J. Embryol. exp. Morph. 73, 297-306, 1983
297
Printed in Great Britain © The Company of Biologists Limited 1983
Segregation of germline granules in early embryos
of Caenorhabditis elegans: an electron microscopic
analysis
By NURIT WOLF1 , JAMES PRIESS1 AND DAVID HIRSH1
From the Department of Molecular, Cellular and Developmental Biology,
University of Colorado
SUMMARY
Using an improved fixation method for electron microscopy, we have found germline
granules in Caenorhabditis elegans embryos shortly after fertilization and prior to the first
cleavage. They are localized in the egg cytoplasm which becomes segregated into the posterior
blastomere at the first cleavage. In the following divisions, the granules continue this pattern
of asymmetric segregation and are ultimately segregated into the germline precursor cell. The
granules are then symmetrically segregated into the germline cells.
INTRODUCTION
'Germline granules' have been observed by both light and electron microscopy
in many organisms from hydra to man (Eddy, 1975). These have been called
'polar granules', 'dense bodies', and lnuages\ Such 'granules' have attracted
attention because they may participate in the determination of cells to become
the germline (Illmensee & Mahowald, 1974; Wakahara, 1977; Wakahara, 1978).
In an electron microscopic study of the nematode Caenorhabditis elegans, Krieg
et al. (1978) observed cytoplasmic structures characteristic of the germline cells
in embryos as early as the 6-cell stage. We have further characterized these
structures by electron microscopy, in order to find the earliest embryonic stage
at which these structures could be identified and to see how they become
segregated into the germline cells.
METHODS
Nematodes
Wild-type C. elegans var. Bristol were grown at 20 °C on Petri dishes with E.
coli 0P50 as a food source as described previously (Brenner, 1974; Hirsch, Oppenheim & Klass, 1976).
1
Authors' address: Department of Molecular, Cellular and Developmental Biology,
University of Colorado, Boulder, CO 80309, U.S.A.
298
N. WOLF, J. PRIESS AND DAVID HIRSH
Preparation of embryos for electron microscopy
Gravid worms were washed off a Petri plate with distilled water and collected
by centrifugation. A concentrated suspension of worms was put in a depression
dish. Degassed 1 % NaOCl (Fisher Scientific Co.) with 0-25 M - K O H was added
for 2-3 min until eggs were released from the partially lysed adults. The eggs
were collected on a Nucleopore Corp. polycarbonate membrane filter (8-0/im)
and rinsed with M9 salt solution. The egg shell was removed from the embryos
by digestion with chitinase (U.S. Biochem. Corp.) according to a procedure
developed by Dr. Paulo Bazzicalupo. The chitinase was first dissolved at 20 mg/
ml in a salt solution of 50 mM-NaCl, 70 mM-KCl, 2-5 mM-MgCl2,2-5 mM-CaCl2,
then centrifuged at 12 000 g. The supernatant fluid was added to the eggs at room
temperature. The eggs were observed under a dissection microscope at X25
magnification. As soon as the egg shell was seen to be removed, the eggs were
transferred to fixative.
Eggs were fixed for 1-2 h at room temperature in 4 % glutaraldehyde in
100 mM sodium phosphate buffer, pH7-4; 2mM-MgCl2. Postfixation was done
with 1% osmium tetroxide in lOOmM-sodium phosphate buffer, pH7-4; for
15 min. The eggs were encased in agar during the water rinse so that several eggs
formed a group that could be sectioned together. Eggs were stained en bloc in
1 % uranyl acetate, dehydrated in ethanol, and embedded in Epon between two
microscope slides.
Sectioning was done on a Porter-Blum MT-2 microtome and sections were
stained with uranyl acetate and lead citrate and viewed in a Hitachi H-600
electron microscope.
Nomenclature
The nomenclature of the early embryonic cells lineages is that of Deppe et al.
(1978).
RESULTS
Fertilization and the early cleavages of C. elegans have been described on a
light microscope level (Schierenberg, 1978; von Ehrenstein & Schienenberg,
1980). We have found the germline granules in the earliest embryos we
examined: the 'pseudocleavage' stage shortly after fertilization and prior to the
first true cleavage. In Fig. 1, these granules can be seen near the posterior pole
of the egg near the male pronucleus. Much vesicular material is present in the
pseudocleavage egg but the darker staining granules can be distinguished. As the
egg and sperm pronuclei move together and fuse to form the zygotic nucleus, the
granules remain at the posterior pole. While they are distributed highly
asymmetrically with respect to the anterior-posterior axis of the embryo at this
stage, they do not show any apparent asymmetry along the left-right or
Germline granules in C. elegans
299
dorsal-ventral axes. During the first cleavage, the granules remain at the posterior pole of the egg and are thus partitioned into the posterior blastomere, PI
(Fig- 2).
In the second cleavage of the living embryo, the PI spindle initially is formed
along the anterior-posterior axis of the egg. However, as cleavage begins, the
spindle rotates in the dorsal-ventral plane so that one daughter, EMSt, is
anterior-ventral and the other, P2, is posterior-dorsal. The germline granules
appear to become localized in the dorsal sector of the PI blastomere prior to the
cleavage of this cell, as seen in Fig. 3. When PI divides, they are partitioned into
the P2 blastomere (Fig. 3B).
P2 will divide into blastomeres C and P3, and P3 will later divide into D and
P4. The C and D blastomeres divide several times to form part of the somatic
tissues of the animal, while P4 divides only once more during embryogenesis into
Z2 and Z3, which form the germline (Kimble & Hirsh, 1979; J. Sulston, personal
communication). The germline granules continue the pattern of asymmetric
segregation described above, and are ultimately contained only in the P4 blastomere (Fig. 4). At the division of P4, they are segregated equally into both Z2
and Z3 (data not shown).
As the uncleaved egg, Po divides and gives rise to the series of blastomeres PI,
P2, P3 and P4, the granules become much larger in size and are found in closer
proximity to the nucleus (compare Figs 3 and 4). Cytoplasmic granules can be
seen in the P3 and P4 blastomeres, even in the living embryo with the light
microscope, and are probably identical to the granules we see with the EM. The
few large granules found in the P3 and P4 blastomeres could come from an
aggregation of the numerous smaller granules we find in the earlier stages, or
perhaps by the differential growth and attrition of certain granules.
The granules are often observed in close approximation to endoplasmic
reticulum and mitochondria, and are not enclosed by membranes. We have not
observed any striking association of the granules with microtubules or filaments
that might suggest a mechanism for their movement.
In the study by Krieg et al. (1978), these granules were seen as 'electron light
cytoplasmic areas'. With our fixation methods, the granules appear as electron
dense spherical bodies of an apparent fibrous nature, similar to the structure of
germline particles described in other organisms (for review, see Beams & Kessel,
1974; and Eddy, 1975). The ultrastructure of the granules themselves offers no
obvious suggestion of a possible function in the embryo.
DISCUSSION
These EM studies have demonstrated that the germline granules of C. elegans
are present and asymmetrically distributed in embryos as early as the pronuclear
stage of development. These granules continue to be segregated into the Plineage throughout the early cleavages.
300
N. WOLF, J. PRIESS AND DAVID HIRSH
Fig. 1. For legend see p. 301.
Germline granules in C. elegans
301
In Drosophila, germline-specific granules are also located at the posterior pole
of the fertilized egg. The nuclei which migrate into this region at the first
cytoplasmic division become the germline cells. In contrast, the germline
granules of C. elegans must be segregated asymmetrically through four divisions
before they are in the definitive germline cells. The granules of Drosophila have
been implicated in determining the nuclei that they surround to become germ
nuclei. If the C. elegans granules have a similar function, either they are not
active in the Po , PI, P2 and P3 blastomeres, or these blastomeres are not competent to respond to the hypothetical 'signal'. In this light, it is perhaps interesting that the granules only begin showing a strong association with the nucleus of
the P4 blastomere, having been dispersed in the cytoplasm of the earlier stages.
Fuchs (1913) noticed granules he termed 'ectosomes' surrounding one of the
asters in the first cleavage of the arthropod, Cyclops vividis. In successive
divisions, these granules were asymmetrically segregated to only one of the
daughter cells, and were finally localized in two of the primordial germ cells with
a lineage pattern identical to that of C. elegans. The association of these granules
with the asters of the cleaving blastomeres suggested an obvious mechanism for
their segregation. As described above, the germline granules of C. elegans are
asymmetrically distributed even before the first spindle is formed. Similarly, the
prelocalization of the granules in the later blastomeres, prior to spindle formation, make an astral involvement in their specific segregation less likely in this
animal. Still, a transient association of the granules with cytoplasmic
microtubules or microfilaments could have been missed in our studies.
Fig. 1. Pronuclear stage of C. elegans embryogenesis. (A) The pseudocleavage has
occurred. The female pronucleus is in the anterior (left) end of the egg and the male
pronucleus resides in the posterior (right) end of the egg. Arrows point to the germline granules. Mag. 2700x. (B) Higher magnification of the posterior end of the egg
in A showing two granules near the male pronucleus. Mag. 8300x. (C) Migration of
the pronuclei towards the middle of the egg. Arrows show the position of the germline granule. Mag. 2700x.
Fig. 2. Germline granules in the 2-cell embryo of C. elegans. (A) The anterior AB
blastomere is on the left and the posterior PI blastomere on the right. The 2-cell
embryo contains germline granules in the posterior end of the PI blastomere, as
designated by arrows. Mag. 2900x. (B) and (C) Higher magnifications of germline
granules in the 2-cell embryo showing the granular morphology of the particles which
are unbounded by membranes. Endoplasmic reticulum and several mitochondria are
visible near the granules. (B) Mag. 23000X; (C) Mag. 33 000x.
Fig. 3. Segregation of the germline granules during the division of the PI blastomere. (A) The PI blastomere (right side of figure) is in anaphase. The granules
(arrows) are concentrated in the region of the PI blastomere that will become the P2
daughter blastomere. Mag. 2700x. (B) The PI blastomere has reached telophase
and the granules (arrows) remain segregated into the region of the cell that becomes
the P2 daughter blastomere. Mag. 2800x.
Fig. 4. Germline granules in the P4 cell of a 24-cell-stage embryo of C. elegans. (A)
The P4 cell, which is the lower right cell of the embryo contains the granules adjacent
to its nucleus. Mag. 9000x. (B) Higher magnification of the P4-cell nucleus with its
flanking granules. Mag. 17000x.
20
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302
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Germline granules in C. elegans
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303
304
N . WOLF, J. PRIESS AND DAVID HIRSH
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Fig. 4. For legend see p. 301.
Germline granules in C. elegans
305
While germline specific granules have apparently not been observed in other
species of nematodes, perhaps due to the difficulties of obtaining adequate
fixation of the early stages, there are striking differences between cytoplasms of
somatic and germline precursor cells during the embryogenesis of the nematode
Ascaris megalocephala (Boveri, 1899). Shortly after the first cleavage of the
fertilized Ascaris embryo, chromosomal diminution occurs in one of the daughter cells; chromosomal fragments are left in the cytoplasm and are subsequently
lost. However, the sister blastomere, PI, which will ultimately produce the
germline cells as in C. elegans, retains the full chromosomal complement. The
potential not to undergo chromosomal diminution is segregated into P o , PI, P2,
P3 and P4 in A. megalocephala just as the germline granules are in C. elegans.
There is evidence that the chromosomal diminution is prevented by the
cytoplasm the germline normally receives; if the first cleavage is altered such that
this cytoplasm is divided between the first two sisters, diminution does not occur
in either cell (Boveri, 1910; Hogue, 1910). Thus there is a clear difference in the
properties of somatic precursor versus germline precursor cells. Though C.
elegans does not appear to undergo chromosomal diminution (Emmons, Klass
& Hirsh, 1979; Sulston & Brenner, 1974) the asymmetric segregation of granules
we observed could conceivably be related to the same basic cytoplasmic difference between the somatic and germline precursor blastomeres.
Krieg et al. (1978) reported seeing 'electron light cytoplasmic areas' in germline precursors in C. elegans embryos from the 6-cell stage onward. These 'areas'
were localized around the nuclei. Thus, the segregation pattern and intracellular
localization of these 'areas' are the same as those of the granules we have observed. Although their morphology is quite different, presumably as a result of the
different fixation procedures, it is likely that those 'areas' represent the same
structures as the granules we have described.
Strome & Wood (1982) recently reported staining particulate cytoplasmic
components of the germline cells and their precursors in C. elegans embryos
using FITC-conjugated rabbit anti-mouse IgG. The location and segregation of
the staining material appears to be identical to that of the granules reported here,
suggesting that the antiserum may be recognizing some component of these
structures. Unfortunately the antiserum does not bind to the particles after
aldehyde fixation, preventing a direct comparison of the particles recognized by
the antiserum and the granules described here (S. Strome, personal communication).
We thank J. Richard Mclntosh for helping develop the improvedfixationand for valuable
discussions. This work was supported by Public Health Service Grant No. 19851.
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