/ . Embryol. exp. Morph. Vol. 41, pp. 101-110, 1977
Printed in Great Britain © Company of Biologists Limited 1977
\Q\
Protein synthesis in the early Drosophila
embryo; analysis of the protein species synthesized
By DAVID B.ROBERTS1 AND GIORGIO GRAZIOSI
From the Genetics Laboratory, Biochemistry Department, Oxford
and Institute of Zoology and Comparative Anatomy,
University of Trieste, Italy
SUMMARY
The soluble proteins were extracted from Drosophila eggs which had been permeabilized
and incubated in medium containing [35S]methionine. These proteins were analysed on
immunoelectrophoresis plates and on SDS polyacrylamide gels both by staining for total
protein and by autoradiography. The radioactive proteins must have been synthesized
during the period of incubation with [35S]methionine. In the period covered by this study
(0-3 h) there was much protein synthesis but no new proteins were synthesized which had
not already been synthesized during oogenesis. We conclude that the considerable protein
synthesis that occurs in early Drosophila development is translated from maternal mRNA
which is activated both by egg deposition and fertilization. Translation of protein from either
masked maternal mRNA, which had not been previously translated, or from mRNA
transcribed from the zygote genome must occur after blastoderm formation.
INTRODUCTION
Many of the proteins synthesized during early development of the sea urchin
or during the maturation of the amphibian oocyte migrate into the cleavage
nucleus or into the nuclei of the blastula (Merriam, 1969; Smith & Ecker, 1970;
Ecker & Smith, 1971; Brothers, 1976). The function of the histone proteins
(Kedes, Gross, Gognett & Hunter, 1969) is known but the function of the
majority of these proteins is unknown although some might be involved in
controlling the activity of genes during early development. One such protein,
synthesized during oogenesis and interacting with the blastula nuclei is the o +
substance (Brothers, 1976).
The importance of the role played by the maternal genome to the normal
early development of Drosophila is well established by the large number of
embryonic mutants showing maternal effects (Bakken, 1973; Rice & Garen,
1975; Gans, Audit & Masson, 1975). In the mature oocyte proteins necessary
for normal embryonic development may be stored as such or as mRNAs
which are translated during development (Goldstein & Snyder, 1973; Zalokar,
1
Author's address: Genetics Laboratory, Biochemistry Department, South Parks Road,
Oxford, 0X1 3QU, U.K.
102
D. B. ROBERTS AND G. GRAZIOSI
1976; Graziosi & Roberts, in preparation). One particular example of maternal
mRNA being involved in embryonic differentiation and possibly with gene
regulation is demonstrated by the behaviour of the posterior polar cytoplasm
and the pole cells. The pole cells are determined by the cytoplasm and differentiate autonomously (Illmensee & Mahowald, 1976; Illmensee, Mahowald &
Loomis, 1976). As soon as the pole cells form they begin to synthesize proteins
at a much greater rate than the later blastoderm cells (Zalokar, 1976). The polar
cytoplasm is characterized by specific protein antigens (Graziosi & Roberts,
1975) and the first proteins synthesized seem to be involved in germ cell determination (Graziosi & Micali, 1974; Graziosi & Marzari, 1976). In other words
it may be that the newly synthesized proteins of the posterior pole are responsible for the developmental pattern of the pole cells.
Before studying the role played by individual proteins in determination at
this early stage we have investigated which major protein species are synthesized
in the first 3 h of Drosophila development and whether the initiation of the
synthesis of these proteins depended on fertilization or egg deposition or
whether it was a continuation of the synthesis of proteins which were synthesized during oogenesis.
MATERIALS AND METHODS
Flies
The Drosophila stock Oregon-R maintained on yeast-cornmeal-agar medium
at 25 °C was used in these studies.
Antisera
The antisera used are described in Table 1. All of the antisera were prepared
in rabbits against crude extracts of soluble proteins, unless indicated to the
contrary, by an intramuscular injection of 5 mg of protein in 1 ml together
with 1 ml of complete Freund's adjuvant (Difco) followed by 3 x fortnightly
injections of the same but with incomplete adjuvant. Rabbits were bled 1 week
after the last injection and immunoglobulin G was prepared from the sera by
the method of Keckwick (1940). Rabbits were given an intramuscular injection
1 week prior to all subsequent bleeds.
Double diffusion and immunoelectrophoresis plates
The plates were prepared, run, washed, dried and stained as described previously (Roberts & Pateman, 1964).
The strategy for comparing extracts on immunoelectrophoresis plates was
as described by Boavida & Roberts (1975).
Acrylamide gels
The sample was prepared by mixing with an equal volume of 0-0625 M tris
buffer pH 8-6 with 2 % SDS and 5 % 2-mercaptoethanol added and boiled for
Protein synthesis in early Drosophila embryo
103
Table 1
Antiserum
number
503
508
514
524
525
539
542
546
547
549
561
562
563
564
565
566
573
G7-4
Antigen used to elicit antibody response
Male fly extract
Fly extract
Larval haemolymph
Cell line GM3
Cell line LI
Embryo
3rd instar larva
Embryo, posterior pole precipitate
Embryo, posterior pole supernatant
Cell line Gm 3 incubated with juvenile hormone
Embryo 0-30 % ammonium sulphate cut
Embryo 30-40 % ammonium sulphate cut
Embryo 40 % +ammonium sulphate cut
Embryo, deoxycholate extract
1st instar larva
Embryo
Cell line Gm 3 + ecdysterone
Fat body (3rd instar larva)
Reference
Roberts, 1971
Roberts, 1971
Boavida & Roberts, 1975
Roberts, 1975
Moir & Roberts, 1976
Graziosi & Roberts, 1975
Roberts, 1971
Graziosi & Roberts, 1975
Graziosi & Roberts, 1975
Moir, 1974
Graziosi & Roberts, 1975
Graziosi & Roberts, 1975
Graziosi & Roberts, 1975
Graziosi & Roberts, 1975
Roberts, 1971
Roberts, 1975
Moir, 1974
Boavida & Roberts, 1975
2 min. This was centrifuged and the appropriate volume of supernatant was
placed on the stacking gel and electrophoresed according to the technique of
Laemmli (1970). The gels were fixed in 50% trichloracetic acid and stained
with Coomassie blue.
Extracts
Eggs were sonicated in 0-05 M pH 7-2 phosphate buffer and the sonicate
was centrifuged at 40 000 # for 20 min. The concentration of protein in the
supernatant was adjusted either by dilution or by freeze drying after dialysis
against distilled water. The protein concentration used in these studies was
generally 30 mg/ml.
Autoradiographs
Dried, stained double diffusion or immunoelectrophoresis plates were
clamped in contact with X-ray film (Kodak B 54) between two pieces of hardboard, wrapped in foil and stored at 4 °C for the appropriate time. The^film
was developed according to the manufacturer's instructions.
Polyacrylamide gels were sliced after fixing and staining and the slices dried
on Whatman's filter paper (No. 1) under vacuum (Jeffreys, 1975). Autoradiographs of the dried gels were prepared as described'above.
104
D. B. ROBERTS AND G. GRAZIOSI
Protein concentration
The protein concentration of the extracts was determined with reference to
a standard curve prepared with bovine serum albumin by the method of Lowry,
Rosebrough, Farr & Randall (1951).
Permeabilization of eggs
The eggs were permeabilized using a modification of the method described
by Limbourg & Zalokar (1973) which allowed hundreds of eggs to be permeabilized at the same time (Graziosi, unpublished results).
RESULTS
Antigens
All antisera were compared using the same antigen extract (0-24 h eggs for
timing see Roberts, 1971) and while the analysis of such a comparison is not
straightforward (Roberts, 1975) we are confident, from a careful examination
of all immuncelectrophoresis plates, that our antisera will detect in excess of
40 different antigens. The differences between the antisera are illustrated by
analysis on double diffusion plates (Fig. 1) but so many precipitin bands are
obscured by the overlay of other bands that this method of analysis was only
used in special cases.
Extracts were made from permeabilized eggs of four different stages (2050 min, 50-90 min, 90-130 min and 130-190 min post-lay) (Graziosi & Roberts,
in preparation) which had been incubated in incorporation medium containing
[35S]methionine. The extracts were adjusted to approximately the same protein
concentration and analysed by immunoelectrophoresis using all antisera.
FIGURES
1-4
Fig. 1. Double diffusion plate showing the different antigens detected in a 0-24 h
embryo extract by different antisera. G = 0-24h embryo extract; A = 539;
B = 503; C = 566; D = 564; E = 524; F = 508 (Table 1).
Fig. 2. (i) Immunoelectrophoresis plate stained for total protein, (ii) An autoradiograph of the above. C = 20-50 min embryo extract (Table 2); A = 561;
B = 573 (Table 1).
Fig. 3. (i) Immunoelectrophoresis plate stained for total protein, (ii) An autoradiograph of the above. C = 20-50 min embryo extract (Table 2); A = 539;
B = 566 (Table 1). The arrow indicates the putative yolk protein band which is
radioactive.
Fig. 4. (i) Double diffusion plate stained for total protein, (ii) An autoradiograph
of the above. B = extract of ovaries hand dissected from female flies; D = extract
of unfertilized eggs; E = 20-50 min embryo extract incubated in incorporation
medium. All extracts were adjusted to the same protein concentration of 30 mg//tml.
A = 508; C = 561 (Table 1). The arrow indicates a protein synthesized by E
(radioactive), not found in D (no reaction of identity) but found in B (reaction
of identity).
Protein synthesis in early Drosophila embryo
.4©
105
106
D. B. ROBERTS AND G. GRAZIOSI
Table 2
Age of embryos
(min)
Dpm in TCA precipitable
material//tg of protein
20-50
50-90
90-130
130-190
350
30
40
27
Staining for total protein showed significant differences between the extracts
but these were quantitative differences, especially involving the proteins we
think are yolk proteins.
The comparison of the autoradiographs of these immunoelectrophoresis
plates is more difficult because of the difference in the amount of [35S]methionine
incorporated into protein at the different ages after fertilization (Table 2). To
overcome this problem the autoradiographs were allowed to develop for
different times but even this was not wholly satisfactory.
The best result, as expected from the incorporation data, came from the
youngest eggs with the heavily labelled proteins. Most of the different antigens
were labelled indicating that they were synthesized during this period (Fig. 2).
A comparison of the autoradiographs of the immunoelectrophoresis plates of
different ages showed that precipitin arcs labelled in the earliest stage were
unlabelled later. This could reflect the difference in incorporation by the
different stages rather than cessation of protein synthesis; however, all of the
antigens labelled at the latest stage were also labelled at the earliest stage,
which suggests that in the period covered by this study there was no considerable synthesis of a new protein in the later stages.
Of particular interest in the earliest stage are a class of proteins which are
electrophoretically heterogeneous (long arc on immunoelectrophoresis plates)
which are detected as strong antigens by antisera directed against egg or embryo
extracts. These proteins are not present in 1st instar larvae nor are they present
in male flies. They are, however, present in female flies but only in the abdomen
and we suspect that these are yolk proteins. Examination of the autoradiographs
shows part of one of these proteins to be radioactive. In other words only
some of this class of immunologically related molecules incorporate [35S]methionine at this time (Fig. 3). It is worth noting that these same bands show some
incorporation even at the later stages studied.
Because of the difficulty in comparing immunoelectrophoresis plates we
compared the treated extracts of newly fertilized eggs with extracts of ovaries
and unfertilized eggs on double diffusion plates. Again the bands of newly
synthesized proteins were detected by autoradiography. All of the major bands
shown on the autoradiographs were present in ovaries and all except one were
present in unfertilized eggs (Fig. 4). This comparison is unambiguous as the
Protein synthesis in early Drosophila embryo
1 2
Fig. 5
3
4
1
107
2
Fig. 6
Fig. 5. SDS polyacrylamide gels stained with Coomassie blue of 1, 20-50 min;
2, 50-90 min; 3, 90-130 min; 4, 130-190 min embryo extracts (Table 2). Bands
A-F are bands which are apparent on an autoradiograph of a section of 1. The
molecular weight markers are G = bovine serum albumin (68000); H = glutamate dehydrogenase (53000); I = lactate dehydrogenase (35000).
Fig. 6. (i) SDS polyacrylamide gel of a preparation of 30-50 min embryos incubated in incorporation medium and stained for total protein with Coomassie
blue, (ii) An autoradiograph of the same. Bands A-F are the bands shown to be
radioactive in Fig. 5.
major bands on the autoradiographs can be seen to curve round in a reaction
of identity with the unlabelled proteins in the ovary and unfertilized egg
extracts. However, some of the less prominent bands are not fully resolved on
these plates and so it is difficult to say whether they cross-react or not.
SDS polyacrylamide gels
Extracts of the four post-fertilization stages were run on SDS polyacrylamide
gels and as can be seen from Fig. 5, there is no significant difference between
them. Autoradiographs of these gels showed bands A-F in the 20-50 min
preparation and only two of these, C and E, in the later stages. This difference
108
D. B. ROBERTS AND G. GRAZIOSI
may again be attributed to the differential incorporation of [35S]methionine by
the different stages.
Figure 6 shows a polyacrylamide gel of an extract of newly fertilized eggs
permeabilized in a later experiment. Many of the bands stained by the protein
stain are also obvious on the autoradiograph and the most prominent bands
on the autoradiographs are those found in the earlier study, A-F. One band in
particular is worth noting, band E. In all gels stained for protein there is a
thick band with molecular weights ranging from 50000 to 56000 daltons. This
we suspect to be the band of yolk protein subunits. Only the lowest molecular
weight form is radioactive in all experiments. Indeed scrutiny of the autoradiograph in Fig. 6 shows that the heavier molecular weight molecules in this band
are notable for the absence of radioactivity. This echoes the finding that only
part of the putative yolk protein precipitin arcs were radioactive.
DISCUSSION
The antisera used in this study detected over 40 antigens in extracts of early
embryos and on SDS polyacrylamide gels stained for protein we counted 60
different bands. Some proteins will be detected by both techniques but some of
the antigens are likely to be heteropolymers and so it is not unreasonable to
assume that by these techniques we are able to analyse the products of about 100
structural genes in the early embryo.
We can only discuss the results obtained so that a positive result means that
a protein is present and a negative result that it is absent, even though it may
be present at a concentration too low to be detected by our techniques. This
proviso will not be repeated.
Comparing the proteins present in extracts of four different stages of early
development by staining for protein we found only quantitative differences
between the extracts. In other words by the first stage studied all the proteins
found in the pre-cellular blastoderm were already present and that by the last
stage studied no new species of protein was synthesized. There is, however,
convincing evidence that protein synthesis occurs during this period (Goldstein
& Snyder, 1973; Zalokar, 1976; Graziosi & Roberts, in preparation).
In order to study which of these proteins were synthesized during this period
we analysed autoradiographs of the immunoelectrophoresis plates and SDS
polyacrylamide gels. Most of the proteins were synthesized during this period
and all of the proteins labelled at the latest stage were also labelled at the
earliest stage. There is no evidence for synthesis of new protein species in the
later periods.
Part of the electrophoretically heterogeneous protein which we think is one
of the yolk proteins was labelled on immunoelectrophoresis plates and the
leading edge of the band we consider to be the yolk protein subunits on SDS
polyacrylamide gels was also labelled. It is surprising that yolk proteins, if
Protein synthesis in early Drosophila embryo
109
they are yolk proteins, are synthesized in the fertilized egg and an alternative
explanation is that these proteins are modified in the egg by the addition of,
at least, methionine.
We found all of the radioactive proteins of the newly fertilized egg in ovaries
and, with one exception, in unfertilized eggs. This suggests that proteins are
synthesized during oogenesis, but not by the oocyte where there is little protein
synthesis (Goldstein & Snyder, 1973) and that after fertilization protein synthesis increases with the major proteins synthesized being the same species as
those synthesized during oogenesis, although not all of the proteins synthesized during oogenesis are synthesized after fertilization. These proteins synthesized by the fertilized egg must be translated from maternal mRNA as
there is no nuclear RNA synthesis during this period (Zalokar, 1976; Graziosi
& Roberts, in preparation).
Goldstein & Snyder (1973) showed an increase in protein synthesis in extracts
of unfertilized eggs over oocyte extracts. This increase is likely to be stimulated
by egg deposition. However, egg deposition alone cannot be responsible for
initiating all of the protein synthesis in newly laid eggs, as we found a protein
in the newly fertilized egg which was not found in unfertilized eggs. Its synthesis
must have been initiated by fertilization. This protein was also found in ovaries
and may represent a labile protein synthesized during oogenesis with synthesis
re-initiated by fertilization but not by egg deposition. It is worth noting that
this protein was detected by most of the antisera used.
These results do not provide evidence for the early synthesis of any new
protein species which might be involved in embryonic determination, although
they do show that many proteins are being translated throughout this period
from maternal mRNA. The synthesis of new protein species either from masked
maternal mRNA or from mRNA transcribed from the zygote genome must
occur after cellular blastoderm formation.
The techniques described here only detect proteins synthesized in comparatively large amounts and so we are unable to draw any conclusions as to the
behaviour of genes coding for proteins only found at low concentrations. Such
proteins are likely to be important in the control of development. No broad
based study will ever be able to analyse these proteins, each will require an
individual analytical technique.
We wish to acknowledge the receipt of a NATO grant which has enabled us to collaborate
over this work. G.G. wishes to acknowledge the support of a Royal Society-Accademia
Nazionale dei Lincei fellowship.
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{Received 26 January 1977, revised 22 March 1977)