J. Embryol. exp. Morph. 73, 297-306, 1983 2 9 7Printed in Great Britain © The Company of Biologists Limited 1983

Segregation of germline granules in early embryosof 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 germlinegranules in Caenorhabditis elegans embryos shortly after fertilization and prior to the firstcleavage. They are localized in the egg cytoplasm which becomes segregated into the posteriorblastomere at the first cleavage. In the following divisions, the granules continue this patternof asymmetric segregation and are ultimately segregated into the germline precursor cell. Thegranules are then symmetrically segregated into the germline cells.

INTRODUCTION

'Germline granules' have been observed by both light and electron microscopyin many organisms from hydra to man (Eddy, 1975). These have been called'polar granules', 'dense bodies', and lnuages\ Such 'granules' have attractedattention because they may participate in the determination of cells to becomethe germline (Illmensee & Mahowald, 1974; Wakahara, 1977; Wakahara, 1978).In an electron microscopic study of the nematode Caenorhabditis elegans, Krieget al. (1978) observed cytoplasmic structures characteristic of the germline cellsin embryos as early as the 6-cell stage. We have further characterized thesestructures by electron microscopy, in order to find the earliest embryonic stageat which these structures could be identified and to see how they becomesegregated 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, Opp-enheim & 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 collectedby centrifugation. A concentrated suspension of worms was put in a depressiondish. Degassed 1 % NaOCl (Fisher Scientific Co.) with 0-25 M - K O H was addedfor 2-3 min until eggs were released from the partially lysed adults. The eggswere collected on a Nucleopore Corp. polycarbonate membrane filter (8-0/im)and rinsed with M9 salt solution. The egg shell was removed from the embryosby digestion with chitinase (U.S. Biochem. Corp.) according to a proceduredeveloped 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 roomtemperature. The eggs were observed under a dissection microscope at X25magnification. As soon as the egg shell was seen to be removed, the eggs weretransferred to fixative.

Eggs were fixed for 1-2 h at room temperature in 4% glutaraldehyde in100 mM sodium phosphate buffer, pH7-4; 2mM-MgCl2. Postfixation was donewith 1% osmium tetroxide in lOOmM-sodium phosphate buffer, pH7-4; for15 min. The eggs were encased in agar during the water rinse so that several eggsformed a group that could be sectioned together. Eggs were stained en bloc in1 % uranyl acetate, dehydrated in ethanol, and embedded in Epon between twomicroscope slides.

Sectioning was done on a Porter-Blum MT-2 microtome and sections werestained with uranyl acetate and lead citrate and viewed in a Hitachi H-600electron 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 alight microscope level (Schierenberg, 1978; von Ehrenstein & Schienenberg,1980). We have found the germline granules in the earliest embryos weexamined: the 'pseudocleavage' stage shortly after fertilization and prior to thefirst true cleavage. In Fig. 1, these granules can be seen near the posterior poleof the egg near the male pronucleus. Much vesicular material is present in thepseudocleavage egg but the darker staining granules can be distinguished. As theegg and sperm pronuclei move together and fuse to form the zygotic nucleus, thegranules remain at the posterior pole. While they are distributed highlyasymmetrically with respect to the anterior-posterior axis of the embryo at thisstage, they do not show any apparent asymmetry along the left-right or

Germline granules in C. elegans 299dorsal-ventral axes. During the first cleavage, the granules remain at the pos-terior 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 formedalong the anterior-posterior axis of the egg. However, as cleavage begins, thespindle rotates in the dorsal-ventral plane so that one daughter, EMSt, isanterior-ventral and the other, P2, is posterior-dorsal. The germline granulesappear to become localized in the dorsal sector of the PI blastomere prior to thecleavage of this cell, as seen in Fig. 3. When PI divides, they are partitioned intothe P2 blastomere (Fig. 3B).

P2 will divide into blastomeres C and P3, and P3 will later divide into D andP4. The C and D blastomeres divide several times to form part of the somatictissues of the animal, while P4 divides only once more during embryogenesis intoZ2 and Z3, which form the germline (Kimble & Hirsh, 1979; J. Sulston, personalcommunication). The germline granules continue the pattern of asymmetricsegregation described above, and are ultimately contained only in the P4 blas-tomere (Fig. 4). At the division of P4, they are segregated equally into both Z2and 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 closerproximity to the nucleus (compare Figs 3 and 4). Cytoplasmic granules can beseen in the P3 and P4 blastomeres, even in the living embryo with the lightmicroscope, and are probably identical to the granules we see with the EM. Thefew large granules found in the P3 and P4 blastomeres could come from anaggregation of the numerous smaller granules we find in the earlier stages, orperhaps by the differential growth and attrition of certain granules.

The granules are often observed in close approximation to endoplasmicreticulum and mitochondria, and are not enclosed by membranes. We have notobserved any striking association of the granules with microtubules or filamentsthat might suggest a mechanism for their movement.

In the study by Krieg et al. (1978), these granules were seen as 'electron lightcytoplasmic areas'. With our fixation methods, the granules appear as electrondense spherical bodies of an apparent fibrous nature, similar to the structure ofgermline particles described in other organisms (for review, see Beams & Kessel,1974; and Eddy, 1975). The ultrastructure of the granules themselves offers noobvious suggestion of a possible function in the embryo.

DISCUSSION

These EM studies have demonstrated that the germline granules of C. elegansare present and asymmetrically distributed in embryos as early as the pronuclearstage of development. These granules continue to be segregated into the P-lineage 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 poleof the fertilized egg. The nuclei which migrate into this region at the firstcytoplasmic division become the germline cells. In contrast, the germlinegranules of C. elegans must be segregated asymmetrically through four divisionsbefore they are in the definitive germline cells. The granules of Drosophila havebeen implicated in determining the nuclei that they surround to become germnuclei. If the C. elegans granules have a similar function, either they are notactive in the Po , PI, P2 and P3 blastomeres, or these blastomeres are not com-petent to respond to the hypothetical 'signal'. In this light, it is perhaps interest-ing that the granules only begin showing a strong association with the nucleus ofthe P4 blastomere, having been dispersed in the cytoplasm of the earlier stages.

Fuchs (1913) noticed granules he termed 'ectosomes' surrounding one of theasters in the first cleavage of the arthropod, Cyclops vividis. In successivedivisions, these granules were asymmetrically segregated to only one of thedaughter cells, and were finally localized in two of the primordial germ cells witha lineage pattern identical to that of C. elegans. The association of these granuleswith the asters of the cleaving blastomeres suggested an obvious mechanism fortheir segregation. As described above, the germline granules of C. elegans areasymmetrically distributed even before the first spindle is formed. Similarly, theprelocalization of the granules in the later blastomeres, prior to spindle forma-tion, make an astral involvement in their specific segregation less likely in thisanimal. Still, a transient association of the granules with cytoplasmicmicrotubules or microfilaments could have been missed in our studies.

Fig. 1. Pronuclear stage of C. elegans embryogenesis. (A) The pseudocleavage hasoccurred. The female pronucleus is in the anterior (left) end of the egg and the malepronucleus resides in the posterior (right) end of the egg. Arrows point to the germ-line granules. Mag. 2700x. (B) Higher magnification of the posterior end of the eggin A showing two granules near the male pronucleus. Mag. 8300x. (C) Migration ofthe pronuclei towards the middle of the egg. Arrows show the position of the germ-line granule. Mag. 2700x.Fig. 2. Germline granules in the 2-cell embryo of C. elegans. (A) The anterior ABblastomere is on the left and the posterior PI blastomere on the right. The 2-cellembryo contains germline granules in the posterior end of the PI blastomere, asdesignated by arrows. Mag. 2900x. (B) and (C) Higher magnifications of germlinegranules in the 2-cell embryo showing the granular morphology of the particles whichare unbounded by membranes. Endoplasmic reticulum and several mitochondria arevisible near the granules. (B) Mag. 23000X; (C) Mag. 33 000x.

Fig. 3. Segregation of the germline granules during the division of the PI blas-tomere. (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 P2daughter blastomere. Mag. 2700x. (B) The PI blastomere has reached telophaseand the granules (arrows) remain segregated into the region of the cell that becomesthe 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 adjacentto its nucleus. Mag. 9000x. (B) Higher magnification of the P4-cell nucleus with itsflanking granules. Mag. 17000x.

20 EMB 73

302 N. WOLF, J. PRIESS AND DAVID HIRSH

V . ^ • . V ^ : . . v V . ^ , - • . • • ' - •••• : , • • - • ' "

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Fig. 2. For legend see p. 301.

Germline granules in C. elegans 303

Fig. 3. For legend see p. 301.

304 N. WOLF, J. PRIESS AND DAVID HIRSH

Fig. 4. For legend see p. 301.

'*,

Germline granules in C. elegans 305While germline specific granules have apparently not been observed in other

species of nematodes, perhaps due to the difficulties of obtaining adequatefixation of the early stages, there are striking differences between cytoplasms ofsomatic and germline precursor cells during the embryogenesis of the nematodeAscaris megalocephala (Boveri, 1899). Shortly after the first cleavage of thefertilized Ascaris embryo, chromosomal diminution occurs in one of the daugh-ter cells; chromosomal fragments are left in the cytoplasm and are subsequentlylost. However, the sister blastomere, PI, which will ultimately produce thegermline cells as in C. elegans, retains the full chromosomal complement. Thepotential not to undergo chromosomal diminution is segregated into Po, 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 thecytoplasm the germline normally receives; if the first cleavage is altered such thatthis cytoplasm is divided between the first two sisters, diminution does not occurin either cell (Boveri, 1910; Hogue, 1910). Thus there is a clear difference in theproperties 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 granuleswe observed could conceivably be related to the same basic cytoplasmic dif-ference between the somatic and germline precursor blastomeres.

Krieg et al. (1978) reported seeing 'electron light cytoplasmic areas' in germ-line precursors in C. elegans embryos from the 6-cell stage onward. These 'areas'were localized around the nuclei. Thus, the segregation pattern and intracellularlocalization of these 'areas' are the same as those of the granules we have obser-ved. Although their morphology is quite different, presumably as a result of thedifferent fixation procedures, it is likely that those 'areas' represent the samestructures as the granules we have described.

Strome & Wood (1982) recently reported staining particulate cytoplasmiccomponents of the germline cells and their precursors in C. elegans embryosusing FITC-conjugated rabbit anti-mouse IgG. The location and segregation ofthe staining material appears to be identical to that of the granules reported here,suggesting that the antiserum may be recognizing some component of thesestructures. Unfortunately the antiserum does not bind to the particles afteraldehyde fixation, preventing a direct comparison of the particles recognized bythe antiserum and the granules described here (S. Strome, personal communica-tion).

We thank J. Richard Mclntosh for helping develop the improved fixation and for valuablediscussions. This work was supported by Public Health Service Grant No. 19851.

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(Received 24 August 1982)