Brief cytochalasin-induced disruption of microfilaments …...Development 108, 159-172(1990) Printed in Great Britain ©The Company of Biologists Limited L990 159 Brief cytochalasin-induced

Development 108, 159-172(1990)Printed in Great Britain © T h e Company of Biologists Limited L990

159

Brief cytochalasin-induced disruption of microfilaments during a critical

interval in 1-cell C. elegans embryos alters the partitioning of developmental

instructions to the 2-cell embryo

DAVID P. HILL* and SUSAN STROMEf

Department of Biology and Institute for Cellular and Molecular Biology, Indiana University, Bloomington, IN 47405, USA

'Current address: Mount Sinai Hospital Research Institute, 600 University Avenue, Toronto, Ontario M5G 1X5, CanadatTo whom reprint requests should be sent

Summary

We are investigating the involvement of the microfila-ment cytoskeleton in the development of early Caenor-habditis elegans embryos. We previously reported thatseveral cytoplasmic movements in the zygote requirethat the microfilament cytoskeleton remain intact duringa narrow time interval approximately three-quarters ofthe way through the first cell cycle. In this study, weanalyze the developmental consequences of brief, cyto-chalasin D-induced microfilament disruption during the1-cell stage. Our results indicate that during the first cellcycle microfilaments are important only during thecritical time interval for the 2-cell embryo to undergo thecorrect pattern of subsequent divisions and to initiate thedifferentiation of at least 4 tissue types. Disruption ofmicrofilaments during the critical interval results inaberrant division and P-granule segregation patterns,generating some embryos that we classify as 'reversepolarity', 'anterior duplication', and 'posterior dupli-cation' embryos. These altered patterns suggest that

microfilament disruption during the critical intervalleads to the incorrect distribution of developmentalinstructions responsible for early pattern formation. Thestrict correlation between unequal division, unequalgerm-granule partitioning, and the generation ofdaughter cells with different cell cycle periods observedin these embryos suggests that the three processes arecoupled. We hypothesize that (1) an 'asymmetry deter-minant', normally located at the posterior end of thezygote, governs asymmetric cell division, germ-granulesegregation, and the segregation of cell cycle timingelements during the first cell cycle, and (2) the integrityor placement of this asymmetry determinant is sensitiveto microfilament disruption during the critical timeinterval.

Key words: C. elegans, microfilaments, partitioning,cytochalasin.

Introduction

One mechanism that has been hypothesized to explainthe generation of cell differences in early embryos isthat developmental instructions are differentially par-titioned to embryonic cells during the early cleavages.Although microsurgical and cleavage-blocking exper-iments in a number of organisms support this hypoth-esis, the nature of the developmental instructions andthe mechanisms by which they are distributed are stillnot well understood (see Davidson, 1986 for review). InCaenorhabdhis elegans, several lines of evidence indi-cate the importance of partitioned maternal infor-mation (for review see Strome, 1989). (1) In cleavage-blocked embryos, expression of lineage-specificmarkers occurs only in the cells that would normallygive rise to the assayed lineage. This implies thatinstructions for the expression of these markers are

partitioned to the correct lineages during early embryo-genesis (Laufer et al. 1980; Cowan and Mclntosh,1985). (2) When the cytoplasmic content of earlyblastomeres is altered, either by extruding cytoplasmfrom the embryo or by mixing cytoplasm from differentblastomeres, the blastomeres' cell cycling times, cleav-age patterns, and developmental fates are altered,implying that cytoplasmic factors are responsible for thebehavior of early blastomeres (Schierenberg, 1985;1988). (3) A recently identified class of mutations,called par (for partitioning-defective) mutations, ap-pear to affect the partitioning of cytoplasmic factorsthat control cell cycling times, cell division patterns,and differentiation pathways of early blastomeres(Kemphues et al. 1988). In this paper, we present aninvestigation of the mechanism of cytoplasmic localiz-ation and the roles of some of the localized factors inearly C. elegans development.

160 D. P. Hill and S. Strome

A number of cytoplasmic rearrangements occur dur-ing the first cell cycle of C. elegans embryos. Theseinclude (1) a series of contractions of the anteriorportion of the embryo, called pseudocleavage, (Hirshetal. 1976), (2) streaming of cytoplasm toward theposterior end of the embryo (Nigon, 1960), (3) concen-tration of foci of filamentous actin in or around theanterior cortex of the embryo (Strome, 1986), (4)segregation of cytoplasmic germ-line or P granules tothe posterior cortex of the embryo (Strome and Wood,1982; Yamaguchi et al. 1983), and (5) movement of themitotic spindle toward the posterior end of the embryo(Albertson, 1984). The zygote subsequently undergoesan unequal division, generating two blastomeres thatdiffer in size, division pattern, and developmental fate(Sulston et al. 1983).

In previous studies, we determined that microfila-ments play a critical role in the cytoplasmic rearrange-ments described above, but that during this early phasemicrofilaments are required only during a short(5-10 min) interval about 3/4 of the way through thefirst cell cycle (Hill and Strome, 1988). In this study, wedemonstrate that disruption of microfilaments duringthe previously defined critical interval has profound andinteresting effects on subsequent development; treatedembryos often generate cells in a reverse polarity orduplicated pattern and fail to generate differentiatedcell types later in development. Our results suggest thatmicrofilaments participate in the segregation of devel-opmental instructions that are responsible for celldivision patterns, cell cycle timing, and P-granule segre-gation patterns. Furthermore, the segregation ofinstructions that control cell division patterns, cell cycletiming, and P-granule segregation appear to becoupled.

Materials and methods

Maintenance of C. elegansWild-type worms were grown at 16°C on NGM plates withEscherichia coli strain OP50 as a food source (Brenner, 1974).

Analysis of the development of embryos 'pulsed' withcytochalasin DThe cytochalasins disrupt the actin cytoskeleton and appear tobe relatively devoid of toxic side-effects in cells (Brenner andKorn, 1979; Schliwa, 1986; Toyama and Toyama, 1988). Wepreviously analyzed and discussed the effects of cytochalasinson the microfilament cytoskeleton and on events in the 1-cellC. elegans embryo (Strome and Wood, 1983; Hill and Strome,1988). For our analysis of embryos 'pulsed' with cytochalasinD (CD), we divided the first cell cycle into three time intervalsdefined by developmental events (Hill and Strome, 1988): (1)early, just after the completion of meiosis until the onset ofegg pronuclear migration; (2) middle, from the onset of eggpronuclear migration until pronuclear meeting; (3) late, frompronuclear meeting until late anaphase. The middle interval isthe 'critical' period, when microfilament disruption results inthe loss of zygotic asymmetry.

To disrupt microfilaments during these intervals, embryoswere dissected on a poly-lysine-coated glass slide from adulthermaphrodite gonads in a drop of egg buffer (EB; 118 ITIM-

NaCl, 40mM-KCl, 3.4mM-CaCl2, 3.4mM-MgCl2, 5mM-Hepes, pH7.4, 50% fetal calf serum; Edgar and McGhee,1986) containing 2^igml~1 CD (Strome and Wood, 1983).Embryos were overlaid with an 18x18 mm glass coverslip,with high vacuum grease applied along two edges to serve as aspacer. The embryos were permeabilized by applying gentlepressure on the coverslip. After exposure of the embryos toCD for the desired period of time, CD was removed bywicking embryonic culture medium (ECM; 90mM-NaCl,50mM-KCl, 4mgml~l hyaluronic acid, 10% fetal calf serum;Laufer et al. 1980) under the coverslip from one side to theother, using a paper towel. Success of initial permeabilizationwas judged by two criteria: (1) the altered appearance of thecytoplasm that is characteristic of CD-treated embryos(Strome and Wood, 1983), and (2) the inhibition of knownmicrofilament-dependent events (Strome and Wood, 1983).After the CD pulse, some embryos were monitored continu-ously throughout the early cell divisions. This was performedat 16°C by Nomarski differential interference contrast mi-croscopy.

The anterior-posterior orientation of embryos was deter-mined by the position of the polar body and by monitoring themigration patterns of the two pronuclei. Whether blastomeresdivided equally or unequally was judged using four criteria:(1) the movement of the cell's nucleus to the cortex and backto the center of the cell, which we have observed to precedeunequal divisions but not equal divisions, in both untreatedand manipulated embryos (Hill, Ph.D. thesis, IndianaUniversity), (2) the movement of the mitotic spindle towardone end of the cell just prior to an unequal cleavage, (3) theposition of the cleavage furrow, and (4) the sizes of the tworesulting blastomeres as judged by focusing through theembryo. Cell divisions that could not be placed with confi-dence into either the 'equal' or 'unequal' categories were notincluded in our analyses. The frequency with which CD-pulsed embryos underwent different patterns of division wascalculated from the first 50 embryos examined, after whichunusually cleaving embryos were preferentially analyzed; >10embryos from each class were analyzed.

After observing the early divisions, embryos were eitherfixed and stained for P granules (see below) or were allowedto develop overnight. For long-term incubation, the coverslipwas gently removed and the embryos were incubated in ECMat 20°C in a humid chamber. The removal of the coverslipincreased embryo viability, presumably by allowing a greaterdegree of gas exchange.

Immunofluorescent staining of P granulesEmbryos were fixed for immunofluorescent staining as de-scribed by Strome and Wood (1982). Slides with embryosattached were frozen in liquid N2, the coverslips wereremoved, and the embryos were fixed in cold methanol,followed by cold acetone, and then air dried. Staining wasperformed on fixed embryos by the procedure of Strome andWood (1982). Fixed embryos were incubated at 4°C with1.5% ovalbumin, 1.5% bovine serum albumin in PBS for30 min and then with K76, a monoclonal antibody directedagainst C. elegans P granules (Strome and Wood, 1983), for12 h at 4°C. Slides were washed with multiple changes of PBS,after which the embryos were incubated in fluorescein-labelled goat anti-mouse secondary antibody (US Biochemi-cals) in PBS for 4h at 4°C and then washed five times in PBS.DAPI was included in the third wash to stain nuclei. Thesamples were mounted in Gelvatol (Monsanto) mountingfluid.

Partitioning in C. elegans embryos 161

Assays for differentiation markersTo assay the differentiation of intestinal cells, living embryoswere observed using either epi-fluorescence microscopy orpolarization microscopy to visualize 'gut granules' (Babu,1974). Gastrulation was indicated by the internal location ofthe intestinal and germ-line progenitor (P-granule staining)cells. Muscle differentiation was analyzed by staining embryoseither with 5.6, a monoclonal anti-myosin antibody kindlyprovided by D. Miller (Miller et al. 1983), or a rabbit anti-paramyosin antiserum prepared by J. Izant and R. Knowlton(Cowan and Mclntosh, 1985). Muscle differentiation couldalso be assessed by monitoring embryos for twitching. Differ-entiation of hypoderm was assayed by staining embryos with amonoclonal antibody, MH27, kindly provided by R. Francesand R. Waterston (Priess and Hirsh, 1986); this antibody isdirected against an antigen located in the belt desmosomes ofhypodermal cells. Hypodermal differentiation could also beassessed by monitoring the smoothing of the surface of theembryo (Priess and Hirsh, 1986).

Photography and microscopyFluorescence microscopy was performed on a Zeiss ICM405inverted microscope equipped with epifluorescence optics anda 40x neofluar objective. Zygotes were examined with515-560 nm epi-illumination for rhodamine fluorescence,450-490 nm epi-illumination to visualize fluorescein, and365 nm epi-illumination to visualize DAPI and autofluor-escent gut granules. Living embryos were monitored byNomarski differential interference contrast microscopy usinga 40x planachromat objective. Photographs were taken usingKodak Tri-X film at ASA 1600. Film was developed usingDiafine two-bath developer (Acufine Inc., Chicago, IL).

Results

Microfilament disruption outside the critical timeinterval does not appear to affect subsequentdevelopmentWe previously reported that brief disruption of micro-filament structure in 1-cell C. elegans embryos eitherbefore the onset of pronuclear migration or afterpronuclear meeting (defined as the early and late timeintervals; see Materials and methods) did not affect anyof the manifestations of asymmetry normally observedin the first cell cycle (pseudocleavage, posterior meetingof the pronuclei, P-granule segregation, asymmetry ofthe mitotic spindle, and the generation of two different-sized blastomeres) (Hill and Strome, 1988). To deter-mine whether or not proper microfilament structure isrequired during the early and late time intervals forcorrect developmental programming, we pulsed 1-cellembryos with the microfilament inhibitor, cytochalasinD (CD), during these time intervals. The developmen-tal fates of the two blastomeres were then analyzed bymonitoring subsequent cell division patterns and byassaying for cell differentiation markers. The blasto-meres divided in the same order and with the samepatterns as the blastomeres in untreated embryos(Figs 1,2). The embryos initiated morphogenesis anddeveloped to at least the 'lima bean' stage of embryo-genesis, and sometimes continued as far as the late'comma' stage (Fig. 3) (for a review of C. elegansembryogenesis, see Wood, 1988). Embryos permeabil-

Fig. 1. Nomarski micrographs showing the cleavageorientations during division of a normal 2-cell C. elegansembryo. Anterior is to the left, and posterior is to the right.(A) The anterior cell (AB) divides first. Its spindle isoriented perpendicularly to the anterior-posterior axis.(B) The posterior cell (PI) divides after the anterior cell. Itundergoes an unequal division along the anterior-posterioraxis. (C) A 4-cell embryo. Bar=10fjm.

ized in culture medium lacking CD also arrested at thelima bean or comma stage, indicating that the inabilityto develop further is not a consequence of CD treat-ment. The CD-treated embryos segregated P granulesproperly, gastrulated, expressed gut-, hypoderm- andmuscle-specific markers correctly (Fig. 3), and twitchedat the late 'comma' stage, indicative of formation of


Fig. 2. Nomarski micrographs showing the cleavageorientations in an embryo pulsed with CD during the earlytime interval. Anterior is to the left, and posterior is to theright. (A) The anterior cell divides first. Its spindle isoriented perpendicularly to the anterior-posterior axis.(B) The posterior cell divides after the anterior cell andalong the anterior-posterior axis. (C) A 4-cell embryo.Bar=10/


(like Po or PI, but not like P2 or P3; Schierenberg,1987) suggesting a reiteration of the Po or PI celldivision pattern.

The third pattern will be referred to as 'anterior

duplication' (Fig. 7). In these embryos, both cells of the2-cell embryo appeared to cleave symmetrically andperpendicularly to the anterior-posterior axis (similarto AB). Embryos of this class are difficult to analyze

Fig. 3. Apparently normaldevelopment of embryos pulsed withCD outside the critical time interval.Panels on the left are untreatedcontrol embryos. Panels on the rightare embryos that were pulsed withCD during the late time interval.Anterior is to the left, and posterior isto the right. (A,B) Nomarskimicrographs of embryos at the late'lima bean' stage of embryogenesis.(C,D) Immunofluorescent staining of Pgranules in 'lima bean' stage embryosshowing two internal, P-granule-positive cells. (E,F) Polarizationmicrographs of the embryos shown inpanels A and B, respectively. Bothembryos contain internal birefringentgut granules. (G,H) Confocal laserscanning micrographs of 'comma' stageembryos stained with MH27, amonoclonal antibody that recognizeshypodermal belt desmosomes.(I,J) Confocal laser scanningmicrographs of 'comma' stage embryosstained with 5.6, a monoclonalantibody against myosin. Bar=10^m.


Fig. 4. Nomarski micrographs showing 'normal' division ofan embryo treated with CD during the critical time interval.Anterior is to the left, and posterior is to the right.(A) 2-cell stage. (B) The anterior cell divided equally andperpendicularly to the anterior-posterior axis, while theposterior cell divided unequally along the anterior-posterioraxis. (C) This embryo continued to develop to a fewhundred cells. Bar=10/«n.

because as the 2-cell embryo divides, the dividing cellsoften rearrange in the eggshell making it difficult todiscriminate between symmetric and asymmetric div-ision, as well as to determine the true cleavage orien-tation. After the rearrangement it is difficult to inter-pret the subsequent cleavage orientations of theblastomeres. However, the P-granule staining patternsdescribed below provide evidence that this 'anteriorduplication' class of embryos exists.

Alterations in cell-cycle timingThe early blastomeres in C. elegans embryos havecharacteristic cell cycling periods (Deppe et al. 1978).

Fig. 5. Nomarski micrographs showing 'reverse polarity'division of an embryo treated with CD during the criticaltime interval. Anterior is to the left, and posterior is to theright. (A) 2-cell stage. (B) The anterior cell dividesunequally along the anterior-posterior axis, while theposterior cell divides equally and perpendicularly to theanterior-posterior axis. (C) The 4-cell embryo resembles a'reversed' control embryo. (D) This embryo developed to afew hundred cells. Note that the surface of the embryo isnot smooth, indicating a failure to correctly differentiatehypoderm. Bar=10jtm.

The sequence of cell divisions probably plays an import-ant role in generating cells in the correct relative spatialpositions within the eggshell (Schierenberg, 1985).Embryos pulsed with CD during the critical timeinterval, but not those pulsed outside the criticalinterval, showed altered cell cycle periods, which oftenled to an altered arrangement of cells in the 4-cell


Fig. 6. Nomarski micrographs showing a 'posteriorduplication' embryo treated with CD during the criticaltime interval. Anterior is to the left, and posterior is to theright. (A) 2-cell stage. (B) The posterior cell dividesunequally along the anterior-posterior axis. (C) Theanterior cell also divides unequally along the anterior-posterior axis. (D) 4-cell stage. The cells have not yetrearranged in the eggshell and remain in a line. (E) Theembryo in panel D continued to divide to a few hundredcells. The focal plane of this embryo is at the embryo'ssurface. Note that it is rough, indicating an absence ofhypodermal differentiation. Bar=10^m.

embryo. In control embryos (exposed to ECM lackingCD), the anterior cell of the 2-cell embryo dividedbetween 2.5 and 5.9min before the posterior cell(mean=4±1.2min). In embryos treated with CD dur-ing the critical interval, we observed variability in the

Fig. 7. Nomarski micrograph showing an 'anteriorduplication' embryo treated with CD during the criticaltime interval. Anterior is to the left, and posterior is to theright. (A) 2-cell stage. (B) Both cells divided equally andperpendicularly to the anterior-posterior axis. This embryoresembles a normal embryo at the 4-cell stage (see Fig. 1C).The assignment of this embryo to the symmetric class wasconfirmed by P-granule staining (Fig. 8D). Bar=10^m.

relative cell cycle timing of the blastomeres of the 2-cellembryo. The anterior cell divided as much as 6.3 minbefore the posterior cell or as much as 5.2min after theposterior cell (mean=0.8±3.1min, where a positivevalue indicates that the anterior cell divided before theposterior cell). The variability of cell cycle timing in the2-cell embryos did not correlate with cell size. That is,treating embryos with CD during the critical intervalcauses the zygote to divide symmetrically or withvariable asymmetry (Hill and Strome, 1988). Thesmaller of the two resulting daughter cells did notalways have the longer cell cycle, as is seen in untreatedembryos (Deppe et al. 1978). These results are consist-ent with the hypothesis of Schierenberg (1984) that cell


cycling time is controlled by the quality of the cytoplasmrather than cell size.

However, after the defects in relative cell cycle timingobserved in the 2-cell embryo generated after CDtreatment, the 4-cell embryo regained normal patternsof relative cell cycling time. That is, after an asymmetricdivision, the smaller of the two daughter cells had alonger cell cycle and, after a symmetric division, the twodaughter cells had approximately equal cell cyclingtimes.

Asymmetric divisions are accompanied byappropriately asymmetric P-granule partitioning

In untreated C. elegans embryos, the asymmetric div-isions of the P-cell lineage are accompanied by thepartitioning of cytoplasmic germ granules, called Pgranules, to the smaller of the two daughter cells(Strome and Wood, 1982). Based on the sensitivity ofP-granule segregation to microfilament inhibitors, thesegregation of P granules is thought to be a microfila-ment-mediated process (Strome and Wood, 1983).When microfilaments are disrupted during the criticaltime interval during the 1-cell stage, P granules aredistributed to both cells of the 2-cell embryo (Hill andStrome, 1988).

To determine the behavior of P granules duringsubsequent divisions, we analyzed the distribution of Pgranules in 4-cell embryos of each of the divisionpatterns described above. In embryos treated with CD,P-granule segregation and asymmetric cell division arecoupled. The most dramatic example of this is seen in'posterior duplication' embryos. In 3/3 'posterior dupli-cation' embryos, both blastomeres of the 2-cell dividedasymmetrically and segregated P granules to the smallcells generated at the ends of the embryo (Fig. 8A). Pgranules were not seen in the two larger internalblastomeres (Fig. 8A). In 6/6 'normal' embryos, onlythe cell that divided asymmetrically partitioned P gran-ules, and did so to its smaller daughter (Fig. 8B). The Pgranules in the cell that divided symmetrically weredistributed to both daughter cells (Fig. 8B), and wereoften faint and perinuclear. In the 2/2 'reverse polarity'embryos in which P-granule staining was seen, Pgranules were observed only in the small anterior celland were not visible in any of the other three cells of theembryo (Fig. 8C). (In two other 'reverse polarity'embryos, P granules were not visible in any cells of the4-cell embryo.) In 2/2 'anterior duplication' embryos, Pgranules were found in all 4 cells of the 4-cell embryo(Fig. 8D). Thus, in a total of 14/16 cases in which a cellunderwent an asymmetric division, P granules weresegregated to the smaller daughter; in the other twocases, no P-granule staining was observed. In 6/10 casesin which a cell underwent a symmetric division, Pgranules were distributed to both daughters; in theother four cases there was no observable P-granulestaining. Therefore, among 20 cells whose daughtersstained positively for P granules, there were no excep-tions to the rule that P granules are segregated to thesmaller daughter during an asymmetric cell division and

Table 1. Expression of differentiation markers inembryos treated with CD during the early, middle and

late intervals in the first cell cyclePulse Expression of markers for:time Embryo class Development Hypoderm Gut Muscle

NoneEarlyLateMiddleMiddle

Middle

Middle

NormalNormalNormalNormalReverse

poianiyPosterior

duplicationAnterior

duplication

NormalNormalNormalVariableAbnormal

Abnormal

-

22/2215/156/10

0*/5

lt/4

—

100%22/2215/159/121/5

0/5

-

22/2215/152/50/5

0/5

-

•0/5 embryos showed staining, but 3/5 had a smooth surface.11/4 embryos showed staining, but 0/5 had a smooth surface.

(One of the embryos whose surface was smooth was lost beforestaining with the hypodermal antibody was complete.)

are distributed to both daughters during a symmetricdivision.

Analysis of tissue differentiation in CD-pulsedembryos

To try to determine whether disruption of microfila-ments during the critical time interval in the 1-cell stageperturbs the segregation of developmental potential,CD-pulsed embryos were allowed to develop for12-14 h, and then a number of tissue-specific andmorphological markers were used to determine whattissue types had differentiated in these treated embryos.Embryos that showed abnormal early cell divisions (5/5'reverse polarity' and 5/5 'posterior duplication' em-bryos) also displayed profound defects in differen-tiation. The embryos continued to divide to severalhundred cells by the end of 14 h, but very few of themdisplayed differentiation markers (Table 1). None ofthe embryos underwent morphogenesis into a lima beanshape or showed any signs of elongation into a worm.Because of the difficulty (described above) in assigningembryos to the 'anterior duplication' class, these em-bryos were not included in the analysis of differen-tiation markers.

Fig. 8. Nomarski and immunofluorescence micrographs ofP-granule staining in embryos from each of the cleavagepatterns observed after a CD pulse in the critical timeinterval. Anterior is to the left, and posterior is to the right.(A) A 'posterior duplication' embryo. Both cells of thisclass of embryos undergo an asymmetric division. In thisembryo, P granules have been segregated to the two endcells (see Fig. 6). (B) A 'normal' embryo. In these embryos,only the posterior cell undergoes an asymmetric division(see Fig. 4). In this embryo, P granules were observed inthe small posterior-most cell and in the two large anteriorcells. (C) A 'reverse polarity' embryo. In these embryosonly the anterior cell undergoes an asymmetric division (seeFig. 5). In this embryo, P granules were observed only inthe anterior-most cell. (D) An 'anterior duplication'embryo. In these embryos, neither of the cells undergoes anasymmetric division (see Fig. 7). In this embryo, P granuleswere seen in all four cells of the embryo. Bar=10//m.


4+


Fig. 9. Nomarski and fluorescence micrographs illustrating aberrant development of two embryos that underwent a 'normal'early division pattern after a CD pulse in the critical time interval. Anterior is to the left, and posterior is to the right.(A) Nomarski micrograph of an embryo containing a few hundred cells that shows no signs of morphogenesis.(B) Polarization micrograph of the embryo in panel A showing birefringent gut granules along the posterior edge of theembryo. (C) Immunofiuorescence micrograph of the embryo in A shows P granules are localized in two cells at the edge ofthe embryo. Panels B and C demonstrate that this embryo failed to gastrulate. (D) Nomarski micrograph of an embryocontaining a few hundred cells that has undergone abnormal morphogenesis. The anterior end of the embryo has failed toelongate correctly. (E) Polarization micrograph of the embryo in D showing that gut-granule-positive cells are in the properlocation in the interior of the embryo. (F) P-granule staining of the embryo shown in D. P granules are localized in twointernal cells. Bar=10//m.

In CD-pulsed embryos that underwent apparentlynormal early cell divisions, a wide variety of differen-tiation 'phenotypes' were observed (Table 1). Twelve'normal' embryos were analyzed. Three embryosshowed apparently normal differentiation of both gutand hypoderm, contained two internal P-granule-stain-ing cells, and appeared to undergo normal developmentto at least the lima bean stage; one of these developedto the comma stage and displayed muscle differen-tiation as assayed by twitching. Two other embryos

showed differentiation of gut and hypoderm, but under-went abnormal morphogenesis; one did not appear tohave gastrulated correctly, while the other had gastru-lated but underwent abnormal elongation (Fig. 9). Thelatter embryo also twitched, suggesting the formation ofmuscle tissues. The remainder of the embryos displayedvarying degrees of differentiation. Of the remainingseven embryos, all were assayed for gut granules, sixwere assayed for the position of P-granule staining cells,and five were assayed for hypodermal differentiation.

Three out of seven failed to express gut granules, andfour showed gut-granule differentiation in the wrongarea of the embryo, presumably due to failure of theintestinal precursor cells to invaginate during gastru-lation (Deppe et al. 1978). Three of these apparentlygastrulation-defective embryos also containedP-granule staining cells that were not positioned inter-nally. The other three embryos assayed for P granulesdid not show any visible staining. Four out of fiveembryos failed to show hypodermal differentiation, asassayed by either the tissue-specific marker or bysmoothing of the surface of the embryo; one embryoshowed some abnormal surface smoothing but did notadopt the typical lima bean shape. These results indi-cate that disruption of microfilaments during the criticaltime interval generally results in the disruption ofdifferentiation in the embryo.

Discussion

The results that we present in this study support thehypothesis that developmental instructions or factorsthat govern cell division and P-granule segregationpatterns and cell cycle timing are partitioned to earlyblastomeres in C. elegans embryos (Wood et al. 1984;Schierenberg, 1985; Schierenberg and Wood, 1985).Furthermore, our results suggest that the microfila-ment-based cytoskeleton plays an important role in thepartitioning process and that microfilament partici-pation is only required during a short period approxi-mately 3/4 of the way through the first cell cycle of theembryo. When microfilaments are disrupted outsidethis critical period and are then allowed to recover, thezygote divides into a normal looking 2-cell embryo, andboth daughter blastomeres behave like the blastomeresin untreated embryos. These embryos continue todevelop apparently normally and express the correcttissue-specific differentiation products at the correctpositions in the embryo. In contrast, when the micro-filament cytoskeleton is disrupted during the criticaltime interval, the zygote often undergoes a symmetricfirst division (Hill and Strome, 1988), and the resultingblastomeres often divide and segregate germ granulesin the pattern characteristic of their sister cell in anuntreated embryo (see Fig. 10 for a schematic sum-mary).

Schierenberg (1985) previously reported that thepotential for a cell to undergo an asymmetric division islocalized at the posterior end of the zygote before thecritical time interval defined by our experiments. Thispotential appears to be passed to the posteriordaughter, PI, and then to the P-cell daughters gener-ated by each unequal cell division. Our results suggestthat after brief disruption of microfilaments during thecritical interval, this potential for unequal division or'asymmetry determinant' can be inherited by either theanterior or posterior cell or in some cases by both cellsof the 2-cell embryo. One model that is consistent withour data is that, during the critical time interval, theasymmetry determinant is stabilized at the posterior


Untreated

CD-pulsed

Fig. 10. Schematic representation of the four cleavagepatterns observed after a CD pulse during the critical timeinterval. Anterior is to the left, and posterior is to the right.The dots illustrate the distribution of P granules in thesetypes of embryos.

Untreated

wild-type

"normal"

"reverse polarity"

• "posteriorduplication"

"anteriorduplication"

Fig. 11. A model describing the behavior of the proposedasymmetry determinant during and after a CD pulse in thecritical time interval. The asymmetry determinant (stippledarea) is localized at the posterior end of wild-type embryos.It is hypothesized to (1) cause cells to undergo an unequalcell division, (2) cause the segregation of P granules to thesmaller daughter, and (3) participate in the segregation offactors that affect cell cycle timing. According to ourmodel, the asymmetry determinant is disrupted or displacedby disruption of the microfilament cytoskeleton during thecritical interval. It then may function in the posterior cell,the anterior cell, or both cells to generate a 'normal','reverse polarity', or 'posterior duplication' embryo,respectively. It may also be destroyed or inactivated bymicrofilament disruption, leading to the 'anteriorduplication' class of embryos.

end of the embryo by microfilaments, and that disrup-tion of microfilaments allows it to shift in position (seeFig. 11). In most cases (62%), disruption of microfila-ments during the critical interval does not disrupt theasymmetry determinant severely enough to cause it to


be inherited by the anterior daughter cell. As a result,the blastomeres of the 2-cell embryo undergo thecorrect pattern of cell divisions. However, in theremaining 38 % of the cases, the asymmetry determi-nant may shift in the cytoplasm or along the membraneof the zygote and become distributed to the anterior orto both blastomeres of the 2-cell embryo. This wouldresult in either the anterior cell or both cells undergoingthe series of asymmetric divisions characteristic of theposterior cell in an untreated embryo. The possibilityalso exists that the asymmetry determinant cannotrecover after its disruption, resulting in neither cellundergoing an asymmetric cell division.

Another possible explanation for the variety of cleav-age patterns that we see in CD-pulsed embryos is thatembryos are actually exposed to slightly different con-centrations of CD. Since our permeabilization pro-cedure is a manual disruption of the eggshell and innervitelline membrane, it is possible that the disruption ismore severe in some cases than in others. This couldlead to variability in the actual concentration of inhibi-tor that each embryo is exposed to. However, we thinkthat this possibility is unlikely, since different concen-trations of CD in the buffer do not lead to variability inthe results, and the concentration of CD that we usedfor most of the experiments leads to an apparentlycomplete disruption of the microfilament cytoskeleton,as judged by rhodamine-phalloidin staining (Hill andStrome, 1988). We also examined the effect of exposingembryos to CD for different periods of time during thefirst cell cycle. As long as the entire critical interval iscontained within the pulse, the embryos display thesame range of phenotypes.

In normal embryos, each germ-line or P cell under-goes an unequal division and partitions P granules tothe small P-cell daughter (Strome and Wood, 1982,1983). Our results provide two pieces of informationabout the relationship between P-granule segregationand unequal divisions: unequal division and unequalP-granule partitioning appear to be coupled, but thepresence of P granules in a blastomere does not insurethat the blastomere will undergo an unequal division.That is, after microfilament disruption during the 1-cellstage, both blastomeres contain P granules (Hill andStrome, 1988). In most cases only one of the twoblastomeres divided unequally, suggesting that P gran-ules do not cause unequal division. However, in allblastomeres that underwent an unequal division, Pgranules had been partitioned to the smaller daughtercell. Similarly, in all blastomeres that underwent asymmetric division, P granules were observed in bothdaughters. The only exceptions to this were two em-bryos that failed to stain for P granules altogether. Thisresult suggests that unequal divisions and P-granulepartitioning are mechanistically coupled. At least twopossibilities exist to explain this coupling: (1) Theasymmetry determinant that participates in positioningthe mitotic spindle is also the target site for P-granulesegregation. (2) The segregation of P granules to theposterior cortex of the embryo may be a necessaryevent in the functioning of the asymmetry determinant.

If this event does not occur, then the spindle does notassume the correct asymmetric position in the cell.

Our data also show that microfilament disruptionaffects cell cycle timing. It has already been proposedthat the division times of C. elegans blastomeres aredetermined by the quality and not the quantity of eachcell's cytoplasm (Schierenberg, 1985). Our results areconsistent with this hypothesis, since we do not observea correlation between cell size and cell cycle timing inthe two unusually sized blastomeres generated by a CD-pulsed zygote. However, after the 2-cell stage, when acell underwent an asymmetric division, the smaller ofthe two cells had a slower cell cycle period, as observedin untreated embryos. These results suggest that theelements that control cell cycle timing are randomlysegregated during the first division after CD treatment,but that during subsequent divisions they are correctlypartitioned during asymmetric cell divisions. This latterfinding is similar to the one described for P granules, inthat the proper segregation of cell cycle control el-ements appears to be coupled to asymmetric division.One attractive hypothesis to explain the coupling ofthese three processes is that they are all controlled bythe asymmetry determinant that confers the ability toundergo unequal division.

The existence of the proposed asymmetry determi-nant may explain the patterns of division and P-granulesegregation observed in C. elegans embryos after centri-fugation during the first cell cycle (Cowan and Mcln-tosh, unpublished). The 2-cell embryos that were ana-lyzed after centrifugation displayed a range of divisionand P-granule segregation patterns similar to those thatwe have described for CD-pulsed embryos. Thus centri-fugation may mechanically displace or disrupt theasymmetry determinant. In addition, the existence ofan asymmetry determinant could explain the pheno-types of the par mutants (see Introduction). At leastsome of the par genes may encode elements of theasymmetry determinant or cytoskeletal components,for instance microfilament-associated proteins, in-volved in positioning it. Interestingly, the embryosproduced by par-3 worms display division patternssimilar to those seen in CD-treated embryos(Kemphues et al. 1988; K. Kemphues, personal com-munication), making par-3 a good candidate for a genethat affects the positioning of the asymmetry determi-nant.

Our results show that disruption of microfilamentsduring the critical time interval leads not only to effectson early division and partitioning patterns, but also tosevere defects in subsequent development of the em-bryo. The severity of these developmental defectsappears to correlate with the severity of the abnormali-ties observed in the 2-cell embryo. A few embryos thatappeared normal at the 2-cell stage underwent appar-ently normal differentiation events and limited morpho-genesis of the embryo. Other 'normal' 2-cell embryosand all of the 'reverse polarity' and 'posterior dupli-cation' embryos displayed defects in differentiation andmorphogenesis. There are several possible explanationsfor our results. (1) Differentiation may be prevented or


extinguished by inhibitory factors that in normal em-bryos are segregated away from the appropriate blasto-meres, but that fail to be partitioned after microfila-ment disruption during the critical interval. Asdiscussed by Cowan and Mclntosh (1985), the presenceof inhibitory factors may also explain the results of theiranalysis of C. elegans blastomeres cleavage-blockedwith cytochalasin B. Although such cleavage-blockedblastomeres are known to contain the potential toexpress multiple differentiation programs, they eitherfailed to express any differentiation markers or onlyexpressed markers for one differentiation program. (2)Microfilament disruption may interfere with the par-titioning of somatic lineage-specific factors that mustreach a critical threshold concentration for activity. (3)Alterations in cell patterning, caused by the alteredpatterns of cell division and altered cell cycle times, mayinterfere with cell-cell interactions and/or the gener-ation of spatial cues that normally influence differen-tiation. (4) Embryos may be generally 'sick' aftermicrofilament disruption. However, such sickness onlyoccurs in embryos treated with CD during the criticalinterval; embryos pulsed with CD outside the criticalinterval develop apparently normally. In any case, ourresults imply that the potential to undergo gut, muscleand hypodermal differentiation is sensitive to microfila-ment disruption during the critical time interval, longbefore these differentiation pathways are initiated.

It has previously been reported that the polarity ofinsect embryos can be perturbed during early embryo-genesis (see Kalthoff, 1979 for review). In many of theresulting double cephalon, double abdomen and re-verse polarity embryos, differentiation appears to occurfairly normally, as if the perturbation has simply alteredbody patterning. The differences between these resultsand ours in C. elegans may be attributable to differencesin the manipulations (CD pulse in C. elegans versusirradiation, centrifugation and cytoplasmic transfer indiptera), or they may reflect fundamental differences inthe mechanisms of cell-fate specification in these organ-isms. In insects, there is good evidence that gradients ofinformation present in the early embryo are responsiblefor specifying axis position and developmental decisions(Driever and Nusslein-Volhard, 1988a, 19886; Stewardetal. 1988). When the distribution of that information isperturbed, differentiation still occurs, but with incor-rect spatial patterning, suggesting that the specificationof cell fates has a great deal of plasticity. As discussedearlier, several lines of evidence suggest that in C.elegans developmental decisions may be specified by avariety of factors, all of which must be partitionedcorrectly at each of the early embryonic divisions. Ourresults suggest that partitioning during the first celldivision is extremely important. Embryos in which thispartitioning is perturbed are not able to regulate, anddevelopment proceeds aberrantly. Our results alsosuggest that microfilaments are important during thecritical time interval for the organization and/or distri-bution of developmental instructions, and that certaininstructions are partitioned as a unit (the instructionsfor asymmetric cell division, P-granule segregation, and

cell cycling times), while others (differentiation instruc-tions) are likely to be separately controlled. This resultsin cells of the early embryo displaying characteristicdivision and P-granule segregation patterns, but failingto display the expected differentiation patterns later indevelopment. These observations suggest that the'mosaicism' thought to exist in the C. elegans egg is notthe result of a single mechanism, but rather results fromseveral different mechanisms, each responsible for asubset of developmental instructions.

We thank Drs Ann Cowan, J. Richard Mclntosh andWilliam B. Wood for their critical reading of the manuscript.This work was supported by Research Grant GM34059 and byPredoctoral Training Grant GM07227-12 from the US PublicHealth Service.

References

ALBERTSON, D. G. (1984). Formation of the first cleavage spindle innematode embryos. Devi Biol. 101, 61-72.

BABU, P. (1974). Biochemical genetics of Caenorhabditis elegans.Mol. gen. Genet. 135, 39-44.

BRENNER, S. (1974). The genetics of Caenorhabditis elegans.Genetics 77, 71-94.

BRENNER, S. L. AND KORN, E. D. (1979). Substoichiometricconcentrations of cytochalasin D inhibit actin polymerization.J. biol. Chem. 254, 9982-9985.

COWAN, A. E. AND MCINTOSH, J. R. (1985). Mapping thedistribution of differentiation potential for intestine, muscle, andhypodermis during early development in Caenorhabditis elegans.Cell 41, 923-932.

DAVIDSON, E. H. (1986). Gene Activity in Early Development, pp.409-524. Academic Press, Orlando.

DEPPE, U., SCHIERENBERG, E., COLE, T., KRIEG, C , SCHMITT, D.,YODER, B. AND VON EHRENSTEIN, G. (1978). Cell lineages of theembryo of the nematode Caenorhabditis elegans. Proc. natn.Acad. Sci. U.S.A. 75, 376-380.

DRIEVER, W. AND NUSSLEIN-VOLHARD, C. (1988a). A gradient ofbicoid protein in Drosophila embryos. Cell 54, 83-93.

DRIEVER, W. AND NUSSLEIN-VOLHARD, C. (19886). The bicoidprotein determines position in the Drosophila embryo in aconcentration-dependent manner. Cell 54, 95-104.

EDGAR, L. G. AND MCGHEE, J. D. (1986). Embryonic expression ofa gut-specific esterase in Caenorhabditis elegans. Devi Biol. 114,109-118.

HILL, D. P. AND STROME, S. (1988). An analysis of the role ofmicrofilaments in the establishment and maintenance ofasymmetry in Caenorhabditis elegans zygotes. Devi Biol. 125,75-84.

HIRSH, D., OPPENHEIM, D. AfoD KLASS, M. (1976). Development ofthe reproductive system of Caenorhabditis elegans. Devi Biol. 49,200-219.

KALTHOFF, K. (1979). Analysis of a morphogenetic determinant inan insect embryo (Smittia Spec, Chironomidae, Diptera). InDeterminants of Spatial Organization (ed. S. Subtelny and I.R.Konigsberg), pp. 97-126. Academic Press, New York.

KEMPHUES, K. J., PRIESS, J. R., MORTON, D. G. AND CHENG, N.

(1988). Identification of genes required for cytoplasmiclocalization in early C. elegans embryos. Cell 52, 311-320.

LAUFER, J. S., BAZZICALUPO, P. AND WOOD, W. B. (1980).

Segregation of developmental potential in early embryos ofCaenorhabditis elegans. Cell 19, 569-577.

MILLER, D. M., ORITZ, I., BERLINER, G. C. AND EPSTEIN, H. F.

(1983). Differential localization of two myosins within nematodethick filaments. Cell 34, 477-490.

NIGON, V., GUERRIER, P. AND MONIN, H. (1960). L'architecture

polaire de l'oeuf et les mouvements des constituants cellulairesau cours des premieres etapes du developpement chez qeulquesnematodes. Bull. Biol. Fr. Belg. 94, 131-202.


PRIESS, J. R. AND HIRSH, D. I. (1986). Caenorhabdiris elegansmorphogenesis: The role of the cytoskeleton in elongation of theembryo. Devi Biol. 117, 156-173.

SCHIERENBERG, E. (1984). Altered cell division rates after laser-induced cell fusion in nematode embryos. Devi Biol. 101,240-245.

SCHIERENBERG, E. (1985). Cell determination during earlyembryogenesis of the nematode Caenorhabditis elegans. ColdSpring Harbor Symp. quant. Biol. 50, 59-68.

SCHIERENBERG, E. (1987). Reversal of cellular polarity and earlycell-cell interaction in the embryo of Caenorhabdilis elegans.Devi Biol. 122, 452-463.

SCHIERENBERG, E. (1988). Localization and segregation of lineage-specific cleavage potential in embryos of Caenorhabditis elegans.Rouxs Arch. Devi Biol. 197, 282-293.

SCHIERENBERG, E. AND WOOD, W. B. (1985). Control of cell-cycletiming in early embryos of Caenorhabditis elegans. Devi Biol.107, 337-354.

SCHLIWA, M. (1986). The Cytoskeleton, An Introductory Survey,pp. 7-17. Springer-Verlag, New York.

STEWARD, R., ZUSMAN, S. B., HUANG, L. H. AND SCHEDL, P.

(1988). The dorsal protein is distributed in a gradient in earlyDrosophila embryos. Cell 55, 487-495.

STROME, S. (1986). Fluorescence visualization of the distribution ofmicrofilaments in gonads and early embryos of the nematodeCaenorhabditis elegans. J. Cell Biol. 103, 2241-2252.

STROME, S. (1989). Generation of cell diversity during earlyembryogenesis in the nematode Caenorhabditis elegans. Int. Rev.Cytol. 114, 81-123.

STROME, S. AND WOOD, W. B. (1982). Immunofluorescencevisualization of germ-line-specific cytoplasmic granules inembryos, larvae, and adults of Caenorhabditis elegans. Proc.natn. Acad. Set. U.S.A. 79, 1558-1562.

STROME, S. AND WOOD, W. B. (1983). Generation of asymmetryand segregation of germ-line granules in early C. elegansembryos. Ceil 35, 15-25.

SULSTON, J. E., SCHIERENBERG, E., WHITE, J. G. AND THOMSON, J.

N. (1983). The embryonic cell lineage of the nematodeCaenorhabditis elegans. Devi Biol. 100, 64-119.

TOYAMA, S. AND TOYAMA, S. (1988). Functional alterations in beta-actin from a KB cell mutant resistant to cytochalasin B. J. CellBiol. 107, 1499-1504.

WOOD, W. B. (1988). Embryology. In The NematodeCaenorhabditis elegans (ed. W. B. Wood), pp. 215-242. ColdSpring Harbor, New York.

WOOD, W. B., SCHIERENBERG, E. AND STROME, S. (1984).

Localization and determination in early embryos ofCaenorhabditis elegans. In Molecular Biology of Development(ed. E. H. Davidson and R. A. Firtel), pp. 37-49. Alan R. Liss,Inc., New York.

YAMAGUCHI, Y., MURAKAMI, K., FURUSAWA, M. AND MIWA, J.

(1983). Germline-specific antigens identified by monoclonalantibodies in the nematode Caenorhabditis elegans. Dev. GrowthDiffer. 25, 121-131.

{Accepted 29 September J989)

Documents

Brief cytochalasin-induced disruption of microfilaments …...Development 108, 159-172(1990) Printed in Great Britain ©The Company of Biologists Limited L990 159 Brief cytochalasin-induced