During gastrulation of a vertebrate embryo, various cellmovements including involution and convergence lead toformation of a gastrula with the three classically defined germlayers and a blueprint of the body plan. In amphibians andfishes, gastrulation is preceded and accompanied by epiboly -a process of vegetal expansion of the blastoderm (Keller, 1980;Trinkaus, 1951). The mechanisms that determine directions ofcell migrations and forces that drive epiboly and gastrulationmovements are still poorly understood. Several features maketeleost embryos attractive for studies of these processes(Trinkaus et al., 1992; Kimmel, 1989). First, due to opticalclarity of certain teleost embryos, cell movements and behaviorcan be observed in intact embryos (Trinkaus et al., 1991;
Warga and Kimmel, 1990). Second, the relatively large size of
Fundulus heteroclitus or zebrafish (Danio rerio) embryosallows for easy experimental manipulations (Ho, 1992).Finally, zebrafish is amenable to genetic analysis (Streisingeret al., 1981).
At the onset of epiboly in teleosts with meroblastic earlycleavages, a syncytial yolk cell is capped by the blastoderm(Fig. 1A). A superficial, single-cell-thick enveloping layer(EVL) is attached by its vegetal rim to the yolk cell, andcovers a mass of deep cells. Three compartments of continu-ous cortical cytoplasm can be distinguished in the yolk cell(Trinkaus, 1992, 1993; Long, 1984). A thin, anuclear yolkcytoplasmic layer (YCL) surrounds the bulk of the yolk masswith the vegetal pole. The external yolk syncytial layer (YSL),located between the YCL and the blastoderm rim, is a rela-
Danio rerio), meroblastic cleavages generatean embryo in which blastomeres cover the animal pole ofa large yolk cell. At the 500-1000 cell stage, the marginalblastomeres fuse with the yolk cell forming the yolksyncytial layer. During epiboly the blastoderm and the yolksyncytial layer spread toward the vegetal pole. We havestudied developmental changes in organization andfunction during epiboly of two distinct microtubule arrayslocated in the cortical cytoplasm of the yolk cell. In theanuclear yolk cytoplasmic layer, an array of microtubulesextends along the animal-vegetal axis to the vegetal pole. Inthe early blastula the yolk cytoplasmic layer microtubulesappear to originate from the marginal blastomeres. Onceformed, the yolk syncytial layer exhibits its own networkof intercrossing mitotic or interphase microtubules. Themicrotubules of the yolk cytoplasmic layer emanate fromthe microtubule network of the syncytial layer.
At the onset of epiboly, the external yolk syncytial layernarrows, the syncytial nuclei become tightly packed andthe network of intercrossing microtubules surroundingthem becomes denser. Soon after, there is a vegetalexpansion of the blastoderm and of the yolk syncytial layerwith its network of intercrossing microtubules. Concomi-tantly, the yolk cytoplasmic layer diminishes and its set ofanimal-vegetal microtubules becomes shorter.
We investigated the involvement of microtubules in
epiboly using the microtubule depolymerizing agent noco-dazole and a stabilizing agent taxol. In embryos treatedwith nocodazole, microtubules were absent and epibolicmovements of the yolk syncytial nuclei were blocked. Incontrast, the vegetal expansion of the enveloping layer anddeep cells was only partially inhibited. The process of endo-cytosis, proposed to play a major role in epiboly of the yolksyncytial layer (Betchaku, T. and Trinkaus, J. P. (1986)Am. Zool. 26, 193-199), was still observed in nocodazole-treated embryos. Treatment of embryos with taxol led to adelay in all epibolic movements.
We propose that the yolk cell microtubules contributeeither directly or indirectly to all epibolic movements.However, the epibolic movements of the yolk syncytiallayer nuclei and of the blastoderm are not coupled, andonly movements of the yolk syncytial nuclei are absolutelydependent on microtubules. We hypothesize that themicrotubule network of the syncytial layer and the animal-vegetal set of the yolk cytoplasmic layer contribute differ-ently to various aspects of epiboly. Models that address themechanisms by which the two microtubule arrays mightfunction during epiboly are discussed.
Microtubule arrays of the zebrafish yolk cell: organization and function during
epiboly
Lilianna Solnica-Krezel and Wolfgang Driever
Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 13th Street, Bldg. 149,Charlestown, MA 02129, USA
2444 L. Solnica-Krezel and W. Driever
BLASTODERM:Deep Cells
Enveloping Layer
Yolk Cytoplasmic Layer
External YSL
Internal YSL
Yolk Syncytial Nuclei
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAA
AAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAA
Internal YSL
External YSL
YCL
A B
AAAAAA
AAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAA
AAAAAAAAA
Internal YSL
YCL
C
AAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAA
AAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAA
AAA
Internal YSL
External YSL
YCL
AAAAAAAAA
AAAAAAAAA
D
E
Internal YSN
Fig. 1. Schematic illustration of the changes in the organization of thecortical cytoplasm of the yolk cell in relation to other cell types in zebrafishembryo during epiboly in normal (A-D) and nocodazole-treated embryos(E). The organization of microtubule networks in the YSL and YCLobserved in this study is indicated by thin lines. Only part of the blastodermis shown to reveal the morphology of the yolk cell. The relative sizes ofelements are not proportional. (A) The late blastula just before the onset ofepiboly (sphere stage, 4.0 h). The blastoderm, composed of the internal deepcells and the superficial enveloping layer (EVL), is positioned atop of thesyncytial yolk cell. The animal surface of the yolk cell underlying theblastoderm is flat. Most of the yolk syncytial nuclei (YSN) are in theexternal yolk syncytial layer (external YSL) vegetal to the blastoderm. Themicrotubules of the external YSL form a network. The organization ofmicrotubules in the internal YSL at this stage of development is not known.The microtubules of the anuclear yolk cytoplasmic layer (YCL) radiate fromthe organizing centers associated with the vegetal-most YSN and arealigned along the animal-vegetal axis. (B) 30% epiboly (4.7h). Theblastoderm covers 30% of the yolk cell that bulged toward the animal pole
taking on a dome shape. The external YSL has contracted and exhibits densely packed YSN and a dense network of microtubules. The externalYSL is partially covered by the expanding vegetally blastoderm. (C) 50% epiboly (5.2h). The blastoderm arrives at 50% of the yolk celllatitude and covers almost completely the YSN which is also migrating vegetally and the YSL microtubule network. Only the YCL with itsarray of the animal-vegetal microtubules is visible vegetally to the blastoderm. (D) 60% epiboly (6.5h). Deep cells cover 60% of the yolk cell.The YSN nuclei are now visible in front of the blastoderm and lead the epibolic movement. The YSN are often stretched along the animal-vegetal axis. The EVL rim is closer to the vegetal pole than the margin of deep cells. The YCL is diminished. (E) An 6.5h embryo (same ageas in D) treated from the sphere stage on with 10
µg/ml nocodazole. Microtubules are absent. The yolk cell acquired the sphere shape. TheYSN are blocked in their movement towards the vegetal pole and do not exhibit elongated shapes. Deep cells move very slowly toward thevegetal pole and almost cover the YSN. Epiboly of the EVL is slower than in control embryos.
2445Microtubule arrays of the zebrafish yolk cell
tively thick belt of cytoplasm populated by the yolk syncytialnuclei (YSN). Beneath the blastoderm is the internal YSLcomprising a thinner cytoplasm also populated by the YSN.Epiboly in Fundulus begins with a contraction of the externalYSL that becomes covered by the expanding blastoderm. Sub-sequently, the YSL and blastoderm expand vegetally, whilethe YCL progressively disappears (Trinkaus, 1993; Wargaand Kimmel, 1990). Studies in Fundulus indicated that theepiboly of the YSL is autonomous, i.e. it can occur in theabsence of the blastoderm (Betchaku and Trinkaus, 1978;Trinkaus, 1951). Moreover, vegetal expansion of the EVLduring epiboly may be driven by the epibolic movements ofthe YSL, to which the EVL is tightly linked at its margin(Betchaku and Trinkaus, 1978). The molecular basis for theexpansion of the YSL is not understood. It has been suggestedthat microfilaments may play a role in this process (Trinkaus,1984). Recently, it has been proposed that epiboly is alsodriven by a pulling force dependent on microtubules presentin the yolk cell (Strähle and Jesuthasan, 1993). However,developmental changes of the yolk cell microtubule organiz-ation and their involvement in distinct aspects of epiboly werenot studied.
Here, we demonstrate that two distinct microtubule arraysexist in the cortical cytoplasm of the zebrafish yolk cell. TheYCL contains an array of microtubules aligned in the directionof epiboly, extending toward the vegetal pole. Once formed,the YSL exhibits its own network of intercrossing interphaseor mitotic microtubules. Our studies demonstrate that thechanges in the configuration of the yolk cell microtubules arestrictly correlated with epibolic movements. Analysis ofepiboly in embryos treated with nocodazole and taxol indicatesthat microtubules are necessary and actively involved inepibolic movements of the syncytial nuclei, and only partiallyrequired for the remaining aspects of epiboly.
MATERIALS AND METHODS
ReagentsReagents were obtained from Sigma Chemical Co. (St Louis, MO),with the following exceptions. Formaldehyde (EM grade) and Ladd-o-lac from LADD (Burlington, VT), glutaraldehyde from Ted Pella(Redding, CA), taxol from Drug Synthesis and Chemistry Branch,Developmental Therapeutics Program, Division of Cancer Treatment,of the National Cancer Institute (Bethesda, MD), and Hoechst 33258from Molecular Probes (Eugene, OR). Antibodies: KMX-1, a mouseIgG monoclonal antibody from Boehringer Mannheim (Germany);DM1A a mouse IgG antibody (ICN Immunobiologicals, Costa Mesa,CA); alkaline phosphatase-conjugated anti-mouse IgG serum andTexas Red-conjugated anti-mouse IgG antibody Jackson ImmunoRe-search Laboratories, Inc. (West Grove, PA). Vectastain ABC EliteMouse IgG kit was from Vector Laboratories, Inc. (Burlington, CA).The sample of a purified mouse tubulin was a generous gift of Dr B.Weinstein (MGH, Boston, MA).
Treatment of embryosWild-type AB strain zebrafish embryos were obtained by single paircrosses. Embryos were collected within the first hour after fertiliza-tion and maintained at 28.5-29.5°C in ‘egg water’ (0.03% InstantOcean salt mix in Millipore MilliQplus deionized water) (Westerfield,1993). From each egg lay 8-cell embryos were selected, ensuring thatthey differed less than 15 minutes with respect to the time of fertil-ization. Staging of embryos was performed as described by Kimmel
et al. (1993). Developmental times are given in hours after fertiliza-tion.
Nocodazole (Methyl-5[2-thienylcarbonyl]1H-benzimidazol-2-yl)-carbamate was prepared as a stock solution of 10 mg/ml in 50%DMSO and diluted to concentrations of 0.5-20 µg/ml. Taxol wasprepared as a 10 mM stock solution in DMSO and diluted to con-centrations of 10-100 µM. In the case of the 50 and 100 µM dilutions,precipitation was observed. Therefore the actual concentrations oftaxol were probably lower. Culture of control embryos in corre-sponding concentrations of DMSO in egg water did not have anyadverse effect on development.
Western blot analysisTotal proteins from 4- and 20-hour-old embryos were resolved in 10%SDS-acrylamide gel and transferred to ImmobilonTMPVDFmembrane (Millipore, Bedford, MA) (Towbin et al., 1979).Immunoblotting was performed as described in Birkett et al. (1985).Two monoclonal antibodies, α-tubulin-specific DM1A (Gard, 1991)and β-tubulin-specific KMX-1 (Birkett et al., 1985), detected a singleband corresponding to protein(s) of apparent molecular mass about50×103 in the samples from zebrafish embryos. The detected zebrafishprotein(s) comigrated with protein(s) recognized by the two anti-bodies in a fraction of purified mouse tubulins (data not shown). Thisindicates that the KMX-1 and DM1A antibodies most likely specifi-cally recognize tubulin in zebrafish.
Whole-mount antibody stainingsThe best preservation of microtubules and structure of the embryowere achieved with the formaldehyde-glutaraldehyde-taxol fix (FGTfix) developed for Xenopus oocytes (Gard, 1991). Embryos were fixedat room temperature or at 28.5°C for 2-4 hours. As in Xenopus (Gard,1991), microtubules were also preserved when taxol was omitted fromthe FGT-fix (data not shown). Staining with the KMX-1 and DM1Aantibodies was performed as described by Gard (1991) with thefollowing modifications. Incubations with antibodies were carried outovernight in a cold room. Washes were performed at room tempera-ture: 1× 5 minutes and 3× 30 minutes. Embryos stained with thesecondary antibody conjugated to Texas Red were dehydrated andmounted in benzyl benzoate-benzyl alcohol (Gard, 1991), in bridgeslides described by Warga and Kimmel (1990), and sealed with Ladd-o-lac. Alternatively, detection of the antigen was carried out using theVectastain Avidin/Biotin/Horseradish peroxidase ABC Elite Systemaccording to standard procedures (Roth et al., 1989). Embryos stainedwith the Vectastain ABC detection system were kept in 100%glycerol. To visualize DNA, embryos stained with antibodies werewashed 4-5 times for 5 to 10 minutes with deionized water, and thenstained for 10 minutes with 0.2 µg/ml solutions of DAPI (4′,6-diamidino-2-phenylindole) or Hoechst 33258. Embryos stained withDNA dyes were washed once with PBS and transferred to 1% w/v p-phenylenediamine, 90% v/v glycerol, 0.1 M Tris-HCl, pH 7.5.
MicroscopyObservation of living embryos was performed either with a dissect-ing microscope Wild, or with a Zeiss Axiophot microscope usingNomarski optics. Embryos stained with fluorescent antibodies orDNA dyes were observed under epifluorescent illumination usingNeofluar objectives on the Axiophot microscope, or using confocalimaging (Bio-Rad MRC 600 attached to a Zeiss Axioscop).
RESULTS
The origin and organization of the yolk cellmicrotubules during cleavage stages Two anti-tubulin monoclonal antibodies, DM1A and KMX-1,were examined for their ability to detect zebrafish tubulins by
2446
western blotting (see Materials and Methods) and by whole-mount immunocytochemistry. In early cleavage-stage embryosthe marginal blastomeres contacting the yolk cell maintain thecytoplasmic bridges with the YCL (Kimmel and Law, 1985a).At these stages, we observed an array of microtubules thatappeared to emerge from the marginal blastomeres into theyolk cell, and extended along the animal-vegetal axis towardsthe vegetal pole. The YCL microtubules persisted even duringsynchronous mitotic divisions of the blastoderm cells (Fig.2A,B, and data not shown).
At the 500-1000 cell stage (3h) the marginal blastomeresfuse with the yolk cell (Kimmel and Law, 1985b) forming theYSL. At this stage the syncytial nuclei were organized in asingle row in the narrow external YSL (Fig. 3A) (Trinkaus,1993; Wilson, 1889). In the late blastula embryos (3.7-4h) theenlarged external YSL usually took shape of a syncytial corona(Long, 1980; Ballard, 1973), with single nuclei in each of thevegetal protrusions of the corona (Fig. 4A). Within individualblastomeres of the late blastula embryos, microtubule arraystypical of animal cells were observed: mitotic spindles,midbodies and interphase arrays emanating from centersadjacent to cell nuclei (Fig. 3B). The yolk cell exhibited twodistinct types of microtubule organization. In the external YSLmicrotubules radiated from centers associated with thesyncytial nuclei both during mitosis (prometaphase in Fig.3B,C) and interphase (see below) forming a network. Duringmetasynchronous mitotic divisions of the YSN (Kane et al.,1992), astral microtubules of adjacent mitotic arrays formed inthe YSL appeared to interdigitate (Fig. 3C). The YSL networkof intercrossing or mitotic microtubules usually filled the entiresyncytial corona (Fig. 3B,D). The YCL exhibited a distinct setof microtubules that extended vegetally from the centersserving also as mitotic poles and associated primarily with themost vegetal YSN (Fig. 3B,D,E). In the YCL these micro-tubules formed a dense array aligned along the animal-vegetalaxis (Fig. 3F). Some of these microtubules appeared to end atvarious latitudes of the yolk sphere while the remaining micro-tubules of the array extended to the vegetal pole where they
met in a concentric fashion (Fig. 3G). In some experimentsmicrotubules were observed to cover only 75-90% of the yolksphere. The YCL microtubules persisted during divisions ofthe YSN when only mitotic spindle microtubules were visiblein the YSL.
Sections of embryos previously stained with anti-tubulinantibodies revealed that the yolk cell microtubules werepresent only in the thin cortical cytoplasm of the YCL andexternal YSL. In the blastoderm, microtubules were staineddeeper than in the yolk cell, in two to four layers of blas-tomeres; however, the internal layers were not stained. Stainingof embryos bisected after fixation additionally revealed micro-tubules in the deeper blastoderm layers and in the internal YSL,while no microtubules were detected in the deeper, yolk-con-taining center of the yolk cell (data not shown). We cannot,however, exclude the possibility that the apparent lack ofmicrotubules in the center of the yolk cell is due to incompletefixation.
L. Solnica-Krezel and W. Driever
Fig. 2. Organization and origin of the yolk cellmicrotubules in 8-cell-stage zebrafish embryos,1.2 hours. Fixed embryos in which microtubuleswere stained with anti-tubulin antibody and ahorseradish peroxidase detection system.Background staining visible in the cytoplasmresults from a nonspecific binding of biotin at thisstages of development. Small arrowheads indicateapproximate borders between the blastomeres (b)and the YCL. The YCL microtubules appear toemerge from the blastomeres and extend alongthe animal-vegetal axis (A,B). In blastoderm cellsmicrotubules are also visible in the cytokineticfurrow (large arrowheads). Bars, 100 µm, animalpole toward the top.
Fig. 3. Organization of the yolk cell microtubules after the formationof the YSL. Immunochemical staining of whole-mount embryosusing the KMX-1 anti-tubulin antibody. The animal pole is towardthe top and vegetal to the bottom, except in G. (A,B,D,F,G) Bright-field image and (C,E) fluorescent confocal image of embryos withanti-tubulin staining. Bars, 100 µm in A,D and 50 µm in E,F,G. Theexternal YSL extends between the vegetal rim of the blastoderm(large arrowhead) and the YCL (small arrowhead). (A) 1000-cell-stage embryo, 3h. The YSL microtubules are organized in one row ofmitotic spindles. (B) At the oblong stage, 3.7 hours and (C-G) at thelate blastula stage (sphere, 4.0 hours), several rows of the YSNundergoing mitosis are present. (B,C) The astral microtubules ofadjacent YSN mitotic arrays interdigitate. (B,D) The YSLmicrotubules fill the entire external YSL, which takes the shape of asyncytial corona. (D,E) The YCL microtubules originate from theorganizing centers associated with the most vegetal YSN. (F) Anarray of the YCL microtubules extending in the animal-vegetaldirection on the lateral side of the yolk cell of the sphere-stageembryo. (G) View of the vegetal pole (vp) of the sphere stageembryo. Microtubules of the YCL converge at the vegetal pole in aconcentric fashion.
2447Microtubule arrays of the zebrafish yolk cell
Changes in the organization of the yolk cellmicrotubules during epibolyEpiboly in zebrafish and Fundulus starts in the late blastula,after cessation of mitotic divisions of the YSN (Kane et al.,1992; Trinkaus, 1993). At the onset of epiboly, in Fundulus,
the external YSL contracts and the YSN become denselypacked (Trinkaus, 1984; 1993). Similarly in zebrafish, startingat the sphere/dome stage, the wide belt of the external YSLnarrowed in the animal-vegetal direction and the syncytialnuclei became increasingly crowded. When the blastoderm
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expanding vegetally, reached 30% of yolk sphere latitude (30%epiboly), the nuclei of the external YSL concentrated near andwere partially covered by the rim of the blastoderm (Fig. 4A-B′). Simultaneously, the microtubule network of the external
YSL became denser (Figs 4C, 1B). By 40% epiboly usuallymost of the YSN were covered by the blastoderm and only anarrow belt of the external YSL with a dense microtubulenetwork was visible below the blastoderm rim (Fig. 4D). At50% epiboly, the blastoderm margin arrives at the equator ofthe embryo and deep cells engage in the gastrulationmovements of involution and convergence (Kimmel andWarga, 1990). At this stage blastoderm cells almost completelycovered the YSN and the associated microtubule network. TheYCL microtubules emerged from below the rim of EVL cells(Figs 5E,F, 1C).
The initial stages of gastrulation are correlated with atransient cessation of epiboly by deep cells (Warga andKimmel, 1990). Consequently, after formation of theembryonic shield, when the EVL covered approximately 65%of the yolk cell, the deep cells lagged in epibolic movements(Fig. 5A,C). At this stage, the YSN, surrounded by theirmicrotubule network, reemerged from below the EVL rimaround the circumference of the embryo and populated up to70% of the yolk cytoplasm (Figs 5A, 1D). Some of the nucleiof the external YSL exhibited variable, elongated shapes (Fig.5A,B). The YSL microtubules surrounded the nuclei formingbasket-like arrangements (Fig. 5B,C). The expansion of theYSN and their associated microtubule network, and theapparent shortening of the YCL microtubules continued untilthe YSL and the blastoderm covered the entire yolk cell (datanot shown).
Organization of microtubules in the internal YSLThe analysis described above revealed changes in the organiz-ation of microtubule arrays in the external YSL and in theYCL. To determine the microtubule configuration in theinternal YSL, the blastoderm cap was removed manually fromfixed embryos. Uncapped yolk cells were stained with the anti-tubulin antibody. Fig. 6 shows the microtubule configurationof the yolk cell from an embryo at 60% epiboly. As in theexternal YSL, microtubules of the internal YSL form a densenetwork surrounding the YSN. Such an organization of theinternal YSL microtubules was observed from the dome stage,until the completion of epiboly.
L. Solnica-Krezel and W. Driever
Fig. 4. Contraction of the external YSL, crowding of the YSN andchanges in the organization of the external YSL microtubule networkduring early stages of epiboly. Animal pole toward the top. Largerarrowheads indicate the rim of blastoderm, while the smallerarrowheads indicate the vegetal margin of the external YSL. (A-B′) Nomarski microscopic images of live embryo. (F) Fluorescent and (C-E) Nomarski images of fixed embryos withanti-tubulin staining that were counterstained with DAPI to visualizeDNA. Bar, 100 µm. (A-B′) Single embryo that exhibits a broadexternal YSL at the beginning of the doming process, 4.1h (A), and anarrow external YSL at 30% epiboly, 4.7h (B). B′ displays a deeperplane of focus of the embryo shown in B, to illustrate crowding ofthe YSN vegetally to the margin of deep blastoderm cells. At 30%epiboly, two rows of crowded YSN are still visible in front of theEVL rim (B) and microtubules of the external YSL form a densenetwork (C). (D) At 40% epiboly, 5h, the microtubule networkassociated with the syncytial nuclei becomes even denser, and isprogressively covered by the blastoderm undergoing epiboly. TheYCL microtubules emerge from this dense network of YSLmicrotubules. (E,F) At 50% epiboly, 5.2 hours, the syncytial nucleiand the associated microtubule network are completely covered bythe expanding vegetally EVL.
2449Microtubule arrays of the zebrafish yolk cell
Microtubule drugs reveal functions of the yolk cellmicrotubules during epibolyTo investigate a potential role of microtubules in epibolicmovements, we studied the effects of the microtubule depoly-merizing agent nocodazole (Lee et al., 1980), and the micro-tubule stabilizing agent taxol (Schiff et al., 1979), on epibolyof the YSN, YSL and blastoderm. Embryos were culturedin the presence of drugs starting at the late blastula (3.7-4 h,Fig. 3D), or 40% epiboly stages. The progress of develop-ment was monitored in treated and control embryos for
the next several hours until the completion of epiboly incontrol embryos. First, at the time when control embryosreached 30 or 40% epiboly, treated embryos were inspectedwith Nomarski optics to determine whether the thickexternal YSL became narrow and the YSN densely packed(Trinkaus, 1993). Second, the epibolic movements during thelater stages of epiboly were monitored by determining thevegetal extent of the YSN, the rim of EVL and of deep cellsin live and fixed embryos. The distribution of YSN and theorganization of microtubules were also assessed in embryos
Fig. 5. Organization of yolk cell microtubules during late stages of epiboly. In all figures the animal pole is at the top. The margin of deep cellsis indicated by large arrowheads, the margin of the EVL by arrows, and the vegetal extent of the external YSL by small arrowheads.Fluorescent (A,B) and Nomarski (C) images of fixed embryos with anti-tubulin and Hoechst 33258 DNA stainings. After the formation of theembryonic shield, (60% epiboly; 6.5 hours), deep cells approach 60% of the yolk sphere latitude, and the EVL rim is ahead of the deep cellsmargin (A,C). The YSN and their associated microtubules are again visible in front of the blastoderm (B,C). At this stage some YSN areextended along the animal-vegetal axis (A,B). The YCL microtubules appear to be continuous with the YSN microtubules (B,C). Bars, 100 µm.
Fig. 6. Organization of microtubules of the internal-YSL in embryos at 60% epiboly. To expose the internal YSL, the blastoderm was removedmanually from fixed embryos which were subsequently stained with anti-tubulin antibodies and a DNA dye. The animal pole is at the top. Bars,100 µm. (A) Fluorescent image of an embryo with anti-tubulin and Hoechst 33258 staining. Lateral view showing the rim of EVL which wasnot completely removed (arrow). The YSN are present in both internal YSL, and also in front of the EVL rim in the external YSL. (B) Bright-field image of a fixed embryo with anti-tubulin staining. Magnified view of a border between the internal and external YSL (i-ysl and e-ysl,respectively) which is demarcated by the residues of the EVL rim (arrow). Microtubules form a dense network in both layers. (C) Confocalimage of microtubules of the internal YSL at the animal pole. A dense network of microtubules surrounds the YSN (n).
2450
fixed and stained with anti-tubulin antibodies and a DNAdye.
(i) Nocodazole inhibits contraction of the YSL andcrowding of the YSN Treatment of the late blastula stage embryos with 5-20 µg/mlnocodazole dramatically affected the organization of micro-tubules, cell divisions, epiboly and gastrulation. In embryosfixed 30 minutes after addition of 10 µg/ml nocodazole, micro-tubule arrays of blastoderm and yolk cell were completely dis-organized (Fig. 7A). After one hour of incubation with noco-dazole, microtubules were no longer detected in the blastodermcells, while only few short microtubules still persisted in theyolk cell (data not shown). Blastoderm cells remained large
throughout the experiment, indicating that cell divisions wereinhibited (Fig. 8). Neither germ ring nor embryonic shield weredetected, indicating that both involution and convergencetowards the dorsal side (Warga and Kimmel, 1990) were eithergreatly repressed or completely stopped.
Distinct aspects of epiboly were variably impaired in theabsence of microtubules. The external YSL remained a widebelt below the blastoderm ring and no crowding of the YSNwas observed (Fig. 7C). Embryos treated at the sphere stage(4h) for 1h with 0.5 µg/ml nocodazole exhibited numerouslong microtubules of the yolk cell (data not shown). In theseembryos, in contrast to the embryos treated with higher con-centrations of nocodazole, the YSN concentrated close to theblastoderm rim (Fig. 7B versus C). These observations are con-
L. Solnica-Krezel and W. Driever
Fig. 7. Effect of nocodazole and taxol on epiboly and on the organization of microtubules. Lateral views of embryos with the animal pole at thetop. The external YSL extends between the vegetal rim of the blastoderm (large arrowhead) and the YCL (small arrowhead). Bars, 100 µm, (b)blastoderm. (A) Immunocytochemical detection of tubulin in sphere-stage (4h) embryos, which have been treated for 30 minutes with 10 µg/mlnocodazole. Microtubule arrays of the blastoderm and yolk cells are completely disorganized (compare with Fig. 3B). (B,C) Effect of differentconcentrations of nocodazole on the initial phase of epiboly. In embryos treated at the sphere stage with 0.5 µg/ml nocodazole crowding of theYSN is reduced but not totally inhibited (B). The crowding of the YSN appears blocked in embryos treated with 10 µg/ml nocodazole (C).(D) Effect of taxol on organization of microtubules and the initial phase of epiboly. 30 minutes after application of 100 µM taxol to sphere-stageembryos, no YSN are visible in front of the blastoderm rim, indicating that the crowding of the YSN took place. A very dense microtubulenetwork is visible vegetal to the blastoderm. (E) Effect of taxol on the organization of yolk cell microtubules, 8 hours after beginning oftreatment (12h of development). Big gaps and bundles of microtubules (black arrow) are apparent in the microtubule array of the YCL.
2451Microtubule arrays of the zebrafish yolk cell
sistent with a function of the yolk cell microtubules in the com-paction of YSN and contraction of the YSL at the beginningof epiboly.
(ii) Nocodazole blocks vegetal migration of the YSN butonly partially inhibits epiboly of the EVL and deep cellsIn the later stages of epiboly, the movements of the YSNtowards the vegetal pole appeared blocked in the nocodazole-treated embryos, while the vegetal expansion of EVL and deep
blastoderm cells were only partially inhibited. At 75% epibolyin control embryos, YSN covered up to 80% of the yolk sphere(Fig. 8A,D). At the same time in treated embryos, the YSNreached only 40% of the yolk sphere latitude and did notexhibit elongated shapes, characteristic to this stage of epiboly(Fig. 8F). The border between the YSL and the YCL was noteasily visible in the nocodazole-treated embryos. Therefore,we could not determine whether movements of other compo-nents of the YSL were also inhibited. The margin of deep cells
Fig. 8. Effects of nocodazole on vegetal movements of the YSN, EVL and deep cells, and on the process of localized endocytosis. Nomarskimicrographs (A-F, G,J), and fluorescent images (H,I,K,L) of live embryos, lateral views with the animal poles toward the top. The margin ofdeep cells (del) is indicated by large arrowheads, the margin of EVL by arrows, and the vegetal extent of the YSN by small arrowheads. Bars,100 µm.(A-F) 4 hours after start of the experiment, 8 hours of development. In control embryo (A), the blastoderm covers 75% of the yolksphere (75% epiboly). The magnified view of the vegetal portion of the same embryo (D), shows that the YSN are already reaching 80% of theyolk sphere latitude. The EVL margin, not in focus on this micrograph, is between the margins of the YSN and deep cells. (B,C,E,F) Embryostreated with 20 µg/ml nocodazole. The margin of deep cells is uneven and reaches 40/45% of the yolk sphere latitude (B,C). The EVL rim is at55% of the yolk sphere latitude (E), and it is now in front of both YSN and deep cells (E,F). In a deeper plane of focus (F), it is apparent thatdeep cells almost completely cover the YSN. (G-L) Visualization of the process of endocytosis at the 50% of epiboly (5h) in an untreatedembryo (G,H,I) and an embryo treated for 1 h with 5 µM nocodazole (J,K,L). Embryos were immersed in a medium containing 1.5% LuciferYellow. Endocytic vesicles form a ring positioned vegetally to the margin of blastoderm (large arrowheads) in both control (H,I) andnocodazole-treated embryos (K,L).
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was uneven, and reached only to 40-50% of the yolk spherelatitude, almost completely covering the YSN (Fig. 8B,F). TheEVL rim arrived, however, at the 55-60% of the yolk spherelatitude, and was vegetal to both the deep cells and the YSN(Figs 8B,E, 1E). The EVL and deep cells of treated embryosmoved very slowly toward the vegetal pole. When controlembryos were at the tail bud stage (9.5h), the EVL rim of thetreated embryos was about 70% of the yolk sphere latitude, andtreated embryos started to degenerate (data not shown).
(iii) Localized endocytosis can occur in the absence ofmicrotubulesThus, in the nocodazole-treated embryos all epibolicmovements of the YSN were either severely inhibited or com-pletely blocked. In contrast, epiboly of EVL was only partiallyrepressed. It has been proposed that epiboly of the EVL ispassive and is driven by epiboly of the surface of the YSL towhich the EVL is tightly linked (Betchaku and Trinkaus,1978). Epiboly of the surface of the YSL has been explainedin part by the process of endocytosis localized during epibolyto the narrow ring of the external YSL, vegetally to the EVLrim (Betchaku and Trinkaus, 1986). To test whether theprocess of endocytosis is stilloccurring in embryos in whichmicrotubules have beendisrupted, embryos treated for 1or 2 hours with 5 or 10 µg/mlnocodazole and control embryoswere incubated in a solution ofLucifer Yellow (Betchaku andTrinkaus, 1986). Fluorescentmicroscopy revealed a ring ofendocytic vesicles localizedvegetally to the blastodermmargin in control embryos (Fig.8G-I), as well as in nocodazole-treated embryos (Fig. 8J-L). Thisobservation suggests that the dis-ruption of microtubules does notequally affect all the aspects ofepiboly in zebrafish embryos,but rather primarily impairsmovements of the YSN.
(iv) Taxol delays all epibolicmovements toward thevegetal pole30 minutes after incubation ofsphere-stage embryos (4h) in100 µM taxol, the YSN werecovered by the blastoderm, andonly a belt of a dense network ofthe YSL microtubules wasvisible vegetal to the blastodermrim (Fig. 7D). Thus the contrac-tion of the YSL was notinhibited. In taxol-treatedembryos both the YSL, and theYCL microtubule arrays had adenser appearance than incontrol embryos (Fig. 7D versus
Fig. 4D), covered the vegetal pole completely and were moreresistant to nocodazole (data not shown). The movements ofthe YSN, EVL and deep cells towards the vegetal pole in thelater stages of epiboly were delayed. In contrast to nocodazoletreatment, in embryos treated with taxol, epibolic movementsof YSN, EVL and deep cells were affected to a similar extent.When control embryos reached 60% epiboly (s.d.±6%, 48embryos examined), treated embryos exhibited only 48%epiboly (s.d.±6%, 46 embryos examined). At the completionof epiboly in control embryos (Fig. 9A, 9h; 96±5%, 23embryos examined), the blastoderm of treated embryosarrived at 82±7% of the yolk sphere latitude (35 embryosexamined; Fig. 9B,C). However, when control embryos wereat the 2-somite stage (10.5h), the embryos treated with taxolalso exhibited two somites and a forming notochord (Fig.9D,E). This indicated that taxol treatment affected onlyepiboly, but not gastrulation or morphogenesis. However,most of the taxol-treated embryos have not completed epibolyat this stage. Instead, they formed abnormally shapedgastrulae, with a portion of the yolk cell protruding from con-tracted blastopore lips (Fig. 9D). Embryos continued devel-opment in the taxol solution for the next several days,
L. Solnica-Krezel and W. Driever
Fig. 9. Effect of 100 µM taxol on later stages of epiboly. Nomarski images of live embryos, animalpole is towards the top. In A,B,D, dorsal is to the right. The margin of deep cells (del) is indicated bylarge arrowheads, the margin of EVL by arrows, and the vegetal extent of the external YSL by smallarrowheads. Bars, 100 µm. (A-C) 5 hours after addition of taxol, 9 hours of development. In controlembryos yolk plug closure takes place (A), whereas the blastoderm of an embryo treated with taxolremains at 60% of the yolk sphere latitude (B,C). (B,C) Micrographs show that the cortical cytoplasmof the yolk cell exhibits gaps or thinnings (g). The YSN are in front of the blastoderm (C). The EVLrim is visible between the YSN and the margin of deep cells (C). (D-F) Taxol-treated embryo 6 hoursafter addition of the drug, 10 hours of development. Similar to control embryos (not shown) theembryonic body is formed (D), and notochord (nt) and the first somites (s) are apparent in the dorsalview (E). The blastoderm still does not cover the whole yolk sphere. (F) shows a magnified view ofthe yolk cell surface which exhibits distortions (small black arrowheads), and gaps or thinnings in thecortical cytoplasm (g).
2453Microtubule arrays of the zebrafish yolk cell
although up to 30% of them exhibited tail defects (data notshown).
Examination of the tubulin staining pattern of taxol-treatedembryos delayed in epiboly revealed bundles of microtubulesand large gaps in the microtubule arrays of the YCL and YSL(Fig. 7E). Examination of live taxol-treated embryos withNomarski optics indicated that the yolk cortical cytoplasmexhibited apparent discontinuities or thinner regions betweenthicker cytoplasmic regions (Fig. 9C,F). The apparent dis-continuities of the YCL observed in live embryos most likelycorresponded to gaps in the microtubule array, with thickcytoplasmic regions corresponding to bundles of micro-tubules.
DISCUSSION
Two distinct microtubule arrays of the yolk cellThis work demonstrates that the yolk cell of the zebrafishembryo is equipped with two distinct microtubule arrays. TheYSL exhibits a network of mitotic or interphase microtubules,while an array of microtubules aligned along the animal-vegetal axis exists in the YCL (Fig. 1A). The developmentalchanges in the organization of these microtubule arrayscorrelate with epibolic movements (Fig. 1A-E). We provideevidence that these microtubule arrays are actively involved inthe epibolic movements of the YSN, and contribute to otheraspects of epiboly.
Yolk cell microtubules during cleavage and blastulastagesDuring early cleavage stages the YCL microtubules appear toemerge from the marginal blastomeres. Most likely micro-tubules extend through large cytoplasmic bridges connectingthese blastomeres with the YCL (Kimmel and Law, 1986a).After formation of the YSL, microtubules of the YCL appearto emanate from the centers associated with the syncytialnuclei. Since these centers also radiate mitotic spindle micro-tubules during the mitotic divisions of the YSN, they probablycorrespond to true microtubule organizing centers. Wepresume that when the marginal blastomeres completely fusewith the yolk cell, generating the YSL (Kimmel and Law,1985b), both nuclei and microtubule organizing centers areintroduced into the yolk cell. Additionally, the animal-vegetalYCL microtubules are likely to exhibit uniform polarity: theminus ends in the blastomeres or in the YSL with plus endspointing toward the vegetal pole.
During the period of mitotic divisions of the YSN, theexternal YSL expands and the YSN spread towards the vegetalpole while maintaining a very regular distribution (Trinkaus,1993 and this work). We found that the astral microtubules ofneighboring spindles in the YSL overlap and interdigitate. Asimilar configuration of interdigitating astral microtubules hasbeen reported for syncytial nuclei undergoing corticalmigration during the 8th and 9th division cycles in theDrosophila embryo (Baker et al., 1993). These authors proposethat microtubule-dependent forces generated by plus-enddirected microtubule motors act between anti-parallel astralmicrotubules of adjacent spindles to push nuclei apart. Theseputative repulsive forces drive nuclei toward the surface (Bakeret al., 1993). A similar array of forces could explain both the
spreading of the YSN and their regular distribution duringmitotic cycles.
Yolk cell microtubules during epibolyOur analysis revealed that the changes in the organization ofthe yolk cell microtubules correlate with both the process ofcrowding of the YSN at the beginning of epiboly, and withtheir subsequent vegetal movements (Fig. 1). These correla-tions could result from the action of a yet different cellularcomponent, such as a microfilament network (Betchaku andTrinkaus, 1978), acting on both the YSN and the microtubules,or on the entire YSL. Alternatively, the correlations couldindicate that microtubule activity is required for epibolicmovements. This alternative is supported by analysis ofembryos treated with nocodazole. In the absence of micro-tubules, the YSL did not contract and the YSN did not becomedensely packed. Further, YSN movement toward the vegetalpole in the later stages of epiboly was greatly inhibited. Addi-tionally, the YSN of the nocodazole-treated embryos did notexhibit elongated shapes observed during epiboly in controlembryos (Fig. 1D,E). This indicated that the tension thatstretches the nuclei along the animal-vegetal axis duringepiboly is reduced or absent from the nocodazole-treatedembryos.
Several observations argue that the inhibition of nuclearmovements in the nocodazole-treated embryos results specifi-cally from the loss of microtubule function. First, some treat-ments with nocodazole that led to inhibition of the nuclearmovements were initiated at the sphere or at later stages ofepiboly, after the cessation of mitotic divisions of the syncytialnuclei (Kane et al., 1992). Thus, observed effects are ratherunlikely to be an indirect consequence of interference with pro-liferation of the YSN. Second, when sphere-stage embryoswere treated with a low concentration of nocodazole such thatdisruption of microtubules was delayed, then the YSN becamedensely packed near the blastoderm rim. Finally, epiboly ofblastoderm proceeded farther than epiboly of the YSN. Wecannot exclude, however, that some aspects of inhibition ofepiboly in nocodazole-treated embryos were secondary to thedisruption of microtubules.
Microtubule depolymerizing drugs are known to inhibitdirectional movements of treated cells (Vasiliev, 1991). Lossof directional movements of deep cells could both explain theimpairment of gastrulation movements (Winklbauer andNagel, 1991), and account for the observed slow vegetalexpansion of deep cells in the embryos treated with nocoda-zole. Epiboly of the EVL was the least affected in the absenceof microtubules. It has been proposed that epiboly of the EVLis accompanied and driven in part by a process of endocytosislocalized vegetally to the blastoderm rim (Betchaku andTrinkaus, 1986). We have demonstrated that localized endo-cytosis does occur in embryos treated with nocodazole and,thus, it could account for the observed expansion of EVL. Thisobservation underscores the requirement for microtubulefunction specifically in YSN movements.
Taxol treatment experiments also support the involvementof microtubules in epiboly. In embryos treated with taxol, thevegetal movements of both the YSN and blastoderm weredelayed to the same degree. The inhibition of epiboly wasspecific, since gastrulation and morphogenesis seemed toprogress normally. In treated embryos, microtubules were
2454
longer than those of untreated embryos, more resistant to noco-dazole, and often microtubule bundles and gaps in microtubulearrays were observed. Thus, inhibition of epiboly is most likelymediated by the effect of taxol on microtubules of the yolk cell.Interphase PtK2 cells treated with taxol form long microtubulebundles. This is accompanied by the loss of centrosome-nucleated microtubules (DeBrabander et al., 1981). Therefore,the changes in the microtubule organization in the yolk cell oftaxol-treated embryos are consistent with effects of taxol oninterphase cells.
How might taxol delay epiboly? Stabilization of micro-tubules by taxol has been proposed to explain the inhibition ofpronuclear movement in sea urchin embryos (Schatten et al.,1982). Since YCL microtubules become progressively shorterduring epiboly, taxol stabilization could interfere with thisprocess. Alternatively, impairment of epiboly could be only asecondary consequence of the increased microtubule stability.
Toward a model of epibolyThe YSL provides the major force in the vegetal spreading ofthe overlaying blastoderm (Trinkaus, 1951; Betchaku andTrinkaus, 1978). The following mechanisms were proposed tobe involved in the epiboly of the YSL by Trinkaus and his col-leagues (Trinkaus, 1984). Microfilaments forming a concentricring in the external YSL were proposed to generate a con-strictive force that would drive expansion of the YSL. Epibolyof the surface of the yolk cell involves expansion of the surfaceof the YSL with simultaneous diminishing of the surface of theYCL. The expanding surface of the YSL would tag along theEVL attached to it. The diminishing of the surface of the YCLis explained by a process of programmed endocytosis takingplace in the area of the external YSL in front of the EVL. Con-current expansion of the YSL surface would be achieved bythe release of the excess of membrane stored in the internalYSL in a form of membranous microvilli.
Recently, it has been proposed that an array of YCL micro-tubules would aid epiboly by pulling the blastoderm toward thevegetal pole (Strähle and Jesuthasan, 1993). The taxol-inducedgeneral delay in epiboly that we observed is reminiscent of thephenotype described for embryos treated with UV or shortpulses of nocodazole, when the YCL microtubules are presentbut somewhat disorganized (Strähle and Jesuthasan, 1993).The above treatments indicate that microtubules contribute togeneral epibolic movements. However, the observation of ablock of YSN movements, with only partial inhibition ofepiboly of the blastoderm and an unaffected localized endocy-tosis in the absence of microtubules, points to a more complexrole of microtubules in epiboly. Therefore, we propose that theepibolic movements of the yolk syncytial nuclei are notcoupled to the process of endocytosis and to epiboly of theblastoderm. Further, epibolic movements of the YSN but notof the blastoderm are absolutely dependent on microtubules.
We hypothesize that both the YSL network and the YCLmicrotubules contribute differently to various aspects ofepiboly, and that they are primarily involved in movements ofthe YSN. Microtubules have been implicated in nuclearmovements in a variety of organisms, based on either mor-phological criteria (Picket-Heaps, 1991; Lloyd et al., 1987),correlation between the organization of microtubules andnuclear movements (Baker et al., 1993), sensitivity of nuclearmovements to anti-microtubule drugs (Picket-Heaps, 1991;
Lutz and Inouye, 1982; Oakley and Morris, 1981), or on agenetic evidence (Huffaker et al., 1988; Morris, 1976). Onepossible mechanism for microtubule-mediated nuclearmigration involves molecules that interact between the micro-tubules and a nucleus generating force to move it along micro-tubules (e.g. Osmani et al., 1990). Such a mechanism,involving the YSL and YCL microtubules and a plus end-directed microtubule motor, could explain epibolic movementsof the YSN. Alternatively, motor molecules working betweeninterdigitating microtubules in the YSL network wouldgenerate a pushing force similar to that used in anaphase B ofmitosis, effectively expanding the YSL and spreading YSN(e.g. Nislow et al., 1992; reviewed in Saunders, 1993).
The YCL animal-vegetal microtubules would act in theexpansion of the margin of the YSL and possibly in transportof cellular components localized in the external YSL. Since thecellular components of the external YSL, like microfilaments,may also be involved in epiboly; interference with the YCLmicrotubules could affect all epibolic movements. Motormolecules, similar to those involved in pulling the spindle poleby astral microtubules (Saunders, 1993), would pull their orga-nizing centers located in the YSL toward the vegetal pole.Coordinate action of the YSL and YCL arrays would result inthe expansion of the whole YSL, whereas action of the YSLnetwork alone at the beginning of epiboly could account forcrowding of the YSN.
Distinguishing between these models will require identifi-cation of the involved motor molecules and their functionalanalysis during epiboly. Additionally, it is important to under-stand the relationship between the microtubule and microfila-ment systems in this process. Since the large syncytial yolk cellproves very accessible to experimental analysis, further studiesof microtubule arrays of the yolk cell in zebrafish can con-tribute to our understanding of the role of the cytoskeleton invertebrate development.
We are thankful to Elisabeth Vogelsang for sectioning embryosstained with anti-tubulin antibodies, and to Dr Rocco Rotello for helpwith western blotting. Special thanks to Dr Robert M. Ezzell forgiving us access to the confocal microscope, and Dr Yimin Ge forinstructions on collection and processing of confocal images. Wewould like to thank our colleagues Derek Stemple, Timothy Burland,Alexander Schier, Eliza Shah, Brant Weinstein, Fried Zwartkruis andDr John Trinkaus for the constructive comments on the manuscript.Part of this work has been presented in an abstract form at theNortheast Regional Developmental Biology Conference in WoodsHole, March 1993. This work has been supported by National Instituteof Health grant HD29761 and by Bristol-Myers Squibb.
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