and ix
ix’ tissue in a diplo-X, ix+ genetic background, describe
the modein which ixfunctions during development. We
mutants ( MCROBERT
and TOMPKINS 1985)
. These indidemonstrate that the loss-of-function phenotype at ix is
cated that haplo-X ix’ homozygotes exhibit decreased
to specifically transform diplo-X individuals into phenolevelsof male courtship behavior, and that, unlike
typic intersexes while leaving haplo-X individuals unafdiplo-X dsx homozygotes, diplo-X i x ’ homozygotes befected. We also show that, while ix’ may have a effect on
have as normal females: they elicit normal courtship
male courtship behavior when assessed quantitativelyvia
behavior from wild-type males and do notcourt either
a courtship index, ix’ males are nonetheless capable of
males or females. Put another way, these results indicate
normal courtship behavior and no other intersex allele,
that although i x and dsx mutants have highly similar
either in homozygous, hemizygousor heteroallelic comeffects on the external morphology of diplo-X adults,
binations, has a demonstrable effect on male courtship
they have dissimilar effects on the diplo-Xnervous sysbehavior. Finallywe demonstrate that, much like the
tem. Furthermore, they suggest that although the ix‘
female-specific function provided by the dsx locus, ix
mutation has no morphological effect on haplo-X
functions autonomously and is required inindividual
adults, it does affect the haplo-Xnervous system. Thus,
diplo-X cells during their development at least until the
these results suggest that the normal function
of i x may
last few cell divisionsboth to setand maintain the female
not only be to act in a pathwaywith dsx to control
pathway. These results are used to address the role of ix
morphology in diplo-Xanimals, butalso that it may act,
with respect to the action of other loci that function
perhaps in a separate pathway (given that i x and dm
within the sex-determination regulatory hierarchy in Ilre
may have different effects on the diplo-Xadultnervous
sqbhila melanogaster.
system), in the nervous system.
intersex in Drosophila
MATERIALSANDMETHODS
Drosophila strains and culture: Crosses wereperformed using standard Drosophila media (cornmeal, yeast, dextrose
and sucrose) seeded with live yeast at 25" and 60% relative
humidity unless specifically noted. Descriptions of mutations
and chromosomes not given below can be found in LINDSLEY
and ZIMM (1992).
A Canton S (CS) strain isogenic for the second and third
chromosomes, obtained from Ian Duncan (Washington University, St. Louis), was used as a wild-type strain.
ix' is a spontaneous ix allele first reported by MORGANet
al. 1943 and employed by KROEGER 1959. ix2is a W-induced
ix allele first reported by MEYER1951,1958.Both are described in detail byBAKER and RIDGE ( 1980). i ~ ~ ' " . ,' ~or
ix3, is an ethylmethane sulfonate (EMS)induced ix allele
obtained from IANDUNCAN.
It was found on the second chro'~
having a fully
mosome of a +/Zn(3LL)P, ss bxd i ~ b 5 female
pigmented sixth abdominal tergite obtained from a cross of
EMS-treated CS iso-2,3 males and T M I , Me/Zn ( 3 L )P , ss bxd
iab5c7females.
O f ( 2R) e n B [ 47E3-6; 48A41 and Of(2R) enA [ 47D3; 48B251 were obtained from MICHAELRUSSELL (University ofAlberta, Edmonton) ; O f ( 2R) en3' [ 48A-48B51 was obtained
from THOMASKORNBERG
(University of California, San Francisco) ; ad, arch,chl, blo, p u j spt, sha, brh, fai and whd were
obtained from the Mid-America Drosophila stock center;
1( 2 ) ZA109 was obtained from the Tubingen stock center;
~ h n " (~=shn2)
~
was obtained from KATHRYNANDERSON (University of California, Berkeley), shnm5-' and shnTD5,2( =shn3)
are (possibly identical) alleles generated by hybrid dysgenesis
obtained from PETERGERGEN
(SUNY, Stony Brook, N Y ) . The
P-strains 7r-2, Y8b51, 8-31-15, 78.16 and ZnbredCage 3 were
obtained from WILLIAM
ENGELS(University of Wisconsin,
Madison). A w+insert, w+xba9, in region 47F ( HAMAet al.,
1990),and
three X-ray-induced w revertants, wxba9x1,
wxba9x3, wxba9x4, were obtained from CHIHIROHAMA(National Institute of Neuroscience, Tokyo). Each of these revertants failed to complement ix point mutations and cytological examination identified deletions having the following
breakpoints: wxba9x1, Df (2R)47E1; 48A1; wxba9x3, Df (2R)47D; 48B5; wxba9x4, Df (2R) 47D5-6; 48A4.
Mutageneses: An F2 screen for hybrid dysgenesis induced
ixalleles was performed (at room temperature, 21 -23") using
the 7r-2, Y8b51, ZnbredCage 3, 8-31-15 and 78.16 P-strains.
Male progeny from a cross of P-strain malesand c n bw virgin
females isogenic for their second chromosome were mated
to y ; Sco/CyO virgin females, and single cn bw/ Cy0 virgin
females test crossed to pr cn ix'/CyO males. In 1050 Znbred
Cage 3 derived, 1050 8-31-15 derived, 1100 78.16 derived
and 2525 Y8b51 derived chromosomes tested, no ix alleles
were found. In 2075 7r-2 derived chromosomes, one ix allele
was identified. This allele was unable to be reverted by a
second dysgenic cross and was present on a recessive-lethal
bearing chromosome. Cytological examination revealed that
the chromosome bore a deficiency, O f ( 2 Rix87i3
)
( 4704-8;
47FlI-18).
An F1 screen for new X-rayinduced ixalleles was performed
(at room temperature, 22 t 2", mean t SD) using a strategy
similar to that by which ix3 was recovered, using 3900 R (Torrex Model150 X-ray machine, 120-KV,5-mA,330 R/min,
1-mm plexiglass filter). It identified 31 females showing a
fully pigmented sixth abdominal tergite in a screen of -9000
animals. All bore second chromosomes which complemented
ax' at 22".
An F2 screen for X-ray induced ix alleles was performed by
crossing at 22 ? 2" c n bw males isogenic for their second
chromosome or CS males isogenic for both second and third
chromosomes and exposed to either 3500 R or 4300 R (using
1651
the above irradiation conditions) to y ; ScO/CyO, Cy
cn
virgin females and test-crossing individual or cn bw/CyO,
Cy p m2 male offspring to pr cn ix'/CyO, Cy p m virgin females. Of 7475 second chromosomes screened, a number of
lines were recovered that appeared to have intersexual + or
cn bw/p cn ix' individuals. Siblings that were ( + or cn bw)
ix'/CyO, Cy p m2were retested at 25", and one line was recovered that bore a ( + ) second chromosome that only weakly
complemented ix' and was recessive lethal. The potential intersex allele on this chromosome was named i x p H , after
P. HUANG(who assisted in the screen). The possibility that
this allele represented a hypomorphic or temperature-sensitive ix allele was investigated by testing it for complementation with other ix alleles and deletions for zx at 18", 25" and
29". The external morphology and fertility of female progeny
of a number of single pair matings (set up in reciprocal directions) of the cross ixpH/SM5, Cy X ix/CyO, Cy were
scored as measures of intersexuality. The + chromosome was
found to bear a temperature-sensitive ix allele (see RESULTS)
,
hereafter referred to as ix4. Cytological examination of this
chromosome revealed a multiply inverted chromosome having the new order: 21 to 22B3-6/42A6-8 to 59E-60B/42Al5 to 22/59E-60B to 60F. This chromosome also shows a constriction in region 47E2-6 that we sometimes see inour reference CS is0 2 - 3 wild-type strain.
Behavioral assays: Male courtship behavior tests and the
determination of courtship indices (C.I.) were carried out
essentially as described by MCROBERTand TOMPKINS
( 1985),
except that very light ether anesthesia was used for the initial
collection of some adults. To assess the existence of qualitative
differences in courtship behavior between haplo-X ix and ix+
flies, the specific behaviors observed were also noted.
Autonomy and time of i x function: To assess the cellular
autonomy of ix and the developmental period during which
it is expressed, clones of homozygous ix tissue were induced
on a wild-type (heterozygous i d / + ) background at various
times during development using X-ray-induced mitotic crossing over. The cell autonomous marker pun ( 2 - 5 5 . 4 , 42E343C3; GARCIA-BELLIDO
and DAPENA1974; LINDsLEY and ZIMM
1992) was used to aid in the detection of clones (Figure 2 ) .
Clones were induced in the progeny of the cross pwn m ix'/
Cy0 X CS, is0 2-3 (set up reciprocally) by irradiation with
1000 R under the conditions described above. The developmental age of the progeny at the time of irradiation was determined as described (GARCIA-BELLIDO
and MERRIAM1971;
BAKER
and RIDGE 1980). Female adults of the genotype
pwn cn ix were then collected, preserved and mounted, and
pwn i x clones scored as described (BAKER
and RIDGE 1980).
+
+/
RESULTS
Localization: We have localized the ix gene by examining the patterns of complementation of ix point mutants with a set of overlapping deletions. The morphological phenotypes seen in diplo-X individuals associated
with the point mutations ix', ix2 and ix3 are complemented by D f ( 2R) en", but are not complemented by
D f ( 2 R ) e n A , Df ( 2 R )e n B , D f ( 2 R )ix87i3,Df ( 2 R ) wxba%l,
Df ( 2R) wxba9x3and Df ( 2R) wxba9x4, which placesthe
intersex locus in cytologcal region between 47E3-6
and 47F18 (Figure 1) . To address whether other loci
whose meiotic map positions are close to that of ix
might also lie in this cytological region, the following
second chromosome loci were tested for their ability
to be complemented by Of(2R) e n B , Df(2R) e n A and
R. A. Chase and B. S. Baker
1652
47Cf
D I E I F 148A1 B I C
I
I
I
I
w+xbs9
Df(2R)en
Df(2R)i~~"~
Df(Zf?)WxbaSxl
Df(2R)wxba9x3
-
Df(2R)wxba9~4
I
-
-
-
~f(2~)e~31
Df(ZR)enA
1
ix+ 40A; 4895
ix- 47D3; 4082-5
ix
47E36: 40A4
ix- 4704-0; 47F11-10
ix- 47E1; 48A1
-
-
ix'
-
I
47F11-10
47E3-6
47D; 4085
ix- 47D5-6; 48A4
I
FIGURE1.-Cytological localization of the ix locus. Lines
indicate chromosomal material present in the deletions.
7 ~+,.xlm 0 .
IS a 7u+ insertion in 47F from which the X-ray induced
w revertants Df ( 2R) ruxba9xl, Df ( 2R) ruxba9x2 and Df ( 2R)ruxba9x3 were obtained by HAMAr / nl. ( 1990).
D f ( 2R) e n 3 ' : arch (60.5), archoid ( a d , 60.7) , bloated
(hlo, 58.5), brownhead (In-h, 6 1 ) , chaetelk ( c h l , 60.8),
faint (fai, 6 1 ) , I ( 2 ) IA109, puff (fluf, 5 8 ) , schnum'
( s h n , 62; using shnIM56,~hn"'~.' and shn")5,2
shavenoid (sha, 6 2 ) , spmatheca ( s p t , 63.3) and withered
( whd, 61) . Of these loci, shn failed to be complemented
by both Df ( 2R) e
n''and Of ( 2R) en" but was complemented by D f ( 2R) m 3 ' . While the threeshn alleles also
failed to be complemented by D f ( 2R) ixX"', they were
complemented by each of the i x point mutations. Thus,
while shn does not appear to be allelic to i x , it does lie
with i x in 47E3-6 to 47F11- 18. The phenotype of shn is
quite differentfrom that of ix: homozygous shn embryos
lack dorsal hypoderm andare embryonic lethal. As
none of the other loci mapped within this region, it
would appear that ix is not part of a complex of known
genes or a tissue-specificallele at a previously identified
locus. Several of the loci examined do map in this general
region:
both
spt and sha are uncovered by
Df ( 2R) e n A and Df ( 2R) en". Based on previous reports,
spl homozygotes display a temperature-sensitive phenotype that affects spermathecae. We suggest that spt is
a hypomorphic and tissue-specific engraikd ( e n , 48A)
allele, as we have found that it fails to complement en',
and spt/en', spt/Df( 2R) e n A , spt/Df ( 2 R )en" and spt/
Df ( 2R) en3' adults show both engraihdwingand spt phenotypes at 25".
Loss-of-function phenotype: Previous characterization of the ix' and i x 2 point mutations by BAKERand
RIDGE(1980) demonstrated that diplo-Xanimals bearing these alleles are transformed into intersexes: both
male and female genital promordia develop simultaneously and cells exhibiting sexually dimorphic characteristics try to implement both male- and female-type pathways simultaneously. To assess whetherthe
loss-offunction phenotype atix corresponds to the phenotype
associated with these previously knownpoint mutations,
) ?
the morphological phenotypes associated with homozygous and hemizygous [ ix/Df ( 2 R )en"] ix', ix2 and ix3
alleles were characterized at IS", 25" and 29" (Table
1 ) . As reported previously by BAKERand RIDGE( 1980)
for ix' and i x 2 homozygotes, haplo-X ix', ix2 and ix'
homozygotes and hemizygoteswere found to be phenotypically normal, fertile males and both haplo-X and
diplo-X animals have normal viability through eclosion
(see also Table 2, row G ) .
In diplo-X animals, a similar range of intersexual
morphological characteristics was observed for each of
the ix alleles at each temperature and independent of
whether the mutation was homozygous or hemizygous
(Table 1 ) . However, within this range of intersexual
characteristics, specific intersexual characteristics were
sometimes seen to a greater or lesser extent: the intersexual phenotype of a particular genotype tended to
be more robust atlower temperatures and, ata specific
temperature, hemizygotes often evidenced a more robust phenotype than homozygotes. More specifically,at
lower temperatures and in hemizygotes, there was a
greater frequency of animals with extensive male-like
pigmentation, relatively larger seventh abdominal tergites ( a female characteristic) and fewer completely
everted genitalia and analia. In animals with incompletely everted genitalia and analia, extensive intersexual genitalia and analia that presumably were too large
to evert properly and be evidenced externally could
be found upon dissection of fresh tissue or following
processing and mounting of the cuticle. At lower temperatures, and to some extent, in hemizygotes, there
was a greater frequency of animals with more development of the phallus apparatus, although in all cases,
the phallus apparatus was reduced when compared with
structures foundin normal males. Thus, within the similar range of intersexual phenotypes observed in all homozygous and hemizygous ix animals, both male- and
female-type morphological features tended to be more
robust at lower temperatures and in hemizygotes.
The effect of temperature and genetic background
onthe
robustness of sexual characteristics is well
known. Indeed, the extent
of pigmentation of the posterior fifth and sixth tergites in wild-type males and females can vary considerably between strains and at differenttemperatures within astrain. As thegenetic
backgrounds of the three-point mutations are potentially quite diverse (their origin is described in MATERIALS AND METHODS), it is likelythat thesubtle differences
in the intersexual phenotypes of these mutants are attributable to differences in genetic background and not
differences in residual ix+ function. Subtle differences
among genotypically identical animals raised at different temperatures are also unlikely to result from different amounts of residual i x f function. At 18", slower
growth and larger cell size may result in the development of larger intersexual genitalia and analia that are
physically hindered from proper eversion. Under such
1653
"
5
b
4
c:
5
and
B. A. Chase
1654
B. S. Baker
TABLE 2
Temperature-sensitive interallelic complementation of ix4
Percent of total progeny
ix heteroallelic combinations or hemizygotes
Balancer
~
Cross"
A. id/Balancer
X
ix4/Balancer
B. i.x?/BalancerX ix4/Balancer
C. i.d/Balancer
X
&/Balancer
D. Df(2R)mB/Balancer X &/Balancer
E. i~~~*~/Balancer
X ix4/Balancer
F. i~~~'j/Balancer
X Df(2R)enB/Balancer
G. ix'/Balancer X Df(2R)enB/Balancer
Temp.
n'
29
25
18
29
25
18
29
25
18
29
25
18
29
25
18
29
25
18
25
18
933
1330
1360
1073
1787
1083
193
104
217
666
2304
924
1097
1815
1423
371
645
725
850
163
% Fertile
Sterile Fertile
Male
Female
Male
female
female
Intersexual
diplo-X
34.8
33.1
31.0
33.0
32.2
27.3
34.2
34.6
36.9
35.6
34.8
30.4
32.6
31.8
31.5
49.6
52.7
45.2
37.8
34.6
32.5
38.2
33.7
32.1
31.4
38.5
33.2
46.2
38.7
38.1
37.2
39.6
34.6
35.6
35.2
50.4
47.3
54.8
32.6
30.7
17.9
15.6
19.3
18.1
19.5
19.7
16.1
10.6
13.8
14.3
16.1
14.3
16.1
16.9
17.6
0
0
0
15.8
20.3
0
0
0
4.1
15.3
13.7
0
0
0
0
9.5
14.9
14.1
15.2
15.4
0
0
0
0
0
0
0.8
1.6
12.8
1.6
0.6
0
0
9.7
0
0.4
0.8
2.5
0.5
0.4
0
0
0
0
0
14.8
12.3
14.5
0.4
0
0
16.6
8.7
0.9
12.0
1.9
0
0
0
0
0
0
0
13.8
14.4
0
0
0
24
90
96
0
0
0
0
81
95
85
97
98
0
0
Replicate crosses of single pair matings were set up at the specified temperature as described in the MATERIALS AND
section. The complete genotypes of the crosses were A, p r cn ix'/CyO X ix4/SM5, reciprocally; B, i.x?/SMl X ix4/
SM5, reciprocally; C, id/SM6u X ix4/SM5, reciprocally; D, w/w;Df(2R)enB/SM5 X +/Y;ix4/SM5 and w/Y; Df(2R)enB/SM5
X +/+; ix4/SM5; E, i~*~"/Cy0
X ix4/SM5, reciprocally; F, + / K i~*~"/Cy0
X w/w;Df(2R)enB/SM5 and +/+; ix87'3/
Cy0 X w/Y; Df(2R)enB/SM5;and G, +/Y;pr cn ix'/CyO X w/w;Df(2R)enB/SM5and +/+; p r cn ix'/CyO X w/Y; Df(2R)of reciprocal crosses (data not shown), pooled
enB/SM5. AS no significant differences were observed from the results
results of reciprocal crosses are presented here. CyO, SM5,SMl and SM6u are balancers which bear the dominant Cy
mutation andwhose complete genotypeis described in the LINDSLEY
and ZIMM (1992). While the Cy mutation doesexhibit
some temperature-dependent expressivity, it was able to be reliably scored in the genetic backgrounds used here.
" n, total no. of progeny scored.
a
METHODS
circumstances, one might expect to see more male-type
aswellas
female-type genital development. Such intersexes are seen (albeit less frequently) at 29",suggesting that the phenotype
itself does not represent
cold sensitivity of mutant i x l , ix2 or ix3 function. It is
possible that the increased
robustness of the intersexual
phenotype in hemizygotes is largely, if not entirely, attributable to effects of genetic backgroundas well. Both
hemizygotes and homozygotes are strongly intersexual:
it is not the kindof transformation that differs between
homozygotes and hemizygotes, but rather the amount
of tissue growth accompanying the transformation. For
this reason, we interpret the similar range of morphological phenotypes found in both hemizygous and homozygous individuals at all temperatures to provide evidence that these alleles represent substantial, if not
complete, loss-of-function mutations. Thus, at themorphological level, a lossof ix+ function results in the
simultaneous realization of both male and female developmental pathways in diplo-X individuals.
Characterization of new intersex alleles: A number
of screens for new ix alleles were performed with the
goal of obtaining P-element-tagged alleles or chromosomal rearrangements useful for more precisely localizing and cloning the ix locus. Although the general results of these screens have been described in MATERIALS
AND METHODS, the characterization of one new allele
has proven especially intriguing and so is described further here. An X-ray-induced allele, i x 4 , was recovered
on a multiply inverted recessive-lethal chromosome that
in complementation tests with other i x alleles, shows
temperature-sensitive complementation(Table
2) .
When i x 4 is hemizygous with the deletion Of ( 2R) e n H
(row D ) or is heterozygous with ix2 (row B) , pronounced temperature-sensitive intersexuality is observed, and when ix4 is hemizygous with the deletion
O f ( 2R)
(row E ) , some temperature-sensitive sterility is observed. At 18", most i x 4 / D f (2R) en' or i x 4 / i x 2
diplo-X animals are fertile females while at increased
temperatures of 25" and 29", there is a decrease in the
frequency of fertile females and a corresponding increase in the appearance of intersexual genitalia and
Drosophila
intersex in
analia. At 25",diplo-Xindividuals may appear externally
as normal females. When mated to wild-typemales,
some of these animals are found to be sterile: even
though they become gravid and if dissected, appear to
have normal appearingunfertilized oocytes, theydo not
oviposit embryos. A different pattern of temperaturesensitive intersexuality is seen in heterozygotes between
ix4 and either ix' or ix3 (rows A and C ) . These diploXanimals appear to be intersexual at all temperatures,
but the extent of intersexuality increases with increasing temperature. At 18", sterile female-like animals are
found that have at most mildly intersexual genitalia:
intact, completely female genitalia and analia with
added small masses of amorphous chitinous material.
At highertemperatures, severe intersexes arefound
with robust intersexual genitalia and analia like those
described in Table 1. These data suggest that fertility
can be taken as a sensitive measure of intersexuality in
diplo-X animals. At all temperatures, haplo-X animals
are phenotypically normal, fertile males.
A @on', the temperature-sensitive intersexuality seen
in different interallelic ix4 diplo-X individuals may either result from temperature-sensitivity of mutant ix
gene product( s ) and/or reduced levels ofix+ function
revealing the temperature sensitivity of the process that
ix+ function is normally involved in. The first explanation is likely, asnot all interallelic ix4 diplo-Xindividuals
are temperature-sensitive. The complementation patterns seen with ix4 may be explained in at least two
ways. First, it is possible that these patterns of complementation result from interactions of the ix4gene product with different amountsor types ofresidual ix' product produced by other ix alleles. With this view, the
qualitatively different phenotypes of diplo-X ix4/
Of ( 2 R )e n B and i x 4 / D f ( 2 R )ix87i3 hemizygotes would
result from the absence ofany ixf function by the
Of ( 2 R )e n B (47E3-6; 48A4) chromosome and theprovision ofsome ix' function by the O f ( 2R) ix8'I3 (47D48; 47F11-18) chromosome, suggesting that the distal
breakpoint of Of ( 2R)
is near or in the ix locus.
Similarly, the qualitatively different phenotypes of
diplo-X i x 2 / i x 4 us. i x ' / i x 4 and ix3/ix4 heterozygotes
might result from some ix+ function provided by the
i x 4 and ix2 chromosomes together that is not provided
in the other heterozygotes. It is possible for example
that each of these are fully null alleles that together
restore some function to a heterodimer.
A second possibility is that the range of phenotypes
seen in different genetic backgrounds results from the
production of a hypomorphic and possibly temperature-sensitive product by i x 4 whose ix+ function is sensitive to genetic background. This might suggest that the
implementation of terminal sexual differentiation is
also sensitiveto genetic background, reminiscent of the
sensitivity ofthe phenotypes produced by defects in the
initial steps in this hierarchy to genetic background
( CLINE1988) .
2~~'~'
1655
This viewof the temperature sensitivity of ix4 may
also offer insight into the failure of several different
mutageneses to provide strong ix alleles. Several of the
screens produced lines that initially gave rise to ix-like
individuals at 22" or 25", but which, on retesting at room
temperature (22"), appeared to complement extant ix
alleles and deficiencies. Hypomorphic mutations sensitive to temperature and genetic background may be
difficult to recover under the conditions utilized in this
study.
Does intemx have a role in sexual behavior in haplo-X
animals? Previous behavioral analysesof haplo-X ix'
homozygotes indicated that although these individuals
are morphologically normal males and show normal
viability and fecundity, they exhibit decreased levels of
male courtship behavior (MCROBERTand TOMPKINS
1985). Thus, contrary to the inferences obtained from
analysis ofthe effect of ix on externalmorphology (suggesting that ix acts solelyin diplo-X individuals) , analysis of extant behavioral data suggests that ix also functions in haplo-Xanimals. As noted above, one concern
with these results is the extentto which they may reflect
background-specific or allele-specific effects.
To address this concern, we have reexamined male
courtship behavior in haplo-X ix', ix', and i x 3 hemizygotes, ix' homozygotes and a number of heteroallelic
combinations (Table 3 ) . Of the genotypes examined,
pr cn ix' homozygotesshowed a decreased average
courtship index relative to CS controls, while other genotypes are notsignificantly different from CS controls.
Wehave
consistently notedthatrepeated
measurements of the courtship index of a single haplo-xindividual bearing a pr cn ix' chromosome span a wide range
of values, including wild-type values. Such individuals
are capable of normal courtship behavior (Table 3 ) ,
but they do not always display it, or display it for the
length of time seen in control strains. In both our data
and that previously gathered ( MCROBERT
and TOMPKINS 1985), this is reflected in the high value of the
standard error of the mean courtship index observed
in haplo-X individuals homozygous for the pr cn ix'
chromosome. Several of the tested genotypes that bear
a deletion of the ix locus exhibit reduced late phases of
courtship, particularly copulation. Although this might
suggest that more extreme phenotypes are observed in
hemizygotes than in homozygotes, this interpretation
is subject to the caution that individuals hemizygous
for ix+ (i.e., iso-2 ( + ) / O f ( 2 R )e n B also show reduced
copulation. It is therefore difficult to ignore the contribution of genetic background and attribute this behavioral difference solely to mutant ix function. When all
of the data are considered in this context, the observation of wild-type courtship index values except in animals homozygousfor thepr cn ix'chromosome suggests
to us that this variation in courtship index values is due
to chromosome-specific background effects and does
not result from altered function at the ix locus itself.
and
B. A. Chase
1656
B. S. Baker
TABLE 3
Sexual behavior of
ix and control haplo-X flies
Response of mature CS
male to test male
Genotype of test
XY male
CS is0 2-3
pr cn ix'/Df(2R)enB
pr cn i d / p cn ix'
i?/Df(2R)enB
pr cn id/i2
id/Df(2R)enB
nn
CI
20
26
2?0
0
-
l ? O
2?0
-
20
-
21
-
pr cn &/id
i2/id
Response of test male to
mature CS virgin female
60
61
40
64
51
nu
CIb
28
26
40
21
25
20
25
24
t6
? 6
-+ 10
77 t 5
53 ? 6
85 ? 3
-+ 7
2 6
Percent of tested males displaying behavior listed'
Genotype of test
male
nu
CS is0 2-3
96
50
ise2 (+)/Df(2R)enB 24
pr cn ix'/Df(2R)enB 60
pr cn ix'/pr cn ix'
39100
89
46
i?/Df(2R)enB
96
23
pr cn zx'/i2
3 6
97
34
id/Df(2R))aB
48 90
21
pr cn ix'/id
96
23
i2/id
a
No
Following and/ extension
Licking
Wing
courtship
Orientation
tapping
vibration
and
orattempted
copulation
Copulation
0
4
15
0
4
0
0
0
100
96
85
100
96 61
100
9771
100 76
100
83
82
4
and/or
86
79
68
38
57
44
72
36
87
80
87
85
76
83
40
7
87
28
65
78
61
n, no. of males whose courtship behavior was tested.
CIS are listed as mean (calculated using the mean of two trials per tested individual) ? SE.
Behaviors displayed by one individual in separate trials are considered jointly inthis analysis.
How does i x act to regulate terminal sexual differenti- after the developmental period that ix+ is required to
ation? The mode by which the ix+ product functions
function there will have a sexual phenotype like that of
during sexual differentiation can, a priori, be considix+ cells,simply because ix+ function had been proered as follows. The ix+ product may function to reguvided normally during the critical developmental pelate the sexual fate ofcells either by acting autonoriod. Consequently, an analysis of ix' cuticular clones
mouslywithin single cells or by acting as a signal
in an ix+ background in sexually dimorphic cuticular
between cells. It may also function either at a single
regions can be used to determine both whether theix'
product functions autonomously as well as the time it
time during development to set their sexual fate or be
is required to function.
required continuously up to a certain developmental
Clones were induced by X-irradiation in pwn n ix/+
stage or throughout the life of the organism to implediplo-X individuals (Figure 2 ) . All clones in sexually
ment and/or maintain their terminally differentiated
dimorphic regions of the abdominal cuticle were identistate. To distinguish among these possibilities, we examfied, independent of their sexual phenotype, by using
ined the morphology of clones of ix' tissue generated
the bristle and hair marker pwn, and then scored as to
in an ix+ background throughout development using
their sexual phenotype. The irradiations were perX-ray-induced mitotic recombination.
formed on staged individuals to induce mitotic exIf the ix+ product functions within single cells in an
changes throughout thelarval and pupal periods. Howautonomous fashion, then tissue in sexually dimorphic
developmental
regions that lack ix+ function during the
period it is normally required should show a phenotype
PWn
ix 1
42E3-43C3
47E3-47F10
like that of similarly positioned cells in an ix' homozy1
I
gote. In contrast,if the ix+ product achieves itsfunction
+
+
by acting as a signal between cells, e.g., as a diffusible
agent, then clones ofcells lacking ix+ function may
FIGURE2.-The arrangement and cytological location of
appear much as their neighbors with ix+ function, as
pwn and i x . Diplo-X pwn i x ' / + animals were exposed to Xthey can be provided with ix+ function from ixf cells
rays as described in MATERIALSAND METHODS to generate pwn
elsewhere in the organism. Clones of tissue lacking ix+
ix' clones in the abdomen andassess the autonomy andtime
of expression of i x .
function introduced into a sexually dimorphic region
-
I
-
1
Drosophila
intersex in
1657
TABLE 4
Analysis of autonomy of the ix locus and the time of expression of ix+ in the abdomen
Clones“
Tergites 5 and 6
Male,
Male,
Time irradiated
3
Prepupariation (hr)
120-94
94-20
20-12
12-0
Postpupariation (hr)
0-9
9-21
22-30
30-41
41 -48
Tergites
2, 3 and 4
Indeterminate,* Female,
No. of
Frequency
abdomens
clones
male
~
of
Frequency of
pwn clones
pwn
not pwn
13
42
10
8
1
0
11
30
0
0
12
13
0
0
67
159
4
16
228
546
32
78
0.10
0.092
0.094
0.077
0.45
0.45
0.19
0.28
2
3
0
5
7
5
14
8
7
0
5
20
22
13
8
53
98
66
56
16
54
67
33
131
160
0.43
0.19
0.36
0.18
0.062
1.56
2.12
3.27
0.72
0.17
2
21
Pwn
Pwn
pwn
Clone size in 20-120 hr prepupal irradiation
No. of bristles
Class
5
4 1
11
Tergites 5 and 6: male pigmentation and pwn
Tergites 5 and 6: female pigmentation and pwn
Tergites 5 and 6: indeterminate pigmentation andpwn
50
Tergites 2, 3 and 4: pwn
+/x
14
13
16
88
~~~
4
2
3
3
10
10
3
9
32
26
~
~~
26
18
5
1
5
1
1
26
4
0
24
~~
~~
~
Progeny of +/+; pwn cn ix’/CyO X
+/+ (CS,is0 2-3) or the reciprocal cross were irradiatedat the indicated ages and
the resulting pwn m ix’/+ progeny scored for clones as described in MATERIALS AND METHODS.
is not feasible to unambiguously determine the sex-typeof a pwn clone that falls within the posterior regions of the fifth
such regions are therefore listed
and sixth tergites that are normally darkly pigmentedpwn
in m i d / + females. Clones falling in
here as “indeterminate.”
a
ever, as the abdominal histoblasts do notdivide during
the larval period ( GARCIA-BELLIDO
and MERRIAM1971)
and as an exchange must be followed by cell division
before a mutation becomes homozygous, the earliest
time a cell could lose ix+ function would have been
near pupariation.
Clones of ix’ tissue arising near the time of pupation
display an id-like pigmentation pattern (Table 4, Figure 3 ) . In animals irradiated 520 hr before pupation,
69% of the pwn clones in the sexually dimorphic regions of the fifth and sixth tergites were associated with
areas ofdarkly
pigmented male-type pigmentation.
That -28% of the pwn clones were not clearly associated with male-type pigmentation is not unexpected.
As described above, diplo-Xhomozygous ix’ individuals
display a wide range of expressivity. The least severe
intersexes appear as sterile females. While the overwhelming majority of diplo-X ix’ homozygotes do display some male-type pigmentation, this pigmentation
does not always extend to the anterior andlateral edges
of the fifth and sixth tergites. As most of the pwn clones
associated withfemale-type pigmentation encompassing morethanthan
one bristle were located in the
regions close to the lateral and anterior margins, they
behave much as do similarly positioned cells in ix’ hc-
mozygotes. Consistent with this view is the observation
that theaverage size ofclones with female-typepigmentation ( 3 . 3 ) in sexually dimorphic regions is less than
those displaying male-type pigmentation (4.4) (Table
4). Although a few large clones displaying female-type
pigmentation were found, these too were located in the
lateral and anterior regions of the fifth tergite. Thus,
clones of ix’ tissue in sexually dimorphic regions of the
abdomen display a phenotype like that of similar cells
in diplo-Xix‘ homozygotes. This suggests that theproduct of the ix locus functions in a cell-autonomous manner in the abdomen, and that it is required in the abdominal histoblasts after the resumption of cell division
at pupariation for the normal sexual development of
the abdominal tergites.
To address when ix has been transcribed sufficiently
to insure normal sexual development of the fifth and
sixth tergites, diplo-Xpwn en ix’/+ animals were staged
and irradiated to inducehomozygous ix’ clones during
the time abdominal histoblasts are dividing. To relate
the frequency of abdominal histoblast cell division to
the temporal requirement for ix+ function in developing sexually dimorphic regions, the frequency of induction of pwn clones in the second through sixth tergites was compared with the frequency of induction of
B. A. Chase and B. S. Baker
1658
FIGURE3.-Examples of male-type pigmentation clones homozygous for pwn ix'in the anterior region of the sixth abdominal
tergite of pwn ix'/+ females. Arrowheads mark areas of male-type pigmentation and pzun bristles. The clones shown here were
found in animals irradiated at 108 2 5 hrs ( a ) , 79 2 5 hrs ( c ) , 39 -t 3 hrs ( b ) and 8 ? 4 hrs ( d ) before pupariation.
male-type pigmentation clones in the sexually dimorphic regions of the fifth and sixth tergites (Figure 4 ) .
During the larval period, when the abdominal histoblasts are mitotically quiescent, there is a relatively
low and constant induction of both pawn and maletype pigmentation clones. From pupariation until 30
or 40 hr of pupal development, when the abdominal
histoblasts are actively dividing, an increase in the frequency of pwn clones is paralleled by an increase in the
relative frequency of male-typepigmentation clones. At
-40 hr of pupal development, the abdominal histoblasts cease their mitotic activity, and the frequency
of pwn and male-type pigmentation clones drops as
clones are no longer inducible. Thus, pwn and male-
0.5
type pigmentation clones can be induced throughout
the period that the abdominal histoblasts are actively
dividing.
Before drawing conclusions regarding when ix+ function is normally required in this tissue, severalfeatures
of the datawarrant comment. First, when the frequency
of clones obtained in populations irradiated before, or
at, the onsetof the resumption of abdominal histoblast
cell division (i.e., in prepupation and early postpupation irradiations) are compared with the frequency of
clones obtained in populations irradiated during the
period when the abdominal histoblasts are close to ceasing cell division (i.e., in the 41- to 48-hr postpupation
irradiations), it is found that clones induced at later
0 male (ix)clones
Hpwn clones
s
0.4
3
Frequency of 0.3
Male Clones
Per Abdomen 0.2
2
Frequency of
pwn clones
per abdomen
1
0.1
0120-94
94-20
20-12
Pre-pupation
Hours
12-0
I
I
0
0-9
9-21
22-30
30-41
41-48
Hours Post-pupation
FIGURE4.-Frequencies of pzm and male-type pigmentation clones produced in the abdomen o f p m i d / + females at different
times during development.
intersex in Drosophila
times show a generally increased frequency of maletype pigmentation patches not associated with pun and
pun clones not associated with male-type pigmentation
(Table 4 ) . This is expected, as clones induced at the
latest times will be smaller. As the determinative event
that commits a cell to produce either abristle or cuticle
occurs two cell divisionsbefore the end of cell division,
clones induced during the last set of mitotic divisions
will be unlikely to encompass both pun and pigmentation markers. Therefore, one can employ all male-type
pigmentation clones to analyzewhen ix+ has functioned sufficiently to provide for normal sexual differentiation.
A second consideration in the analysis of these data
is that male-type pigmentation clones not marked with
pwn can arise as a consequence of exchanges between
pwn and i x ' . Although some non-pun male-typepigmentation clones may result from such exchanges, it is
unlikely that all such clones do. The small clones induced attimes when the abdominalhistoblasts are close
to ceasing cell division (i.e., the 41- to 4&hr postpupation irradiations) areless likelyto include bothpigmentation and bristle or hair markers. In particular, some
regions of the cuticle (e.g., the posterior portion of the
sixth tergite) lack hairs, and as small male-typepigmentation clones in these regions are less likely to include
a bristle that wouldallow them to be recognized as
being pun, few such clones will exhibit a pwn phenotype.
The fact that the temporal production of male-type
pigmentation (i.e., ix') clones in sexually dimorphic
regions of the fifth and sixth tergites strongly parallels
that of the frequency of abdominal histoblast cell division, as indicated by the frequency ofpwn clones
throughout the second through sixth abdominal tergites, suggests to us that ixf function is needed very
late, most likelythroughout the period
of cellular proliferation, in order to specify the normal sexual fate of
this tissue. Put anotherway, these data indicate that ix+
is unlikely to have been transcribed sufficiently prior to
the cessation of cell division to provide for normal sexual differentiation in this tissue.
DISCUSSION
Prior analysesof ix' and ix2 by BAKER and
RIDGE
( 1980) had suggested that the ix locus is required to
repress male differentiation functions in diplo-X animals. By demonstrating that the null phenotype ofix
is to transform diplo-Xindividuals into intersexes where
both male and female developmental pathways are simultaneously realized, we confirm this suggestion.
Analysisof additional ix alleles has also clarified the
role of ix in haplo-X animals. When assessed morphologically, ix2and ix3behave as (at least substantial) lossof-function alleles at ix. Nonetheless, as either homozygotes or hemizygotes, they show levels
of haplo-Xcourt-
1659
ship behavior like that of control haplo-X flies. This
suggests that the ix+ product is not normally required
to function in haplo-X individuals, but rather is required only in diplo-Xindividuals. When these analyses
are considered together with the results ofprevious
epistatic analyses of ix' and ix2 with d m , tra, and tra-2
(BAKER
and RIDGE 1980) and the observation that the
transcriptional profile of dsx is unaltered in heterozygous ix'/ix2, ix1/ix3 and ix2/ix3 genetic backgrounds
( NAGOSHI
et al. 1988) , ixwould appear to be positioned
within the sexdetermination regulatory hierarchy at
the same level as,or subsequent to, dsx. Analyses of the
behavior of ix' in clones of tissue inan ix+background
alsosuggest that, like the female-specific product of
d m + ( DSXF), the product of ixf is required to be present at least until the end of cell proliferation for the
repression of male-type differentiation functions in individual cells.
Although these analyses confirm that ix+ provides at
least one regulatory function in addition to DSXF that
is required to repress male-type cellular differentiation
in diplo-Xanimals, it is unclear whether this regulatory
function acts in conjunction with, acts in parallel to, or
lies downstream of, that provided by DSXF. The data
presentedherecannot
formally distinguish between
three alternative pathways: in one pathway, ix is under
the controlof tru and tra-2 directly and either produces
a product that acts with DSXF or a product that acts in
parallel to that of DSXF. In a second pathway, DSXF
acts via ix to achieve some aspect of the repression of
male-type cellular differentiation. In a third pathway,
the relationship between ix and dsx is like that between
tra-2 and tra: ix+ produces a product constitutively but
it only functions in the presence of DSXF.The issue of
potential branches at this level of the regulatory pathway governing the implementation of terminal sexual
differentiation will achieve resolution via molecular
analyses of the products of these loci and their interactions.
Previous analysis of the sex-specific regulation of the
yolk protein genes YP1 and Y P 2 by the tru-2 and dsx loci
has provided a detailed model for the action of these
loci during terminal sexual differentiation (BELOTEet
al. 1985; BURTISand BAKER 1989; BURTISet al. 1991;
COSHIGANOand WENSINK
1993). Here, both of the
DSXMand DSXF polypeptides are capable of binding
in vitro to the same three sites in a Yp regulatory element, the fat body enhancer (FBE). However, the proteins are thought to have different effects whenbound,
so that YP production is stimulated in diplo-xindividuals, but repressed in haplo-X individuals ( BURTISet al.
1991; COSHIGANO
and WENSINK
1993).While DSXFcan
activate FBE-directedtranscription in diplo-X individuals, DSXM represses transcription ( COSHIGANOand
WENSINK
1993). It has been proposed that thedifferential activation of YP transcription in diplo-X animals
may alsoinvolve the interaction of additional gene
1660
B. A. Chase and B. S. Baker
products with DSXF, in particular that of ix+ ( BURTIS
et al. 1991; COSHIGANO
and WENSINK1993). In this
scenario, the ix+ product would presumably act as a
positive regulatory factor to further stimulate gene expression. It has been further suggested that at least in
some situations, the ix+ product may be present in limiting amounts (JURSNICH and BURTIS1993), which in
turn limits the effectiveness ofDSXFfunction. This view
would be consistent with observations that in diplo-X
ix mutants, reduced hemolymph levels ofWs are found
( BOWNESand NOTHIGER1981) . As these latter observations were made in intersexual individuals, and the
amount of W synthesis may be dependent on thetypes
and amounts of tissues present, it is not feasible to infer
a directrole for ix+ in YP transcription from these studies alone. Analyses ofW transcription in phenotypically
female i x 4 hemizygotes placed at restrictive temperature after eclosion indicate that YP transcription is unaffected under these conditions (B. A. CHASEand R. R.
RISLEY, unpublished data). This would suggestthat the
product of ix+ may not be required to achieve high
levels of YP transcription.
At least formally, the view that the product of ix+ is
a positive regulator of gene expression is in contrast to
that obtained from the genetic analysis of ix presented
here. Our results suggest that theabsence of ixfunction
allows male-specific functions to be derepressed, and
point to the normal role of the ix+ product being that
of a repressor of gene expression. Clearly, if the ix+
product acted as a negative regulator of a negative regulator, it would appear that ix+ acted as a positive regulator of W expression. However, havingevidence in hand
that W transcription is activated by DSXF,one must be
cautious about the role of the ix+ product. Although
the ix+ product may function like previously identified
transcriptional regulators that arecapable of either positive or negative regulatory action dependent on their
interactions with other gene products, theix+ product
may not regulate YP transcriptional activation. It is possible that DSXF may interact only with other tissue-specific (and potentially nonsex-specific) regulatory factors to activate female-specifictranscription at the FBE.
If this is the case, sexual differentiation would appear
to be implemented by the differential utilization of the
terminally positioned sex-determination regulatory loci
ix and dsx in (potentially) parallel pathways. Investigations at a molecular level should lead to a finer understanding of the role of the ix+ product in such regulatory processes.
The questions raised in consideration of this model
illustrate the needto understand morefully the identity
and regulation of the spectrum of targets which the
dsx and ix regulatory genes act upon. The range of
phenotypes associated with ectopic expression of DSXM
(JURSNICH and BURTIS1993) reinforce the widely held
view that the productsof the regulatory loci acting during terminal sexual differentiation function by regulat-
ing the gene activity ofnumerous targets in the context
of other regulatory processes. It will be of considerable
interest to build on the extremely insightful analyses of
the regulation of the yolk protein loci to understand
how the dsx and ix loci function to achieve sex-specific
(or perhapssexdifferential) expression as multiple
regulatory pathways converge to control target gene
expression in different cellular contexts.
We thank IANDUNCAN,
CHIHIROHAMA,TOMKORNBERG,
MICHAEL
KATHRYN ANDERSON and WILLIAM
ENGELS
RUSSEIL,PETERGERGEN,
for providing fly stocks, our colleagues in the BAKERlaboratory for
helpful discussions, CARRIE GARRET-ENGELE
and KATERINA MARKOP ~ U L O Ufor insightful comments on the manuscript and GUENNET
BOHMfor providing food forthe flies. This work has been supported
by American Cancer Society and National Institutes of Health postdoctoral grants to B.A.C., a National Institutes of Health grant to
B.S.B. and a grant from the University Committee on Research at
the University of Nebraska-Omaha to B.A.C.
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Communicating editor: R. E. DENELL