/. Embryo!, exp. Morph. Vol. 33, 4, pp. 1003-1011, 1975
Printed in Great Britain
1003
Restoration of the capacity to form pole cells
in u.v.-irradiated Drosophila embryos
By RICHARD WARN 1
From the MRC Laboratory of Molecular Biology, Cambridge
and the Genetics Laboratory, Department of Biochemistry, Oxford
SUMMARY
Injection of pole plasm into u.v.-irradiated posterior poles of early Drosophila embryos
leads to the restoration of the capacity to form pole cells in nearly half of the recipients. The
effect is specific, since cytoplasm from the anterior tip has no such result. In most cases only
a small number (between 1 and 5) of discrete pole cells are formed. However, a large number
of pole cell fragments with or without nuclei occur. Occasionally pole cells were formed
outside the area of the originally irradiated pole plasm. This happened when material was
injected more anteriorly than usual. Thus polar cytoplasm contains some factor(s) necessary
for the formation of pole cells.
INTRODUCTION
The pole plasm of insect embryos has long been believed to contain special
substances required to influence the activity of the nuclei which subsequently
enter it (Hegner, 1914). In Drosophila the pole cells which are formed from the
posterior region of the egg are precociously budded off f h prior to the cellularization of the rest of the embryo. They have a characteristic morphology
which is very different from that of the other cells which are subsequently
formed. Thus, even at the time of their formation, pole cells have certain
characteristics which mark them off from the cells surrounding them. The cells
which come to contain pole plasm have a precisely determined fate. The germ
cell line comes only from these cells.
Destruction of pole cells by u.v.-irradiation (Geigy, 1931; Aboim, 1945) led
to the absence of sex cells in the adult gonads and showed that regulation of
this region of the embryo did not occur, even early in development. More recent
studies (Poulson & Waterhouse, 1960; Hathaway & Selman, 1961; Warn,
1972; Graziosi & Micali, 1974) have shown that the loss or destruction of pole
plasm in the newly fertilized egg leads to a failure of pole cell formation and
subsequently to adults with sterile gonads.
So far little is known of the nature of pole cell determinants, although it has
been hypothesized (Mahowald, 1968) that they are contained as maternal mRNA
1
Author's address: Genetics Laboratory, Department of Biochemistry, South Parks Road,
Oxford 0X1 3QU, U.K.
1004
R. WARN
within the polar granules. These organelles are characteristic of the pole cells of
Drosophila (Huettner, 1923). In order to identify the constituents of pole plasm
which are necessary for its specific biological effects it is necessary to have a
direct assay for these effects. Smith (1966) first used this kind of assay system to
demonstrate the biological properties of the germinal plasm of Rana pipiens.
In this paper the results of injecting the pole plasm into irradiated posterior
poles of early-stage Drosophila embryos are described. The results agree well
with the recent papers of Okada, Kleinman & Schneiderman (19746) and
Illmensee & Mahowald (1974).
MATERIALS AND METHODS
Stocks and collection of eggs
Eggs were collected from standard collecting boxes containing about
10000 flies, 4-8 days old. To obtain approximately synchronized eggs, it was
necessary to pre-feed the flies for at least 1 h with collecting dishes containing
an agar medium rinsed with a solution of 7 % acetic acid and smeared with
a thin layer of yeast. Eggs were then collected at 10 min intervals using similar
collecting dishes filled with yeast agar medium topped with live yeast but
without acetic acid.
U.v.-irradiation
The chorion was first removed mechanically. The eggs were lifted out of the
collecting dish, immobilized on double-sided 'Scotch' tape, and dried for
2-3 min in a current of warm air from a hair drier. The chorion then became
rather brittle and could be cracked open with a mounted needle, freeing the
embryo.
U.v.-irradiation was carried out with the short-wavelength region of a Mineralite UVSL-15 lamp (Ultra-violet Products Inc.), lacking the filter. This gives
out light with a wavelength of 254 nm. The method of irradiation was based
on the technique of Graziosi & Micali (1974). A block of agar (3 % in Becker's
saline and blackened by the addition of powdered charcoal) 1 mm thick was
placed on a microscope slide and cut in two. The eggs were oriented with their
posterior poles upwards along the cut surface of one half of the agar block,
and the two halves were then fitted together. The embryos were placed 30 mm
from the u.v. source. Prior to each experiment the output of the u.v. lamp was
checked with a dosimeter (Black-Ray, Ultra-violet Products Inc.). A dose of
3000 ergs/mm2, delivered over 15 sec, was adopted. At this dose a high proportion of embryos did not show any generalized irradiation damage, but it is
also well above the limit where any pole cells were seen to form (1000 ergs/mm2).
The total time to prepare the eggs and carry out the irradiation was about
10 min.
U.v.-irradiated Drosophila embryos
1005
Micro-injection
Micro-injection was carried out using a glass micropipette held in a Singer
'micro-dissector', and attached to an Agla syringe (as described by Elsdale,
Gurdon & Fischberg, 1960). The pipettes were prepared from BDH hard glass
capillary tubes. After an initial pulling-out over a small Bunsen burner, the
pipettes were tapered to a final external diameter of about 10 fim and an internal
diameter of about 5 /tm and had a very short tip. Smoothing of this tip was
accomplished by melting the edge against a heated wire and carefully pulling it
away. (A detailed description of the method of drawing and smoothing micropipettes is given by Gurdon, 1974.) The whole shaft of the pipette was filled with
paraffin oil so as to transmit small changes in pressure without delay. However,
as injections were carried out in paraffin oil, a tiny bubble of air had to be
introduced into the very tip to permit identification of its position.
Transfer of cytoplasm
A group of about 15 embryos was irradiated while ca. 10 donor eggs were left
drying on the 'Scotch' tape. The irradiated embryos were transferred to a
Falcon Petri dish and dried for 6 min at 25 °C in a calcium chloride desiccator.
This reduced the turgor pressure so that cytoplasm could be introduced without
loss of fluid. Similarly, the donor embryos were dechorionated and desiccated.
After this period liquid paraffin (Boots' Liquid Paraffin) was poured into the
dishes and the embryos examined under transmitted light. Following the
observations of Imaizumi (1954) it was possible to determine the age of early
embryos accurately and all stages older than 20 min were discarded. The remaining irradiated embryos were divided into two lots; one group was transferred into another Petri dish to act as recipients whilst the others served as
controls. The donor embryos were embedded in deep grooves to hold them
firm, prior to the addition of the irradiated recipients which were placed in
shallower hollows to allow easier subsequent handling.
Between 0-1 and 0-2 nl of fluid (which represents about 1 % of the egg volume)
was removed from directly under the vitelline membrane at the posterior pole.
As the cytoplasm was slowly sucked out the angle of the pipette was changed
slightly so that surrounding pole plasm and not the underlying yolky cytoplasm
was taken up. It was then introduced into the recipient embryo at the dorsal
margin of its pole plasm, so as to introduce new material without disturbing
unnecessarily the organization of the original pole plasm. Any embryos where
cytoplasm exuded from the injection wound after the operation were removed,
as were all the donor embryos. In general, cytoplasm from one donor was
injected into one recipient. The whole operation took about 30 min. A control
series, injected with cytoplasm from the anterior tip of the egg, was carried out
in exactly similar fashion. After injection, the embryos were cultured at room
temperature in darkness to avoid the possibility of photoreactivation. At three
1006
R. WARN
Table 1. Results of cytoplasmic transfer into the posterior poles of
irradiated embryos o/"Drosophila
Treatment of
irradiated embryos
A. Injected with posterior pole plasm
Control, no injection
B. Injected with anterior tip cytoplasm
Control, no injection
With
pole cells
Without
pole cells
18
0
0
0
21
22
16
16
Table 2. Results of each pole plasm transfer experiment
(from first line of Table 1)
Experiment no.
Total no. embryos injected
No. embryos with pole cells
No. embryos lacking
pole cells
No. pole cells in each
positive case
1
2
3
4
5
6
7
8
9
4
3
1
4
1
3
5
4
1
4
1
3
3
2
1
4
2
2
7
2
5
4
0
4
4
3
1
5
1
43
1
—
2
5
2
—
5
4
3
3
1
1
—
3
2
—
—
—
—
—
—
—
31
3
2
hours of development they were removed and sections were cut to look for the
presence or absence of pole cells.
Histology
After removal from the oil the embryos were placed for 30 min in a formaldehyde-alcohol-acetic acid mixture (with proportions of 6:16:1 plus 30 parts
water) to fix the exterior of the eggs, thereby reducing their fragility. They were
then punctured with a fine glass micro-needle and full-strength solution (lacking
the water) was substituted. To this 0-5 % Triton X detergent had been added.
Sayles, Procunier & Browder (1973) found that this detergent was successful
in making eggs permeable, and a combination of Triton X and puncture resulted
in very good fixation of embryos. After impregnation with Paraplast (Sherwood
Medical Industries Inc.), sections were cut at 6/tm and stained with Heidenhain's iron haematoxylin.
RESULTS
In Tables 1 and 2 a summary of the principal findings has been drawn up.
A high percentage (nearly half) of embryos injected with pole plasm contained
morphologically normal pole cells, whereas the embryos injected with anterior
tip cytoplasm as well as the irradiated controls showed no sign of pole cell
U.v.-irradiated Drosophila embryos
1007
Fig. 1. Posterior pole region of an unmanipulated mid-blastema embryo. x2200.
b.n., blastema nucleus; p.c, pole cell; p.g., polar granules.
hn
Fig. 2. Posterior pole of a mid-blastema stage embryo lacking pole cells after
irradiation of this region at about the 1st cleavage stage, x 1900. b.n., blastema
nucleus; v.m., vitelline membrane.
1008
R. WARN
Fig. 3. Mid-blastema embryo resulting from an egg irradiated 15 min after fertilization. Pole plasm was injected at the 4-8 cell stage, x 1900. n.p.c, morphologically
normal pole cell; a.c, abnormally formed cell; b.n., blastema nucleus.
Fig. 4. Mid-blastema embryo. This was irradiated 15 min after fertilization and
injected with anterior tip cytoplasm. xl300. p.p., posterior pole; b.n., blastema
nucleus.
U.v.-irradiated Drosophila embryos
1009
formation. The difference is seen in Figs. 1-4. It proved necessary to define
what constituted a pole cell because a spectrum of effects was seen in embryos
which received pole plasm. As shown in Fig. 1, a typical pole cell is circular in
cross-section in contrast to the columnar epithelial cells of the blastoderm. The
ratio of cytoplasm to nucleus is much less, and in suitable preparations polar
granules can be seen. Pole cell nuclei are spherical and the chromatin stains
fairly densely as a clump, while the somatic nuclei form ovoid structures with
a prominent nucleolus attached to a less densely staining strand of nucleoplasm.
In general only a small number (1-5) of fully formed pole cells were observed
(cf. the last line of Table 2). However, quite a large number of cellular fragments, with or without nuclei, were seen. Such fragments differed from the
developing epithelial cells because they were not included in the blastoderm cell
layer. Furthermore, the general structure and nuclear morphology were similar
to those of pole cells. Because of this, the results given in Tables 1 and 2 are
a minimum estimate of the effects of the injected pole plasm. Two embryos
injected with pole plasm contained fragments but no normally formed pole
cells. They were included among the negative results in Table 1. Fig. 3 shows
one of these cell-like fragments on the left-hand side. The nucleus appears
fairly well developed but there is only a small amount of cytoplasm, which is,
in fact, still attached to the main mass of the egg. In contrast, four normal
pole cells are present in the centre of the field.
Two cases of transfer of posterior pole plasm into irradiated embryos provided more spectacular results. In one 40 pole cells were formed, and in the
other 31. These numbers are just below the bottom end of the spread of numbers
of pole cells in unmanipulated embryos (41-75, average 58).
Not all embryos contained pole cells solely in the region of the posterior
pole. In two cases pole cells were found in positions some way removed from
the posterior pole. In both embryos the injection wound was close to the site of
formation of the pole cells, suggesting that the material forming the pole cells
does not migrate far from the point of its introduction.
How constant are the results of pole plasm transfers ? Nine separate experiments were carried out involving 43 recipient embryos. Thirty-nine of these
developed, of which 18 (46%) contained normally formed pole cells. The
success rate per experiment varied, but not significantly, and in most cases at
least two or three positive results were obtained. Only one experiment gave no
positive results. The two embryos with large numbers of pole cells came from
different experiments.
DISCUSSION
The above experiments show that pole plasm has a specific capacity to induce
pole cell formation in irradiated posterior poles and sometimes also in more
anterior regions of the embryo. Because of the two cases where pole cells were
formed in more anterior regions of the embryo, pole plasm can act outside the
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R. WARN
area of the posterior pole. Its activity is unaffected by its position in the embryo.
This result has also been found by Ulmensee & Mahowald (1974) as described
below. Pole plasm transfer constitutes the transfer of specific 'determinants'
because a particular cell type, as judged by the criteria described, is formed
from nuclei which can be assumed to be totipotent (Zalokar, 1971; Ulmensee,
1972). However, there are other types of cytoplasmic transfer experiment in
Drosophila (Garen & Gehring, 1972; Okada, Kleinman & Schneiderman,
1974o) where the factor restored promotes further general development rather
than the appearance of a new cell type. In these cases, the molecules required
to continue development cannot be considered as 'determinants'.
The results described in this paper depend on morphological criteria to
identify the presence or absence of pole cells. Two recent papers (Okada et ah
19746; Ulmensee & Mahowald, 1974) have shown that pole cells resulting from
cytoplasmic transfer can differentiate to form mature gametes. Okada et ah
(19746) achieved such results by a similar procedure to that described in this
paper, dissecting the adults that emerged to examine the state of the gonads.
Ulmensee & Mahowald (1974) injected the pole plasm into the anterior tips of
embryos of the same age as the donor and observed the subsequent formation
of cells very similar to pole cells. Transfer of these cells into the posterior pole
of blastoderm stages of different genotype yielded a few imagines which gave
offspring carrying the genetic markers of the transferred 'pole' cells.
Although the results described from the present work do not show that the
cells formed as a result of pole plasm transfer have all the properties of pole cells,
they do provide a third independent demonstration of the biological effects of
transferring such cytoplasm in Drosophila. Furthermore, examination of embryos at the earliest stage when specific effects due to injection of pole plasm
can be seen provides a simple and sure test. More than 90 % of all embryos
develop to this stage after manipulation. However, during later development
a high proportion of embryos irradiated or injected at the posterior tip die
(Warn, 1972). Thus examination of embryos for the earliest effects of injection
of fractions would be a preferable method of assaying the biological activity
of the constituents of pole plasm such as polar granules.
I would like to thank Dr J. B. Gurdon for much help and advice during the course of this
study and also Dr D. B. Roberts and Dr G. Graziosi for their help and critical reading of
the manuscript.
U.v.-irradiated Drosophila embryos
1011
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{Received 20 November 1974)
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