J. Embryol. exp. Morph. 75, 241-257 (1983)
241
Printed in Great Britain © The Company of Biologists Limited 1983
Expression of the yolk-protein genes in the mutant
doublesex dominant (dsxD) of Drosophila
melanogaster
By MARY BOWNES 1 , MAUREEN DEMPSTER AND
MAIREARAD BLAIR
From the Department of Molecular Biology, University of Edinburgh
SUMMARY
Adult flies mutant for doublesex dominant (dsxD) are intermediate in phenotype between
males and females. The dsxD mutation acts in the heterozygous state to transform only flies
with two X chromosomes into intersexes, XY flies are unaffected by the mutation. Yolkprotein synthesis, which normally occurs in the ovaries and fat bodies of females, but not in
males unless stimulated with 20-hydroxy-ecdysone, is reduced. The dsxD fat body synthesizes
less yolk proteins throughout adult life, and the gonads rarely make yolk proteins. Using
cloned yolk-protein genes as probes for measuring transcript levels we have shown that expression of these genes in dsx° is regulated both transcriptionally and post-transcriptionally.
We suggest that the dsxD locus regulates the expression of the yolk-protein genes from within
the fat body cells and does not operate by modulating ecdysteroid titres in the adults.
INTRODUCTION
The three yolk polypeptides of Drosophila melanogaster are synthesized by
the fat body and the ovarian follicle cells of adult females (Bownes & Hames,
1978; Brennan, Warren & Mahowald, 1980). Each polypeptide is coded for by
a single-copy gene located on the X chromosome (Barnett, Pachl, Gergen &
Wensink, 1980). Their expression is regulated in isolated female abdomens by
juvenile hormone and 20-hydroxy-ecdysone (Jowett & Postlethwait, 1980), and
males can be induced to express the yolk-protein genes by high levels of
20-hydroxy-ecdysone (Postlethwait, Bownes & Jowett, 1980; Bownes, 1982).
Whether or not the yolk-protein genes are expressed in adults is correlated with
the expression of a series of autosomal genes affecting sex determination. Mutations in these genes (intersex, transformer, transformer-2 and doublesex) alter the
expression of many genes such that XX flies, which would normally follow a
female developmental pathway, can become phenotypic males (pseudomales) or
intersexual, and XY flies, which would normally follow a male developmental
pathway, can become intersexual (Baker & Ridge, 1980). All intersexual flies
1
Author's address: Department of Molecular Biology, University of Edinburgh, King's
Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
242
M. BOWNES, M. DEMPSTER AND M. BLAIR
have circulating yolk proteins in the haemolymph, and all pseudomales lack yolk
proteins regardless of the X and Y chromosome constitution (Postlethwait et al.
1980; Bownes & Nothiger, 1981).
In our previous studies we found that the quantities of yolk proteins circulating
in the haemolymph were low in intersexual flies compared to wild-type females,
even in cases where there were two X chromosomes present and thus reduced
levels were not due to XY flies having only half the number of genes available
for transcription (Bownes & Nothiger, 1981). We have investigated the expression of the yolk-protein genes in one of the sex mutants, doublesex dominant
(dsxD) which when heterozygous causes XX, but not XY, flies to develop an
intersexual phenotype, in an attempt to understand the mechanism by which the
sex genes regulate the expression of the YP genes.
We have measured yolk-protein synthesis in adults during maturation and the
levels of transcripts coding for the yolk proteins during adult development and
in various tissues. The results presented in this paper lead us to propose that the
dsxD gene affects the expression of the YP genes in the fat body from the time
of eclosion and that no feedback mechanisms operate involving circulating yolkprotein levels. We propose that post-transcriptional control mechanisms affect
the level of expression of the yolk-protein genes and as a consequence of this the
transcripts of the yolk-protein genes are translated less frequently into polypeptides in vivo in dsxD than in wild-type flies.
MATERIALS AND METHODS
Maintenance of stocks
Flies were maintained on a standard yeast, cornmeal, sugar, and agar medium
at 25 °C. Wild-type control flies were an Oregon R (OrR) strain and the sex
mutant used was dsxD as a +/T (1;3) OR60, /TM6/dsxD Sb e stock (for mutations and symbols see Lindsley & Grell, 1968) which allows identification of
dsx° adults by morphological markers. The dsxD allele was selected for these
experiments because dsxD affects flies with 2 X chromosomes so there are no
problems of reduced numbers of copies of the yolk-protein genes as there are in
XY flies and because this stock is healthy and viable. Several populations of
dsxD and OrR were maintained.
The mutant flies used were dsxD/ + , XX and are referred to throughout as
dsxD adults or intersexes.
Preparation of tissues
When RNA was to be isolated from individual tissues, or when tissues were
cultured in vitro, they were dissected into a Ringer's solution (Chan & Gehring,
1971). These tissues were either cultured in Ringer's or Grace's medium (Grace,
1962) or placed in RNA-extraction buffer (see below) and then stored at - 2 0 °C
Yolk-protein-gene expression in dsxD
243
or extracted immediately as appropriate. For preparing various tissues the gut
and Malpighian tubules were separated at the joint of the thorax and abdomen
and at the genitalia, gonads were separated at the genitalia. The head and thorax
were taken intact, and the remainder was the abdominal body walls.
Analysis and quantitation of yolk proteins
Flies were injected with 0-2 fA of [35S]methionine and maintained for 4h at
25 °C. The haemolymph of each set (8-10 flies) was then collected into 50 /xl
Laemmli buffer
(Laemmli, 1970) containing 1% SDS, 0-1%
2-mercaptoethanol, 10% glycerol, and 0-05M-Tris HC1 pH6-8. The flies were
also placed in 50 /il Laemmli buffer, vortexed, and the cuticle and insoluble parts
removed by centrifugation. The samples were then heated to 90 °C for 15min,
ljUl of each sample was T.C.A.-precipitated and the amount of label incorporated was measured by liquid scintillation counting. Equal numbers of counts
of each sample (the exact number varied from gel to gel) were loaded onto a
7 %-20 % polyacrylamide gradient slab gel and run overnight (Laemmli, 1970).
The gels were then prepared for fluorography using the technique of Bonner &
Laskey, 1974.
The resulting autoradiographs were scanned with a densitometer and the areas
under the yolk-protein bands were calculated. This method is useful for comparison of yolk-protein synthesis between samples.
In vitro culture of tissues
Tissues were cultured in 20/^1 of Ringer's or Grace's medium supplemented
with 4/^1 of [35S]methionine for 4h. Cells and medium were separated by
centrifugation and either Laemmli buffer or antibody-precipitation buffer was
added to each sample. The cells were broken by vortexing and the debris was
removed by centrifugation. Samples were either analysed directly as above, or
were first precipitated with anti-yolk protein antibody before separation using
SDS polyacrylamide gel electrophoresis.
Precipitation with anti-YP antibody
The specificity of the antibody has been described previously (Bownes &
Nothiger, 1981) and the precipitations were carried out in the same way.
Isolation of RNA
RNA was extracted from tissues and whole flies by homogenization in RNAextraction buffer (2 mM-MgCl2,0-5 % SDS and 10 mM-Tris/HCl, pH 7-5). They
were then subjected to multiple rounds of extraction against a phenol/chloroform mix (6:1, by volume) by vortexing. The organic layers were re-extracted
at least once before being discarded. The final aqueous layer was subjected to
ethanol precipitation (2-5 volumes ethanol plus 0-1 volume of 3 M-sodium acetate
244
M. BOWNES, M. DEMPSTER AND M. BLAIR
pH6-0) at - 2 0 °C overnight. The resulting precipitated nucleic acids were
washed in ethanol and again precipitated (with 0-6 ml ethanol plus 0-3 ml 0-1 MNaCl 0-01 M-Hepes buffer pH 7-0) at —20 °C overnight, and dried under vacuum.
The resulting RNA was stored as an aqueous stock solution.
Translation of RNA in a cell-free translation system
Samples of RNA, either total RNA samples or polyA+ and polyA" fractions
separated using an oligo-dT column, were translated in the rabbit reticulocyte
cell-free translation system as described by Pelham & Jackson (1976).
Resulting proteins were precipitated with anti-yolk-protein antibody as in
Isaac &Bownes (1982).
Preparation of 32P-labelled YF'-probes
2/ig of an equimolar mixture of pYPl, pYP2, and pYP3, which are plasmids
containing one copy of a yolk-protein DNA sequence (details of the plasmids
used can be found in Barnett et al. 1980), was nick translated to approximately
10 7 c.p.m.//ig DNA (method adapted from Maniatis, Jeffrey & Kleid, 1975).
Unincorporated nucleotides were removed using a Sephadex G-50 column.
Measurement of YP-RNA levels
There was some translation of yolk proteins from the polyA" fraction in the
rabbit reticulocyte lysate cell-free translation system. Although this was minor
compared to the polyA"1" fraction it was not clear that this would always be the
case in experiments, thus we used total extracted nucleic acids for these experiments, quantitating YP-RNA levels.
RNA was separated on formaldehyde. The gels were 1-2 % agarose, 20 mMMOPS, 5mM-sodium acetate, lmM-EDTA and 2-2M-formaldehyde pH7. The
RNA samples were dried down and dissolved in 20/il 50% formamide, 2-2 Mformaldehyde, 20mM-MOPS, 5mM-sodium acetate and lmM-EDTA.
Bromophenol blue and Ficoll were added to the samples. The gels were run at
200 V for 1 to 2h and the RNA was transferred by blotting for 12-15 h onto
nitrocellulose previously equilibrated with 20xSSC. For dot blots the RNA was
spotted directly onto nitrocellulose which has been equilibrated with 20 x SSC
and dried under a lamp. The nitrocellulose was then baked for 2 h at 80 °C under
vacuum (Thomas, 1980).
After prehybridization for 8-20 h at 37 °C in 50% (vol/vol) formamide,
5 x SSC, 50mM-sodium phosphate pH6-5, sonicated denatured salmon sperm
DNA at 250/ig/ml and 1 x Denhardts (0-02 % BSA, 0-02 % Ficoll and 0-02 %
P.V.P), the filters were hybridized to the labelled probe in four parts
prehybridization buffer and one part 50 % (weight/vol) dextran sulphate for 20 h
at 37 °C. The filters were washed with four changes of 2 x SSC, 0-1 % SDS for
5mins each at room temp followed by two changes 0-lxSSC, 0-1 % SDS for
Yolk-protein-gene
expression in dsx D
245
15min at 50 °C, and then exposed to X-ray film for varying lengths of time at
—70°C. Some RNA is lost in the transfer method since, when similar quantities
of RNA were either spotted directly onto nitrocellulose or transferred to it by
blotting and the filters hybridized in the same probe and exposed to X-ray film for
the same period of time, the signal was much stronger from the spots.
Consequently we used the dot blot method for RNA quantitation in these experiments.
By dotting variable amounts of RNA onto nitrocellulose and carrying out
hybridizations and autoradiography we found that if we measured the area x
density of the resulting spot this was proportional to the amount of RNA loaded
(Fig. 1) between 2 and 5 /ig RNA. We have therefore used this method to measure
the amount of YP-RNA in the total RNA samples and we always loaded a 5 jUg
RNA and a 2-5 fig RNA spot. The results can then be compared between samples
though we do not have a direct measure of the precise number of YP-RNA
5 •
2
3
4
5
7-5
10
^g of female RNA
Fig. 1. Increasing amounts of RNA were dotted onto nitrocellulose and hybridized
to 32P-labelled pYPl, pYP2 and pYP3. The filter was exposed to X-ray film and the
area and density of the dots on the resulting autoradiographs were measured. The
graph shows four repeats of this experiment using RNA isolated from different populations of females and hybridized to different probes.
246
M. BOWNES, M. DEMPSTER AND M. BLAIR
2
Before hydrolysis
After hydrolysis
Fig. 2. Dot blot of 5 jUg samples of female RNA before and after alkaline hydrolysis.
molecules present. This technique does not distinguish hnRNA from mRNA.
Hydrolysis of RNA
To remove RNA from samples and discover the background hybridization
level to DNA extracted along with the RNA, alkaline hydrolysis was used. 5 fig
samples of RNA were placed in 45 /A water and 5 [A 5 N-NaOH. After heating to
60 °C for 2h the solutions were neutralized with 250^1 of 300mM-NaCl, 60 mMTris base and 140mM-HCl, and precipitated with ethanol as described above.
Fig. 2 shows the results of a dot hybridization with 5/ig of female RNA before
and after hydrolysis. A weak signal remains due to the DNA in the samples. This
gives approximately the same level of hybridization as 5/^g of male RNA. Males,
therefore, have undetectable levels of YP transcripts and hybridization to the
controls of each filter provides a measure of background hybridization to DNA.
RESULTS AND DISCUSSION
Morphology of dsxD adults
Adults carrying the dsxD mutation are intersexual in phenotype. Exactly which
sexual morphological characters are shown varies between individuals. The
intersexual flies tend to be similar in size to females rather than males. The sex
combs, normally present on the male prothoracic leg but absent from the female
prothoracic leg, are poorly formed and the pigmentation of the posterior
abdominal segments is much darker and more solid than in a female. The external genitalia are also intermediate between those of males and females (Fig. 3).
The gonads of males and females are shown in Fig. 3, along with an example
from a dsxD adult. The gonads are of an intersexual type, often very rudimentary,
but tending to be 'male-like' more frequently than 'female-like' with structures
resembling deformed accessory glands. It is very rare to observe any ovarian cell
Fig. 3. A-C Genitalia and D-F gonads from wild-type males and females and dsxv
intersexual flies. Size bars represent 0-1 mm. A and D, female; B and E, dsxD; C and
F, male.
Yolk-protein-gene expression in dsxD
.T-
B
VI
247
248
M. BOWNES, M. DEMPSTER AND M. BLAIR
Table 1. Distribution of yolk-protein (YP) transcripts in the ovaries and fat bodies
of wild-type adult flies.
Age from eclosion
(h)
% YP transcripts
in ovary
% YP transcripts
in fat body
0
0
100
100
24
46
55
54
45
48
21
19
79
81
72
21
31
79
69
96
28
8
72
92
120
65
47
35
53
0
The results of two experiments are given in each case.
types but occasionally we saw 'nurse-cell-like' cells and one or two flies in over
100 analysed had defective yolky oocytes present.
dsxD flies lack a major site of yolk-protein synthesis
The levels of transcripts coding for the yolk proteins in the ovary as compared
to the fat body (body walls) were measured using dot blots (see Materials and
Methods). Whole body-wall preparations from the head, thorax and abdomen
were used for these experiments. Although this means that there are other cell
types present the tissue loss dissecting fat body free of the cell walls would be too
great. The results in Table 1 show the distribution of transcripts in two groups
of wild-type flies. The contribution of the ovary, once oocytes are developing,
ranges from as low as 8 % to as high as 65 % of the total yolk-protein transcripts
in a fly. This high variability is not really surprising because the ovarian
contribution will depend upon the number of oocytes present in stages 8 to 11
(King, 1970) since only these stages synthesize yolk proteins (Isaac & Bownes,
1982; Brennan, Weiner, Goralski & Mahowald, 1982). The average ovarian
contribution of approximately 35 % of the total yolk-protein transcripts agrees
well with results obtained by translating polysomal RNA from ovaries and fat
bodies in a cell-free translation system (Isaac & Bownes, 1982) and with
measurement of yolk-protein poly A + RNA (Brennan et al. 1982).
We analysed the distribution of yolk-protein transcripts from 72 h-old females
in more detail (Fig. 4). The results were: gut and Malpighian tubules 0 %, ovaries
Yolk-protein-gene expression in dsxD
head and
thorax
gut
abdominal
body
wall
249
ovary
Fig. 4. Dot blot of 5;ug samples of RNA extracted from various tissues of a single
group of flies. (OrR females.)
40 %, head and thorax 30 %, and abdominal body wall 30 %. Expression of the
yolk-protein genes is clearly tissue limited as well as sex limited.
The very small gonads from dsxP flies give very poor yields of RNA. When
transcripts coding for yolk proteins from the gonads were measured they were
rarely significantly above the male background. Occasionally we have detected
very small amounts of yolk-protein transcripts in these samples, presumably
from some of the rare female-like gonads. Clearly dsxD flies lack one of the major
sites of yolk-protein synthesis which may account, at least in part, for the reduced
levels of the yolk-protein synthesis detected in these flies.
Yolk-protein synthesis in dsxD and wild-type adults
To establish how similar rates of yolk-protein synthesis are between the fat
bodies of wild-type and dsxD flies we labelled the proteins with [35S]methionine
for 4-5 h periods at eclosion and at daily intervals thereafter until the adults were
5 days old. The [35S]methionine was injected into the haemolymph of groups of
ten flies, and at the end of the labelling period haemolymph was collected. We
analysed how much newly-synthesized yolk protein was present in the
haemolymph as compared to total newly synthesized haemolymph proteins. This
should account for most of the yolk proteins synthesized by the fat body. The
yolk proteins synthesized by the follicle cells do not circulate in the haemolymph
but pass directly into the oocyte (Srdic et al. 1979). During the 5 h period some
of the yolk proteins synthesized in the fat body could be transported into the
oocytes of wild-type females, but not dsxD flies which lack ovaries. This experiment may therefore slightly underestimate fat-body synthesis of yolk proteins in
the wild-type flies.
It can be seen from Fig. 5 that at every stage there is always considerably less
newly-synthesized yolk protein circulating in dsxD compared to wild-type adult
haemolymphs. The results of several experiments are listed in Table 2. The
proportion of yolk proteins synthesized in the labelling period is variable between
250
M. BOWNES, M. DEMPSTER AND M. BLAIR
YPs{
YPs
0
24
48
72
96
120h
Fig. 5. Autoradiograph of newly-synthesized haemolymph proteins separated by gel
electrophoresis. Samples were collected from flies of varying ages. YPs, Yolk
polypeptides; HI, haemolymph protein 1; $ = female; <jf = intersex, dsxD; h = age
in hours.
groups of flies, but is always strikingly reduced in dsxD. There is another
haemolymph polypeptide, marked HI in Fig. 5, which continues to be
synthesized as efficiently in dsxD as in wild-type, suggesting that yolk-protein
synthesis is selectively reduced in the dsxD flies.
It was possible that dsxD flies instead of secreting the yolk proteins were
retaining them in the fat body. All the tissues of the flies were therefore analysed
by SDS polyacrylamide gel electrophoresis and the example in Fig. 6 shows that
there is not a large retention of yolk proteins in dsxD tissues.
There appears to be a genuine reduction in yolk-protein synthesis in the fat
body rather than a failure of secretion. Since the low level of yolk-protein
synthesis in dsxD is observed from early adult life it presumably does not result
from a feedback mechanism operating because there are no ovaries to sequester
the yolk proteins and prevent their accumulation in the haemolymph. The lack
of ovarian yolk-protein synthesis in dsxD flies does not, therefore, account for the
reduced levels of yolk-protein synthesis in the whole flies, synthesis being also
reduced in the fat bodies.
Yolk-protein-gene expression in dsxD
251
Table 2. Newly-synthesized yolk-proteins in the haemoloymph of wild-type (OrR)
and dsx° adults
Age of adults
from eclosion
(h)
% newly synthesized YPs
in total haemolymph
dsxD
OrR
„
. dsxD
Ratio
QrR
24
49
40
46
14
6
17
5
0-28
0-15
0-36
48
40
62
59
12
10
12
7
0-28
0-15
0-20
72
62
31
51
8
15
16
4
0-13
0-48
0-31
96
61
55
53
8
3
7
6
0-13
0-17
0-20
120
57
17
39
9
3
7
4
0-16
0-15
0-18
For each time-point the results of various experiments are given separately.
Accumulation of transcripts coding for the yolk proteins
To establish whether the reduced levels of yolk-protein-gene expression in
dsxD resulted from reduced transcript levels in these adults, we isolated RNA
from whole adults at various ages after eclosion and measured the transcripts by
hybridization to a mixed probe containing cloned DNA coding for each of the
three yolk polypeptides. We used several populations of flies for these experiments so that variability between groups of flies could be taken into consideration. In all, five populations were used for various experiments and the variability in accumulation of yolk-protein transcripts was quite marked between these
groups of flies and with the age of the adults. The results of some of these
experiments (those using probes of similar specific activity (approximately
1 x 10 7 c.p.m.)) are shown in Fig. 7.
Initially the dsxD flies have considerably fewer transcripts coding for the yolk
proteins than wild-type flies. Generally the ratio of dsxD to OrR levels was
between 0-01 and 0-3. The fact that levels are low from the beginning of adult
life again suggests that there is no feedback mechanism operating to reduce
252
M. BOWNES, M. DEMPSTER AND M. BLAIR
}YPs
0
24
48
72
96
120
9
Fig. 6. Autoradiograph of newly-synthesized proteins in the tissues offliesat various
ages. $ = female; d" = male; ^ = intersex, dsxD; YPs = yolk polypeptides.
transcription of the yolk-protein genes as the quantities of yolk proteins increase
in the haemolymph. After several days, however, this pattern changes and dsxD
flies have accumulated yolk-protein transcripts to levels close to the wild-type
level and in one population there was several times more than in the wild-type
flies (see Fig. 7).
The mechanism bringing about this increased accumulation of transcripts
coding for the yolk proteins in mature dsxD flies is not known. The genes could
be more actively transcribed, the polyploidy of the fat body cells could increase
Yolk-protein-gene expression in dsxD
253
30
24
48
72
hours after eclosion
96
120
Fig. 7. Graphs of area x density of dot, as a measure of yolk-protein transcript levels
plotted against the age of the flies. Results from three populations are shown.
O---O, dsxD population 1; A - - - A, dsxD population 2; D---D, dsxD population
3; # — - • , OrR population 1; •
A, OrR population 2; •
• , OrR population 3.
thus increasing the number of genes available for transcription, or the stability
of the RNA could be increased. However, whatever the mechanism used in vivo,
these transcripts are not translated efficiently into proteins since actual fat-body
synthesis of the yolk proteins does not increase at this time.
The RNA from the dsxD and OrR adults was separated on formamide,
formaldehyde gels, transferred to nitrocellulose and hybridized to the cloned
EMB75
254
M. BOWNES, M. DEMPSTER AND M. BLAIR
[4,
YPs
wild-type
ds>P
Fig. 8. Autoradiograph of yolk-polypeptide synthesis in the rabbit reticulocyte
lysate cell-free translation system from 5 fig samples of dsxD and OrR RNA from
96 h, population 1. The translated proteins were antibody precipitated before being
separated by gel electrophoresis.
yolk-protein genes. The RNA was intact and of the same size in both mutant and
wild-type flies (data not shown). The gels were not, however, of a resolution
sufficient to distinguish whether the introns had been processed from these
transcripts. They are about 70 base pairs in length (Hung & Wensink, 1981).
Thus it was possible that much of this RNA was not suitable for translation into
proteins. We translated 5 /ig samples of the RNA in the rabbit reticulocyte cellfree translation system and found that the RNA from dsxD flies at 96 h (when
maximum transcripts were detected) translated just as efficiently as the wild-type
RNA; (the ratio of yolk-protein transcripts in OrR:dsxD was 1:1-15 and the ratio
of yolk proteins synthesized in the cell-free translation system was 1:1-14). A
similar pattern of yolk polypeptides was observed when the translation products
were precipitated with anti-yolk-protein antibodies and analysed by gel
electrophoresis (Fig. 8). Thus there seems to be a mechanism in vivo whereby
these accumulated yolk-protein transcripts are not efficiently translated.into
proteins, but this does not appear to be due to changes in the mRNA which
prevent it from being efficiently translated in vitro.
Yolk-protein-gene expression in dsxD
255
GENERAL DISCUSSION
As might be predicted from many studies of egg-laying rates in Drosophila, the
expression of the yolk-protein genes, which code for the major egg components,
is very variable. Studies on the levels of expression of these genes under various
experimental conditions must therefore be interpreted with caution, and the
variability between groups of flies and with age must always be considered.
Despite these problems we can make a number of conclusions from our studies
about yolk-protein-gene expression in the intersexual, dsxD, adults. These flies
lack one of the major sites of yolk-protein synthesis, the ovary, and the fat body
synthesizes and secretes into the haemolymph considerably reduced levels of
yolk proteins compared to wild-type females. The reduced protein synthesis is
initially reflected in the reduced accumulation of RNA transcripts coding for the
yolk proteins, however when the flies are 3-5 days old, transcripts build up in
dsx° and sometimes exceed wild-type levels. We therefore propose that the
actual expression of the genes is regulated at both transcriptional and posttranscriptional levels.
We need to consider the mechanism by which the mutated dsx gene affects
transcription of the yolk-protein genes. One way which might explain both these
results and the induction of yolk-protein synthesis in isolated abdomens with
ecdysteroids (Jowett & Postlethwait, 1980) would be that the dsx locus
modulates titres of steroid hormones in the flies and that females would have a
high titre, males a low titre and intersexual flies an intermediate titre. However,
experiments measuring ecdysteroid titres in adult males and females detect no
such differences. (Handler, 1982; Bownes, Diibendorfer & Redfern, unpublished). These experiments must be extended to analyse haemolymph levels of
active 20-hydroxy-ecdysone, and we cannot, therefore, be sure that there are
no differences in hormones between males and females. The other aspects
of the sexual phenotype, such as bristle patterns and other morphological
characteristics affected by the sex genes, are controlled in a cell-autonomous
fashion (Baker & Ridge, 1980). It seems likely, therefore, that the dsxD mutation
exerts its effect on expression of yolk-protein genes from within the fat-body
cells. The normal wild-type allele of dsx would therefore, by its activity or
inactivity, lead to the yolk-protein genes being transcribed in female but not in
male fat-body cells.
Ecdysteroids are required for the metamorphosis of the fat body, and for
synthesis of yolk proteins in newly-emerged females (Jowett & Postlethwait,
1980; Diibendorfer & Eichenberger-Glinz, 1980), but not for continued yolkprotein synthesis in mature females (Bownes, 1982). It is possible that the effect
of dsxD on expression of yolk-protein genes occurs at the time of fat-body
metamorphosis, and that different (qualitatively or quantitatively) hormone
receptors are present in male and female fat-body cells so that there is a differential response to the same levels of hormone. There is no evidence either for or
256
M. BOWNES, M. DEMPSTER AND M. BLAIR
against this idea, and it is entirely possible that the wild-type dsx genes act to
regulate the expression of the yolk-protein genes via a pathway totally independent of ecdysteroids and their receptors, and they may well exert their effect
throughout adult life rather than just during fat-body development.
At present we can only speculate upon how the wild-type dsx allele can affect
the expression of the yolk-protein genes and our experiments give no indication
of what is abnormal in dsxD mutants such that they have altered sexual characteristics, which includes altered yolk-protein synthesis. However, now that we
know that the yolk-protein genes are regulated by the sex genes we can begin to
investigate how this is achieved in vivo. Experiments designed to unravel the
relationship between the sex genes, ecdysteroids and the yolk-protein genes are
in progress.
This research was supported by the Medical Research Council. We are grateful to Pieter
Wensink for providing us with pYPl, pYP2 and pYP3, Judy Chisholm for typing the
manuscript and Graham Brown for printing the photographs.
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(Accepted 2 February 1983)