DEVELOPMENTAL

BIOLOGY

Mutations

142,203-215 (1990)

in the par Genes of Caenorhabditis elegans Affect Cytoplasmic Reorganization during the First Cell Cycle COLLEEN KIRBY,’ Section

of Genetics

MEREDITH

and Development,

KLJSCH,’ AND KENNETH Cornell

Accepted

July

University, Ithaca,

KEMPHUES New

York

14853

23, 1990

A dramatic reorganization of cytoplasm occurs during the first cell cycle in embryos of the nematode, Cae-norhabditis elegans. We present here the results of a quantitative study of some of the events during this reorganization in wild-type embryos and in par mutant embryos. The par mutations define a set of genes required for cytoplasmic. localization in early embryos. We show that par mutations lead to defects in several events of the reorganization. Mutations in all four of the par genes we studied lead to defects in pseudocleavage and asymmetric redistribution of cortical microfilaments. In addition, some of the par mutations affect streaming of cytoplasm, migration of the pronuclei, and asymmetric shortening of the embryo. We propose that the major function of the par genes might be to orchestrate this initial reorganization of cytoplasm. 0 1990 Academic Press, Inc.

ments during the first cell cycle. Exposure to cytochalasin D, as a pulse during a critical period (from about 0.7 to 0.85 of the first cell cycle), or continuously starting before or during that period, alters or blocks all aspects of cytoplasmic reorganization including the localization of P granules to the posterior (Hill and Strome, 1988; Strome and Wood, 1983). This reorganization appears to be required for establishing embryonic anterior-posterior polarity and may also be involved in localization or activation of cytoplasmic determinants. Pulses of cytochalasin during the period of sensitivity for P granule localization also prevent the asymmetric placement of the first cleavage spindle (Hill and Strome, 1988), and often alter subsequent cleavage patterns (Hill and Strome, 1990). Cytoplasmic movements during the first cell cycle might also be responsible for localizing or activating determinants of the intestinal lineage. Cytoplasm mixing experiments have demonstrated that the posterior blastomere of a two-cell embryo (the Pl blastomere) contains determinants specifying intestinal differentiation (Schierenberg, 1985). These determinants, presumably present in the one-cell embryo, may need to be activated during the first cell cycle. Embryos that are cleavage blocked by continuous exposure to cytochalasin from the two-cell stage often synthesize intestine-specific molecular markers, while cleavage-blocked one-cell embryos do not (Cowan and McIntosh, 1985; Edgar and McGhee, 1986; Laufer et ah, 1980). Thus, the microfilament-mediated events of cytoplasmic reorganization, or the first cleavage itself, are required for the expression of the intestinal lineage potential. One approach to the study of cytoplasmic localization is the isolation and analysis of mutations that are defec-

INTRODUCTION

Many animal embryos undergo dramatic reorganizations of cytoplasm during the first cell cycle. Although the function (or functions) of the reorganizations has not been firmly established in every case, they appear to be necessary for localizing or activating cytoplasmic determinants, maternally provided factors conferring lineage-specific or region-specific developmental properties (reviewed in Davidson, 1988). Such a reorganization of cytoplasm occurs midway through the first cell cycle in embryos of the nematode Caenm-habditis elegans. The visible features of the cytoplasmic reorganization include anterior cortical contractions, streaming of cytoplasm from anterior to posterior, a prominent partial cleavage near the center of the embryo (pseudocleavage), and asymmetric migration of the pronuclei to meet in the posterior (Albertson, 1984; Hirsh et al., 1976; Nigon et ab, 1960). These movements are roughly coincident with a transient increase in the relative concentration of microfilaments in the anterior cortex (Strome, 1986) and the localization of germ-linespecific granules (P granules) to the posterior cortex (Strome and Wood, 1982,1983). The function of P granules is unknown, but their segregation pattern is consistent with the behavior of proposed germ-line determinants. It is unclear which, if any, of the visible cytoplasmic changes are required for localization of P granules. All of the events of the cytoplasmic reorganization as well as localization of P granules require intact microfilai These authors contributed alphabetically.

equally to this analysis and are listed

203

0012-1606/90 $3.00 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

204

DEVELOPMENTAL

BIOLOGY

tive in cytoplasmic partitioning in the early cleavage stages. Hermaphrodites homozygous for mutations in four maternally expressed genes, par-l, par-2, par-3, and par-h, produce embryos that often have equal first cleavages, altered subsequent cleavage patterns and cell lineages, and defects in localization of P granules (Hirsh et ah, 1985; Kemphues et ab, 1988). Differentiation of the intestine and the germ line seems particularly sensitive to mutations in the par genes. These phenotypes have been interpreted as indicating that the par genes are required for proper cytoplasmic localization (Kemphues et ab, 1988). We have examined the role of the par genes in this reorganization in an effort to better understand the relationships of the observed events of the first cell cycle to each other and to the localization or activation of cytoplasmic determinants. If the cytoplasmic reorganization acts to localize determinants, we thought it likely that some of the par mutations would exhibit defects in reorganization; analysis of such defects would provide insight into the mechanism of action of the par genes and might help to establish functional relationships between early embryonic events. In this paper, we report that mutations in all four par genes lead to gene-specific defects in at least two aspects of cytoplasmic reorganization: pseudocleavage and transient redistribution of cortical microfilaments. We also report evidence for a functional relationship between cytoplasmic streaming and the transient redistribution of microfilaments. We propose that the par genes are exerting their primary effect during the first cell cycle. MATERIALS

Strains

and Culture

AND

METHODS

Conditions

C. elegant var. Bristol (N2 is the wild-type designation) was cultured according to the procedures of Brenner (Brenner, 1974). This paper conforms to standard C. elegans nomenclature (Horvitz et al., 1979). Many of the mutant alleles used were obtained from the Caenorhab ditis Genetics Center. par mutations used in the study were par-l(b274, it32)V, par-2(it5ts, it.49, it53) III, par3(e2074, it54, it62, it7l)III, and par-4(it33, it47ts) V (Kemphues et ab, 1988) (D. Morton, N. Cheng, C. Kirby, and K. Kemphues unpublished results). Because the par mutations are maternal-effect lethal, the par mutant phenotype is expressed only in the progeny of homozygous par mothers. As a shorthand, we will often refer to mutant embryos of homozygous par mothers simply as par embryos or par mutants throughout this report. Maternaleffect lethal, nonconditional par mutations were maintained as double mutants with closely linked morphological markers in cis to the par mutation and with a

VOLUME

142,199O

chromosome carrying a par+ allele in trans. par-l mutations were balanced by nT1 unc(n754) (IV;V), a translocation that suppresses crossing over on the right arm of LGIV and the left arm of LGV (Ferguson and Horvitz, 1985). par-3 mutations were homozygous and balanced by the free duplication sDp3 (Rosenbluth et al., 1985) or were balanced by qC1 dpy-19(e1259) gZp-l(q339) (Austin and Kimble, personal communication). Other mutations are described by (Hodgkin et ah, 1988). Except for animals carrying defective par-2, daf-7, or par-4(it&7ts) alleles, nematodes were grown at 20°C. par-2 daf-?‘/++hermaphrodites were maintained at 25°C to promote dauer formation. daf-7(el372ts) is a temperature-sensitive mutation which leads to constitutive dauer formation when animals are grown at 25°C (Riddle, et al, 1981). To obtain 25°C embryos from either daf-7 or daf-7par-2 animals, dauers were picked at 25°C transferred to 16°C for 2 days, and then returned to 25°C for 12-18 hr before embryos were used. Animals homozygous for mutations in par-2(it5ts) and parh(it47ts) were maintained at 16°C and transferred to 25°C as young larvae to obtain mutant embryos in the next generation. Strong par Mutations

The phenotypes

are Likely

to be Null Mutations

exhibited by the mutations paror it&?), par-3(it71), and par-4(it33) are likely to represent complete loss-of-function of the gene product (null phenotype). These tentative assignments of null phenotypes are based on criteria discussed by Herman (Herman, 1988) and briefly summarized below for each gene. All par mutations are completely recessive. par-l(b274). 10 par-l alleles have been recovered at a frequency of 2.5 x 10e4, close to the frequency expected for loss-of-function mutations at typical C. elegans loci (D. Morton, M. ROOS, and K. Kemphues, in preparation). All 10 alleles exhibit full expressivity and their phenotypes are similar. We have isolated a deletion spanning the locus, itDf2, and have found that par-l+ hemizygotes exhibit no detectable phenotype, and that par-l(b.274) hemizygotes are indistinguishable from par-l(b274) homozygotes in viability, fecundity, and maternal-effect lethal phenotype. par-Z(it5.3). 12 par-2 alleles have been isolated at a frequency of 2.5 X 10p4, similar to the frequency for loss-offunction mutations (N. Cheng, D. Morton, in preparation). These mutations can be placed in an allelic series based on expressivity of the maternal-effect lethal phenotype; it53 and it49 are among those alleles with the strongest expressivity. Heterozygotes for par-2+ and a deletion that removes or at least is broken in or near par-2 have no obvious phenotype. Heterozygotes for it53 l(b274), par-2(it53

KIRBY,KUSCH,ANDKEMPHUES

Cytophmic

and this deletion exhibit no nonmaternal-effect phenotypes and have the same degree of maternal-effect lethality as it53 homozygotes. (The deletion strain was lost before we could test it in trans to i&49). Heterozygotes for this deletion and a weakly expressed par-2 allele (e.2030) have the same intermediate level of expression of the maternal-effect lethality as do it53/e2030 or it49/e2030 heterozygotes (N. Cheng and K. Kemphues, in preparation). par3(it71). There are five par-3 alleles. it71 is one of the three strongly expressed alleles (it62 is another), and is an amber mutation (C. Kirby and K. Kemphues, unpublished). par-h(it33). We have isolated 11 par-4 alleles at a frequency of 2.9 x 10m4. it33 is among those mutations with the strongest expressivity. Heterozygotes for par-4+ and a deletion that removes or is at least broken in or near par-4 have no obvious phenotype. Heterozygotes of par,4(it33) and this deletion are indistinguishable in phenotype from par-h(it33) homozygotes, and heterozygotes of this deletion with a weak par-4 allele (it.57) show a phenotype indistinguishable from it33/it57 heterozygotes (D. Morton, M. Roos, and K. Kemphues, in preparation). Construction of Double Mutants par-l(b274) v par-3(it62)111 double homozygotes were obtained as Lon Rol segregants from the balanced strain ro&(sc8) par-l(bWh)/nTl unc(n754); lon-l(e185) par-3(it62)/qCl dpy-lg(el2.59) g1p-l(q339). par-2(it5ts) par-3(it62) double homozygotes were obtained as Lon segregants from the strain par-2(it5ts) Zen-l(e185) par3(it62)/par-2(it5ts)++. Because a few Lon animals might not be homozygotes for par-3 due to crossing over between ion-1 and par-3, we gathered data only from

early embryos taken from mothers producing some embryos with the par-3 cleavage pattern at the two-cell stage (par-3 is epistatic to par-2 for this phenotype) (N. Cheng and K. Kemphues, in preparation). Videotaping and Measuring Early Embryos

Embryos were dissected from young gravid hermaphrodites into water. Early pronuclear-stage embryos were transferred via a pulled microcapillary pipet to a polylysine-coated microscope slide. To insure that embryos were not deformed by pressure from the coverslip, coverslips were supported by petroleum jelly rings surrounding the embryos. The slides were transferred to a Zeiss universal microscope for viewing at room temperature between 21.5 and 23°C. This exposure to a temperature slightly lower than 25°C at the time of viewing had no discernible effect on the phenotypes of par-2(it5ts) and par-/t(it47ts) when compared to nonconditional par-2 and par-4 alleles. Embryos were viewed

Reorganization

in C elegans

205

with a 63X oil immersion lens and were videotaped at l/16 or l/12 real time using either an NEC model VCTL50E or a Panasonic Model AG6050 time-lapse video recorder equipped with a time/date generator. To obtain spatial measurements, an acetate sheet was taped to the video monitor and several tracings of each embryo were taken, including, whenever possible, the following times: start of recording, maximal pseudocleavage constriction, pronuclear conjunction, pronuclear envelope breakdown, completion of first cleavage furrow. For each tracing the time was recorded. The following measurements were taken from the acetate sheets: eggshell length, eggshell width, embryo length, transverse distance between pronuclei, distance between pronuclei and anterior and posterior eggshell, distance between the anterior and posterior plasma membrane and the eggshell, distance from lateral eggshell to the furthest point of pseudocleavage constriction, and placement of maximal pseudocleavage relative to the anterior eggshell. All measurements were normalized to eggshell length or width as appropriate and expressed as percentages prior to being pooled. To determine the time from fertilization to pronuclear meeting, embryos were observed in utero. Young adult hermaphrodites were anesthetized for 15-20 min in a solution of 0.1% tricaine, 0.01% tetramisole in water, mounted on agar pads (Sulston and Horvitz, 1977), and videotaped as described above. Wild-type embryos fertilized within mothers anesthetized in this manner develop normally and are perfectly viable (S. Sprunger and K. Kemphues, personal communication). We defined fertilization as the time the oocyte enters the spermatheca. Statistical comparisons were done using unpaired t tests. Percentages were adjusted using an arcsine transformation prior to being compared. Fixation

of Embryos and MicroJilament Staining

The fixation and staining procedures were modifications of those used by Strome (1986). Embryos were cut out of young gravid hermaphrodites into water. Early embryos were transferred to polylysine-coated slides, and the water was replaced by two changes of fixation buffer. Fixation buffer was prepared fresh daily as follows: 0.225 g paraformaldehyde was suspended in 5 ml water at 60°C for approximately 1 hr with occasional mixing. Eighty microliters 1 N NaOH was added followed by 5 ml of 0.25 M phosphate buffer, pH 7.4. To 0.95-1.0 ml of this solution was added 40 ~1 Triton X-100 (5%). The buffer was adjusted to maintain a solution isoosmotic with the embryos by adding from 0 to 50 ~1 of water so that embryos neither swelled nor shrank upon permeabilization.

206

DEVELOPMENTALBIOLOGY

As rapidly as possible (within 2-5 min) after the addition of fixative, embryos were permeabilized. Slides with embryos were mounted on a Zeiss inverted microscope equipped with Nomarski optics and a 32X longdistance working lens. A drawn-out glass microneedle attached to a Leitz microinjection apparatus was used to apply just enough pressure to embryos to crack their vitelline membranes, rendering the embryos permeable to fixative. Embryos were lightly fixed for 20 min at room temperature and washed with two changes of 0.125 Mpotassium phosphate buffer, pH 7.4, for at least 10 min each. To specifically stain F-a&in (Strome, 1986; Wieland, 1977; Wulf et ah, 1979) embryos were incubated with 6 units/ml rhodamine phalloidin (R-ph; Molecular Probes, Inc.) in 0.125 M phosphate buffer, pH 7.4, and 0.2% Triton X-100. Fixed embryos were usually left overnight at 4°C before R-ph staining but could be stained immediately or left in phosphate buffer at 4°C for at least 3 days without a noticeable effect on staining. Stained embryos were washed briefly in phosphatebuffered saline (PBS), pH 7.2, chromosomes were stained with diamidinophenylindole (DAPI) at a concentration of 1 x 1O-4 mg/ml in PBS for 1 min and slides were washed once with PBS. Some embryos were stained with DAPI before R-ph. Embryos were mounted for fluorescence microscopy in 1 mg/ml phenylenediamine (Sigma) in 80% glycerol, 20% PBS, pH 8.5. Stained embryos were viewed through a Zeiss Photomicroscope III equipped for epifluorescence. Photographs were taken on Kodak Plus X film at ASA 400 (DAPI) or at ASA 800 or 1600 (rhodamine) and developed in HC-110 at dilution B for 7.5 min at 20°C. Due to variability in staining intensity from day to day and to our use of an automatic exposure meter, the apparent brilliance of the microfilament staining in the micrographs does not necessarily reflect absolute microfilament concentrations. The extent of intense microfilament staining (the microfilament “cap”) was measured as a function of position along the anterior-posterior axis with a millimeter ruler from contact prints and normalized to egg length. When possible, the cap length for individual embryos was calculated as the average of the length measured at both the upper and lower cortices. The data were evaluated using an unpaired t test as described above. RESULTS

Summary of Wild-Type Development

The major

events of the first cell cycle of wild-type C. have been described in detail (Albertson, 1984; Hirsh et ak, 1976; Nigon et al., 1960) and are summarized below and depicted in the first row of Fig. 1 and the first column of Fig. 3. In C. elegans, oocytes elegans embryos

VOLUME 142,1990

arrest in late prophase of meiosis I and complete meiosis after fertilization. The completion of meiosis II occurs about 60% of the way through the first cell cycle and is roughly coincident with the establishment of a permeability barrier by the newly formed vitelline membrane. At this time, pronuclei form and multiple anterior cortical contractions occur (Fig. la). Coincident with the appearance of these contractions, subcortical cytoplasm can be observed to stream from anterior to posterior. As the minor cortical contractions diminish, but while strong cytoplasmic streaming continues, a single large cortical contraction called the pseudocleavage appears (Fig. lb). As the pseudocleavage forms, the female pronucleus begins its migration to meet the male pronucleus in the cell’s posterior hemisphere. The pseudocleavage reaches its maximum extent near the time when the female pronucleus migrates through the channel formed by the pseudocleavage furrow (Fig. lc). By this time, cytoplasmic streaming is no longer apparent. During or just after passage of the pronucleus, the pseudocleavage begins to disappear and the anterior cortex relaxes toward the posterior (Figs. lc-le). The joined pronuclei then move toward the cell’s center, fuse, and enter mitosis (Fig. le). The first mitotic spindle is initially positioned symmetrically near.,khe cell’s center but then migrates posteriorly so that the first cleavage creates blastomeres of unequal size: a larger, anterior blastomere (called AB) and a smaller posterior blastomere (called Pl) (Fig. 3g, 3i, and 3k).

Analysis of Early Events in Living par Embryos

We began our study with a thorough time-lapse video analysis of the first cell cycle in wild-type and par mutant embryos. Because embryos from stages prior to the second meiotic division are osmotically sensitive, continuous observations from the time of fertilization must be made in utero. Unfortunately, embryos in utero are often unfavorably oriented for microscopy, making precise measurements impossible. As a compromise, we carried out our analysis in two steps. First, we video-recorded embryos from the earliest time they would survive outside the mother to obtain precise spatial measurements. As a second step, we determined the time from fertilization to pronudear meeting in utero to establish a reference point for timing. We included in our analysis mutations that are most likely to represent the null phenotype (See Materials and Methods). These alleles are par-l(b274), par-2(it53), par-3(it62, it71), and par-4(it33). However, for some analyses, we used the temperature-sensitive alleles par2(itsts) and par-h(it47ts). At restrictive temperature the embryonic phenotypes of these mutations are indistin-

KIRBY, KUSCH, AND KEMPHUES

Cytoplasmic

Reorganization

in C. elegans

FIG. 1. Nomarski micrographs of wild-type and par mutant embryos undergoing cytoplasmic reorganization. series of a single embryo. Anterior is to the left. (a-e) Wild type. (f-j) par-l(b274). (k-o) par-2(it53). (p-t) par-3&Z). represents 10 w.

guishable from the mutant phenotypes of the putative null alleles. We have attempted to be conservative in evaluating our data. Because of variability inherent in these types of observations (e.g., slight differences in temperature, growth conditions, and genetic background), it is difficult to rule out sampling error as a source of observed differences. We will limit our discussion to differences in timing that are greater than 1 min (about l/50 of the total time measured) or, for spatial measurements, differences greater than 1%) and that have P values of less than 0.05 in comparison with wild type in t tests.

Each row is a chronological (u-y) par-h(it47ts). Bar in (a)

scribed below and shown in Figs. 1 and 2 and Tables 1 and 2. N2 embryos show a consistent placement of the pseudocleavage at about 51% of egg length and a maximal

Pseudocleavage is Abnormal in par Embryos

Although other events in the first cell cycle are abnormal in embryos mutant in different par genes (discussed below), pseudocleavage is consistently abnormal in mutants of all four genes. The exact nature of the pseudocleavage abnormality is gene specific as de-

FIG. 2. Superimposed tracings of several wild-type and par embryos taken at time of maximal pseudocleavage. Embryos are aligned at 50% egg length. (a) wild type (b) par-l(b274) (c) par-d(it53) (d) par3(it62)

(e) par4(it47ts).

208

DEVELOPMENTALBIOLOGY VOLUME 142,199O TABLE 1 par-2 embryos also exhibit pseudocleavages with rePLACEMENTANDEXTENTOFPSEUDOCLEAVAGEFURROW duced extent (30% of egg width) that are positioned on No. of embryos

N.2 par-l(b274) par-Z(it53) par-3(it62) paM(it71)

par-h(it47ts) par-h(it33) par-l(b274);

par-2(&ts)

par-3(it62) par-3(&2)

Placement”

16 16 13 14 6 19 5 7 8

51 + 45 + 56 + 56 f 52 f 52 -t 47 rt 53 f 5’7 f

2.2 2.8” 4.3” 8.2” 8.2 5.2 7.3” 6.3 8.6

Extentb 36 30 30 34 27 21 25 28 35

f 6.6 + 6.2d + 7.7” f 9.3 f 6.6d f 5.1” + 3.3d r 7.0 +- 5.2

a Average position f standard deviation of maximal pseudocleavage as percentage of egg length (anterior = 0%). * Average size of constriction of maximal pseudocleavage as percentage of egg width f standard deviation. c P d 0.0005 in unpaired t test comparing pur embryos with wildtype embryos. (Unless indicated otherwise, P > 0.05). d 0.0005 < P G 0.01. “0.01

< P < 0.025.

constriction of about 36% of egg width (Fig. 2a). The furrow is initially positioned more posteriorly at about 55% egg length but moves anteriorly to its final position as it reaches maximal constriction. The furrow persists for about 2 min after it reaches maximal constriction, then disappears at roughly the same time that the pronuclei meet (data not shown). par-l embryos show a consistent anterior placement of the pseudocleavage furrow (Figs. If-lj; Fig. 2b) at about 45% of egg length at maximal and a reduced extent of pseudocleavage constriction (30% of egg width). In other respects pseudocleavage is like wild type. TABLE

average slightly more posteriorly than wild type (56% of egg length) (Figs. lk-lo; Fig. 2~). The more posterior placement seems to be due, at least in part, to inconsistent directed anterior movement of the furrow as it reaches maximal constriction. par-3 embryos are extremely variable in extent, placement, and directed anterior movement of the pseudocleavage furrow (Fig. 2d). The embryo in Figs. lp and lq shows a pseudocleavage configuration often seen in par3 embryos but only rarely observed in wild type: the furrow is deeper on one side of the embryo. The differences between it62 and it?‘1for the mean values for placement and extent are not significant (P > 0.05), but rather reflect the variability of the process in par-3 embryos. As described above for par-2 embryos, there is general failure in the directed anterior movement of the furrow. Furrow movement occurs in most embryos but the direction and extent are variable. par-4 embryos have a nearly normal pseudocleavage placement and movement but have a very small constriction of only about 21% of egg width (Fig. 2e). In addition to defects in extent, position, and directed anterior movement, the time of maximal pseudocleavage constriction in par-2 and par-3 embryos occurs earlier than in wild type, relative to the time of pronuclear meeting (Table 2, column B). However, the overall duration of pseudocleavage is not changed in these mutants. It is also possible that pseudocleavage occurs early relative to fertilization in par-4 embryos as a consequence of the shortening of the time between fertilization and pronuclear meeting. 2

TIMINGOFEARLYDEVELOPMENTALEVENTSAT~~~C A Genotype N2 par-l(b274) par-Z(it53) par-3(it62)

par-3(it71) par-Q(it47t.s)

par-4(&T) par-l(b274); par-3(it62)’ par-2(i&s)par-3(it62)

Fertilization to meeting in utero 40 42 40 40

f + + k

1.5” (7)b 2.0 (6) 2.1 (8) 3.0 (8) n.d. 35 f 3.4” (6) 36 f 4.0d (5) n.d. n.d.

B Pseudocleavage to meeting 1.4 f 1.8 + 2.9 f 5.7 + 5.4 f 1.8 f 2.3 f 6.1 f

0.6 (16) 0.8 (16) 1.0” (13) 1.4” (14) 1.7” (6) 0.5d (19) 0.8d (5) 0.8 (7)

5.1 * 1.2

(8)

C Meeting to fusion 4.2 4.0 4.4 4.4 4.9 4.0 4.0 4.6 4.0

f f f + k f f k f

0.6 (16) 0.5 (16) 0.6 (13) 0.8 (12) 0.5d (6) 1.0 (19) 0.5 (5) 0.6 (7) 0.5 (8)

D

E

Fusion to 1st cleavage

Fertilization to 1st cleavage

3.5 -c 0.6 (14) 3.7 f 0.7 (16) 4.7 f 0.5” (13) 3.8 f 0.5 (14) 4.7 f 0.8” (6) 3.9 + 0.6” (18) 4.1 f 0.3d (5) 4.1 + 0.6 (7) 4.4 + 0.6 (8)

47.7” 49.7 49.1 48.2

QMean times are given in minutes with standard deviation indicated. b The numbers in parentheses are the numbers of embryos measured. ‘P =S0.005 in unpaired t test comparing par embryos with wild-type embryos. (Unless indicated otherwise, P > 0.05). d 0.01 < P d 0.05. e Calculated as the sum of the means in columns A, C, and D. jAverages for these embryos were not statistically compared to wild type.

42.9 44.1

KIRBY, KUSCH, AND KEMPHUES

Cytoplasmic

Because all of the par mutants exhibited gene-specific alterations in pseudocleavage but none eliminated pseudocleavage completely, we examined pseudocleavage in double mutants to see if the mutations would show synergism. We examined embryos from mothers of genotype par-l; par-3 and par-2 par-d In both cases, for the parameters we measured, the phenotypes of the double mutants were no more severe than either of the single mutants (Tables 1 and 2). In fact, based on the parameters that showed significant differences between the single mutants, position of pseudocleavage for par-l and par-??,and timing of maximal constriction for all three mutants, par-3 appears to be epistatic to both par-l and par-2. Timing of Early Developmental Events

In Table 2 we show the average time in minutes at 22” between fertilization and pronuclear meeting, between maximal pseudocleavage and pronuclear meeting, between pronuclear meeting and pronuclear fusion, and between pronuclear fusion and first cleavage for wildtype and par mutant embryos. Summing the times in columns A, C, and D gives an estimate of the total time from fertilization to first cleavage (column E). With the exception of par-h, the total time of the first cell cycle in the par embryos is not appreciably different from wild type. The shortening of the first cell cycle in par-4 is not due to an overall increase in developmental rate but rather is due specifically to a shortening of the time between fertilization and pronuclear meeting. This could be a direct consequence of the smaller average size of par-4 embryos. (The embryo shown in Figs. lu-ly is an extreme example of this smaller size.) Since the pronuclei have a shorter distance to travel they meet earlier. One other possibly significant deviation from wild type is a slowing in the time between pronuclear fusion and first cleavage in par-2 and in par-3(it71). Other Deviaticms from Wild Type

We also noted deviations from wild type in some but not all par mutants in three other aspects of cytoplasmic reorganization: cytoplasmic streaming, position of meeting of pronuclei, and asymmetric changes in embryonic length. Although it is difficult to quantitate, we made careful comparisons of the pattern and strength of cytoplasmic streaming of wild-type and par mutant embryos (N> 15 for each gene). We found that streaming was absent in par-d, par-l; par-3, and par-2 par-3 embryos, weak in par-2 embryos, but apparently normal in par-l and par-4 embryos. Because there is a striking asymmetry in the migration of the pronuclei in wild type (Figs. la-ld) we wished

Reorganization

209

in C elegans

TABLE 3 POSITION OF PRONUCLEAR MEETING AND RELATIVE SHORTENING OF EMBRYOS

Genotype N2 pwI(b274) pw2(it53) parx‘S(it62) par-S(it7l) par-h(it47ts)

par-4(i#3) par-l(b274); paM(it62) puP2(ittits) paH(it62)

n

(Wd (1’3)

Position of pronuclear meeting”

(5) (7)

59 f

3.6

13 f 2.1

69 f 17

(8)

56 f

3.3g

15 f 2.2

56 f 21

(6) (19)

3.4 5.8 3.7” 2.4e 3.2f

13 13 14 14 14

f f f f f

3.3 4.0 1.6 4.8 3.0

Anterior shortening”

70 68 62 60 59 74 64

(13) (14)

?I f f f +-

Embryo shorteningb

84 79 69 60 64 68

-+ 12 of- 6.4 f 13’+ 12’

+- 10

12 f 3.2

f

8.1g

14 ?I 3.6

f 1g8 -f- 19f ‘71 f 18

a Position is given as mean percentage of egg length from the anterior + standard deviation (anterior = 0%). ’ Calculated as [(maximal embryo length - embryo length at pronuclear fusion)/maximal embryo length] X 100. ’ Percentage of shortening contributed by anterior shortening; calculated as described under Materials and Methods. d Number of embryos measured. e P < 0.0005 (Unless indicated otherwise, P > 0.05). f0.0005 < P G 0.005. B 0.005 < P < 0.025.

to determine if the par mutations have any effect on the position at which the pronuclei meet. We noted only a slight, if any, difference from wild type in par-l and par-4 (note, however, the large variability in par-Q) but more striking differences in par-2, par-3, and the double mutants (Table 3). In these latter mutants, the anterior migration of the paternal pronucleus is greater than that in the wild type, so that the nuclei tend to meet more medially. In our observations of wild-type embryos we had noted an additional asymmetric movement. As the pseudocleavage disappears, there is a reduction in embryo length of about 13% (compare Fig. lb to Fig. le). This appears to be, in large part, a consequence of the release of the pseudocleavage furrow as cytoplasm flows into egg space from which it was excluded by the furrow. Interestingly, this change in egg length is greater in the anterior. In three of the par mutants, the reduction in egg length occurs more symmetrically. This is most striking in par-3 (Figs. 1s and It) and double mutants of par-3 but also occurs in par-2 and par-4 embryos to a lesser extent (Table 3). Cortical Microjilament Embryos

Distribution

in Wild-Type

Strome (1986) characterized microfilament staining patterns relative to developmental time in one-cell C.

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DEVELOPMENTAL BIOLOGY

elegans embryos and found that cortical microfilaments become asymmetrically distributed during the latter portion of the first cell cycle. In order to verify that our staining procedures were working properly and to obtain a wild-type baseline with which to compare mutant embryos, we reexamined microfilament staining in wild-type embryos. As shown in Fig. 3, our results confirm the findings of Strome. Just after formation of the pronuclei (Fig. 3a), the earliest stage we examined, brightly staining microfilament foci along with a fainter microfilament network are visible distributed throughout the embryo’s cortex (Fig. 3b). During pronuclear migration (Fig. 3c), the distribution of microfilament foci begins to appear asymmetrical (Fig. 3d) with the anterior more brightly stained than the posterior. It is not possible to determine whether existing microfilament foci are redistributed or whether new foci are created in the anterior region as foci are removed from the posterior hemisphere. This asymmetry may be due only to the microfilament foci as the fiber network does not appear to change during the one-cell stage. However, we would be unable to distinguish small differences in microfilament staining in the fiber network. The pseudocleavage furrow usually disappears during fixation, but when preserved, it is brightly stained. By the time of pronuclear meeting and fusion (Fig. 3e), most of the microfilament foci are found in the anterior region of the embryo’s cortex (Fig. 3f), although some embryos display an additional and much smaller accumulation of microfilament foci at the posterior pole (Fig. 3f). In effect, the embryo appears to have a brightly stained cap with a distinct anterior-posterior boundary perpendicular to the cell’s long axis. For simplicity, we will refer to this brilliantly stained anterior region as the microfilament “cap” or simply the cap. The cap generally persists through metaphase (Figs. 3g and 3h); however, occasional embryos do not have distinct caps at metaphase. Those without caps look similar to anaphase embryos (Figs. 3i and 3j) in which the foci are almost completely dispersed throughout the embryo’s cortex. While a faint asymmetry is often apparent at anaphase, the distinct cap of earlier stages has disappeared. By first cleavage (Fig. 3k) the distribution of cortical microfilaments again appears relatively uniform with foci and fibers evenly distributed along the embryo’s surface (Fig. 31).

Abnormal Cortical Micro&lament Embryos

Distribution

in par

We found that mutations in the par genes also lead to gene-specific perturbations of the asymmetric distribution of cortical microfilaments in one-cell embryos. We analyzed microfilament distribution in embryos from

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FIG. 3. Nomarski and fluorescence micrographs of wild-type embryos. The column on the left shows a chronological series of a live embryo. The column on the right shows the cortices of embryos at comparable developmental stages that have been fixed and stained with rhodamine-conjugated phalloidin. (a, b) early pseudocleavage. (c, d) late pseudocleavage. (e, f) pronuclear meeting. (g, h) metaphase of first mitosis. (i, j) anaphase of first mitosis. (k, 1) telophase of first mitosis. Bar in (1) represents 10 )L.

mothers homozygous for par-l(b274), par-2(it49), par3(it62, it71), and par-4(it33). Microfilament distribution was examined in par embryos ranging in developmental stage from pronuclear

KIRBY,

KUSCH,

AND

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Cytoplasmic

formation to first cleavage. In all the par mutants examined, the transient asymmetric localization of microfilament foci to the anterior of the embryo was abnormal. The exact nature of the mutant phenotype, however, differed for each par gene. In some mutants cortical microfilaments were not distributed asymmetrically at any time during the one-cell stage and in others, defects were found in cap size or cap orientation, or both. At the earliest stages of pronuclear migration all the embryos looked similar to wild type, with dispersed cortical microfilament foci and fibers. By pronuclear meeting, however, distinct abnormalities were apparent in the mutant embryos, as shown in Fig. 4 and Table 4 and described below. By anaphase all par mutant embryos exhibited cortical microfilament staining that was indistinguishable from wild type. In those mutants which displayed microfilament asymmetries, the relative timing of cap formation did not differ from wild type. par-l embryos have smaller caps than wild-type embryos (Fig. 4c and Table 4). Measurements of 44 par-l embryos showed a mean cap size of 36 + 6%, whereas the N2 cap measured 41 + 6%. In addition to being smaller than wild type, the cap in about 50% of the par-l embryos appeared to be askew (Fig. 4d). par-2 embryos frequently had no cap (Table 4 and Fig. 4f). When caps were present, they were larger than those found in wild type (Fig. 4e). In addition, in these embryos, the difference in staining intensity between anterior and posterior was less pronounced than in the wild type. Mean cap size in par-2 embryos was 65 + 8%. Almost all par-3 embryos examined at pronuclear meeting and metaphase lacked caps (Table 4); the cortex generally appeared uniformly covered with microfilament foci and fibers (Fig. 4g). Only one par-3 embryo at the pronuclear meeting stage of development displayed a faint asymmetry. In addition, we never saw a cap at any other time during the first cell cycle. Double mutant par-&par-3 and par-2 par-3 embryos also lacked caps (Table 4). In par-4 mutants, the caps looked similar to wild type except that they covered a significantly larger fraction of the embryo’s cortex. par-4 embryos had distinct, brightly stained caps (Fig. 4b) with a mean size of 53 ?I 6%.

For ease of manipulation, each of the embryos carrying a par mutation also carries a mutation in a closely linked visible marker (rol-4, daf-7, km-l, or dpy-21). As a control, we examined cortical microfilament distribution in one-cell embryos with mutations only in marker genes. Microfilament distribution was not distinguishable from wild type in any of these mutant embryos except in daf-7 embryos raised at 25°C. At 25°C daf-7 embryos had caps with a mean size (n = 17) of 45 + 5% versus 41 t- 6% for wild-type embryos raised at 20°C

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211

and the difference between daf-7 and wild type was statistically significant (P < 0.005). When daf-7 was raised at 20°C however, mean cap size (n = 18) was identical to wild type. DISCUSSION

We have undertaken a quantitative study of cytoplasmic reorganization during the first cell cycle in wildtype and par mutant embryos in order to gain a better understanding of the mode of action of the par genes in this reorganization. Table 5 summarizes our results, comparing par mutant embryos to wild-type embryos for six major events of the first cell cycle. In general, our results indicate that the par gene products are required for several aspects of cytoplasmic reorganization during the first cell cycle, most notably pseudocleavage, cytoplasmic streaming, and the transient asymmetric distribution of microfilaments. This raises the possibility that the sole function of the par genes might be to orchestrate this initial reorganization of cytoplasm. This idea is consistent with unpublished findings that the temperature-sensitive periods of par-2(it5ts) and par4(it57ts) begin during oogenesis and end by the two-cell stage (N. Cheng, D. Morton, and K. Kemphues, in preparation). Mutations in the par genes were previously shown to produce phenotypes indicating that the genes play a role in cytoplasmic localization. P granule localization, cleavage pattern and rate, and specification of the intestine and germ line lineages are defective in par mutants (Kemphues et al, 1988). The manner of expression of these defects, however, is gene specific. For example, although all par mutants are defective in early cleavage patterns, par-l, par-i?, and par-3 mutants cleave abnormally at all early divisions while par-4 cleaves normally at the first cleavage but abnormally at subsequent divisions. par-l, par-,$, and strong par-3 (C. Kirby, unpublished) mutations cause failure to localize P granules, while all par-2 mutant embryos retain some capacity for localizing granules (N. Cheng and K. Kemphues, in preparation). The differences do not appear to be due to quantitative differences in expression of the mutant phenotype; all alleles at each locus share characteristics that distinguish them from alleles at other loci. Because the defects we found in pseudocleavage and cortical microfilament redistribution are also gene specific, we searched for correlations among the defects. Such a search does not reveal any simple relationships. For example, there is no correlation between cortical microfilament distribution and the placement of the first cleavage spindle in the par mutants. In wild-type embryos the spindle is positioned asymmetrically and first cleavage produces two blastomeres of unequal size.

DEVELOPMENTAL BIOLOGY

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FIG. 4. Rhodamine-conjugated phalloidin staining of wild-type and paparembryos at pronuclear (e, f) par-Z(it49). (g) par-3(&Z). Bar in (g) represents 10 p.

meeting. (a) wild type. (b) par-4(it33). (c, d)

par-l(bZ74).

Mutations in par-l, par-2, or par-3 lead to symmetrical spindle placement. In these mutants, microfilament distribution can be either uniform, as in par-2 and par-3 embryos, or the asymmetry can be more pronounced

than that in wild type, producing embryos with smaller caps such as those found in par-l mutants. par-4 one-cell embryos have wild-type spindle placement and blastomeres that are wild type in relative sizes, but cortical

KIRBY, KUSCH, AND KEMPHUES

Cytoplasmic

TABLE 4 SIZE OF MICROFILAMENT CAP IN WILD-TYPE AND par EMBRYOS Embryos without caps

Gene (allele) N2 (wild type) par-l(b.U.&)

4 (6%) 0 (0%)

Embryos with caps

Cap size”

59

41 f 6

44

36 _+ 6'

24

65 f gb

par-3(it71)

15(38%) 48(98%) 11 (100%)

par-h(it33) par-l(bZ7h); par-3(it62) par-2(it5ts) par-3(&Z)

5 (11%) 11 (100%) 14 (100%)

46

pad(it49)

par-3(&Z)

1

-

0

53 +- 6'

0 0

-

a Cap size is expressed as the mean percentage of egg length f the standard deviation and was calculated as described under Materials and Methods. *P G 0.0005.

microfilament caps that cover a larger proportion of the cortex than those in wild-type embryos. Our data argue counter to one possible relationship. Strome (1986) has suggested that cortical microfilament foci could participate in a contractile network in which contractions at the cell’s anterior occur as the foci become concentrated in the embryo’s anterior cortex. These contractions could push cytoplasmic components such as P granules toward the posterior. Hill and Strome (1988) presented circumstantial evidence counter to this model. Our results are also inconsistent with this model. In par embryos, failure to localize P granules to the embryo’s posterior does not correlate with failure to localize microfilament foci to the cell’s anterior region. In wild-type one-cell embryos cortical microfilaments become asymmetrically distributed concomitant with localization of P granules. In par-l and par-4 embryos, P granules are not localized but microfilaments are asymmetrically distributed. In par-Zmutant

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in C elegans

embryos, most P granules are localized to the posterior in the first cell cycle (Kemphues et al, 1988) (N. Cheng and K. Kemphues, in preparation), but cortical microfilaments are either weakly localized or not localized in par-2 mutant embryos. Thus, the microfilament cap is not solely responsible for localizing P granules to the posterior. We also considered the possibility that the microfilament cap is functionally related to pseudocleavage. In par-l mutants there is a correlation: both the pseudocleavage furrow and the microfilament cap are located more anteriorly than in wild type. In the other par mutants, however, this correlation does not hold. In par-4 embryos, the position of pseudocleavage is not different from wild type in spite of the presence of a larger than normal cap, and par-2 and par-3 embryos still undergo pseudocleavage in the absence of any microfilament asymmetry. Thus, the microfilament cap is not required for pseudocleavage. The correlation between lack of a cap and the premature achievement of maximal constriction in par-2 and par-3 embryos, however, might mean that the cap or a process leading to its formation play some role in the pseudocleavage process. Our data do indicate that the microfilament cap is related to the cytoplasmic streaming seen during the pseudocleavage period. Mutations preventing cap formation also block streaming, mutations leading to weak caps lead to weak streaming, and mutations that do not block cap formation do not block streaming. Streaming does not seem to require the presence of the cap, however. In our observations of wild-type embryos, we noted that embryos in early pseudocleavage (when streaming is seen in most embryos) had no detectable cap, while nearly all embryos at pronuclear meeting have caps although streaming has stopped by then. The biological significance of differences in cortical microfilament cap size is not clear. Although caps ex-

TABLE 5 SUMMARY OF PHENOTYPES OF par EMBRYOS DURING THE FIRST CELL CYCLE Genotype of embryos

Time from fertilization to 1st cleavage”

Pseudocleavage Timing

Placement

Extent

Position of pronuclear meeting

Asymmetric embryonic shortening

Cytoplasmic streaming

par-l par-2

normal normal

normal early

anterior posterior

weak weak

normal medial

normal symmetric

normal weak

par-3 par-4 par-l; par-3 par-l?; par-3

normal shortened n.d. n.d.

very early unclear very early very early

variable variable variable variable

normal* very weak weak normal

medial normal” medial medial

symmetric symmetric symmetric symmetric

absent normal absent absent

QAll characteristics are described relative to wild-type b Normal for it62, weak for it71. ’ Normal for it47 medial for it%?.

embryos.

Transient microfilament redistribution present; abnormal variably present; abnormal absent present; abnormal absent absent

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DEVELOPMENTAL BIOLOGY

tended on average to 41% of egg length in wild-type embryos, our data set included individuals with cap sizes of 25 and 45% indicating that normal development is probably compatible with a large range of cap sizes. In addition, microfilament cap size may be sensitive to the temperature at which an embryo is developing, since in control experiments with daf-7 embryos, the mean cap size was 4% larger in 25°C embryos than in 20°C embryos. Two other aspects of cytoplasmic reorganization are affected by mutations in some, but not all, of the par genes. Pronuclei meet more toward the center of the embryo in par-2 and par-3 embryos. This defect is also seen in cytochalasin-treated embryos (Hill and Strome, 1988; Strome and Wood, 1983). We interpret this to mean that in wild-type embryos there is a microfilament-dependent interaction between the posterior cortex and the male pronucleus or its centrosomes that prevents their movement away from the pole. A second aspect of the reorganization affected in some par mutants is the embryonic shortening that accompanies the relaxation of the pseudocleavage furrow. In wild-type embryos the length of the embryo decreases by about 13% during the first cell cycle. This shortening is strikingly asymmetric, occurring almost entirely in the anterior. In par-Z, par-3, and par-4 embryos, the amount of shortening is the same as wild type but is more symmetrical. We interpret the asymmetric shortening in wild type to mean either that the anterior cytoplasm is more fluid or that the posterior cortex is more rigid. In either case, the posterior of the embryo may have a more highly ordered cytoskeletal structure. This structure is weakened by mutations in par-Z, par-3, and par-4 Surveying all of the parameters we measured reveals a similarity in the phenotypes of par-2 and par-3 that is not apparent in their phenotypes after the first cell cycle. Mutations in both genes lead to reduction or absence of cortical microfilament asymmetry, reduction or failure in cytoplasmic streaming, premature achievement of maximal pseudocleavage, and loss of asymmetry in pronuclear meeting and embryonic shortening. These similarities may mean that the wild-type par-2 and par3 gene products function via a common process. In summary, mutations in all four par genes lead to gene-specific defects in pseudocleavage and anterior microfilament cap formation, and mutations in some par genes lead to other defects in cytoplasmic behavior during the first cell cycle. Our results indicate that anterior microfilament asymmetry is not directly related to localization of P granules to the posterior, but that there is a functional relationship between microfilament cap formation and cytoplasmic streaming. In general, however, there is no simple correlation between the pseudocleavage defects and microfilament cap abnormalities,

VOLUME 142.1990

or between these defects and defects in cleavage pattern, P granule localization, and intestinal differentiation. One possible explanation for the apparent complexity of the relationships between these events is that the par mutations are partial loss-of-function mutations. While we cannot rule this possibility out with strictly genetic data, all of our data indicate that the mutations we analyzed are null alleles (see Materials and Methods). Another possible explanation is that the par genes have overlapping functions. If this is the case, we might expect double mutants to show synergism, and exhibit a phenotype that is worse than either single mutant. We saw no indication of synergism in the two double mutants we examined; other multiple mutant combinations may prove more informative. A third explanation is that the results of our study reflect the complexity of the process of cytoplasmic reorganization. The par genes could represent only a few of the gene products involved in the reorganization and each could exert its influence by modifying normal cytoskeletal functions. Absence of the par gene activities does not eliminate these cytoskeletal functions, but alters them so that normal embryonic asymmetries are not established. We thank Diane Morton and Niansheng Cheng for their contributions of mutant par-2 and par-l alleles for this study and for sharing unpublished results; Diane Morton, Diane Shakes, and Willie Mark for critical reading of the manuscript; and Mike Roos and Chris Coupe for technical assistance. This work was supported by NIH Grant GM33763. C. Kirby was supported by NIH Genetics Training Grant GM0’7617. Some nematode strains used in this study were provided by the Caenorhabditis Genetics Center, which is supported by Contract Number NOl-AG-9-2113 between the National Institutes of Health and the Curator of the University of Missouri. REFERENCES ALBERTSON, D. (1984). Formation of the first cleavage spindle in nematode embryos. Dev. Biol 101,61-72. BRENNER, S. (1974). The genetics of Caenorhabditti elegant. Genetics 77,71-94. COWAN, A. E., and MCINTOSH, J. R. (1985). Mapping the distribution of differentiation potential for intestine, muscle, and hypodermis during early development in Caenorhabditis elegans. Cell 41,923-932. DAVIDSON, E. (1988). “Gene Activity in Early Development.” Academic Press, New York. EDGAR, L., and MCGHEE, J. D. (1986). Embryonic expression of a gut specific e&erase in Caewrhabditis elegans. Dev. Biol. 114,109-118. FERGUSON,E., and HORVITZ, H. R. (1985). Identification and characterization of 22 genes that affect the vulva1 cell lineages of the nematode Caenorhabditis elegans. Genetics 110, 17-72. HERMAN, R. K. (1988). Genetics. In “The Nematode Caenorhabitis Elegans” (W. B. Wood, Ed.), pp. 17-45. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. HILL, D. P., and STROME, S. (1988). An analysis of the role of microfilaments in the establishment and maintenance of asymmetry in Caenorhabditis elegant zygotes. Deu. Biol. 125,75-84.

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