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Formation of the first cleavage spindle in nematode embryos
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Formation of the First Cleavage Spindle in Nematode Embryos DONNA G. ALBERTSON Laboratory of lWol.eoular Biology, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, England Received November 27, 1982;accepted in revised fwm August 2, 1983 The distribution of microtubules and microtubule organizing centers in the events leading up to the establishment of the first asymmetric cleavage furrow in nematode embryos was followed using indirect immunofluorescence of antibodies to tubulin. Oocytes arrest in meiotic prophase then undergo two meiotic reduction divisions after fertilization. At both of these divisions barrel-shaped spindles were observed. Initially a single microtubule organizing center was seen adjacent to the sperm pronucleus following fertilization in Caewrhabditis elegant, but later two sperm asters were distinguished. These increased in size as the egg pronucleus migrated toward the sperm pronucleus and reached maximum size, with fascicles of microtubules extending to the cortex, once the pronuclei had become juxtaposed. The first cleavage spindle formed following rotation and migration of the juxtaposed pronuclei back toward the center of the embryo. The distribution of microtubules in a temperature-sensitive mutant that fails in both pronuclear migration and rotation was also examined. Asters in the mutant embryos at the nonpermissive temperature contained only short microtubules suggesting that the morphology of the asters is important for directing the movement of the pronuclei. In Panagrellus redivivus sperm asters were not detected by anti-tubulin staining until the female pronucleus had migrated to the centrally placed sperm pronucleus. Asters then increased in size and formed the first cleavage spindle.
INTRODUCTION
metric cleavage furrow in C. elegant and Panagrellus redivivus.
Nematodes develop by an essentially invariant cell lineage (Sulston and Horvitz, 1977; Sulston et aL, 1982). Frequently, daughter cells with different developmental potentials are formed by an asymmetric cleavage, such that the sister cells differ in size as well as fate. Thus the eccentric location of a cleavage spindle in a cell is an early indicator that the cell division will be determinative. The mechanisms by which the cleavage furrow is positioned such that a cell is unequally partitioned may be studied in the asymmetric first cleavage of nematode embryos. Following fertilization the embryos of some nematodes display a series of well-defined movements that bring the pronuclei together and position them in the zygote such that first cleavage is asymmetric. This process has been analyzed in great detail by microcinematography up to the four-cell stage in Caenorhabditis elegant (var. Bergerac) and other nematodes (Nigon et a& 1960). More recently similar observations were made on C. eZegune(var. Bristol) and compared to mutants defective in early development (Hirsh et a& 1976; Deppe et al, 1978; von Ehrenstein and Schierenberg, 1980; Schierenberg et al, 1980; Wood et al, 1980; Denich, 1982). In this paper indirect immunofluorescence of antibodies to tubulin has been used to study the distribution of microtubules and microtubule organizing centers in the events leading up to the establishment of the first asym-
MATERIALS
AND
METHODS
Nematode Strains and Gwyth Cultures of the wild-type N2 strain of C. elegans (var. Bristol) were maintained at 20°C as described by Brenner (1974). Animals with the temperature-sensitive mutation b.Z4.& (zyg-9)11 (Wood et d, 1980) were maintained at 15°C. To obtain xvg-9 embryos grown at the restrictive temperature, plates of larvae were transferred to 25°C. Panagrellus redivivus was kindly provided by David Hooper, Rothamsted Experimental Station, Harpenden, Herts, England, and has been maintained in the laboratory since 1976. Indirect Immunc&orescence Staining of Embqos Young gravid adults were picked and placed in a 5to lo-p1 drop of buffer on a subbed slide (Gall and Pardue, 1971) then cut in half at the vulva so that embryos were released. A 12 X 12-mm coverslip was placed gently on top of the drop so that embryos were not crushed and the slide placed on dry ice for at least 10 min. Coverslips were removed and the slides were fixed in methanol at -20°C for 2 min and acetone at -20°C for 4 min. The slides were then passed through a series of alcohols and two changes of phosphate-buffered saline (0.15 MNaCl, 0.02 M KCl, 0.01 M sodium phosphate, pH 7, PBS). The excess PBS was wiped off the slide, leaving a drop over 61
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the specimen to which the monoclonal antibody to tu- rescence were observed in oocytes from C. elegans and bulin YL1/2 or YOL1/34 (Kilmartin et a,!, 1982) was P. redivivus. In C. elegans staining was seen throughout added for 1 hr at 20°C. Slides were rinsed in two changes the oocytes in the proximal arm of the ovary (Fig. la), of PBS then FITC-anti-rat IgG (Miles, Slough, England) was added for 20 min at 20°C and the slides rinsed again. Some preparations were also stained with Hoechst 33258. Specimens were observed with a Zeiss RA microscope equipped with both epi- and transillumination using the Planapo 40X objective, so that FITC and Hoechst 33258 fluorescence might be viewed simultaneously or separately. The staining patterns of C. elegans embryos that were observed with YLV2 and YOL1/34 were indistinguishable. In the course of this work several hundred wild-type and mutant embryos were observed. Eighty wild-type and 70 xyg-9 embryos were photographed. Light Microscopzl of Living Embqos Embryos were dissected from hermaphrodites and mounted on agar slabs for viewing by Nomarksi differential interference contrast optics (Sulston and Horvitz, 1977). Pronuclear migration and first cleavage was analyzed in detail from video recordings of 10 wild-type and 10 xgg-9 embryos. Electron Microscopll The preparation of embryos for electron microscopy has been described previously (Albertson and Thomson, 1982). Twenty embryos, fixed at various times from fertilization through first cleavage have been sectioned. They were surveyed by reconstruction from electron micrographs taken of approximately every 15th serial section. Areas of interest were selected for more detailed study. RESULTS
1. Completion oj* Meiosis Nematode oocytes are arrested in prophase of meiosis and when meiosis resumes two polar bodies are formed at one pole of the ellipsoidal embryo (Nigon and Brun, 1955; Nigon et al, 1960). In C. elegans the oocyte nucleus migrates distal to the spermatheca before the oocyte passes from the proximal arm of the ovary into the spermatheca. Fertilization takes place at the opposite end of the oocyte as it begins to enter the spermatheca. Generally, metaphase I and II take place at the future anterior end of the C. elegans embryo, but infrequently embryos may be found with the polar bodies at the posterior end of the embryo. These develop normally (J. Sulston, personal communication). In contrast, in another nematode, P. redivivus, the polar bodies are located posteriorly. Different distributions of anti-tubulin immunofluo-
FIG. 1. Anti-tubulin staining of meiosis in C. eleguns hermaphrodites. (a) Oocytes in the proximal arm of the hermaphrodite gonad in diakinesis. The most mature oocyte is at the bottom of the line of oocytes. (b) Barrel-shaped meiotic spindle in an embryo. The cytoplasmic staining is underexposed so that the structure of the spindle may be seen. X1290.
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FIG;. 2. Anti-tubulin staining of meiosis in P. redivivus females. (a) Proximal arm of the ovary of P. redivivus. (b) Metaphase I. (c) Barrelshape td metaphase II spindle (arrow) and first polar body in an embryo. (d) Same embryo shown in (c). but stained with Hoechst 33258 to show the centrally located sperm nucleus. Individual chromosomes may also be seen on the metaphase II plate (arrow) and in the first polar body. x1200.
while in P. redivivus the immunofluorescent staining was concentrated around the oocyte nuclei (Fig. 2a). In both species the meiotic spindles were barrel-shaped and microtubules appeared to originate from several foci at the spindle poles (Figs. lb and 2b and c). The polar bodies of C elegant did not stain with anti-tubulin, whereas the first polar body in P. redivivus displayed the characteristic pattern shown in Fig. 2c and a midbody stained prominently with anti-tubulin following the completion of the meiotic divisions (Fig. 9). 2.
Pronuclear Migration and Fmatim Cleavage Spindle in C elegans
of the First
Video recordings were made of developing embryos from the time of the meiotic divisions through first cleavage. Detailed observations on the events that take
place following fertilization have been described for C elegans var. Bergerac and other nematodes (Nigon et aL, 1960) and for C! elegans var. Bristol (Hirsh et aL, 1976; von Ehrenstein and Schierenberg, 1980). Only certain points relating to pronuclear migration will be discussed here. Upon completion of meiosis II the pronuclei reappear and then increase in diameter. The sperm pronucleus is apposed to the posterior surface of the embryo while the egg pronucleus is situated at the anterior end. The egg pronucleus then begins to migrate over a distance of approximately 30 pm directly toward the sperm pronucleus, passing through a pseudocleavage furrow that constricts the embryo. The rate of movement increases as the egg pronucleus approaches the sperm pronucleus (Fig. 3). The sperm pronucleus migrates over a distance of approximately 7 pm at a rate of 4 pm/min toward the center of the embryo in the later stages of
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FIG. 3. Pronuclear migration in C elegant. The distance of the sperm (0) and egg (m) pronuclei from the posterior end of the egg was plotted and the internuclear distance (A) was calculated from tracings of a video recording of a fertilized embryo. The juxtaposed pronuclei (0) migrated back toward the center of the embryo where the first cleavage spindle developed. The anterior aster (0) remained stationary while the posterior aster (0) migrated posteriorly and began to rock at the last time point plotted. Time is in minutes before first cleavage.
the egg pronuclear migration (Fig. 3). The path taken fashion except that the aster at the polar body end of by the egg pronucleus is in a direct line toward the the embryo rocked from side-to-side and migrated toward the polar body end of the embryo. Upon completion sperm pronucleus and may mean that the two pronuclei are mutually attracted. A plot of the distance between of cytokinesis a two-cell embryo was produced with the the centers of the pronuclei during migration is also polar bodies adjacent to the smaller cell. The pattern of cleavages then continued in characteristic fashion shown in Fig. 3. Centrosomes, which become visible in the light mi- for the large and the small cell. croscope just before the two pronuclei meet, lie to either side of the sperm pronucleus, facing the egg pronucleus. 3. Formation of the Spem Asters in C. elegans The juxtaposed pronuclei and centrosomes migrate at The distribution of tubulin containing structures in a rate of about 2 pm/min toward the center of the embryo and rorate 90” such that the centrosomes come to fixed embryos was examined by indirect immunofluolie along the long axis of the embryo. The first cleavage rescence of antibodies to tubulin. Prior to migration of spindle forms here. No zygote nucleus is formed, the the pronuclei, when the egg pronucleus and the sperm pronuclear membranes breaking down only at prometa- pronucleus lie at opposite ends of the embryo, anti-tuphase. First cleavage is preceded by migration of the bulin staining revealed a small discrete point of stain posterior aster further posterior during anaphase and within a background of anti-tubulin staining that is characteristic of oocytes (Fig. 4a). A single centrosome by the side-to-side rocking of the aster. The development of a single C. elegans embryo in which lying between the sperm pronucleus and the surface of the polarity of the first cleavage was reversed with re- the embryo has been identified in electron micrographs spect to the polar bodies was recorded from meiosis II. of serially sectioned embryos of this age (Fig. 5a). The sperm pronucleus behaved differently in this emAs development proceeds, two microtubule organizing bryo, as it was first seen after the completion of meiosis centers may be distinguished. These grow in size and and formation of the egg pronucleus in the same end migrate from the cortical side of the sperm pronucleus of the embryo as the polar bodies and the egg pronucleus. to the interior side of the sperm pronucleus, facing the Both pronuclei increased in diameter and then the egg egg pronucleus (Figs. 4b and c). In electron micrographs pronucleus migrated to the sperm pronucleus, which of serially sectioned embryos at this stage the sperm remained stationary. Pseudocleavage furrows were ob- asters were similar in morphology to prophase centroserved at the opposite end of the embryo during mi- somes seen in older embryonic cells. They are composed gration. The juxtaposed pronuclei migrated to the center of centrioles surrounded by amorphous pericentriolar of the embryo where first cleavage took place in a normal material (Fig. 5b). When the egg pronucleus had passed
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FIG. 4. Indirect immunofluorescence staining of tubulin during formation of the first cleavage spindle in wild type C elegans embryos. (a) Single sperm aster adjacent to the male pronucleus before migration begins. (b) Growing sperm asters viewed from the surface of a tilted embryo and showing the extensive cytoplasmic network of tubulin. (c) Sperm asters facing the migrating egg pronucleus. (d) Pronuclei before juxtaposition. The male pronucleus had moved away from the edge of the embryo and fasicles of microtubules can be seen that extend from the asters toward the posterior cortex (bottom) of the embyro. (e) Pronuclear juxtaposition. (f) Pronuclear rotation. (g) Prometaphase of first cleavage. (h) Metaphase of first cleavage. The posterior aster (bottom) is displaced to the right suggesting that the spindle was rocking at the time of fixation. (i) Early anaphase. X1200.
through the pseudocleavage coursing around and between distinguished (Fig. 4d).
furrow, microtubules the pronuclei could be
The asters continue to grow as pronuclear migration proceeds, reaching a maximum size once the pronuclei are juxtaposed (Fig. 4e). Just prior to that time they
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FIG. 5. Morphology of sperm asters during pronuclear migration. (a) Sperm aster before migration begins at approximately the stage shown in Fig. 4a. The centrosome (arrow) lies between the male pronucleus (nu) and the outer membrane of the embryo. (b) One of the pair of sperm asters (arrow) lying adjacent to the male pronucleus (nu) and facing the egg pronucleus, which had migrated into the pseudocleavage furrow at the time of fixation. (c) A sperm aster lying adjacent to one of the juxtaposed pronuclei (nu) prior to rotation. (d) Section through a sperm aster and a pronucleus (nu) at the time of pronuclear juxtaposition. A fascicle of microtubules (arrow) extends toward the cortex. (a-c), X25,000. (d), X15,000.
become visible in the light microscope (Nigon et aL, 1960). ing the centrioles and the osmiophilic pericentriolar The asters at the time of pronuclear juxtaposition were material appeared to be less dense (Fig. 5~). Fasicles of distinct from the other stages. Cytoplasmic constituents microtubules extended to the cortex (Fig. 5d). appeared to be excluded from a larger volume surroundAfter rotation the first cleavage spindle develops
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FIG. 6. First cleavage in a zyg-9 embryo grown at the nonpermissive temperature. A series of tracings were made from video recordings of developing zyg-9 embryos at the nonpermissive temperature. Time is given relative to first cleavage. (a) Time: -6 min. Beginning of pseudocleavage. (b) Time: -5 min. Pseudocleavage. The asters (stippled areas) were first seen at this time. (c) Time: -4 min. Pseudocleavage furrow has regressed and asters have increased in size. (d) Time: -1 min. First cleavage. The nuclear membrane of the egg pronucleus appears to break down when the sperm pronucleus enters mitosis. (e) Time: 0 min. Telophase. Three cleavage furrows have been initiated. (f) Time: 7 min. Three cells were formed. The cleavage furrow passed through the egg pronucleus and two nuclei reformed. The arrow indicates that one of the nuclei derived from the egg pronucleus later migrated over to lie next to the daughter nucleus formed by the mitotic division of the sperm pronucleus.
around the pronuclei (Figs. 4f and g). The prometaphase and metaphase asters appeared to be of similar morphology (Figs. 4g and h), but by early anaphase the anterior aster was larger (Fig. 4i).
4 Formation of the First Cleavage Swindle in qg-9 Temperature-sensitive mutants of C elegans defective in early embryogenesis have been isolated and partially characterized (Hirsh et al., 1976; Schierenberg et al, 1980; Wood et al., 1980; Denich, 1982). Some of these have defects in the events leading up to the formation of the first cleavage spindle. Observations on one of these mutants are of interest here. The development of xyg-9 embryos at the nonpermissive temperature was studied with video recordings
(Fig. 6). Following the completion of meiosis the egg pronucleus failed to migrate toward the sperm pronucleus. The first cleavage spindle formed around the stationary sperm pronucleus in the absence of rotation. Shortly after the breakdown of the sperm pronuclear membrane the egg pronuclear membrane also broke down. Initially, cytokinesis resulted in the formation of three cells, but later one furrow relaxed. Following cleavage each cell might contain from zero to three nuclei. The nuclei were derived from the mitotic division of the sperm pronucleus, the reformation of the egg pronucleus, sometimes the division of the egg pronucleus apparently due to a cleavage furrow passing through it, and sometimes by the resorption of the second polar body into the embryo.
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FIG. 7. Indirect Immunofluorescence staining with anti-tubulin during the formation of the first cleavage spindle in zyg-9 embryos at 25°C. (a) Sperm aster showing the unstained center of the aster and short astral rays. (b) Bright field of embryo in (a). (c) Sperm asters. (d) Bright field of embryo in (c). The large arrow indicates the position of the egg pronucleus which failed to migrate and the small arrow the position of the sperm pronucleus. (e) First cleavage perpendicular to the long axis of the embryo. The arrow indicates the position of the egg pronucleus. (f) Bright field of the embryo in (e). (g) First cleavage asters and an accumulation of polymerized tubulin which may be centered over the egg pronucleus. (h) Bright field of the embryo in (g). The arrow indicates the beginning of a cleavage furrow which forms in these embryos in addition to one between the asters to produce three cells at first cleavage. X1200.
Anti-tubulin staining of fixed embryos of xvg-9 at the nonpermissive temperature revealed that although there appeared to be many microtubules in the aster, they were short and failed to contact distant parts of the cortex (Fig. 7). One- and two-cell embryos of xyg-9 grown at the nonpermissive temperature were reconstructed from electron micrographs (Fig. 8). Abnormalities seen in the four mutant embryos included: the displacement of the second polar body to the interior of the embryo (two embryos), more than one nucleus per cell (4 embryos), either regression or incomplete formation of the first cleavage furrow (one embryo), and excessive accumulations of membranous vesicles (three embryos). Cen-
trioles were present in asters (four embryos), but the density of pericentriolar material appeared to be reduced in some embryos. 5.
Fmatim of the First Cleavage Spindle in P. redivivus
Although the pattern of early embryonic cell cleavages of P. redivivus closely resembles that observed in C. elegant (J. Sulston, personal communication), the events leading up to the formation of the first cleavage spindle appear to be different. At metaphase II the sperm pronucleus is found in the center of the embryo (Fig. 2d). Sperm asters could not be seen by anti-tubulin staining
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FIG. 8. Reconstruction of one- and two-cell zyg-9 embryos grown at the restrictive temperature. (a) One-cell embryo at telophase of first cleavage. Cleavage had occurred along the short axis of the embryo as indicated by the position of the asters (stippled areas). There were large amounts of endoplasmic reticulum or vesicular material surrounding the centrioles. The small telophase nuclei that were found between the asters probably derive from the mitotic division of the sperm pronucleus, whereas the telophase nuclei between the polar body (PB) and one of the asters are probably from the egg pronucleus. (b) Two-cell embryo. One cell contained one nucleus and the other three. (c) Twocell embryo. The smaller cell contained four nuclei together and the other cell a single nucleus and the second polar body. (d) One-cell embryo. Cytokinesis had failed or the cleavage furrow had regressed as suggested by a transverse furrow across half of the embryo. All four nuclei were in prometaphase and eight asters (stippled areas) were present. The polar body (PB) was found in the center of the embryo.
at this time, although centrioles adjacent to the sperm pronucleus were seen in electron micrographs of P. v-ediwivus embryos at metaphase II (D. Albertson and J. N. Thomson, unpublished observations). Upon completion of metaphase II the female pronucleus reformed and migrated toward the sperm pronucleus. Two microtubule organizing centers were first distinguished by immunofluorescence once the two pronuclei were sideby-side. These increased in size to form the first cleavage spindle.
DISCUSSION
The meiotic maturation divisions of mammalian oocytes are characterized by a barrel-shaped spindle. Centrioles are absent from the broad ends of the spindle and instead regions of fibrous material act as foci for microtubules (Calarco, 1972, Szollosi, 1972; Zamboni, 1972). In oocytes of C elegans and P. red&iw the meiotic spindles are also barrel-shaped and lack centrioles as demonstrated by reconstruction of electron micrographs of serial sections (D. G. Albertson and J. N. Thomson,
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staining of P. redivivus embryos. (a-c) Juxtaposition of pronuclei FIG. 9. Anti-tubulin metaphase II can be seen in (a) and (b). (d) Prometaphase of first cleavage. X1200.
and growth
of asters. The midbody
remaining
after
unpublished observations). In C elegant males and her- comparable to those described in sea urchins (Schatten, 1981). In these organisms experimental manipulations maphrodites the metaphase I and II spindles in primary and secondary spermatocytes are not barrel-shaped and with drugs that disrupt microtubules and microfilaments have suggested that the microtubules of the growing centrioles are present in the asters (D. G. Albertson and J. N. Thomson, unpublished observations). Cen- sperm aster mediate pronuclear migration (see Schatten, trioles are contributed to the embryo by the sperm. 1981, for references), possibly through assembly and Electron micrographs of sectioned sperm from C elegans disassembly of tubulin subunits. Furthermore, in favorable specimens bundles of microtubules connecting show that the centrioles are embedded in the material that surrounds the chromatin (Wolf et a& 1978). Fol- the migrating female pronucleus and the sperm aster lowing fertilization the centrioles remain adjacent to could be distinguished by indirect immunofluorescence of antibodies to tubulin (Bestor and Schatten, 1981). the sperm pronucleus. Metaphase I and II in nematode embryos appear to Microtubules appear to be involved in other nuclear mitake place independently of the events determining the grations, since in syncytia formed by virus-infected baby future anterior and posterior poles of the embryo, since hamster kidney cells nuclei move along bundles of miin C. elegans polar bodies may be found at either end crotubules and intermediate filaments (Wang et aL, of the embryo. On the other hand, the observation that 1979), and in Aspergillus nidulans it has been demonthe sperm pronucleus is situated at the future posterior strated that functional /3-tubulin is required for nuclear end of the embryo suggests that the position of the movement (Oakley and Morris, 1980). sperm pronucleus at or before pronuclear migration may The distribution of tubulin containing structures in determine or may reflect the polarity of the embryo. C. elegans suggests that the sperm pronucleus could be The rates of pronuclear movements in C. elegans are pushed or pulled away from the periphery of the embryo
DONNA G. ALBERTSON
First
by the developing sperm asters, since microtubules radiating from the asters to the surface can be seen in Fig. 4d. However, the migration of the egg pronucleus does not appear to involve microtubules directly even though the egg pronucleus appears to be attracted to the sperm pronucleus (and hence also to the sperm asters positioned to either side of it), since no bundle of microtubules could be distinguished that radiated from the asters to the egg pronucleus in the initial stages of migration. In P. redivivus embryos the egg and sperm pronuclei come together prior to the development of the sperm asters, a further suggestion that nematode pronuclei do not migrate along the astral rays. Alternatively, if microtubules are involved in egg pronuclear migration, they may be very labile or very few in number. It is also possible that asters might indirectly influence the migration of the egg pronucleus in C. elegant. At the time of pronuclear rotation and centration in C. elegant the asters were large and fascicles of microtubules extended to all regions of the periphery of the zygote (Fig. 3~). Therefore the position of the asters is such that they could direct these movements of the pronuclei. Also, pronuclear rotation and centration may require that the astral rays be large, since in xvg-9 embryos at the nonpermissive temperature the microtubules were short and centration and rotation did not occur. The reorientation of the asters along the long axis of the egg might be accomplished by slippage of the large asters into a more favorable conformation in the ellipsoidal shell. Alternatively, a specific interaction between the asters and either cytoplasmic constituents (Nigon et aL, 1960) or the cortex might result in rotation. Studies on the in vitro assembly and disassembly of microtubules suggest some possible agents. For example, a polysaccharide, isolated from the cortex of unfertilized sea urchin eggs causes inhibition of microtubule assembly or promotes disassembly by binding to microtubuleassociated proteins (Naruse and Sakai, 1981). Also, membrane phospholipids have been shown to bind microtubule protein and inhibit assembly (Reaven and Azhar, 1981). The asymmetric position of the first cleavage furrow results from the posterior migration of the posterior aster in anaphase. At this time the asters differ in size (Fig. 4i) and one can speculate that a specific interaction with the cortex might selectively block polymerization or even depolymerize microtubules to provide the smaller posterior aster. It may be that the depolymerization of microtubules in the posterior aster results in instabilities that promote the oscillations of the posterior aster while the anterior one remains tethered by the spherical array of astral microtubules. Alternatively, the difference in the size and later the difference in shape (Nigon et aL, 1960) of the centrosomes may reflect
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some intrinsic difference in the capacity of the two centrosomes to act as microtubule organizing centers. Some of these speculations may be clarified by further analysis of the morphology of asters in mutants which display defects at or before first cleavage and by analysis of other nematodes that are natural variants of the process of pronuclear migration observed in C elegana I thank E. Hedgecock for conversions, N. Thomson for electron microscopy, and K. Buck for help with photography. D.G.A. was a Thomas C. Usher Research Fellow. REFERENCES ALBERTSON,D. G., and THOMSON,J. N. (1982). The kinetochores of Caerwr~bditti elegana Chromosome 86,409-428. BESTOR,T. H., and SCHATTEN,G. (1981). Anti-tubulin immunofluorescence microscopy of microtubules present during the pronuclear movements of sea urchin fertilization. Da. Bill 88,80-91. BRENNER,S. (1974). The genetics of Caenor habditk elegana Genetics 77.71-94. CALARCO,P. (1972). The kinetochore in oocyte maturation. In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 65-86. Univ. Park Press, Baltimore. DENICH, K. T. R. (1982). “Cell Lineages and Developmental Defects of Temperature-Sensitive Embryonic Arrest Mutants of the Nematode Caenorhabditis elegans: The Giittingen Set.” Ph.D. Thesis, University of Manitoba, Winnipeg, Manitoba. DEPPE,U., SCHIERENBERG, E., COLE,T., KRIEG, C., SCHMIT&D., YODER, B., and VONEHRENSTEIN,G. (1978). Cell lineages of the embryo of the nematode Gwrwrhabditis ekgans. Proc. Natl. Acad Sci USA 75, 376-380. VONEHRENSTEIN,G., and SCHIERENBERG, E. (1980). Cell lineages and ekgana and other nematodes. In development of CaenorMitis “Nematodes as Biological Models,” Vol. 1. “Behavioral and Developmental Models” (B. M. Zuckerman, ed.), pp. 1-71. Academic Press, New York. GALL, J. G., and PARDUE,M. L. (1971). Nucleic acid hybridization in cytological preparations. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 21, pp. 470-480. Academic Press, New York. HIRSH, D., OPPENHEIM,D., and KLASS, M. (1976). Development of the reproductive system of Coenorhobditis elegans. Dev. BbL 49, 200219. KILMARTIN,J. V., WRIGHT,B., and MILSTEIN,C. (1982). Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J. CeU Biol 93, 576-582. NARUSE, H., and SAKAI, H. (1981). Evidence that a polysaccharide from the cortex of sea urchin egg inhibits microtubule assembly through its binding to microtubule-associated proteins. J. Biochem 90,581-587. NIGON,V., GUERRIER,P., and MONIN,H. (1960). L’Architecture polaire de l’oeuf et les mouvements des constituents cellulaires au tours des premieres etapes du developpement chez quelques nematodes. Bull. Biol Fr. Belg. 93,131-202. NIGON, V., and BRUN, J. (1955). L’evolution des structures nucleaires dans l’ovogenese de Coew/&cZitia elegans Maupas (1900). Chromoma 7, 129-169. OAKLEY, B. R., and MORRIS, N. R. (1980). Nuclear movement is &tubulin-dependent in AspergiUus nidulans CeU 19,255-262. REAVEN,E., and AZHAR, S. (1981). Effect of various hepatic membrane fractions on the role of membrane phospholipids. J. CeU BtiL 89, 300-308.
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in mammals. In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 47-64. Univ. Park Press, Baltimore. WANG, E., CROSS,R. K., and CHOPPIN,P. W. (1979). Involvement of microtubules and lo-nm filaments in the movement and positioning of nuclei in syncytia. J. CeU BioL 83,320-337. WOLF,N., HIRSH, D., and MCINTOSH,J. R. (1978). Spermatogenesis in males of the free-living nematode, Ca.enmMitti ekgans. J. Ultrmtmt. Res. 63, 155-169. WOOD,W. B., HECHT, R., CARR, S., VANDERSLICE,R., WOLF, N., and HIRSH, D. (1980). Parental effects and phenotypic characterization of mutations that affect early development in Caenorhabditis &guns Lkv. Biol
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ZAMBONI,L. (1972). Comparative studies on the ultrastructure of mammalian oocytes. In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 5-45. Univ. Park Press, Baltimore.
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Formation of the First Cleavage Spindle in Nematode Embryos DONNA G. ALBERTSON Laboratory of lWol.eoular Biology, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, England Received November 27, 1982;accepted in revised fwm August 2, 1983 The distribution of microtubules and microtubule organizing centers in the events leading up to the establishment of the first asymmetric cleavage furrow in nematode embryos was followed using indirect immunofluorescence of antibodies to tubulin. Oocytes arrest in meiotic prophase then undergo two meiotic reduction divisions after fertilization. At both of these divisions barrel-shaped spindles were observed. Initially a single microtubule organizing center was seen adjacent to the sperm pronucleus following fertilization in Caewrhabditis elegant, but later two sperm asters were distinguished. These increased in size as the egg pronucleus migrated toward the sperm pronucleus and reached maximum size, with fascicles of microtubules extending to the cortex, once the pronuclei had become juxtaposed. The first cleavage spindle formed following rotation and migration of the juxtaposed pronuclei back toward the center of the embryo. The distribution of microtubules in a temperature-sensitive mutant that fails in both pronuclear migration and rotation was also examined. Asters in the mutant embryos at the nonpermissive temperature contained only short microtubules suggesting that the morphology of the asters is important for directing the movement of the pronuclei. In Panagrellus redivivus sperm asters were not detected by anti-tubulin staining until the female pronucleus had migrated to the centrally placed sperm pronucleus. Asters then increased in size and formed the first cleavage spindle.
INTRODUCTION
metric cleavage furrow in C. elegant and Panagrellus redivivus.
Nematodes develop by an essentially invariant cell lineage (Sulston and Horvitz, 1977; Sulston et aL, 1982). Frequently, daughter cells with different developmental potentials are formed by an asymmetric cleavage, such that the sister cells differ in size as well as fate. Thus the eccentric location of a cleavage spindle in a cell is an early indicator that the cell division will be determinative. The mechanisms by which the cleavage furrow is positioned such that a cell is unequally partitioned may be studied in the asymmetric first cleavage of nematode embryos. Following fertilization the embryos of some nematodes display a series of well-defined movements that bring the pronuclei together and position them in the zygote such that first cleavage is asymmetric. This process has been analyzed in great detail by microcinematography up to the four-cell stage in Caenorhabditis elegant (var. Bergerac) and other nematodes (Nigon et a& 1960). More recently similar observations were made on C. eZegune(var. Bristol) and compared to mutants defective in early development (Hirsh et a& 1976; Deppe et al, 1978; von Ehrenstein and Schierenberg, 1980; Schierenberg et al, 1980; Wood et al, 1980; Denich, 1982). In this paper indirect immunofluorescence of antibodies to tubulin has been used to study the distribution of microtubules and microtubule organizing centers in the events leading up to the establishment of the first asym-
MATERIALS
AND
METHODS
Nematode Strains and Gwyth Cultures of the wild-type N2 strain of C. elegans (var. Bristol) were maintained at 20°C as described by Brenner (1974). Animals with the temperature-sensitive mutation b.Z4.& (zyg-9)11 (Wood et d, 1980) were maintained at 15°C. To obtain xvg-9 embryos grown at the restrictive temperature, plates of larvae were transferred to 25°C. Panagrellus redivivus was kindly provided by David Hooper, Rothamsted Experimental Station, Harpenden, Herts, England, and has been maintained in the laboratory since 1976. Indirect Immunc&orescence Staining of Embqos Young gravid adults were picked and placed in a 5to lo-p1 drop of buffer on a subbed slide (Gall and Pardue, 1971) then cut in half at the vulva so that embryos were released. A 12 X 12-mm coverslip was placed gently on top of the drop so that embryos were not crushed and the slide placed on dry ice for at least 10 min. Coverslips were removed and the slides were fixed in methanol at -20°C for 2 min and acetone at -20°C for 4 min. The slides were then passed through a series of alcohols and two changes of phosphate-buffered saline (0.15 MNaCl, 0.02 M KCl, 0.01 M sodium phosphate, pH 7, PBS). The excess PBS was wiped off the slide, leaving a drop over 61
0012-1606/34 $3.00 Copyright All rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
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the specimen to which the monoclonal antibody to tu- rescence were observed in oocytes from C. elegans and bulin YL1/2 or YOL1/34 (Kilmartin et a,!, 1982) was P. redivivus. In C. elegans staining was seen throughout added for 1 hr at 20°C. Slides were rinsed in two changes the oocytes in the proximal arm of the ovary (Fig. la), of PBS then FITC-anti-rat IgG (Miles, Slough, England) was added for 20 min at 20°C and the slides rinsed again. Some preparations were also stained with Hoechst 33258. Specimens were observed with a Zeiss RA microscope equipped with both epi- and transillumination using the Planapo 40X objective, so that FITC and Hoechst 33258 fluorescence might be viewed simultaneously or separately. The staining patterns of C. elegans embryos that were observed with YLV2 and YOL1/34 were indistinguishable. In the course of this work several hundred wild-type and mutant embryos were observed. Eighty wild-type and 70 xyg-9 embryos were photographed. Light Microscopzl of Living Embqos Embryos were dissected from hermaphrodites and mounted on agar slabs for viewing by Nomarksi differential interference contrast optics (Sulston and Horvitz, 1977). Pronuclear migration and first cleavage was analyzed in detail from video recordings of 10 wild-type and 10 xgg-9 embryos. Electron Microscopll The preparation of embryos for electron microscopy has been described previously (Albertson and Thomson, 1982). Twenty embryos, fixed at various times from fertilization through first cleavage have been sectioned. They were surveyed by reconstruction from electron micrographs taken of approximately every 15th serial section. Areas of interest were selected for more detailed study. RESULTS
1. Completion oj* Meiosis Nematode oocytes are arrested in prophase of meiosis and when meiosis resumes two polar bodies are formed at one pole of the ellipsoidal embryo (Nigon and Brun, 1955; Nigon et al, 1960). In C. elegans the oocyte nucleus migrates distal to the spermatheca before the oocyte passes from the proximal arm of the ovary into the spermatheca. Fertilization takes place at the opposite end of the oocyte as it begins to enter the spermatheca. Generally, metaphase I and II take place at the future anterior end of the C. elegans embryo, but infrequently embryos may be found with the polar bodies at the posterior end of the embryo. These develop normally (J. Sulston, personal communication). In contrast, in another nematode, P. redivivus, the polar bodies are located posteriorly. Different distributions of anti-tubulin immunofluo-
FIG. 1. Anti-tubulin staining of meiosis in C. eleguns hermaphrodites. (a) Oocytes in the proximal arm of the hermaphrodite gonad in diakinesis. The most mature oocyte is at the bottom of the line of oocytes. (b) Barrel-shaped meiotic spindle in an embryo. The cytoplasmic staining is underexposed so that the structure of the spindle may be seen. X1290.
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63
FIG;. 2. Anti-tubulin staining of meiosis in P. redivivus females. (a) Proximal arm of the ovary of P. redivivus. (b) Metaphase I. (c) Barrelshape td metaphase II spindle (arrow) and first polar body in an embryo. (d) Same embryo shown in (c). but stained with Hoechst 33258 to show the centrally located sperm nucleus. Individual chromosomes may also be seen on the metaphase II plate (arrow) and in the first polar body. x1200.
while in P. redivivus the immunofluorescent staining was concentrated around the oocyte nuclei (Fig. 2a). In both species the meiotic spindles were barrel-shaped and microtubules appeared to originate from several foci at the spindle poles (Figs. lb and 2b and c). The polar bodies of C elegant did not stain with anti-tubulin, whereas the first polar body in P. redivivus displayed the characteristic pattern shown in Fig. 2c and a midbody stained prominently with anti-tubulin following the completion of the meiotic divisions (Fig. 9). 2.
Pronuclear Migration and Fmatim Cleavage Spindle in C elegans
of the First
Video recordings were made of developing embryos from the time of the meiotic divisions through first cleavage. Detailed observations on the events that take
place following fertilization have been described for C elegans var. Bergerac and other nematodes (Nigon et aL, 1960) and for C! elegans var. Bristol (Hirsh et aL, 1976; von Ehrenstein and Schierenberg, 1980). Only certain points relating to pronuclear migration will be discussed here. Upon completion of meiosis II the pronuclei reappear and then increase in diameter. The sperm pronucleus is apposed to the posterior surface of the embryo while the egg pronucleus is situated at the anterior end. The egg pronucleus then begins to migrate over a distance of approximately 30 pm directly toward the sperm pronucleus, passing through a pseudocleavage furrow that constricts the embryo. The rate of movement increases as the egg pronucleus approaches the sperm pronucleus (Fig. 3). The sperm pronucleus migrates over a distance of approximately 7 pm at a rate of 4 pm/min toward the center of the embryo in the later stages of
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FIG. 3. Pronuclear migration in C elegant. The distance of the sperm (0) and egg (m) pronuclei from the posterior end of the egg was plotted and the internuclear distance (A) was calculated from tracings of a video recording of a fertilized embryo. The juxtaposed pronuclei (0) migrated back toward the center of the embryo where the first cleavage spindle developed. The anterior aster (0) remained stationary while the posterior aster (0) migrated posteriorly and began to rock at the last time point plotted. Time is in minutes before first cleavage.
the egg pronuclear migration (Fig. 3). The path taken fashion except that the aster at the polar body end of by the egg pronucleus is in a direct line toward the the embryo rocked from side-to-side and migrated toward the polar body end of the embryo. Upon completion sperm pronucleus and may mean that the two pronuclei are mutually attracted. A plot of the distance between of cytokinesis a two-cell embryo was produced with the the centers of the pronuclei during migration is also polar bodies adjacent to the smaller cell. The pattern of cleavages then continued in characteristic fashion shown in Fig. 3. Centrosomes, which become visible in the light mi- for the large and the small cell. croscope just before the two pronuclei meet, lie to either side of the sperm pronucleus, facing the egg pronucleus. 3. Formation of the Spem Asters in C. elegans The juxtaposed pronuclei and centrosomes migrate at The distribution of tubulin containing structures in a rate of about 2 pm/min toward the center of the embryo and rorate 90” such that the centrosomes come to fixed embryos was examined by indirect immunofluolie along the long axis of the embryo. The first cleavage rescence of antibodies to tubulin. Prior to migration of spindle forms here. No zygote nucleus is formed, the the pronuclei, when the egg pronucleus and the sperm pronuclear membranes breaking down only at prometa- pronucleus lie at opposite ends of the embryo, anti-tuphase. First cleavage is preceded by migration of the bulin staining revealed a small discrete point of stain posterior aster further posterior during anaphase and within a background of anti-tubulin staining that is characteristic of oocytes (Fig. 4a). A single centrosome by the side-to-side rocking of the aster. The development of a single C. elegans embryo in which lying between the sperm pronucleus and the surface of the polarity of the first cleavage was reversed with re- the embryo has been identified in electron micrographs spect to the polar bodies was recorded from meiosis II. of serially sectioned embryos of this age (Fig. 5a). The sperm pronucleus behaved differently in this emAs development proceeds, two microtubule organizing bryo, as it was first seen after the completion of meiosis centers may be distinguished. These grow in size and and formation of the egg pronucleus in the same end migrate from the cortical side of the sperm pronucleus of the embryo as the polar bodies and the egg pronucleus. to the interior side of the sperm pronucleus, facing the Both pronuclei increased in diameter and then the egg egg pronucleus (Figs. 4b and c). In electron micrographs pronucleus migrated to the sperm pronucleus, which of serially sectioned embryos at this stage the sperm remained stationary. Pseudocleavage furrows were ob- asters were similar in morphology to prophase centroserved at the opposite end of the embryo during mi- somes seen in older embryonic cells. They are composed gration. The juxtaposed pronuclei migrated to the center of centrioles surrounded by amorphous pericentriolar of the embryo where first cleavage took place in a normal material (Fig. 5b). When the egg pronucleus had passed
DONNA G. ALBERTSON
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FIG. 4. Indirect immunofluorescence staining of tubulin during formation of the first cleavage spindle in wild type C elegans embryos. (a) Single sperm aster adjacent to the male pronucleus before migration begins. (b) Growing sperm asters viewed from the surface of a tilted embryo and showing the extensive cytoplasmic network of tubulin. (c) Sperm asters facing the migrating egg pronucleus. (d) Pronuclei before juxtaposition. The male pronucleus had moved away from the edge of the embryo and fasicles of microtubules can be seen that extend from the asters toward the posterior cortex (bottom) of the embyro. (e) Pronuclear juxtaposition. (f) Pronuclear rotation. (g) Prometaphase of first cleavage. (h) Metaphase of first cleavage. The posterior aster (bottom) is displaced to the right suggesting that the spindle was rocking at the time of fixation. (i) Early anaphase. X1200.
through the pseudocleavage coursing around and between distinguished (Fig. 4d).
furrow, microtubules the pronuclei could be
The asters continue to grow as pronuclear migration proceeds, reaching a maximum size once the pronuclei are juxtaposed (Fig. 4e). Just prior to that time they
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FIG. 5. Morphology of sperm asters during pronuclear migration. (a) Sperm aster before migration begins at approximately the stage shown in Fig. 4a. The centrosome (arrow) lies between the male pronucleus (nu) and the outer membrane of the embryo. (b) One of the pair of sperm asters (arrow) lying adjacent to the male pronucleus (nu) and facing the egg pronucleus, which had migrated into the pseudocleavage furrow at the time of fixation. (c) A sperm aster lying adjacent to one of the juxtaposed pronuclei (nu) prior to rotation. (d) Section through a sperm aster and a pronucleus (nu) at the time of pronuclear juxtaposition. A fascicle of microtubules (arrow) extends toward the cortex. (a-c), X25,000. (d), X15,000.
become visible in the light microscope (Nigon et aL, 1960). ing the centrioles and the osmiophilic pericentriolar The asters at the time of pronuclear juxtaposition were material appeared to be less dense (Fig. 5~). Fasicles of distinct from the other stages. Cytoplasmic constituents microtubules extended to the cortex (Fig. 5d). appeared to be excluded from a larger volume surroundAfter rotation the first cleavage spindle develops
DONNA G. ALBERTSON
First Cleavage Spindle Formation
67
FIG. 6. First cleavage in a zyg-9 embryo grown at the nonpermissive temperature. A series of tracings were made from video recordings of developing zyg-9 embryos at the nonpermissive temperature. Time is given relative to first cleavage. (a) Time: -6 min. Beginning of pseudocleavage. (b) Time: -5 min. Pseudocleavage. The asters (stippled areas) were first seen at this time. (c) Time: -4 min. Pseudocleavage furrow has regressed and asters have increased in size. (d) Time: -1 min. First cleavage. The nuclear membrane of the egg pronucleus appears to break down when the sperm pronucleus enters mitosis. (e) Time: 0 min. Telophase. Three cleavage furrows have been initiated. (f) Time: 7 min. Three cells were formed. The cleavage furrow passed through the egg pronucleus and two nuclei reformed. The arrow indicates that one of the nuclei derived from the egg pronucleus later migrated over to lie next to the daughter nucleus formed by the mitotic division of the sperm pronucleus.
around the pronuclei (Figs. 4f and g). The prometaphase and metaphase asters appeared to be of similar morphology (Figs. 4g and h), but by early anaphase the anterior aster was larger (Fig. 4i).
4 Formation of the First Cleavage Swindle in qg-9 Temperature-sensitive mutants of C elegans defective in early embryogenesis have been isolated and partially characterized (Hirsh et al., 1976; Schierenberg et al, 1980; Wood et al., 1980; Denich, 1982). Some of these have defects in the events leading up to the formation of the first cleavage spindle. Observations on one of these mutants are of interest here. The development of xyg-9 embryos at the nonpermissive temperature was studied with video recordings
(Fig. 6). Following the completion of meiosis the egg pronucleus failed to migrate toward the sperm pronucleus. The first cleavage spindle formed around the stationary sperm pronucleus in the absence of rotation. Shortly after the breakdown of the sperm pronuclear membrane the egg pronuclear membrane also broke down. Initially, cytokinesis resulted in the formation of three cells, but later one furrow relaxed. Following cleavage each cell might contain from zero to three nuclei. The nuclei were derived from the mitotic division of the sperm pronucleus, the reformation of the egg pronucleus, sometimes the division of the egg pronucleus apparently due to a cleavage furrow passing through it, and sometimes by the resorption of the second polar body into the embryo.
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FIG. 7. Indirect Immunofluorescence staining with anti-tubulin during the formation of the first cleavage spindle in zyg-9 embryos at 25°C. (a) Sperm aster showing the unstained center of the aster and short astral rays. (b) Bright field of embryo in (a). (c) Sperm asters. (d) Bright field of embryo in (c). The large arrow indicates the position of the egg pronucleus which failed to migrate and the small arrow the position of the sperm pronucleus. (e) First cleavage perpendicular to the long axis of the embryo. The arrow indicates the position of the egg pronucleus. (f) Bright field of the embryo in (e). (g) First cleavage asters and an accumulation of polymerized tubulin which may be centered over the egg pronucleus. (h) Bright field of the embryo in (g). The arrow indicates the beginning of a cleavage furrow which forms in these embryos in addition to one between the asters to produce three cells at first cleavage. X1200.
Anti-tubulin staining of fixed embryos of xvg-9 at the nonpermissive temperature revealed that although there appeared to be many microtubules in the aster, they were short and failed to contact distant parts of the cortex (Fig. 7). One- and two-cell embryos of xyg-9 grown at the nonpermissive temperature were reconstructed from electron micrographs (Fig. 8). Abnormalities seen in the four mutant embryos included: the displacement of the second polar body to the interior of the embryo (two embryos), more than one nucleus per cell (4 embryos), either regression or incomplete formation of the first cleavage furrow (one embryo), and excessive accumulations of membranous vesicles (three embryos). Cen-
trioles were present in asters (four embryos), but the density of pericentriolar material appeared to be reduced in some embryos. 5.
Fmatim of the First Cleavage Spindle in P. redivivus
Although the pattern of early embryonic cell cleavages of P. redivivus closely resembles that observed in C. elegant (J. Sulston, personal communication), the events leading up to the formation of the first cleavage spindle appear to be different. At metaphase II the sperm pronucleus is found in the center of the embryo (Fig. 2d). Sperm asters could not be seen by anti-tubulin staining
DONNA G. ALBERTSON
First Cleavage Spindle Formation
69
FIG. 8. Reconstruction of one- and two-cell zyg-9 embryos grown at the restrictive temperature. (a) One-cell embryo at telophase of first cleavage. Cleavage had occurred along the short axis of the embryo as indicated by the position of the asters (stippled areas). There were large amounts of endoplasmic reticulum or vesicular material surrounding the centrioles. The small telophase nuclei that were found between the asters probably derive from the mitotic division of the sperm pronucleus, whereas the telophase nuclei between the polar body (PB) and one of the asters are probably from the egg pronucleus. (b) Two-cell embryo. One cell contained one nucleus and the other three. (c) Twocell embryo. The smaller cell contained four nuclei together and the other cell a single nucleus and the second polar body. (d) One-cell embryo. Cytokinesis had failed or the cleavage furrow had regressed as suggested by a transverse furrow across half of the embryo. All four nuclei were in prometaphase and eight asters (stippled areas) were present. The polar body (PB) was found in the center of the embryo.
at this time, although centrioles adjacent to the sperm pronucleus were seen in electron micrographs of P. v-ediwivus embryos at metaphase II (D. Albertson and J. N. Thomson, unpublished observations). Upon completion of metaphase II the female pronucleus reformed and migrated toward the sperm pronucleus. Two microtubule organizing centers were first distinguished by immunofluorescence once the two pronuclei were sideby-side. These increased in size to form the first cleavage spindle.
DISCUSSION
The meiotic maturation divisions of mammalian oocytes are characterized by a barrel-shaped spindle. Centrioles are absent from the broad ends of the spindle and instead regions of fibrous material act as foci for microtubules (Calarco, 1972, Szollosi, 1972; Zamboni, 1972). In oocytes of C elegans and P. red&iw the meiotic spindles are also barrel-shaped and lack centrioles as demonstrated by reconstruction of electron micrographs of serial sections (D. G. Albertson and J. N. Thomson,
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staining of P. redivivus embryos. (a-c) Juxtaposition of pronuclei FIG. 9. Anti-tubulin metaphase II can be seen in (a) and (b). (d) Prometaphase of first cleavage. X1200.
and growth
of asters. The midbody
remaining
after
unpublished observations). In C elegant males and her- comparable to those described in sea urchins (Schatten, 1981). In these organisms experimental manipulations maphrodites the metaphase I and II spindles in primary and secondary spermatocytes are not barrel-shaped and with drugs that disrupt microtubules and microfilaments have suggested that the microtubules of the growing centrioles are present in the asters (D. G. Albertson and J. N. Thomson, unpublished observations). Cen- sperm aster mediate pronuclear migration (see Schatten, trioles are contributed to the embryo by the sperm. 1981, for references), possibly through assembly and Electron micrographs of sectioned sperm from C elegans disassembly of tubulin subunits. Furthermore, in favorable specimens bundles of microtubules connecting show that the centrioles are embedded in the material that surrounds the chromatin (Wolf et a& 1978). Fol- the migrating female pronucleus and the sperm aster lowing fertilization the centrioles remain adjacent to could be distinguished by indirect immunofluorescence of antibodies to tubulin (Bestor and Schatten, 1981). the sperm pronucleus. Metaphase I and II in nematode embryos appear to Microtubules appear to be involved in other nuclear mitake place independently of the events determining the grations, since in syncytia formed by virus-infected baby future anterior and posterior poles of the embryo, since hamster kidney cells nuclei move along bundles of miin C. elegans polar bodies may be found at either end crotubules and intermediate filaments (Wang et aL, of the embryo. On the other hand, the observation that 1979), and in Aspergillus nidulans it has been demonthe sperm pronucleus is situated at the future posterior strated that functional /3-tubulin is required for nuclear end of the embryo suggests that the position of the movement (Oakley and Morris, 1980). sperm pronucleus at or before pronuclear migration may The distribution of tubulin containing structures in determine or may reflect the polarity of the embryo. C. elegans suggests that the sperm pronucleus could be The rates of pronuclear movements in C. elegans are pushed or pulled away from the periphery of the embryo
DONNA G. ALBERTSON
First
by the developing sperm asters, since microtubules radiating from the asters to the surface can be seen in Fig. 4d. However, the migration of the egg pronucleus does not appear to involve microtubules directly even though the egg pronucleus appears to be attracted to the sperm pronucleus (and hence also to the sperm asters positioned to either side of it), since no bundle of microtubules could be distinguished that radiated from the asters to the egg pronucleus in the initial stages of migration. In P. redivivus embryos the egg and sperm pronuclei come together prior to the development of the sperm asters, a further suggestion that nematode pronuclei do not migrate along the astral rays. Alternatively, if microtubules are involved in egg pronuclear migration, they may be very labile or very few in number. It is also possible that asters might indirectly influence the migration of the egg pronucleus in C. elegant. At the time of pronuclear rotation and centration in C. elegant the asters were large and fascicles of microtubules extended to all regions of the periphery of the zygote (Fig. 3~). Therefore the position of the asters is such that they could direct these movements of the pronuclei. Also, pronuclear rotation and centration may require that the astral rays be large, since in xvg-9 embryos at the nonpermissive temperature the microtubules were short and centration and rotation did not occur. The reorientation of the asters along the long axis of the egg might be accomplished by slippage of the large asters into a more favorable conformation in the ellipsoidal shell. Alternatively, a specific interaction between the asters and either cytoplasmic constituents (Nigon et aL, 1960) or the cortex might result in rotation. Studies on the in vitro assembly and disassembly of microtubules suggest some possible agents. For example, a polysaccharide, isolated from the cortex of unfertilized sea urchin eggs causes inhibition of microtubule assembly or promotes disassembly by binding to microtubuleassociated proteins (Naruse and Sakai, 1981). Also, membrane phospholipids have been shown to bind microtubule protein and inhibit assembly (Reaven and Azhar, 1981). The asymmetric position of the first cleavage furrow results from the posterior migration of the posterior aster in anaphase. At this time the asters differ in size (Fig. 4i) and one can speculate that a specific interaction with the cortex might selectively block polymerization or even depolymerize microtubules to provide the smaller posterior aster. It may be that the depolymerization of microtubules in the posterior aster results in instabilities that promote the oscillations of the posterior aster while the anterior one remains tethered by the spherical array of astral microtubules. Alternatively, the difference in the size and later the difference in shape (Nigon et aL, 1960) of the centrosomes may reflect
Cleavage
Spindle
Forwmtim
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some intrinsic difference in the capacity of the two centrosomes to act as microtubule organizing centers. Some of these speculations may be clarified by further analysis of the morphology of asters in mutants which display defects at or before first cleavage and by analysis of other nematodes that are natural variants of the process of pronuclear migration observed in C elegana I thank E. Hedgecock for conversions, N. Thomson for electron microscopy, and K. Buck for help with photography. D.G.A. was a Thomas C. Usher Research Fellow. REFERENCES ALBERTSON,D. G., and THOMSON,J. N. (1982). The kinetochores of Caerwr~bditti elegana Chromosome 86,409-428. BESTOR,T. H., and SCHATTEN,G. (1981). Anti-tubulin immunofluorescence microscopy of microtubules present during the pronuclear movements of sea urchin fertilization. Da. Bill 88,80-91. BRENNER,S. (1974). The genetics of Caenor habditk elegana Genetics 77.71-94. CALARCO,P. (1972). The kinetochore in oocyte maturation. In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.), pp. 65-86. Univ. Park Press, Baltimore. DENICH, K. T. R. (1982). “Cell Lineages and Developmental Defects of Temperature-Sensitive Embryonic Arrest Mutants of the Nematode Caenorhabditis elegans: The Giittingen Set.” Ph.D. Thesis, University of Manitoba, Winnipeg, Manitoba. DEPPE,U., SCHIERENBERG, E., COLE,T., KRIEG, C., SCHMIT&D., YODER, B., and VONEHRENSTEIN,G. (1978). Cell lineages of the embryo of the nematode Gwrwrhabditis ekgans. Proc. Natl. Acad Sci USA 75, 376-380. VONEHRENSTEIN,G., and SCHIERENBERG, E. (1980). Cell lineages and ekgana and other nematodes. In development of CaenorMitis “Nematodes as Biological Models,” Vol. 1. “Behavioral and Developmental Models” (B. M. Zuckerman, ed.), pp. 1-71. Academic Press, New York. GALL, J. G., and PARDUE,M. L. (1971). Nucleic acid hybridization in cytological preparations. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 21, pp. 470-480. Academic Press, New York. HIRSH, D., OPPENHEIM,D., and KLASS, M. (1976). Development of the reproductive system of Coenorhobditis elegans. Dev. BbL 49, 200219. KILMARTIN,J. V., WRIGHT,B., and MILSTEIN,C. (1982). Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J. CeU Biol 93, 576-582. NARUSE, H., and SAKAI, H. (1981). Evidence that a polysaccharide from the cortex of sea urchin egg inhibits microtubule assembly through its binding to microtubule-associated proteins. J. Biochem 90,581-587. NIGON,V., GUERRIER,P., and MONIN,H. (1960). L’Architecture polaire de l’oeuf et les mouvements des constituents cellulaires au tours des premieres etapes du developpement chez quelques nematodes. Bull. Biol Fr. Belg. 93,131-202. NIGON, V., and BRUN, J. (1955). L’evolution des structures nucleaires dans l’ovogenese de Coew/&cZitia elegans Maupas (1900). Chromoma 7, 129-169. OAKLEY, B. R., and MORRIS, N. R. (1980). Nuclear movement is &tubulin-dependent in AspergiUus nidulans CeU 19,255-262. REAVEN,E., and AZHAR, S. (1981). Effect of various hepatic membrane fractions on the role of membrane phospholipids. J. CeU BtiL 89, 300-308.
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