1303Development 122, 1303-1312 (1996)Printed in Great Britain © The Company of Biologists Limited 1996DEV3365

Segregation of germ granules in living Caenorhabditis elegans embryos: cell-

type-specific mechanisms for cytoplasmic localisation

Steven N. Hird1,*, Janet E. Paulsen2,† and Susan Strome2

1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK2Department of Biology, Jordan Hall, Indiana University, Bloomington, IN 47405, USA

*Present address: Department of MCD Biology, Porter Biosciences Building, Campus Box 347, University of Colorado, Boulder, CO 80309-0347, USA†Present address: Genetics Institute, 87 CambridgePark Drive, Cambridge, MA 02140, USA

Germ granules are ribonucleoprotein particles that arethought to function in germline specification in inverte-brates and possibly in vertebrates. In Caenorhabditiselegans, these structures, termed P granules, are parti-tioned to the germline P cells during the early embryonicdivisions. By injecting a fluorescently labelled anti-P-granule antibody into the C. elegans germline syncitium,we followed P-granule segregation in live embryos usinglaser-scanning confocal microscopy. We show that, in earlyP cells (P0 and P1), P-granule partitioning is achievedprimarily by their migration through the cytoplasmtowards the site of formation of the germline daughter cell.

A different mechanism appears to operate in later P cells(P2 and P3): P granules associate with the nucleus and movewith it toward the site of formation of the germlinedaughter cell, where they are then deposited. At eachdivision, there is also disassembly or degradation of thoseP granules that remain in the cytoplasm destined for thesomatic daughter cell. Microfilaments, microtubules andthe product of the gene mes-1 are required for the normalpattern of P-granule segregation in P2.

Key words: Caenorhabditis elegans, P granules, cytoplasmiclocalisation, in vivo confocal fluorescence microscopy, germ granule

SUMMARY

INTRODUCTION

The eggs of many organisms display asymmetric distributionsof determinants. These molecules are differentially partitionedto different daughter cells during the early embryoniccleavages and then specify the fates of the cells that containthem. The mechanisms by which determinants becomelocalised are poorly understood at present. We are investigat-ing localisation mechanisms using Caenorhabditis elegans Pgranules as a model system.

P granules are cytoplasmic factors that contain poly(A)+

mRNA and are visible both by electron microscopy and byindirect immunofluorescence (Strome and Wood, 1982, 1983;Wolf et al., 1983; Seydoux and Fire, 1994). They are synthe-sised in the germline of hermaphrodites and males and passedon to offspring via the oocyte. The cell that gives rise to thegermline in C. elegans, P4, is formed by a series of four asym-metric, stem-cell-like divisions of the fertilised egg; eachdivision produces a somatic precursor cell and a germline Pcell (Fig. 1) (Deppe et al., 1978; Sulston et al., 1983). P0, P1,P2, and P3 partition P granules to the cortical and cytoplasmicdomains of the mother cell that are destined for the germline-cell daughter, resulting in inheritance of the P granules by P4.P4 divides once during embryonic development, distributing Pgranules to its daughters, Z2 and Z3. During postembryonicdevelopment, Z2 and Z3 proliferate to give rise to the germline.P granules are associated with the nuclei of all undifferentiated

germ cells throughout larval and adult development (Stromeand Wood, 1982). Structures that are morphologically similarto P granules are involved in determination of the germline inDrosophila melanogaster (reviewed in Lehmann, 1992); Pgranules in C. elegans might serve the same function.

By visualising P granules in fixed embryos, it has beenshown that their localisation in the fertilised egg occurs duringa specific interval in the first cell cycle and is disrupted by themicrofilament inhibitor cytochalasin D (Strome and Wood,1983; Hill and Strome, 1988). In addition, treatment withcytochalasin D during this interval often causes the firstdivision to be symmetric, producing daughters with altereddivision axes and cell-cycle timing (Hill and Strome, 1990).Mutations in the maternal-effect par genes (for partitioning-defective) also disrupt P-granule localisation in the fertilisedegg, as well as altering the axes of the early cleavages and thefates of blastomeres (Kemphues et al., 1988; Kirby et al., 1990;Morton et al., 1992; Levitan et al., 1994; Cheng et al., 1995).Considered together, these results have led to the proposal thatactin microfilaments and the par gene products mediate thelocalisation of P granules and developmental determinantsduring a critical interval in the first cell cycle. Furthermore,some mutations that disrupt P-granule segregation also disruptthe localisation of the putative determinant SKN-1 (Bowermanet al., 1993). This also suggests that common mechanisms areused to localise a variety of cytoplasmic factors in C. elegansembryos.

1304 S. N. Hird, J. E. Paulsen and S. Strome

Fig. 1. (A) The germ-cell lineage in C. elegans. At each division, the daughter cellon the left side of the figure is anterior and the daughter on the right is posterior. Thegerm cells are shown in bold. The thick black lines connecting P cells schematicallyrepresent the passage of P granules. Note that P2 and P3 segregate P granulesanteriorly and generate their germline daughters anteriorly (Schierenberg, 1987).(B-J) Nomarski images of the germ-cell divisions. Anterior is left, dorsal is up.(B) An embryo at pseudocleavage. The maternal pronucleus (o) is migrating towardthe sperm pronucleus (sp) at the posterior. The pseudocleavage furrow is visible.

(C) The pronuclei have met and thecentrosomes (arrowheads) rotated so thatthey are aligned along the anteroposterioraxis. (D) The first division produces ABat the anterior and P1 at the posterior. TheP1 centrosomes (arrowheads) rotate ontothe anteroposterior axis. (E) The P1spindle (arrowheads mark poles)becomes skewed along the dorsal-ventralaxis by the division of AB. (F) At thefour-cell stage, P2 is dorsal-posterior,EMS is ventral-anterior. The position ofthe unrotated P2 centrosomes, which arenot in the plane of focus, is indicated byarrowheads. (G) The P2 centrosomes(arrowheads) rotate onto the divisionaxis, pulling the P2 nucleus ventrally.(H) The spindle in P2 (arrowheads markpoles) is established along the dorsal-ventral axis. (I) The germ cell P3 isproduced on the ventral side, whilst C isgenerated dorsally. (J) P3 divides in asimilar way to P2, producing P4 ventrallyand D dorsally. Scale bar, 10 µm.

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In this paper, we address how P granules are localised byasking the following. (1) Is the localisation of P granules dueto granule migration toward the cytoplasm/cortex inherited bythe next P cell, or due to stabilisation of granules in this regionand destabilisation of granules in the region inherited by thesomatic cell? (2) Do all P cells use the same mechanism of P-granule segregation? This seemed especially important toinvestigate in P2 and P3, which switch the polarity of their seg-regation axis relative to P0 and P1 (Fig. 1) (Schierenberg,1987). (3) In mutant embryos that display mispartitioned Pgranules, which aspects of P-granule localisation are affected?

To answer these questions, we developed a means of directlyvisualising P granules in live embryos. This was done by flu-orescently labelling an anti-P-granule monoclonal antibodyand injecting the fluorescent antibody into the germlinesyncitium of adult hermaphrodites. Embryos produced byinjected hermaphrodites contain fluorescent P granules andthese were examined by laser scanning confocal microscopyduring the early cleavages. Our results show that, in P0 and P1,P-granule localisation is achieved by a combination ofpolarised migration and localised degradation. P2 and P3 utilisea different strategy to localise P granules; this involvestransport of many of the granules attached to the nucleus andalso localised cytoplasmic degradation. Embryos mutant for

the maternal-effect sterile gene mes-1 (Capowski et al., 1991;Strome et al., 1995) show defects in nuclear behavior in P2,which results in mispartitioning of P granules.

MATERIALS AND METHODS

Strain maintenanceCaenorhabditis elegans was cultured as described in Brenner (1974).The strains used were the wild-type N2 variety Bristol, mes-1(bn7ts)and pgl-1(ct131). mes-1(bn7ts) was maintained at the permissive tem-perature of 15°C, while the other strains were maintained at 20°C.

Preparation of fluorescein-conjugated anti-P-granuleantibodyThe anti-P-granule monoclonal antibody OIC1D4 (Strome and Wood,1983), an IgG, was purified by affinity chromatography on Protein A-Sepharose CL-4B (Pharmacia) according to Hudson and Hay (1980).Purified OIC1D4 was conjugated to fluorescein according to Goding(1976), with the following modifications. Purified OIC1D4 at 2-4mg/ml was dialysed against 0.05 M sodium phosphate pH 8.0 for 1-2 hours. An appropriate volume of 5(and 6)-carboxyfluorescein suc-cinimidyl ester (Molecular Probes, Eugene, OR) at 5 mg/ml in DMSOor DMF was added dropwise to the antibody solution, to achieve afluorochrome to antibody ratio of between 40 and 60 µg fluorochrome

1305Nematode P-granule segregation

to 1 mg antibody. The reaction was incubated for 2 hours at roomtemperature with continuous stirring. Unreacted fluorochrome wasseparated from fluorochrome-conjugated antibody on Sephadex G-25(Pharmacia) that had been equilibrated with PBS, pH 7.4 (150 mMNaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4). The conju-gated antibody, which was easily identified by its color, was collectedin 1-2 ml of the void volume of the column.

The antibody conjugate was analysed for the presence of unreactedfluorochrome according to Kreis and Birchmeier (1980). The approx-imate molar ratio of fluorochrome to protein was calculated asdescribed by The and Feltkamp (1969). A conjugate with an F/P ratioof 7.3 was used in all experiments.

MicroinjectionPurified conjugated OIC1D4 was dialysed against injection buffer (5mM KCl, 0.1 mM KH2PO4) overnight at 4°C. Microneedles (ClarkeElectromedical GC120 F-15, pulled on a Sutter puller) were backfilledby capillary action with the dialysed conjugate at 1-2 mg/ml. The filledneedles were mounted on a MPM-10/ DC3001 piezo-translator micro-manipulator (World Precision Instruments) attached to a Zeiss Axiovertinverted microscope equipped with DIC optics. The germline syncitiumof gravid hermaphrodites was then injected using the protocol developedfor transformation by Fire (1986). Injected wild-type worms wereincubated at 15°C for 15 hours before their embryos were collected.Injected mes-1(bn7ts) worms were incubated for 9 hours at 25°C.

Microscopy The injected worms were dissected in egg salts (118 mM NaCl, 40 mMKCl, 3.4 mM MgCl2, 3.4 mM CaCl2, 5 mM Hepes, pH 7.4; Edgar andMcGhee, 1986), and the embryos were transferred to a poly L-lysine-coated (Sigma) coverslip with clay spacers. The coverslip was invertedover a slide and sealed with Vaseline to prevent desiccation. The slidewas mounted on a Zeiss Axiovert equipped with an Argon laser-scanning confocal scanhead (MRC-500 prototype built in the MRC Lab-oratory of Molecular Biology Mechanical Workshop) and DIC optics.A single confocal section was collected at the intermediate scan speed(1 second/scan) every 30, 60 or 90 seconds at 20°C (25°C for mes-1embryos) using a 63× oil immersion lens. The Argon laser light wasattenuated by neutral density filters (Oriel) to give the lowest amount ofillumination necessary for a satisfactory fluorescent signal. A transmis-sion detector allowed the collection of simultaneous fluorescence andDIC images of the embryos. Image sequences were animated on a PowerMacintosh computer using NIH Image version 1.5, written by WayneRasband at the US National Institutes of Health, and available from theInternet by anonymous FTP from zippy@nimh.nih.gov. Imagesequences were then assembled into Quicktime movies. Representativemovies may be downloaded from the World Wide Web at the followingURL: http://sunflower.bio.indiana.edu/~sstrome/_videos/videos.html

Treatment of embryos with inhibitor solutionsCytochalasin D and nocodazole (both from 5 mg/ml DMSO stocksolutions) were diluted in embryonic growth medium (Edgar andWood, 1993) to a concentration of 10 µg/ml. Embryos were dissectedfrom hermaphrodites as described above and pipetted onto a polyly-sine-coated coverslip with clay spacers. A drop of trypan blue (1mg/ml in egg salts) was placed over the embryos for 2 minutes, andwashed off using egg salts (the trypan blue increases absorption ofthe laser light by the eggshell). The inhibitor solution was placed overthe embryos, and the coverslip was mounted on a slide as describedabove. The eggshells of individual embryos were permeabilised asdescribed previously using 440 nm light from a nitrogen-pumpedcoumarin dye laser (Laser Science Inc.) mounted on a Zeiss Axioplanmicroscope (Hyman and White, 1987).

Indirect immunofluoresenceTo visualise P granules, embryos were fixed and stained with OIC1D4as described in Albertson (1984).

RESULTS

Background informationThe aspects of early embryogenesis relevant to this paper arebriefly reviewed here and shown in Fig. 1 (Nigon et al., 1960;Hirsh et al., 1976; Albertson, 1984; Kirby et al., 1990; Hirdand White, 1993). The fertilised C. elegans embryo iscontained within an elongated eggshell. The oocyte and spermpronuclei form at the future anterior and posterior end, respec-tively, of the embryo (Fig. 1B), after which a period of cyto-plasmic reorganisation takes place. During this period internalcytoplasm streams posteriorly, while cortical cytoplasmstreams anteriorly, and the oocyte pronucleus migrates towardthe sperm pronucleus. P granules become localised in theposterior half of the embryo during this interval, where theyare concentrated around the cortex. These events occur at thesame time as a process called pseudocleavage in which afurrow forms and regresses (Fig. 1B). After cytoplasmicstreaming ceases, the sperm-contributed centrosomes of thepronuclear complex rotate 90 degrees so that they are alignedalong the anteroposterior axis (Fig. 1C). Centrosome alignmentalong the anteroposterior axis sets the mitotic spindle up alongthe axis of P-granule segregation and is a feature of each P-cell division (Albertson, 1984; Hyman and White, 1987;Hyman, 1989). The first division is unequal, producing thesomatic founder cell AB at the anterior, and the smallergermline cell P1 at the posterior (Fig. 1D). P1 inherits all of theP granules.

P granules also become localised to the posterior of P1. P1then divides unequally to produce the somatic daughter EMSon the ventral-anterior side and the germline daughter P2 at theposterior pole of the embryo (Fig. 1E,F). P2 inherits all of theP granules.

Unlike P0 and P1, P2 segregates P granules anteriorly, andgenerates its germline daughter P3 anteriorly and its largersomatic daughter C posteriorly (Fig. 1A). This reversal in seg-regation and division polarity is most easily seen in ‘partialembryos’, which have been released from the constraints of theeggshell. In the intact embryo, the constraints of the eggshelland neighbouring blastomeres force the P2 division to occuralong the dorsal-ventral axis (Fig. 1G,H). During alignment ofthe P2 centrosomes onto this axis, the ventral-most centrosomeis pulled via its astral microtubules toward a specific site in theventral cortex of P2. This causes a ventrally directed movementof the nucleus-centrosome complex (Fig. 1F,G) (Hyman andWhite, 1987). P granules are segregated to the ventral side ofP2, where P3 is born (Fig. 1H,I). P3 reiterates this polarity-reversed segregation and cleavage pattern, passaging the Pgranules ventrally to the primordial germ cell P4 (Fig. 1J).

Visualisation of P granules in living embryosWe have investigated the timing and mechanism(s) of P-granule segregation in living embryos. The anti-P-granulemouse monoclonal antibody OIC1D4 (Strome, 1986) was con-jugated to fluorescein and injected into the syncitial gonads ofadult hermaphrodites. Fluorescent structures that lookedidentical in size to P granules and displayed the same locali-sation pattern were seen in the germline of injected hermaph-rodites (data not shown) and in their embryos (see below).

In order to prove that the fluorescent structures were indeedP granules, rather than, for example, aggregates of unbound

1306 S. N. Hird, J. E. Paulsen and S. Strome

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EFig. 2. Pairs of confocal images (A-D) and simultaneous Nomarskiimages (a-d) of an early one-cell embryo containing tagged Pgranules. Anterior is left. (A,a) The P granules are initiallydistributed throughout the cytoplasm, but begin to move posteriorlybefore the oocyte pronucleus (o) starts to migrate toward the spermpronucleus (sp). The arrowhead indicates one particular granule.(B,b) The granule indicated in A was tracked every 30 seconds overthe next three minutes, during which time it moved posteriorly(arrowhead indicates postion of granule after three minutes).(C,c) By the time the pronuclei have met, the vast majority of Pgranules are close to the posterior cortex. The arrow indicates a Pgranule that remains at the anterior. (D,d) At the first division, all theP granules are inherited by P1; no P granules are seen in the anteriorcell AB. (E) The paths followed by four individual tagged P granulesin the same embryo. The dots indicate the positions of the granules at30-second intervals. The granules moved from anterior to posterior.Scale bar, 10 µm.

antibody, we repeated the injections using hermaphroditeshomozygous for the maternal-effect mutation pgl-1(ct131).Fixed P granules in such hermaphrodites and their embryoslack OIC1D4 immunoreactivity, but do stain with other anti-P-granule antibodies (Wood et al., 1984; I. Kawasaki and S.S.,unpublished data). When we injected fluorescent OIC1D4antibody into pgl-1(ct131) mothers, no granular staining wasvisible in their embryos (data not shown). This shows that thefluorescent granules in wild-type embryos result from the invivo binding of the injected OIC1D4 antibody to P granules.

Localisation of P granules in P0

We followed fluorescently tagged P granules in 22 one-cellembryos (Fig. 2). Prior to pseudocleavage, the P granuleswere dispersed throughout the cytoplasm, as described pre-viously (Strome and Wood, 1982, 1983). During pseudo-cleavage, almost all the P granules in the cytoplasm movedtoward the posterior pole, at an average rate of 3.97±0.8µm/min (n=19 granules; 6 embryos) (Fig. 2E). Similar rateswere observed when labelled embryos were observed usinga silicon-intensified camera and continuous illumination (datanot shown). Upon reaching the posterior, they often appearedto move anteriorly for some distance along the cortex. Thesemovements occurred coincident with the posteriorly directedstreaming of central cytoplasm and anteriorly directedstreaming of cortical material described previously (Nigon etal., 1960; Hird and White, 1993).

As pseudocleavage and pronuclear migration ended, in16/22 cases all of the P granules were associated with thecortex of the posterior half of the embryo and were inheritedby the posterior daughter cell P1. In the remaining 6/22 cases,a few P granules at the extreme anterior of the pre-pseudo-cleavage embryo did not move posteriorly during pseudo-cleavage, but instead remained anterior (Fig. 2C). There is littlecytoplasmic streaming visible in this region of the embryoduring pseudocleavage (Hird and White, 1993). The anterior Pgranules progressively disappeared during mitosis, so that nonewere detectable in the anterior cell AB at the time of its birth(Fig. 2D). We suggest that this depletion of anterior P granulesresulted from disassembly or degradation of P granules in theanterior, non-germline cytoplasm of the embryo. However,since the P granules at this stage were rather small and poorlyvisible (compared to later stages; see below), we were unableto follow individual granules without photobleaching their flu-orescence significantly. Therefore, we cannot rule out the pos-sibility that the few granules that remained anterior afterpseudocleavage dropped out of the plane of focus and thenmoved posteriorly between the end of the normal localisationperiod and the commencement of cytokinesis.

We conclude that the localisation of P granules in the one-cell embryo is achieved primarily by their movement throughthe cytoplasm toward the posterior cortex. There may also bedisassembly or degradation of any stray P granules that remainin the anterior of the embryo prior to division. The localisationof P granules in living embryos, as described here, occursduring the same interval as defined by previous immunoflu-orescence studies of fixed embryos (Strome and Wood, 1983;Rose et al., 1995).

Localisation in P1

We monitored P-granule segregation in 24 two-cell embryos

(Fig. 3). Following the first division, most of the tagged Pgranules were located around the periphery of P1, except at theboundary with its sister cell AB (Fig. 3A). Therefore, themajority of P granules were already localised to the cortex,many to the cortex destined for P2.

1307Nematode P-granule segregation

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Fig. 3. Pairs of confocal images (A-D) and simultaneous Nomarskiimages (a-d) of a two-cell embryo containing tagged P granules.Anterior is left, ventral is up. (A,a) The embryo is shownimmediately after the first division. The P granules in P1 are mostlyconcentrated round the cortex, but a few remain in the cytoplasm(arrowhead). (B,b) The nucleus in P1 has migrated to the posterior ofthe cell, and the P granules are localised around the periphery ofP1.(C,c) The P1 spindle is established along the anteroposterior axis.The AB spindle skews somewhat along the dorsal-ventral axis as itgrows (arrowheads indicate spindle poles). At this time, the Pgranules begin to disappear from the extreme anterior of P1. Note thenuclear fluorescence marked by a star in C. This appears just prior tonuclear membrane breakdown (NMB) and persists for about 1minute. Transient nuclear fluorescence is seen at this time in all liveC. elegans cells that contain fluorescently labelled compounds(S.N.H., unpublished) and is a convenient marker for the approach ofmitosis. (D,d) As P1 divides, the P granules are inherited by P2. Scalebar, 10 µm.

Two patterns of P-granule movement were evident in P1immediately after its birth: the few cytoplasmic P granulesmoved posteriorly through the cytoplasm and eventuallybecame associated with the cortex, and P granules locatedclose to the cortex often moved anteriorly along the cortex. Theeventual result was that all the P granules became located closeto the cortex of P1, except for the region of contact with AB(Fig. 3B). The P-granule movements in P1 occurred at the sametime as yolk granules flow posteriorly through the cytoplasmand anteriorly along the cortex (Hird and White, 1993). The

pattern of P-granule movement in P1 was similar to that in P0,although the extent of segregation was not as dramatic in P1 asin P0.

P1 becomes somewhat extended along the anteroposterioraxis as a result of skewing of the anaphase spindle in thedividing AB cell (Fig. 3c). At the same time, P granules pro-gressively disappeared from the most anterior region of P1(Fig. 3C). Again, this may have resulted from disassembly ordegradation of the P granules in this vicinity. The extension ofthe P1 spindle along the anteroposterior axis appeared to pushthis depleted region further anteriorly and, as a result, the Pgranules eventually remained only around the posterior regionof the cell, which is inherited by P2 at cytokinesis (Fig. 3D).

In summary, P-granule localisation in P1 appears superfi-cially similar to this process in P0. However, at the time of P1’sbirth, most of the P granules are already localised to the site offormation of P2. In P1, there is a limited amount of P-granulemovement visible in the cytoplasm and cortex, and an apparentdepletion of P granules in the anteriormost region of the cell.

Localisation in P2 and P3

We followed tagged P granules in 20 four-cell embryos.Initially, the P granules were present throughout the cytoplasmof P2. However, they assumed a striking perinuclear locationimmediately before the rotation of the P2 centrosomes. As aresult, confocal sections through the centre of the nucleusrevealed a bright ring of P granules encircling it (Fig. 4A). Thisis also visible when embryos at this stage are fixed and thenstained with OIC1D4 (Fig. 4E). Only P granules that were inthe immediate vicinity of the nucleus became associated withit in this way; those distant from the nucleus remained in thecytoplasm.

As one of the P2 centrosomes migrated onto the divisionaxis, the nucleus moved toward the ventral side of P2 next toEMS (Fig. 4B). This brought the perinuclear P granules intothe cytoplasm that would be inherited by P3. P granules werethen deposited near the ventral pole of the spindle at nuclearmembrane breakdown (NMB) (Fig. 4C). This process of per-inuclear association of P granules, followed by ventrallydirected nuclear migration, was repeated in P3, partitioning themajority of P granules to P4 (12 embryos followed; data notshown). In P4, P granules remained associated with the nucleusand were eventually distributed to both its daughters, the pri-mordial germ cells Z2 and Z3, later in embryogenesis (Stromeand Wood, 1982).

Disassembly/degradation of P granules also seems to takeplace in both P2 and P3. Immediately before NMB in P2 andP3, the more dorsal side of the nucleus became depleted of Pgranules (Fig. 4B). This could be due to disassembly or degra-dation of granules on the dorsal side of the nucleus, disassoci-ation of granules from the dorsal side of the nucleus, ormovement of perinuclear P granules to the ventral side of thenucleus. Depletion of P granules from the dorsal side of the P2nucleus was accompanied in 16/20 cases by the progressiveloss of the non-perinuclear P granules that remained in themore dorsal cytoplasm destined for the somatic precursor cellC. In the remaining 4/20 cases, a few P granules remained inthe dorsal cytoplasm inherited by C. Similarly, in P3, stray Pgranules were sometimes seen in the cytoplasm inherited by D(3/12 cases).

To summarise, it appears that a different mechanism is used

1308 S. N. Hird, J. E. Paulsen and S. Strome

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Fig. 4. Pairs of confocal images (A-D) and simultaneous Nomarskiimages (a-d) of a four-cell embryo containing tagged P granules.Anterior is left, ventral is down. (A,a) A perinuclear ring of Pgranules forms in P2 immediately prior to centrosome rotation. Notethat the mass of individual P granules appears to have increasedcompared to the two earlier divisions, as described previously(Strome and Wood, 1982). (B,b) As the P2 centrosomes (arrowheads)rotate, the nucleus is displaced ventrally toward EMS. P granulesbecome depleted from the dorsal side of the P2 nucleus. (C,c) AtNMB, the P granules are deposited at the ventral pole of the P2spindle (arrowheads indicate spindle poles). (D,d) As P2 divides, theP granules are inherited by P3. (E) The perinuclear ring is also seenin a fixed embryo stained with OIC1D4 and visualised by indirectimmunofluorescence. Scale bar, 10 µm.

to segregate P granules in P2 and P3 from that used in P0 and P1.This involves the association of P granules with the nucleus andthe movement of the P-granule-coated nucleus toward the siteof formation of the next P cell. This mechanism may act inconcert with disassembly/degradation of P granules on the dorsalface of the nucleus and in the dorsal cytoplasm of P2 and P3.

Mutations in the mes-1 gene disrupt P-granulesegregation in P2

Embryos produced by hermaphrodites homozygous for null

mutations in the maternal-effect gene mes-1 develop intosterile adults (Capowski et al., 1991). This is due to transfor-mation of the primordial germ cell P4 into a muscle precursor,like its somatic sister cell D (Strome et al., 1995). This trans-formation in cell fate is preceded in P2 and P3 by defects inboth P-granule segregation and in the orientation of divisionaxes. P2 (in some embryos) and P3 (in most embryos) mispar-tition P granules to both their P-cell daughter and their somaticdaughter cell (Strome et al., 1995). We examined the basis ofthis segregation defect by monitoring tagged P granules inembryos from mes-1(bn7ts) mothers raised at the restrictivetemperature (referred to here as Mes-1 embryos).

In Mes-1 embryos, P granules in P2 assumed a perinuclearlocalisation, as in wild-type embryos (Fig. 5A). However, cen-trosome alignment and the concomitant ventral migration ofthe nucleus did not take place. As a result, the nucleusremained in the centre of the cell. Furthermore, there was nodepletion of P granules from the dorsal side of the nucleus priorto NMB. Instead, either the posterior face of the nucleusbecame depleted of P granules (Fig. 5B) or, less frequently,neither face became depleted. As NMB was initiated and thespindle formed, the P granules were released to the anterior(13/18 cases) (Fig. 5C) or both sides (5/18 cases) of the spindle,instead of at one pole of the spindle. Although there was nocentrosome rotation, neighbouring dividing cells physicallyconstrained the P2 cell in such a way that the spindle was estab-lished along the dorsal-ventral axis, as in wild-type embryos.The cytokinetic furrow that bisected the spindle passed throughthe mass of P granules, distributing them to both P3 and C (Fig.5D). P granules often assumed a perinuclear localisation inboth P3 and C (Fig. 5E), and were distributed in turn to theirdaughters, namely P4/D and Ca/Cp, where they again becameperinuclear, forming four rings of P-granule staining (Fig. 5F).

To conclude, Mes-1 embryos fail to partition P granules tothe ventral region of P2 due to the absence of nuclear migrationand the subsequent failure of the dorsal face of the nucleus tobecome depleted of P granules. This results in the P granulesbeing deposited on one or both sides of the spindle, instead ofat one spindle pole after NMB.

Inhibitor studiesThe posterior segregation of P granules in P0 was previouslyfound to depend on the microfilament cytoskeleton, but not onthe microtubule cytoskeleton (Strome and Wood, 1983; Hilland Strome, 1988). Our analysis of P granules in livingembryos suggests that, in P2 and P3, centrosome alignment andthe accompanying migration of the nucleus play a major role

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Fig. 5. Pairs of confocal images (A-D) and simultaneous Nomarskiimages (a-d) of a mes-1(bn7) four-cell embryo containing tagged Pgranules. Anterior is left, ventral is down. (A,a) P granules havebecome associated with the nucleus. (B,b) At NMB, the P granulesare deposited close to the anterior of P2; no centrosome rotation hasoccurred, and the nucleus is not displaced ventrally (i.e. towardEMS). (C,c) The spindle (arrowheads indicate spindle poles) formsin P2, and the P granules lie to one side of it. (D,d) The cytokineticfurrow bisects the spindle and passes through the mass of P granules,partitioning them to both P3 and C. (E) P granules adopt aperinuclear location in both P3 and C. (F) P granules are partitionedto P4, D, Ca and Cp, where they also adopt a perinuclear location.Note that faint granular staining is seen in many somatic cells. Thisis also seen in many late-stage wild-type embryos containingfluorescent OIC1D4 antibody, and in wild-type embryos fixed andstained with preparations of OIC1D4 that give very bright P-granulestaining by indirect immunofluorescence. The late somatic-cellgranules are not stained by other anti-P-granule antibodies and arenot visible in pgl-1(ct131) mutant embryos containing fluorescentOIC1D4 (unpublished observation). This suggests that later somaticcells contain the P-granule component(s) recognised by OIC1D4.Scale bar, 10 µm.

in P-granule segregation. Centrosome alignment in the P cellsis dependent on both microfilament and microtubule function(Hyman and White, 1987). Therefore, we investigated whetherP-granule segregation in P2 requires microfilament function,microtubule function, or both.

Microfilament inhibitionFour-cell embryos were laser-permeabilised in a solution of 10µg/ml of the microfilament inhibitor cytochalasin D. This con-centration blocks P-granule segregation in one-cell embryosand also blocks centrosome rotation and cytokinesis (Stromeand Wood, 1983; Hyman and White, 1987). In all of the 15cases followed, P granules became perinuclear, as in untreatedembryos (Fig. 6A). Due to the inhibition of centrosomealignment, the nucleus did not move ventrally, but remainedcentral, as in Mes-1 embryos. The P granules reproduciblybecame depleted from the posterior side of the nucleus prior toNMB (Fig. 6B), rather than from the dorsal side as in untreatedembryos. Again, this is reminiscent of the situation described

above in Mes-1 embryos. As NMB occurred and the spindleformed along the dorsal-ventral axis (again presumably due toconstraints from the eggshell and neighbouring blastomeres),the P granules were deposited along the anterior periphery ofP2 and thus along the side of the spindle (Fig. 6C). Otherregions of the cell were devoid of P granules; as in untreatedembryos, this may result at least in part from disassembly ordegradation of the granules. Although cytokinesis did notoccur in the presence of cytochalasin D, it was neverthelessclear that the P granules had been mislocalised relative to thespindle. Therefore, microfilaments are required for proper P-granule localisation in P2. Moreover, cytochalasin D appearsto phenocopy the P-granule localisation defect seen in Mes-1embryos at this stage.

Microtubule inhibitionTo inhibit microtubule function, 15 four-cell stage embryoswere permeabilised in a solution of the microtubule inhibitor,nocodazole, at 10 µg/ml. This concentration is sufficient toblock P2 centrosome rotation and attenuate the mitotic spindle;no long astral microtubules are visible, but short microtubulesmay remain associated with the centrosomes (Strome andWood, 1983; Hyman and White, 1987). As in untreatedembryos, P granules formed a perinuclear ring (Fig. 7A), butthere was no ventral movement of the nucleus due to the inhi-bition of centrosome rotation. P granules then became depletedfrom the dorsal side of the nucleus prior to NMB (Fig. 7B).The depletion often appeared more pronounced than inuntreated embryos, and seemed to involve the progressivereduction in size (over a two-minute interval) of individualgranules on the dorsal side of the nucleus. Granules in thedorsal cytoplasm also shrank gradually and eventually disap-peared. The observation that the whole dorsal population of Pgranules progressively and simultaneously disappeared fromthe same plane of focus strongly suggests that a disassembly/degradation mechanism is operating in this cell.

As NMB was initiated and the attenuated spindle formed innocodazole-treated P2 cells, there was a general flow of cyto-plasmic material toward the ventral side of the cell. Similar

1310 S. N. Hird, J. E. Paulsen and S. Strome

ABa

EMS

ABp

P2

A

B

C c

a

b

Fig. 6. Pairs of confocal images (A-C) and simultaneous Nomarskiimages (a-c) of a cytochalasin D-treated four-cell embryo containingtagged P granules. Anterior is left, ventral is down. (A,a) P granulesattach to the P2 nucleus, as in untreated embryos. There is nocentrosome rotation and hence the nucleus is not displaced towardEMS. (B,b) P granules become depleted from the posterior face ofthe P2 nucleus prior to NMB. (C,c) As the spindle forms (arrowheadsindicate spindle poles), the P granules are deposited along theanterior margin of P2, rather than in the ventral domain. Scale bar,10 µm.

A

B

C c

b

a

ABa

EMS

ABp

P2

*

Fig. 7. Pairs of confocal images (A-C) and simultaneous Nomarskiimages (a-c) of a nocodazole-treated four-cell embryo containingtagged P granules. Anterior is left, ventral is down. (A,a) P granulesattach to the P2 nucleus, but no centrosome rotation takes place.(B,b) P granules become depleted from the dorsal face of the P2nucleus. (C,c) The P granules are swept ventrally by the cytoplasmicstreaming that takes place during nocodazole-inhibited cytokinesis(arrow indicates direction of streaming; star indicates position ofattenuated mitotic spindle). Scale bar, 10 µm.

cytoplasmic streaming is seen in other embryonic blastomerestreated with nocodazole, and is thought to result from thepolarised activation of the cytoskeletal machinery thatnormally brings about cytokinesis (Hird and White, 1993). TheP granules present in the cytoplasm at NMB appeared to beswept by this flow toward the ventral side of P2, and thereforewere artificially localised to the cytoplasm that would beinherited by P3 in a normal cleavage (Fig. 7C). This streamingwas clearly an effect of drug treatment and, therefore, not anormal component of the localisation mechanism in untreatedembryos. However, the observation that the depletion of Pgranules from the dorsal side of the P2 nucleus proceedednormally in nocodazole-treated embryos shows that thisprocess does not require intact microtubules.

To summarise, the ventral movement of the nucleus andassociated shell of P granules in P2 requires both microfila-ments and microtubules, whilst the subsequent depletion of Pgranules specifically from the dorsal face of the nucleusrequires microfilaments but not microtubules.

DISCUSSION

We have investigated the mechanism of segregation of the

germline-specific P granules in the early C. elegans embryo.These factors have been tagged in living embryos by injectinga fluorescently labelled anti-P-granule monoclonal antibody intothe germline of adult hermaphrodites. The fluorescent granulesare segregated to P cells during the early cleavages, and areabsent in a mutant in which the P granules are not recognisedby the monoclonal antibody. This confirms that the tagged struc-tures are indeed P granules, and that binding of the antibody doesnot disrupt their segregation. Our results show that P-granulelocalisation is achieved primarily through the directedmovement of P granules toward the site of formation of thegermline daughter cell. However, the mechanism of movementin P0 and P1 is different from that in P2 and P3. We have alsopresented evidence that disassembly or degradation of Pgranules takes place in the cortical and cytoplasmic domains thatwill be inherited by the somatic daughter. These results illustratehow determinants can be localised to specific cells.

Cell-type-specific mechanisms for localising PgranulesOne surprising result of this study is that different P cells use

1311Nematode P-granule segregation

different mechanisms to localise P granules to the site offormation of the next P cell. In this section, we compare thesemechanisms, and propose why they may differ.

In the one-cell embryo, localisation results primarily from Pgranules in the cytoplasm migrating toward the posterior cortexat approximately 4 µm/min. At the same time as the P granulesmove, general cytoplasmic material streams posteriorly atapproximately the same rate (4-7 µm/min) (Nigon et al., 1960;Hird and White, 1993). Both streaming and P-granule locali-sation are disrupted by microfilament inhibitors (Strome andWood, 1983; Hird and White, 1993). These observationssuggest that P granules are swept posteriorly by the cytoplas-mic streaming in the fertilised egg. The trapping of P granulesat the posterior in this model could result from the presence ofspecific P granule attachment sites in the posterior cortex.Alternatively, the P granules may move posteriorly along anarray of polarised cytoplasmic actin cables. However, we donot believe this to be the case as there is no evidence for suchan array in the embryo at this time.

P granules do not display dramatic migration in P1. This maybe because the vast majority of P granules are already locatedaround the periphery of the cell when P1 is born and many aretherefore located in the region of the cell that will be inheritedby P2. Further restriction of P granules to the cortex destinedfor P2 is accomplished primarily by depletion of P granulesfrom the regions of the cortex destined for EMS. This depletiontakes place just prior to mitosis in P1 and may result from dis-assembly/degradation of P granules (see next section).

P-granule localisation in P2 and P3 is achieved by theapparent attachment of the P granules to the nucleus, followedby movement of the nucleus toward the ventral side of P2; thismovement is a result of centrosome rotation. As far as we areaware, this is a novel mechanism for the localisation of cyto-plasmic factors during embryogenesis. Why do P2 and P3 usea different P-granule localisation mechanism than P0 and P1?P0 and P1 segregate P granules posteriorly, while P2 and P3segregate them anteriorly in partial embryos, which corre-sponds to the ventral side of intact embryos. We propose thatthe nuclear migration mechanism may have been adopted tobring about this reversal of P-granule segregation polarity. Thishypothesis can be tested by following P-granule segregation inother nematode species that do not undergo a reversal ofpolarity in P2 and P3, such as Cephalobus (Skiba and Schieren-berg, 1992). This experiment was not included in the presentstudy, because the monoclonal antibody used here does notcross-react with Cephalobus P granules (B. Goldstein, personalcommunication).

Evidence for localised disassembly or degradationof P granulesEach P-cell division is immediately preceded by the disap-pearance of P granules that remain in the cytoplasm or cortexdestined for the somatic daughter. In P2 and P3, there is also adepletion of P granules from the dorsal face (the ‘somatic’side) of the nucleus. We believe that in all cases this resultsfrom the progressive disassembly or degradation of individualP granules. The proposal that P granules are disassembled ordegraded in somatic cytoplasm is not without precedent. Infixed embryos stray P granules are sometimes seen in C andD, the somatic sisters of P2 and P3, respectively (Strome andWood, 1982). However, P granules are not seen in the

daughters of C and D (Strome and Wood, 1982). Analysis offixed embryos also revealed that stray P granules are occa-sionally seen in the anterior of late one-cell embryos (Rose etal., 1995). However, they are not observed in the somaticdaughter cell, AB (Strome and Wood, 1982).

These observations suggest that, prior to division, thecytoplasm of each P cell is polarised with respect to its abilityto maintain P granules. Specifically, there could be a gradientof P-granule-stabilising activity, or an oppositely orientedgradient of destabilising activity, along the segregation axis ofeach P cell. This would allow properly localised P granules tobe maintained and would cause stray P granules in futuresomatic cytoplasm to be destroyed prior to division. Interest-ingly, P granules are not visible until the four-cell stage in par-1 (Guo and Kemphues, 1995), par-4 and some par-3 mutantembryos (S. N. H. and K. J. Kemphues, unpublished obser-vations) and then are visible in multiple cells. Thus, these pargene products may be involved in the normal regulation orlocalisation of the stabilising/destabilising activity. PAR-1 (aserine/threonine kinase) is localised to the periphery of eachP cell in the region where P granules become localised (Guoand Kemphues, 1995), while PAR-3 (a novel protein) showsa complementary distribution (Etemad-Moghadam et al.,1995). Thus, these two PAR proteins are located in appro-priate positions to localise or regulate a P-granule stabilisingor destabilising activity.

Cellular asymmetry in P2

Microfilaments are required for the one-cell embryo togenerate asymmetries, both in the distribution of P granulesand in the placement of the mitotic spindle (Strome and Wood,1983). In contrast, microfilaments are not required for P2 todisplay asymmetry, but are required to orient the segregationaxis correctly: in cytochalasin-treated embryos, P granules inP2 become aberrantly localised to the anterior region of thecell, apparently as a consequence of depletion of granules fromthe posterior side of the nucleus and the posterior cytoplasm.Thus, microfilaments are needed for the normal pattern ofventral localisation and dorsal depletion of P granules. Micro-filaments are also required for the rotation of the P2 centro-somes onto the segregation axis (although in their absence thespindle is constrained in such a way that it forms approxi-mately along the normal axis) (Hyman and White, 1987). Themicrofilament-dependent rotation of the P2 centrosomes maybe responsible for shifting the P-granule segregation axis to thecorrect orientation.

Insights into mes-1 defectsOur results show that the mes-1 product is required to bringabout centrosome rotation exclusively in the P cells thatdisplay polarity reversal, namely P2 and P3. Furthermore, thestriking similarity between the effects of mutations in mes-1and the effects of cytochalasin treatment on P2 suggests thatthe mes-1 product may interact with the microfilamentcytoskeleton. As discussed above, the failure in centrosomerotation could account for the altered pattern of P-granule seg-regation in Mes-1 embryos (Strome et al., 1995) and shouldalso lead to an altered pattern of division. In fact, Mes-1embryos that have been freed from the constraints of theeggshell display defects in the pattern of the P2 division (E.Schierenberg and S.S., unpublished results). However, in intact

1312 S. N. Hird, J. E. Paulsen and S. Strome

Mes-1 embryos, constraints from the eggshell and surroundingcells cause the P2 mitotic spindle to assume an approximatelynormal dorsal-ventral orientation in the absence of centrosomerotation, just as in cytochalasin-treated P2. This results in theincorrectly localised P granules being distributed to bothdaughter cells.

It is worth noting that, in living Mes-1 embryos, P-granulemis-segregation was observed in all 18 P2 divisions that weremonitored, whereas only 29% of fixed Mes-1 embryoscontained mis-segregated P granules in C. This difference mayreflect a difference in the timing of observation, living embryosbeing monitored through the cell cycle and division of P2 andfixed embryos being scored at later stages. This observationmay indicate that in most Mes-1 embryos, C still retains theability to disassemble/degrade P granules.

We thank Julie Ahringer, Susan L. Brown, Bob Goldstein, JonathanHodgkin, Matthew Freeman, and John White for helpful commentson the manuscript. S. N. H. was supported by the Medical ResearchCouncil. J. P and S. S. were supported by NIH grant GM34059.

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(Accepted 17 January 1995)