/. Embryol exp. Morph. Vol. 57, pp. 79-93, 1980
79
Printed in Great Britain © Company of Biologists Limited 1980
Polar granules and pole cells in the embryo
of Calliphora erythrocephala: ultrastructure
and [3H]leucine labelling
By ANDERS LUNDQUIST 1 AND HADAR EMANUELSSON 1
From the Zoophysiological Institute, University of Lund, Sweden
SUMMARY
The polar granules in Calliphora undergo a gradual fragmentation during early cleavage,
but reaggregate after pole-cell formation. Autoradiographic analysis showed that the pole
cells in Calliphora acquire a higher [3H]leucine label than the rest of the embryo during the
blastoderm stage. Such an increased label was not seen in the pole plasm before pole-cell
formation or in the pole cells during gastrulation. Electron microscopic autoradiography
revealed that the polar granules are substantially labelled during the blastoderm stage. At
the same time, characteristic nuclear blebs appear in the pole cells. The observations are
consistent with the hypothesis that polar granules contain maternal messenger RNA, which
is released and translated into proteins.
INTRODUCTION
The idea that the egg contains cytoplasmic determinants has long been held
in developmental biology. It was most successfully applied to the determination
of the germ cells experimentally studied in chaetognath.es, amphibians, nematodes, and insects (reviewed by Beams & Kessel, 1974; Eddy, 1975).
The pole plasm is the hindmost cytoplasmic region in the fly egg. It contains
determinants required for germ-cell differentiation (reviewed by Mahowald,
1977; Agrell & Lundquist, 1973; Counce, 1973). The polar granules are specific
organelles found in the pole plasm, but absent from other parts of the egg.
The pole cells bud off from the posterior pole during cleavage after the nuclei
have reached the surface of the egg syncytium. They enclose the pole plasm with
the polar granules. The pole cells are the sole progenitors of the adult germ cells.
If the pole plasm of the Drosophila egg is damaged or destroyed, no pole cells
are formed (e.g. Geigy, 1931; Poulson & Waterhouse, 1960; Hathaway &
Selman, 1961; Graziosi & Micali, 1974) and if it is transplanted to an incapacitated posterior pole or to ectopic loci, potentially functional pole cells are formed
there (Illmensee & Mahowald, 1974, 1976; Okada, Kleinman& Schneiderman,
1974; Warn, 1975; Illmensee, Mahowald & Loomis, 1976). Thus, it is well
1
Authors' address: Zoophysiological Institute, University of Lund, Helgonavagen 3B,
S-223 62 Lund, Sweden.
6-2
"80
A. LUNDQUIST AND H. EMANUELSSON
established that some factor necessary for pole cell formation resides in the pole
plasm.
The polar granules in Drosophila contain RNA (Mahowald, 1962, 1971;
Counce, 1963). They begin to lose their RNA after fertilization, and the RNA
is no longer cytochemically detectable by the blastoderm stage when the pole
cells have stopped dividing (Mahowald, 1971). During pole-cell formation, the
polar granules in Drosophila are fragmented and surrounded by polysomes
(Counce, 1963; Mahowald, 1968). Therefore, the suggestion that the polar
granule RNA is a long-lived maternal messenger RNA, which is translated into
germ-cell-determining proteins (Mahowald, 1968). Light microscopy has shown
that the polar granules are fragmented during pole-cell formation also in
Calliphora (Noack, 1901; Alleaume, 1971) and they undergo conspicuous ultrastructural changes in Coelopa (Schwalm, Simpson & Bender, 1971). As the polar
granules in higher dipterans seem to be active when the pole cells appear, it was
suggested that they are the factor causing pole-cell formation, but the direct
evidence is uncertain.
The available general studies on the protein synthesis in the fly egg disagree
about the pole plasm. In Musca, an increased label was found in the pole plasm
before pole-cell formation (Pietruschka & Bier, 1972). In Drosophila, labelling
increased only in the early pole cells (Zalokar, 1976). In this study, the amino
acid labelling of Calliphora pole cells and polar granules was examined with
~both light and electron microscopic autoradiography. Moreover, some ultrastructural features of polar granules and pole cells in Calliphora are described
for the first time.
MATERIALS AND METHODS
The culture of the flies {Calliphora erythrocephala Meig.), the collection of the
•eggs, the development of the embryos at 20 °C, and the treatment of eggs for
electron microscopy were earlier described in detail (Lundquist & Emanuelsson,
1979). The eggs were fixed with 2-5 % glutaraldehyde and 4 % formaldehyde in
cacodylate buffer, pH 7-2 (Karnovsky, 1965), and postfixed with 1 % OsO4.
Eggs for autoradiography were permeabilized according to a method modified
after Limbourg & Zalokar (1973). Dechorionized eggs (Lundquist & Emanuelsson, 1979) were placed on a piece of moist tissue paper (Kleenex) inside a small
cup of stainless-steel screen. The cup was immersed in n-octane for 15 sec. The
octane was thoroughly blotted off, and any remaining octane was evaporated
for 15 sec in a humid air stream. The cup was immersed in incubation medium,
.and the eggs were covered with another piece of tissue paper. The cup was then
taken up, partly drained, and incubated suspended in a moist chamber at 20 °C
for 30 or 60 min. In this way, the eggs and a thin layer of medium were sandwiched between two layers of tissue paper, thus ensuring adequate oxygen
.supply and least mechanical damage to the eggs. Shaw's medium (Shaw, 1956),
-which is suitable for Calliphora embryo culture (Davis, Krause & Krause, 1968),
Polar granules in Calliphora
81
Table 1. The condition of permeabilized Calliphora eggs
immediately after incubation in Shaw's medium
Number of eggs
Incubation Age of the eggs
time (min) at fixation (h)
0
Normal
Possibly*
abnormal
Clearly!
abnormal
Total %
H
20
3
0
23
1
20
19
0
0
0
12
12
0
23
0
23
30
1
54
7
73
12
2
62
7
5
74
3
30
3
9
42
4
1
54
0
55
60
24
6
12
42
H
66
48
6
12
21
24
6
4
34
31
42
0
45
4-V
3
Immediately after incubation, the eggs were fixed with Bradley-Carnoy solution (3 parts
absolute ethanol, 1 part glacial acetic acid, 4 parts chloroform). Whole-mounts were prepared,
essentially according to Agrell (1962).
21
31
41
* Delayed development of the nuclear sphere or an unusually large number of vitellophages.
t Generally abnormalities of the nuclear sphere or the blastoderm.
% Some eggs either did not develop or aborted after a few cleavage divisions. These eggs
are probably unfertilized and were not included.
was used. The medium contained 7-4 MBq/ml (= 200/*Ci/ml) L-[4,5- 3 H]leucine (2-1 TBq/mmol ( = 5 7 Ci/mmol); Radiochemical Centre, Amersham).
The leucine content of the medium was about 300 times lower than the natural
leucine content in fly eggs (Chen, Hanimann & Briegel, 1967). This is probably
an important reason why a comparatively high radioactivity in the medium and
a long incubation are necessary to ensure adequate labelling. On the other hand,
the retention of free labelled leucine (Peters & Ashley, 1967) would be reduced
because of the resulting low specific activity in the endogenous leucine pool and
the prolonged incubation. But under the present conditions, acid-insoluble label
of amino acids in the Drosophila egg is nearly half the total label (Limbourg &
Zalokar, 1973; Zalokar, 1976). Therefore, such retention should present no
problem. Eggs of Calliphora incubated for 60 min with [3H]leucine (1-85 MBq/
ml) during oxygen shortage did not show autoradiographically-detectable label.
Tables 1 and 2 record the development of the permeabilized eggs. Eggs of all
stages develop normally in the medium for 1 h, but eggs permeabilized and
incubated during cleavage do not hatch. Possibly, cleavage eggs receive mechanical injuries, which manifest only at a later stage. The eggs lose some turgor
during permeabilization, and this makes them more sensitive to mechanical
82
A. LUNDQUIST AND H. EMANUELSSON
Table 2. The development of permeabilized Calliphora eggs
after incubation in Shaw's medium
Number of eggs
Age of the
eggs after
incubation
in medium
Treatment
Permeabilized,
30 min in
medium,
paraffin oil
(h)
2±
3
3±
3i
3±
4
Dechorionized,
paraffin oil
Dechorionized
—
—
—
—
—
—
A
Eggs
PigSegwithout
hatched mented mented pigmentalarvae* embryosf embryosj
tion§
Un-
Hatched
larvae
Total
0
19
28
61
64
20
69
0
2
3
5
2
8
13
21
36
43
15
19
44
12
55
18
7
3
2
11
3
25
25
19
16
13
17
3
61
3
10
7
20
101
74
88
324
87
359
6
1
3
1
24
3
4
5
2
11
1
2
12
8
38
7
29
100
100
400
100
395
3
2
1
101
100
100
100
100
100
100
Permeabilized eggs were incubated in Shaw's medium for 30 min and transferred to paraffin oil in a
water-saturated oxygen atmosphere. The results were scored a few hours after the end of the normal
hatching period. The values were similar when the eggs were incubated in medium for 60 min except for
a decrease in the number of 'hatched larvae' and a corresponding increase in the number of 'segmented
embryos'. The unpermeabilized control eggs were only decborionized. They were transferred to paraffin
oil during the blastoderm stage and treated as above or allowed to develop on moist filter paper in air.
The hatching percentage was always higher after the latter treatment.
* Otherwise apparently normal.
t Usually with minor segmental defects or abnormal mouth hooks.
% Usually with grossly abnormal segmentation or no visible segments. The cuticular pigmentation
appears shortly before hatching.
§ Unfertilized eggs included.
FIGURE 1
Light-microscopic autoradiographs of pole plasm and pole cells in Calliphora eggs
labelled with [3H]leucine for 30 min. (A) Posterior pole with pole plasm during
intravitelline cleavage (60 min). (B) Pole-cell buds (120 min). (C) Pole cells during
the cellularization of the blastoderm (180 min). (D) Pole cell with polar granules
(arrows) in the yolk region near the posterior midgut invagination. This is phase II
of pole-cell migration. Early germ-band-elongation stage (270 min). PC, Pole cell;
PMI, posterior midgut invagination; Y, yolk. Notice the heavy label of the pole
cells in the blastoderm. See also Table 3. Section thickness: 1 //,m. Exposure time:
27 days.
Polar granules in Calliphora
83
84
A. LUNDQUIST AND H. EMANUELSSON
damage before they have acquired a firmer structure by the cellularization of
the blastoderm. The Calliphora egg should be more susceptible to such damage
than the smaller Drosophila egg.
After incubation, eggs for autoradiography were fixed for 2 h at room
temperature with Karnovsky fixative (six changes). The vitelline membrane was
removed in the fixative after 1-1-5 h. The embryos were washed with cacodylate buffer at + 4 °C overnight (at least five changes), postfixed with 1 %
OsO4 in the same buffer, bulk-stained with 0-5 % uranyl acetate and 1 % phosphotungstic acid, and embedded in Vestopal W.
Thin sections for light microscopic autoradiography (1 pm) were covered with
liquid nuclear emulsion (Ilford K2) according to the dipping method and exposed for 6-27 days. The auto radiograms were developed in Kodak D 19
(5 min, 20 °C), rinsed in distilled water (10 sec), fixed in Kodak F24 (6 min,
20 °C), and stained with Richardson's azure II.
Ultrathin sections for electron microscopic autoradiography were vacuumcoated with carbon, covered with a monolayer of nuclear emulsion (Ilford L4)
according to the loop method, and exposed for 4-24 weeks. The preparations
were developed in Kodak D 19 (2 min, 20 °C), rinsed in distilled water (30 sec),
fixed in 15% Na2S2O3 (3 min, 20 °C), and finally washed in distilled water
(2 min). Examination was made with a Philips EM 300 electron microscope at
the Zoological Institute, University of Lund.
RESULTS
At 20 °C the Calliphora egg has completed nine intravitelline synchronous
nuclear divisions 90 min after egg-laying, when the dividing nuclei reach the
periphery of the egg. The remaining four nuclear divisions are partially synchronous and occur in the syncytial blastoderm (Lundquist & Emanuelsson,
1979). The pole cells bud off during the second or third blastodermal division
(Fig. IB). They proliferate at the posterior end during the ensuing cellularization of the blastoderm (from 150 min; Fig. 1C) and most of them are brought
into the egg interior with the posterior midgut invagination during gastrulation
(between 240 and 270 min; Fig. ID). Pole cells migrate into the yolk region in
FIGURES 2 AND 3
Electron microscopic autoradiographs of polar granules in Calliphora eggs labelled
with [3H]leucine for 30 min. The stages are: (2A) Intravitelline cleavage (60 min).
(2B) Pole-cell formation (120 min). (3 A) Cellularization of the blastoderm (180 min).
(3B) Early germ-band elongation (270 min). dMVB, Dark multivesicular body;
ER, endoplasmic reticulum; N, nucleus; M, mitochondrion; PG, polar granule.
Notice that the polar granules are fragmented during pole-cell formation and
heavily labelled during the cellularization of the blastoderm. Exposure time: 20
weeks (2A), 9 weeks (2B, 3 A), and 24 weeks (3B).
Polar granules in Calliphora
FIGURE 2
85
86
A. LUNDQUIST AND H. EMANUELSSON
FlGURE 3
Polar granules in Calliphora
87
Table 3. Autoradiographic [sH]leucine labelling of the pole plasm
and the pole cells in Calliphora eggs
Egg no.
Age of the
eggs (min)*
Stage
1
60
Pole plasm during early
0
0
0
0
cleavage
0
0
0
120
Pole-cell buds
150
Pole cells in the
+°
0
0
syncytial blastoderm
++
++
++°
++
180
Pole cells during the cellularization of the blastoderm
210
Pole cells during the cellular++
++°
++°
++°
ization of the blastoderm
240
Pole cells at the onset of
0
+°
+°
+°
gastrulationf
0
0
270
Pole cells during the early
germ-band-elongation stage
The grain density over the pole cells was compared with that of the adjacent blastoderm
cells: 0, similar grain density; + , higher grain density; + + , much higher grain density;
°, some pole cells (*S 50%) with no increased grain density.
* The eggs were labelled for 30 min in medium containing [3H]leucine (74 MBq/ml =
200/tCi/ml) and fixed at (he indicated age.
t The eggs were often weakly labelled at this stage.
two phases, before and after gastrulation (reviewed by Counce, 1973; for
Calliphora, see Alleaume, 1971).
The polar granules in Calliphora consist of a network of dense material.
During early cleavage, most of them are irregular in shape and relatively large
(up to 1 /<m), sometimes with annular profiles (Fig. 2A). Sometimes, they are
chained together. Subsequently, they become smaller. During pole-cell formation, small profiles (less than c. 0-5 /im) are gathered in a perinuclear zone
(Fig. 2B). Towards the end of the nuclear cleavage period, large granules
reappear. Later, very large circular or annular profiles become predominant
(up to 2/MTO, although small profiles are still seen at gastrulation, especially
near the nucleus (Fig. 3).
Pieces of endoplasmic reticulum are often very close to the polar granules of
the pole cells (Figs 2, 3). Extranuclear annulate lamellae (Lundquist & Emanuelsson, 1979) are present in the pole-cells (Fig. 4B), but almost absent from the
blastoderm. Dark multivesicular bodies were regularly found in the pole plasm
and the pole cells (Fig. 2B).
Characteristic blebs are seen on the nuclear envelope in the pole cells of the
blastoderm (Fig. 4). Such a bleb consists of an out-pocketing of the outer
nuclear envelope membrane. It contains a circular membrane-enclosed profile
filled with electron-dense material and is often associated with a nuclear pore.
The blebs are first seen in the early pole cells, where they are very frequent
88
A. LUNDQUIST AND H. EMANUELSSON
during the cellularization of the blastoderm. They are only occasionally observed
at the late blastoderm stage and in the early gastrula. They are never seen outside
the pole cells.
The light-microscopic autoradiographs display an even distribution of silver
grains over the pole plasm and the pole cells after [3H]leucine labelling. This
applies to all stages. Both the nucleus and the cytoplasm are labelled. The grain
density over the pole plasm or the pole cells compared with the adjacent part of
the egg was judged semiquantitatively (Table 3; Fig. 1). No difference was
observed before pole-cell formation, but thereafter, the pole cells acquire a more
intense label. This difference disappears at the late blastoderm stage and in the
early gastrula. The highest grain density recorded in the egg was that over the
pole cells during the early cellularization of the blastoderm.
For electron-microscopic autoradiography, a 30 min pulse with [3H]leucine
was generally used (Figs 2, 3). Both 30 min and 60 min pulses were made during
the cellularization of the blastoderm, but the grain distributions were not
detectably different. Silver grains over the polar granules are present at all
stages, but the polar-granule label follows the general pole-cell label. There is
an approximately even distribution of label between the polar granules and
other parts of the cytoplasm and between cytoplasm and nuclei. The polar
granules are thus heavily labelled during the cellularization of the blastoderm.
The silver grains are often seen over the periphery of the granules. The label
of the pole cell nuclei is often found close to the nuclear envelope, and silver
grains are sometimes seen near nuclear blebs.
DISCUSSION
In the CalHphora egg the time course of fragmentation and reaggregation of
the polar granules parallels the rise and fall in amino-acid labelling of the pole
cells. The peak in polar-granule fragmentation during pole-cell formation precedes the peak in pole-cell labelling by about one hour. That applies also to
Drosophila, except that no clear-cut decrease in pole-cell label compared to other
cells was detected in ovo (Mahowald, 1968; Zalokar, 1976). The results of Allis,
Underwood, Caulton & Mahowald (1979) do not contradict the interpretation
that at least a slight decrease occurs in cultured Drosophila pole cells. Because
RNA synthesis in the pole cells is very low or absent in both species (Zalokar,
1976; Lamb & Laird, 1976; Lundquist & Emanuelsson, preliminary results),
FIGURE 4
Nuclear blebs in the pole cells of CalHphora eggs during the cellularization of the
blastoderm (180 min). Each bleb consists of an evagination of the outer nuclear
membrane and contains a membranous vesicle. AL, Annulate lamella; B, bleb; N,
nucleus; NE, nuclear envelope; M, mitochondrion; PG, polar granule. (A)Blebbing
in an egg labelled with [3H]leucine. Exposure time: 9 weeks. (B) Bleb in an unlabelled
egg. The black dot is an artifact.
Polar granules in Calliphora
89
90
A. LUNDQUIST AND H. EMANUELSSON
the amino-acid label should be attributed to translation of stored messenger
RNA. In Calliphora, the polar granules are substantially labelled with [3H]leucine, especially peripherally, when the label of the pole cells is high. In this
respect, by showing a label comparable to the ground plasm, polar granules
differ from the somatic inclusions: the yolk region shows less label than the
periplasm in Calliphora (unpublished) and Drosophila (Zalokar, 1976). The
correlation between polar-granule fragmentation and amino-acid labelling is
compatible with the idea (Mahowald, 1968, 1977) that stored mRNA is released
from the fragmented polar granules and later translated into proteins.
The changes in amino-acid label are interpreted as local changes in protein
synthetic activity. Alternatively, there is an exceedingly large, transient change
in the leucine pool size or the permeability of the pole cells only. This is not
likely. Moreover, pole cells acquire increased label only some time after their
formation. As pointed out by Zalokar (1976), this indicates that the newly formed
pole cells continue to share their amino-acid pools with the blastoderm. Some
translocation of label to the polar granules may occur. But since the total
amount of polar-granule material apparently does not increase before gastrulation (Rabinowitz, 1941; Counce, 1963), such translocation is unlikely to be of
major importance.
The vesicle-containing nuclear blebs of the pole cells have not previously been
recorded. Interestingly, the blebbing coincides with the increased amino-acid
labelling in the pole cells and silver grains were sometimes seen near the blebs.
But more evidence is difficult to obtain, as it is not yet possible to shorten the
labelling pulse.
The polar granules are thought to be responsible for pole-cell formation.
When the polar granules in the Drosophila egg are removed from the pole plasm
by centrifugation, no pole cells are formed at the posterior pole. However, the
dislodged polar granules do not give rise to pole cells in other parts of the egg
(Imaizumi, 1958; Jazdowska-Zagrodziriska, 1966). Obviously, direct evidence is,
as yet, lacking.
In some lower dipterans, certain chromosomes are eliminated from all somatic
nuclei during cleavage, whereas the full chromosome set is retained in the pole
cells, i.e. in the germ line. In gall midges, the polar granule material seems to
protect the pole cells from chromosome elimination, but it is not needed for
pole cells to form (Geyer-Duszyiiska, 1959; Nicklas, 1959; Bantock, 1970).
Chromosome elimination is not known in higher dipterans, but somatic loss of
chromatin does occur. In Calliphora and Drosophila, a specific terminal chromosome fragment is lost in the early embryo, presumably from all somatic nuclei.
In Calliphora, such fragments appear during the last three nuclear divisions of
the syncytial blastoderm and in the gastrula (Melander, 1963). Pseudochiasmata
such as those believed to precede this chromosome diminution were previously
observed in Drosophila. They probably affect the X chromosome (Rabinowitz,
1941). As a second working hypothesis, we tentatively suggest that the polar
Polar granules in Calliphora
91
granules prevent chromosome diminution in the pole cells of Calliphora and
Drosophila. Chromosome diminution occurs after polar-granule fragmentation
and coincides with the increased [3H]leucine label and the nuclear blebbing in
the pole cells. Moreover, a unified hypothesis explaining the function of the
polar granules in all dipterans should be appealing. The hypothesis is testable,
because it offers cytological markers. The tools needed to determine the function
(or functions) of the polar granules might soon become available. As yet, cell
fractions enriched in polar granules have been isolated (Allis, Waring &
Mahowald, 1977; Waring, Allis & Mahowald 1978) and culture methods for pole
cells have recently been designed (Allis, Underwood, Caulton & Mahowald, 1979).
Grants from the Magnus Bergvall Foundation and the Swedish Natural Science Research
Council supported this work. We express our gratitude to Mrs Annagreta Petersen for invaluable technical aid, to Miss Inger Norling for printing the electron micrographs, and to
Mrs Marianne Andersson for typing the manuscript.
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(Received 6 November 1979, revised 16 January 1980)
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