/. Embryol. exp. Morph. Vol. 25, 2, pp. 237-246, 1971
237
Printed in Great Britain
Influence of the RNA
content on oogenesis in the bobbed mutants
of Drosophila melanogaster
By JAG MOHAN 1
From the Department of Molecular Biology, University of Brussels,
Rhode St Genese, Belgium
SUMMARY
Developing egg chambers of Drosophila melanogaster (wild-type and bobbed mutants) have
been examined for their nucleic acid content by cytophotometric methods. No differences
were observed in the total DNA and RNA content of the egg chambers at all stages between
the bobbed mutants and the wild type. It is shown that the process of oogenesis in bobbed
females is prolonged, and that this prolongation occurs at all the stages of oocyte development.
Since the ovaries of the bobbed females synthesize less rRNA per unit time, it is likely that
this prolongation allows the egg chambers of the bobbed females to normalize their RNA
content. When they achieve a given RNA content, they proceed to the next stage of development.
INTRODUCTION
The bobbed syndrome in Drosophila (slow development, short bristles and
etching of the abdominal tergites) is caused by a reduction in the amount of the
DNA complementary to ribosomal RNA (Ritossa, Atwood & Spiegelman,
1966). In most cases, the severity of the bobbed phenotype is inversely proportional to the amount of DNA complementary to ribosomal RNA (rDNA).
Ritossa et al. (1966) proposed that the basis of the bobbed phenotype could be
due to a limitation of rRNA synthesis. Following this proposal, we have recently
shown (Mohan & Ritossa, 1970) that the rate of rRNA synthesis is reduced in
ovaries of bobbed flies which have rDNA content lower than 0-130 %. These
flies lay eggs at a reduced rate, but of normal RNA content. Ovaries from phenotypically wild flies with rDNA contents of 0-180 %, 0-370 % and 0-580 % show,
instead, the same rate of rRNA synthesis and lay eggs at a normal rate and of
normal RNA content. In spite of the reduced rate of rRNA synthesis, bobbed
flies (both males and females) have the same RNA/DNA ratios as the phenotypically wild flies. The normalization of the rRNA content probably occurs
due to the delayed development of the bobbedflies.On the basis of these results,
we proposed that the normal ribosome content has to be achieved in tissues most
1
Author's address: Institute of Animal Genetics, University of Edinburgh, West Mains
Road, Edinburgh EH9 3JN, Scotland, U.K.
16-2
238
J. MOHAN
relevant for the development of the animal. Other less relevant tissues or cells
can develop even with subnormal amounts of ribosomes; they will lead to the
mutated phenotype. The purpose of the present paper is to present results
concerning this proposal. To do so, we have measured cytophotometrically the
RNA and DNA contents of developing egg chambers stage by stage, as classified by King, Rubinson & Smith (1956). Uridine incorporation into RNA has
also been followed by autoradiography. We have been particularly interested in
knowing whether or not the developing oocytes of bobbed flies can proceed to
their next stage of development without having a normal content of ribosomes.
Our results will show that this is clearly not the case; additional data indicates
that there is a general delay of all the stages of the oogenetic process.
MATERIALS AND METHODS
(a) Drosophila stocks. Wild-type Canton S was obtained from the University
of Pavia. g2tybblC(\)DX, yf was from Pasedena; wabbxlBsY and In(\)
sRyscur*cv v / W ere from Oak Ridge National Laboratory. XYL. Ys (108-9),
Y su(wa)waYLYslyvf
was obtained from the University of Rome. These
Drosophila stocks were kept in different genotypic combinations so that the
bobbed flies could be generated in one or two crosses. The stocks were never
kept in the bobbed condition as such because of the labile nature of this locus.
So the bobbed flies were obtained prior to the experiments by crossing parents
carrying both bb+ and bb~ chromosomes. The flies which first emerged were not
strongly bb and they were usually discarded. The flies emerging later were
strongly bb and were employed for the experiments. Only heterozygous flies
were employed in these experiments because homozygous flies of various strains
have a tendency to retain mature eggs and sometimes contain non-functional
egg chambers (David & Merle, 1968).
The percentage of rDNA is given in brackets after each genotype in the following description. Biochemical techniques regarding the estimation of rDNA
content have already been described (Mohan & Ritossa, 1970). Flies having
rDNA content lower than 0-130 % are phenotypically bobbed.
(b) Cytohgicalprocedures. Seven-day-old mated flies unless otherwise specified
were employed. Ovaries were dissected in a drop of Ringer solution. When
twenty-five ovaries had been collected in this way, they were fixed in Serra
solution (ethyl alcohol, 6 parts; formaldehyde, 3 parts; acetic acid, 1 part) for
30 min, dehydrated in a series of alcohol and embedded in paraffin wax. Sections
of 10 fi thickness were always used.
DNA staining was done with the standard Feulgen method. The acid hydrolysis was, however, carried out in 5 N-HC1 at room temperature, since this gives
more intense staining (De Crosse & Aiello, 1966) than hydrolysis at 55-60 °C in
1 N-HC1. RNA staining was done with the gallocyanin chrome alum method
(Sandritter, Kiefer & Rick, 1966). A Barr and Stroud integrating microdensitoSC*L,
2
RNA content in oogenesis of bobbed mutants
239
meter was used to measure the nucleic acids quantitatively. Control slides treated
with DNase (0-25 mg/ml at pH 6-0 containing 0-03 M-SO 4 Mg for 3 h at 37 °C)
and RNase (0-5 mg/ml at pH 60 for 2 h at 37 °C) were employed for all experiments.
(c) Autoradiography. Five-day-old flies of various genotypes were fed on
a radioactive medium containing 5 g dead yeast, 2 g corn, 1 g sugar and 0-2 ml
of 10Ci/ml [3H]uridine (1 Ci/mM). The flies were removed after 2 days and
sections of the ovaries were prepared as described before. Slides were covered
with K2 llford emulsion and exposed for 10 days. The sections were stained with
methyl green pyronin.
Whole mounts of 0-, 1-, 3- and 7-day-old ovaries were done for both Feulgen
reaction and aceto-orcein squashes.
RESULTS AND DISCUSSION
The development of an egg chamber has been subdivided into a series of
14 stages by King et al. (1956). However, this development is a continuous event
and its separation into a series of discrete stages was made by relying on multiple
morphological criteria. The characteristics used by King et al. (1956) have been
employed to allocate the egg chambers to the various stages. A chamber showing
Table 1. DNA and RNA content in the first ten stages of developing egg
chambers in three genotypes
Genotypes
bb+/XYL.Ysbb
t/g*ty
(0100)
(0-275)
Stages
DNA
RNA
1
2
3
4
5
6
7
8
9
10
16 ±2-6
25 ±4-3
53 ±6-7
68 ±10-9
72 ±8-6
89 ± 12-5
95 ±9-6
101 ±11-3
108 ±10-8
32 ±4-7
36 ±3-8
51 ±4-5
72 ±7-3
86 ±7-7
95 ±9-2
103 ±9-3
105 ±8-5
116+11-7
103± 11-4
121 ± 12 3
sciscs/XYL.Ysbb
DNA
12 ± 0-9
15 ± 1-3
26 ±2-8
45 ±4-8
75 ±9-3
73 ±6-5
86 ±8-3
103 ±8-6
95 ±11-3
98 ±10-4
RNA
21 + 3-1
28 ±3-3
35 ±4-4
53 ±5-8
82 ±6-3
81 ±10-5
95 ± 11 -8
106 ±12-4
117 ±6-8
114 ±8-7
(0090)
DNA
14 ±1-8
19 ±2-1
38 ±4-2
62 ±6-5
74 ±7-2
77 ±6-5
78 ±6-5
96 ±8-3
8 9 ± 11 •5
95 ±10- 6
RNA
25 ±2-3
37 ±3-5
42 ±4-6
69 ±6-2
78 ±7-3
90 ±8-4
103 ±11-5
117 ±2-5
98 ± 12-8
123 ±9-5
All figures are given in arbitrary units. DNA was measured by Feulgen staining and RNA
by the gallocyanin chrome alum method after treatment with DNase. The figures are means
( ± S . D . ) of at least ten measurements in each case. Measurements have been done on sections
passing through the middle of the nuclei. Total RNA has been measured but in the description
extrapolation has been made to rRNA. This is considered valid, firstly, because a major
fraction (90-95%) of the ovaries' RNA is ribosomal and, secondly, no difference was seen to
exist in the synthesis of transfer RNA between the bobbed and the wild genotypes (Mohan
& Ritossa, 1970).
240
J. MOHAN
characteristics of two consecutive stages is placed at the stage which includes
the majority of the morphological criteria observed for that chamber.
The DNA and RNA content of developing egg chambers was measured
cytophotometrically in three genotypes and the results are shown in Table 1. To
overcome technical difficulties, the DNA and RNA content was measured on
whole egg chambers (only in the first six stages) and not on individual nuclei.
It has not been considered worth while to measure the nucleic acid content of
the egg chambers in the last four stages during which the nurse cells have started
*
Fig. 1. Autoradiographs of sections 10/* thick of bb+/bb+ (0-330) 7-day-old ovaries.
(A) Three egg chambers of stages 1, 3 and 4. The grains are distributed over all
areas, x 2400.
(B) An egg chamber of stage 7. Most of the grains are seen in the regions adjoining
the neighbouring cells. This pattern of grain distribution is not so obvious before this
stage, x 2800.
(C) An egg chamber of stage 9. The pattern of grain distribution is similar to the one
observed in (B). There is a continuity of grains from the nurse cell cytoplasm to the
ooplasm. Small arrow marks the nurse cell and the large one the oocyte. x 2160.
B
RNA content in oogenesis of bobbed mutants
241
to degenerate. It is obvious from Table 1 that a gradual increase takes place until
the 5th stage in the DNA content in all the three genotypes. Since the measurements were made on 10 [i thick sections in all cases, the figures do not correspond
exactly to the levels of ploidy. The same seems to be the case for the RNA content in the first four stages, but no difference can be seen between 5th and 6th
stage. This is probably due to the fact that the volume at these stages has increased without any apparent change in the quantity of RNA per unit volume.
Although these figures cannot be used for absolute purposes, they are of value
for comparison between the various genotypes. Regarding the DNA content at
these stages, one can say confidently that there is no difference between the
various genotypes. The minor differences found could be due to the fact that,
at the same stage, there are differences in size since each of these stages lasts
for a long time. The differences observed between the RNA contents also are of
the same order of magnitude and should thus be regarded as insignificant.
RNA synthesis in developing oocytes of all stages has also been followed by
autoradiography in various genotypes. As shown by the controls with RNase
digestion, most of the tracer in sections is present in the form of RNA. One can
see, from Figs. 1 and 2, that RNA synthesis occurs through all stages of oocyte
development, but that its intensity varies between different stages. There are
more grains over the nurse-cell nuclei than over the oocyte nucleus; but the
oocyte cytoplasm also contains many grains. Similar observations have already
been made by King & Burnett (1959), Sirlin & Jacob (1960) and by Zalokar
(1960) for wild-type Drosophila. In all the genotypes, the radioactivity of the
cytoplasm, both in the nurse and egg cells, rises continuously. The label moves
along the streams of the ooplasm which are continuous with the nurse cell
cytoplasm, indicating a flow of RNA towards the oocyte (Figs. 1, 2). No difference was seen in the amount of grains present in the cytoplasm of oocytes having
different genotypes. In some sections, more tracer is found moving towards the
oocyte from the nurse cells in bobbed females, suggesting that the nurse cells
contribute comparatively more to the developing oocytes in the bobbed females
than in the wild type.
At all stages of egg-chamber development, no difference was observed in the
amount of RNA between the bobbed and the wild type; yet we know from our
previous biochemical studies that the bobbed ovaries synthesize less rRNA per
unit time. Therefore, one would like to know how the normalization of RNA
content takes place. To get an insight into this, we have examined some physiological and morphological parameters both in bobbed and phenotypically wild
flies. The results are presented in Table 2. There are no differences between the
wild and bobbed flies in mean ovariole number and mean number of egg chambers
per ovariole, but clear-cut differences are seen in other parameters. Egg production
is nearly the same in the two phenotypically wild genotypes (bb+IXYL. Ysbb and
bb+lg2ty), while it is severely reduced in the three other genotypes (Table 2). The
two other measurements (daily rate of egg chamber production and total
242
J. MOHAN
Fig. 2. Autoradiographs of sections of wa bbl/XYL. Ysbb (0-100) 7-day-old ovaries.
(A) Left egg chamber is in stage 7 and the right one in stage 8; one can see more
clearly an accumulation of grains on the adjoining parts of the nurse cells in the right
egg chamber, x 3600.
(B) An egg chamber of stage 10. Continuity of grains from nurse cell to oocyte is
observed as described earlier for wild type. Small arrow marks the nurse cell and the
large one the oocyte. x 3190.
RNA content in oogenesis of bobbed mutants
243
Table 2. Some physiological and morphological parameters in ovaries
of various genotypes*
Genotypes
Mean daily egg production
Mean ovariole no.
Daily rate of egg chamber
production
Mean no. of egg chambers per
ovariole
Total duration of egg chamber
in various stages (h)
wabbx\
gHy
XYL.Ysbb
(0-120)
(0100)
sc*sc8/
XYL.Ysbb
(0090)
bb+l
bb+l
XYL.Ysbb
(0-275)
gHy
(0-255)
77-6
33-4
2-32
71-5
35-3
203
46-7
29-6
1-58
38-9
32-6
119
30-5
37-3
0-82
7-21
7-05
6-98
7-36
7-03
75
83
wabbxl
149
106
206
* Egg production has been measured from 4th to 8th day of life. Other calculations are
based on Feulgen-stained whole mounts of 7-day-old ovaries. The growth duration of egg
chamber from stage 1 to 14 is obtained by dividing the mean number of these chambers in an
ovariole by their daily rate of production.
Table 3. Time spent (h) in various stages by egg chambers of various genotypes*
Genotypes
Stages
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Total
L
bb+l
XY .Ysbb
(0-275)
gHy
(0-255)
gHy
(0100)
XY .Y bb
(0-120)
s^sc8/
XY .Ysbb
(0090)
9-23
9-53
8-85
923
3-38
7-20
7-88
4-65
5-47
4-37
0-75
1-80
0-83
1-87
75
8-80
10-21
9-55
11 04
2-99
7-55
9-63
5-98
5-64
5-48
0-66
2-74
1-25
1-49
83
12-61
13 04
13 04
9-33
413
11-34
12-19
7-21
7-84
6-89
2-44
2-65
1-27
201
106
1609
14-16
14-30
13-86
8-79
16-99
18-63
11 03
12-81
11-62
5-22
2-24
119
209
149
19-36
18-75
1916
17-92
9-89
23-07
26-37
18-33
19-57
18-13
6-80
3-91
2-47
2-47
206
bb+l
wabbxj
L
s
L
* Feulgen-stained whole-mounts of 7-day ovaries were employed. Total time spent in
14 stages by an oocyte is taken from Table 2. The duration of each stage is estimated by
multiplying the whole growth period by the percentage frequency of each. (See Table 4.)
244
J. MOHAN
duration of egg chamber in various stages, which are obviously related to egg
production) are also affected in the bobbed genotypes
(wabb1IXYL.Ysbb,
a
x
L
s
w bb jgHy, sc*s(*lXY . Y bb).
Our results show that the growth duration of the egg chamber from stage
1 to 14 is prolonged in the bobbed flies and that the degree of prolongation
depends upon the severity of the altered phenotype. To know whether this
prolongation is caused by a delay at a particular stage or occurs in all stages, we
have analysed the time duration of the egg chambers at various stages of
Table 4. Stage distribution of egg chambers as a function of age in ovaries of
various genotypes o/Drosophila melanogaster*
Genotypes
L
s
bb+ /AT .Y bb
(0-275)
Age (days)
Stages
0
1
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
16-9
17-1
15-1
13-6
10-9
121
14-3
0
12-6
13-3
12-5
14-3
6-5
13-4
11-6
6-3
1-5
1-3
0-4
3-2
1-3
1-8
119
0
0
0
0
0
0
7
12-3
12-3 12-7
121 11-8
11-6 12-3
3-8
8-7
4-5
9-6
11-6 10-5
80
7-8
5-8
1-2
2-1
11
2-0
sc*scs/XYL.Ysbb
(0090)
Age (days)
wabbllg2ty
(0- 100)
Age (days)
6-2
7-3
5-8
10
2-4
1-1
2-5
0
1
3
17- 7 16-7 13-9
17- 7 16-7 14-3
17- 5 16-4 14-3
13- 6 12-7 12-4
11- 3 9-6
7-8
10- 1 91
8-7
12- 1 11-4 116
0
5-6
8-9
0
1-3 4-8
0
0-5 2-8
0
0-5
0
0
0
0
0
0
0
0
0
0
7
0
1
3
7
8-9
8-7
8-6
7-8
5-3
12-5
13-1
9-3
16-7
16-7
17 1
14-6
11-8
10-7
12-4
0
0
0
0
0
0
0
15-1
151
15-7
13-6
10-8
9-7
110
6-8
2-2
12-4
12-4
12-6
10-6
5-5
11-8
11-6
8-9
9.4
91
9-3
8-7
4-8
11-2
12-8
8-9
0
0
0
8-4
3-7
1-8
0-3
0
0
0
0
9-5
8-8
3-3
1-9
1-2
1-1
9-8
80
4-3
1-5
1-4
0-8
* All figures are given as percentages. Feulgen-stained whole mounts were employed for
3-and 7-day ovaries and aceto-orcein squashes for 0-and 1-day ovaries. Calculations are based
on counts of nearly 500 egg chambers for 0-, 1- and 3-day ovaries and more than 1000 egg
chambers for 7-day ovaries of each genotype.
development. The results of this analysis, carried in five genotypes, are presented in Table 3. The two phenotypically wild strains bb+/XYL.Ysbb and
bb+/g2ty have a duration period of 75 and 83 h respectively; these figures are in
agreement with those presented by other authors (King, 1957; David & Merle,
1968). The figures for each stage are also in agreement with those given by these
authors for the phenotypically wild genotypes. For all the bobbed genotypes, the
time duration of the egg chamber is prolonged in all the fourteen stages and not
in any particular stage. On the basis of these results, one can predict that the
egg-laying should start later in the bobbed females than in the wild ones. Such
a prediction is confirmed by our results (Table 4). At the time of emergence, both
RNA content in oogenesis of bobbed mutants
245
wild and bobbed flies contain egg chambers in the first seven stages of development; however, their frequency varies between the different genotypes: oocytes,
in all 14 stages of development, start to appear in 1-day-old females of the wild
type, while in bobbed females they appear at stage 10 after 1 day, stage 12 after
3 days and at all the 14 stages only later.
It is clear from our results that the developing egg chambers of bobbed females
proceed to their next stage of development only after they have synthesized
a given amount of ribosomes. This is obtained by a general delay through all
stages of oocyte development in the bobbed mutants. The degree of prolongation
in the oogenetic processes depends upon the severity of the altered phenotype
in the bobbed females, which start to lay eggs later than the wild-type females.
This retardation allows them to accumulate ribosomes and to build up an
efficient machinery for egg formation.
RESUME
Influence de la teneur en RNA sur Voogenese chez les
mutants 'bobbed' de Drosophila melanogaster
La teneur en acides nucleiques des oocytes et des cellules nourricieres, de Drosophila
melanogaster, en developpement: type sauvage et mutants 'bobbed', a ete suivie par des
methodes cytophotometriques. On n'observe pas de differences dans la teneur en DNA ni
en RNA des oocytes et des cellules nourricieres a tous les stades etudies, entre les mutants et
les individus de type sauvage. On sait que le processus de 1'oogenese, chez les femelles bobbed
est prolonge et que cette prolongation se manifeste a tous les stades du developpement des
oocytes. Comme les ovaries des femelles bobbed synthetisent moins de rRNA par unite de
temps, il est vraisemblable que cette prolongation permet aux oocytes et aux cellules nourricieres des femelles bobbed de normaliser leur teneur en RNA. Quand el les atteignent une
teneur donnee en RNA, el les precedent au stage suivant du developpement.
I am grateful to Prof. J. Brachet for his valuable suggestions and interest in the work. I was
partially supported by an International Cell Research Organisation fellowship. This work has
been carried out under the Euratom Contract 007.61.10 ABIB.
REFERENCES
DAVID, J. & MERLE, J. (1968). A revaluation of the duration of egg chamber stages in oogenesis
of Drosophila melanogaster. Drosoph. Inf. Serv. 40, J 22-123.
DE CROSSE, J. J. & AIELLO, N. (1966). Feulgen hydrolysis: effect of acid and temperature.
/. Histochem. Cytochem. 14, 601-604.
KING, R. C. (1957). Oogenesis in adult Drosophila melanogaster. II. Stage distribution as
a function of age. Growth 21, 95-102.
KING, R. C. & BURNETT, R. G. (1959). An autoradiographic study of uptake of tritiated
glycine, thymidine, and uridine by fruit fly ovaries. Science, N.Y. 129, 1674-1675.
KING, R. C, RUBINSON, A. C. & SMITH, R. I. (1956). Oogenesis in adult Drosophila melanogaster. Growth 20, 121-157.
MOHAN, J. & RITOSSA, F. M. (1970). Regulation of ribosomal RNA synthesis and its bearing
on the bobbed phenotype of Drosophila melanogaster. Devi Biol. 22, 495-512.
RITOSSA, F. M., ATWOOD, K. C. & SPIEGELMAN, S. (1966). A molecular explanation of the
bobbed mutants as partial deficiencies of the 'ribosomal' DNA. Genetics, Princetown 54,
819-834.
246
J. MOHAN
W., KIEFER, G. & RICK, W. (1966). Gallocyanin chrome alum. In Intd. Quant.
Cytochem. pp. 295-326 (ed. G. L. Wied). New York and London: Academic Press.
SIRLIN, J. L. & JACOB, J. (1960). Cell function in the ovary of Drosophila. II. Behaviour of
RNA. Expl Cell Res. 20, 283-293.
ZALOKAR, M. (1960). Sites of ribonucleic acid and protein synthesis in Drosophila. Expl Cell
Res. 19, 184-196.
SANDRITTER,
{Manuscript received 2 November 1970)