Development 120, 2443-2455 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
2443
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
SUMMARY
In zebrafish (Danio rerio), meroblastic cleavages generate
an embryo in which blastomeres cover the animal pole of
a large yolk cell. At the 500-1000 cell stage, the marginal
blastomeres fuse with the yolk cell forming the yolk
syncytial layer. During epiboly the blastoderm and the yolk
syncytial layer spread toward the vegetal pole. We have
studied developmental changes in organization and
function during epiboly of two distinct microtubule arrays
located in the cortical cytoplasm of the yolk cell. In the
anuclear yolk cytoplasmic layer, an array of microtubules
extends along the animal-vegetal axis to the vegetal pole. In
the early blastula the yolk cytoplasmic layer microtubules
appear to originate from the marginal blastomeres. Once
formed, the yolk syncytial layer exhibits its own network
of intercrossing mitotic or interphase microtubules. The
microtubules of the yolk cytoplasmic layer emanate from
the microtubule network of the syncytial layer.
At the onset of epiboly, the external yolk syncytial layer
narrows, the syncytial nuclei become tightly packed and
the network of intercrossing microtubules surrounding
them becomes denser. Soon after, there is a vegetal
expansion of the blastoderm and of the yolk syncytial layer
with its network of intercrossing microtubules. Concomitantly, the yolk cytoplasmic layer diminishes and its set of
animal-vegetal microtubules becomes shorter.
We investigated the involvement of microtubules in
epiboly using the microtubule depolymerizing agent nocodazole and a stabilizing agent taxol. In embryos treated
with nocodazole, microtubules were absent and epibolic
movements of the yolk syncytial nuclei were blocked. In
contrast, the vegetal expansion of the enveloping layer and
deep cells was only partially inhibited. The process of endocytosis, proposed to play a major role in epiboly of the yolk
syncytial layer (Betchaku, T. and Trinkaus, J. P. (1986)
Am. Zool. 26, 193-199), was still observed in nocodazoletreated embryos. Treatment of embryos with taxol led to a
delay in all epibolic movements.
We propose that the yolk cell microtubules contribute
either directly or indirectly to all epibolic movements.
However, the epibolic movements of the yolk syncytial
layer nuclei and of the blastoderm are not coupled, and
only movements of the yolk syncytial nuclei are absolutely
dependent on microtubules. We hypothesize that the
microtubule network of the syncytial layer and the animalvegetal set of the yolk cytoplasmic layer contribute differently to various aspects of epiboly. Models that address the
mechanisms by which the two microtubule arrays might
function during epiboly are discussed.
INTRODUCTION
Warga and Kimmel, 1990). Second, the relatively large size of
Fundulus heteroclitus or zebrafish (Danio rerio) embryos
allows for easy experimental manipulations (Ho, 1992).
Finally, zebrafish is amenable to genetic analysis (Streisinger
et al., 1981).
At the onset of epiboly in teleosts with meroblastic early
cleavages, 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, and
covers a mass of deep cells. Three compartments of continuous cortical cytoplasm can be distinguished in the yolk cell
(Trinkaus, 1992, 1993; Long, 1984). A thin, anuclear yolk
cytoplasmic layer (YCL) surrounds the bulk of the yolk mass
with the vegetal pole. The external yolk syncytial layer (YSL),
located between the YCL and the blastoderm rim, is a rela-
During gastrulation of a vertebrate embryo, various cell
movements including involution and convergence lead to
formation of a gastrula with the three classically defined germ
layers and a blueprint of the body plan. In amphibians and
fishes, gastrulation is preceded and accompanied by epiboly a process of vegetal expansion of the blastoderm (Keller, 1980;
Trinkaus, 1951). The mechanisms that determine directions of
cell migrations and forces that drive epiboly and gastrulation
movements are still poorly understood. Several features make
teleost embryos attractive for studies of these processes
(Trinkaus et al., 1992; Kimmel, 1989). First, due to optical
clarity of certain teleost embryos, cell movements and behavior
can be observed in intact embryos (Trinkaus et al., 1991;
Key words: teleost, yolk syncytial layer, morphogenetic movements,
nocodazole, taxol
2444 L. Solnica-Krezel and W. Driever
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BLASTODERM:
Deep Cells
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Yolk
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Yolk
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External
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External YSL
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Fig. 1. Schematic illustration of the changes in the organization of the
cortical cytoplasm of the yolk cell in relation to other cell types in zebrafish
Internal YSN
embryo during epiboly in normal (A-D) and nocodazole-treated embryos
(E). The organization of microtubule networks in the YSL and YCL
observed in this study is indicated by thin lines. Only part of the blastoderm
is shown to reveal the morphology of the yolk cell. The relative sizes of
elements are not proportional. (A) The late blastula just before the onset of
epiboly (sphere stage, 4.0 h). The blastoderm, composed of the internal deep
cells and the superficial enveloping layer (EVL), is positioned atop of the
syncytial yolk cell. The animal surface of the yolk cell underlying the
blastoderm is flat. Most of the yolk syncytial nuclei (YSN) are in the
external yolk syncytial layer (external YSL) vegetal to the blastoderm. The
microtubules of the external YSL form a network. The organization of
microtubules in the internal YSL at this stage of development is not known.
The microtubules of the anuclear yolk cytoplasmic layer (YCL) radiate from
the organizing centers associated with the vegetal-most YSN and are
aligned along the animal-vegetal axis. (B) 30% epiboly (4.7h). The
blastoderm 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 external
YSL is partially covered by the expanding vegetally blastoderm. (C) 50% epiboly (5.2h). The blastoderm arrives at 50% of the yolk cell
latitude and covers almost completely the YSN which is also migrating vegetally and the YSL microtubule network. Only the YCL with its
array 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 animalvegetal 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 age
as in D) treated from the sphere stage on with 10 µg/ml nocodazole. Microtubules are absent. The yolk cell acquired the sphere shape. The
YSN are blocked in their movement towards the vegetal pole and do not exhibit elongated shapes. Deep cells move very slowly toward the
vegetal pole and almost cover the YSN. Epiboly of the EVL is slower than in control embryos.
Microtubule arrays of the zebrafish yolk cell 2445
tively thick belt of cytoplasm populated by the yolk syncytial
nuclei (YSN). Beneath the blastoderm is the internal YSL
comprising a thinner cytoplasm also populated by the YSN.
Epiboly in Fundulus begins with a contraction of the external
YSL that becomes covered by the expanding blastoderm. Subsequently, the YSL and blastoderm expand vegetally, while
the YCL progressively disappears (Trinkaus, 1993; Warga
and Kimmel, 1990). Studies in Fundulus indicated that the
epiboly of the YSL is autonomous, i.e. it can occur in the
absence of the blastoderm (Betchaku and Trinkaus, 1978;
Trinkaus, 1951). Moreover, vegetal expansion of the EVL
during epiboly may be driven by the epibolic movements of
the YSL, to which the EVL is tightly linked at its margin
(Betchaku and Trinkaus, 1978). The molecular basis for the
expansion of the YSL is not understood. It has been suggested
that microfilaments may play a role in this process (Trinkaus,
1984). Recently, it has been proposed that epiboly is also
driven by a pulling force dependent on microtubules present
in the yolk cell (Strähle and Jesuthasan, 1993). However,
developmental changes of the yolk cell microtubule organization and their involvement in distinct aspects of epiboly were
not studied.
Here, we demonstrate that two distinct microtubule arrays
exist in the cortical cytoplasm of the zebrafish yolk cell. The
YCL contains an array of microtubules aligned in the direction
of epiboly, extending toward the vegetal pole. Once formed,
the YSL exhibits its own network of intercrossing interphase
or mitotic microtubules. Our studies demonstrate that the
changes in the configuration of the yolk cell microtubules are
strictly correlated with epibolic movements. Analysis of
epiboly in embryos treated with nocodazole and taxol indicates
that microtubules are necessary and actively involved in
epibolic movements of the syncytial nuclei, and only partially
required for the remaining aspects of epiboly.
MATERIALS AND METHODS
Reagents
Reagents were obtained from Sigma Chemical Co. (St Louis, MO),
with the following exceptions. Formaldehyde (EM grade) and Laddo-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 33258
from Molecular Probes (Eugene, OR). Antibodies: KMX-1, a mouse
IgG monoclonal antibody from Boehringer Mannheim (Germany);
DM1A a mouse IgG antibody (ICN Immunobiologicals, Costa Mesa,
CA); alkaline phosphatase-conjugated anti-mouse IgG serum and
Texas Red-conjugated anti-mouse IgG antibody Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Vectastain ABC Elite
Mouse 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 embryos
Wild-type AB strain zebrafish embryos were obtained by single pair
crosses. Embryos were collected within the first hour after fertilization and maintained at 28.5-29.5°C in ‘egg water’ (0.03% Instant
Ocean salt mix in Millipore MilliQplus deionized water) (Westerfield,
1993). From each egg lay 8-cell embryos were selected, ensuring that
they differed less than 15 minutes with respect to the time of fertilization. Staging of embryos was performed as described by Kimmel
et al. (1993). Developmental times are given in hours after fertilization.
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 was
prepared as a 10 mM stock solution in DMSO and diluted to concentrations of 10-100 µM. In the case of the 50 and 100 µM dilutions,
precipitation was observed. Therefore the actual concentrations of
taxol were probably lower. Culture of control embryos in corresponding concentrations of DMSO in egg water did not have any
adverse effect on development.
Western blot analysis
Total proteins from 4- and 20-hour-old embryos were resolved in 10%
SDS-acrylamide gel and transferred to ImmobilonTMPVDF
membrane (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 single
band corresponding to protein(s) of apparent molecular mass about
50×103 in the samples from zebrafish embryos. The detected zebrafish
protein(s) comigrated with protein(s) recognized by the two antibodies in a fraction of purified mouse tubulins (data not shown). This
indicates that the KMX-1 and DM1A antibodies most likely specifically recognize tubulin in zebrafish.
Whole-mount antibody stainings
The best preservation of microtubules and structure of the embryo
were achieved with the formaldehyde-glutaraldehyde-taxol fix (FGT
fix) developed for Xenopus oocytes (Gard, 1991). Embryos were fixed
at room temperature or at 28.5°C for 2-4 hours. As in Xenopus (Gard,
1991), microtubules were also preserved when taxol was omitted from
the FGT-fix (data not shown). Staining with the KMX-1 and DM1A
antibodies was performed as described by Gard (1991) with the
following modifications. Incubations with antibodies were carried out
overnight in a cold room. Washes were performed at room temperature: 1× 5 minutes and 3× 30 minutes. Embryos stained with the
secondary antibody conjugated to Texas Red were dehydrated and
mounted in benzyl benzoate-benzyl alcohol (Gard, 1991), in bridge
slides described by Warga and Kimmel (1990), and sealed with Laddo-lac. Alternatively, detection of the antigen was carried out using the
Vectastain Avidin/Biotin/Horseradish peroxidase ABC Elite System
according to standard procedures (Roth et al., 1989). Embryos stained
with the Vectastain ABC detection system were kept in 100%
glycerol. To visualize DNA, embryos stained with antibodies were
washed 4-5 times for 5 to 10 minutes with deionized water, and then
stained for 10 minutes with 0.2 µg/ml solutions of DAPI (4′,6diamidino-2-phenylindole) or Hoechst 33258. Embryos stained with
DNA dyes were washed once with PBS and transferred to 1% w/v pphenylenediamine, 90% v/v glycerol, 0.1 M Tris-HCl, pH 7.5.
Microscopy
Observation of living embryos was performed either with a dissecting microscope Wild, or with a Zeiss Axiophot microscope using
Nomarski optics. Embryos stained with fluorescent antibodies or
DNA dyes were observed under epifluorescent illumination using
Neofluar objectives on the Axiophot microscope, or using confocal
imaging (Bio-Rad MRC 600 attached to a Zeiss Axioscop).
RESULTS
The origin and organization of the yolk cell
microtubules during cleavage stages
Two anti-tubulin monoclonal antibodies, DM1A and KMX-1,
were examined for their ability to detect zebrafish tubulins by
2446 L. Solnica-Krezel and W. Driever
Fig. 2. Organization and origin of the yolk cell
microtubules in 8-cell-stage zebrafish embryos,
1.2 hours. Fixed embryos in which microtubules
were stained with anti-tubulin antibody and a
horseradish peroxidase detection system.
Background staining visible in the cytoplasm
results from a nonspecific binding of biotin at this
stages of development. Small arrowheads indicate
approximate borders between the blastomeres (b)
and the YCL. The YCL microtubules appear to
emerge from the blastomeres and extend along
the animal-vegetal axis (A,B). In blastoderm cells
microtubules are also visible in the cytokinetic
furrow (large arrowheads). Bars, 100 µm, animal
pole toward the top.
western blotting (see Materials and Methods) and by wholemount immunocytochemistry. In early cleavage-stage embryos
the marginal blastomeres contacting the yolk cell maintain the
cytoplasmic bridges with the YCL (Kimmel and Law, 1985a).
At these stages, we observed an array of microtubules that
appeared to emerge from the marginal blastomeres into the
yolk cell, and extended along the animal-vegetal axis towards
the vegetal pole. The YCL microtubules persisted even during
synchronous mitotic divisions of the blastoderm cells (Fig.
2A,B, and data not shown).
At the 500-1000 cell stage (3h) the marginal blastomeres
fuse with the yolk cell (Kimmel and Law, 1985b) forming the
YSL. At this stage the syncytial nuclei were organized in a
single row in the narrow external YSL (Fig. 3A) (Trinkaus,
1993; Wilson, 1889). In the late blastula embryos (3.7-4h) the
enlarged external YSL usually took shape of a syncytial corona
(Long, 1980; Ballard, 1973), with single nuclei in each of the
vegetal protrusions of the corona (Fig. 4A). Within individual
blastomeres of the late blastula embryos, microtubule arrays
typical of animal cells were observed: mitotic spindles,
midbodies and interphase arrays emanating from centers
adjacent to cell nuclei (Fig. 3B). The yolk cell exhibited two
distinct types of microtubule organization. In the external YSL
microtubules radiated from centers associated with the
syncytial nuclei both during mitosis (prometaphase in Fig.
3B,C) and interphase (see below) forming a network. During
metasynchronous mitotic divisions of the YSN (Kane et al.,
1992), astral microtubules of adjacent mitotic arrays formed in
the YSL appeared to interdigitate (Fig. 3C). The YSL network
of intercrossing or mitotic microtubules usually filled the entire
syncytial corona (Fig. 3B,D). The YCL exhibited a distinct set
of microtubules that extended vegetally from the centers
serving also as mitotic poles and associated primarily with the
most vegetal YSN (Fig. 3B,D,E). In the YCL these microtubules formed a dense array aligned along the animal-vegetal
axis (Fig. 3F). Some of these microtubules appeared to end at
various latitudes of the yolk sphere while the remaining microtubules of the array extended to the vegetal pole where they
met in a concentric fashion (Fig. 3G). In some experiments
microtubules were observed to cover only 75-90% of the yolk
sphere. The YCL microtubules persisted during divisions of
the YSN when only mitotic spindle microtubules were visible
in the YSL.
Sections of embryos previously stained with anti-tubulin
antibodies revealed that the yolk cell microtubules were
present only in the thin cortical cytoplasm of the YCL and
external YSL. In the blastoderm, microtubules were stained
deeper than in the yolk cell, in two to four layers of blastomeres; however, the internal layers were not stained. Staining
of embryos bisected after fixation additionally revealed microtubules in the deeper blastoderm layers and in the internal YSL,
while no microtubules were detected in the deeper, yolk-containing center of the yolk cell (data not shown). We cannot,
however, exclude the possibility that the apparent lack of
microtubules in the center of the yolk cell is due to incomplete
fixation.
Fig. 3. Organization of the yolk cell microtubules after the formation
of the YSL. Immunochemical staining of whole-mount embryos
using the KMX-1 anti-tubulin antibody. The animal pole is toward
the top and vegetal to the bottom, except in G. (A,B,D,F,G) Brightfield image and (C,E) fluorescent confocal image of embryos with
anti-tubulin staining. Bars, 100 µm in A,D and 50 µm in E,F,G. The
external YSL extends between the vegetal rim of the blastoderm
(large arrowhead) and the YCL (small arrowhead). (A) 1000-cellstage embryo, 3h. The YSL microtubules are organized in one row of
mitotic spindles. (B) At the oblong stage, 3.7 hours and (C-G) at the
late blastula stage (sphere, 4.0 hours), several rows of the YSN
undergoing mitosis are present. (B,C) The astral microtubules of
adjacent YSN mitotic arrays interdigitate. (B,D) The YSL
microtubules fill the entire external YSL, which takes the shape of a
syncytial corona. (D,E) The YCL microtubules originate from the
organizing centers associated with the most vegetal YSN. (F) An
array of the YCL microtubules extending in the animal-vegetal
direction on the lateral side of the yolk cell of the sphere-stage
embryo. (G) View of the vegetal pole (vp) of the sphere stage
embryo. Microtubules of the YCL converge at the vegetal pole in a
concentric fashion.
Microtubule arrays of the zebrafish yolk cell 2447
Changes in the organization of the yolk cell
microtubules during epiboly
Epiboly 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 densely
packed (Trinkaus, 1984; 1993). Similarly in zebrafish, starting
at the sphere/dome stage, the wide belt of the external YSL
narrowed in the animal-vegetal direction and the syncytial
nuclei became increasingly crowded. When the blastoderm
2448 L. Solnica-Krezel and W. Driever
Fig. 4. Contraction of the external YSL, crowding of the YSN and
changes in the organization of the external YSL microtubule network
during early stages of epiboly. Animal pole toward the top. Larger
arrowheads indicate the rim of blastoderm, while the smaller
arrowheads 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 with
anti-tubulin staining that were counterstained with DAPI to visualize
DNA. Bar, 100 µm. (A-B′) Single embryo that exhibits a broad
external YSL at the beginning of the doming process, 4.1h (A), and a
narrow external YSL at 30% epiboly, 4.7h (B). B′ displays a deeper
plane of focus of the embryo shown in B, to illustrate crowding of
the YSN vegetally to the margin of deep blastoderm cells. At 30%
epiboly, two rows of crowded YSN are still visible in front of the
EVL rim (B) and microtubules of the external YSL form a dense
network (C). (D) At 40% epiboly, 5h, the microtubule network
associated with the syncytial nuclei becomes even denser, and is
progressively covered by the blastoderm undergoing epiboly. The
YCL microtubules emerge from this dense network of YSL
microtubules. (E,F) At 50% epiboly, 5.2 hours, the syncytial nuclei
and the associated microtubule network are completely covered by
the expanding vegetally EVL.
YSL became denser (Figs 4C, 1B). By 40% epiboly usually
most of the YSN were covered by the blastoderm and only a
narrow belt of the external YSL with a dense microtubule
network was visible below the blastoderm rim (Fig. 4D). At
50% epiboly, the blastoderm margin arrives at the equator of
the embryo and deep cells engage in the gastrulation
movements of involution and convergence (Kimmel and
Warga, 1990). At this stage blastoderm cells almost completely
covered the YSN and the associated microtubule network. The
YCL microtubules emerged from below the rim of EVL cells
(Figs 5E,F, 1C).
The initial stages of gastrulation are correlated with a
transient cessation of epiboly by deep cells (Warga and
Kimmel, 1990). Consequently, after formation of the
embryonic 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 their
microtubule network, reemerged from below the EVL rim
around the circumference of the embryo and populated up to
70% of the yolk cytoplasm (Figs 5A, 1D). Some of the nuclei
of the external YSL exhibited variable, elongated shapes (Fig.
5A,B). The YSL microtubules surrounded the nuclei forming
basket-like arrangements (Fig. 5B,C). The expansion of the
YSN and their associated microtubule network, and the
apparent shortening of the YCL microtubules continued until
the YSL and the blastoderm covered the entire yolk cell (data
not shown).
expanding vegetally, reached 30% of yolk sphere latitude (30%
epiboly), the nuclei of the external YSL concentrated near and
were partially covered by the rim of the blastoderm (Fig. 4AB′). Simultaneously, the microtubule network of the external
Organization of microtubules in the internal YSL
The analysis described above revealed changes in the organization of microtubule arrays in the external YSL and in the
YCL. To determine the microtubule configuration in the
internal YSL, the blastoderm cap was removed manually from
fixed embryos. Uncapped yolk cells were stained with the antitubulin antibody. Fig. 6 shows the microtubule configuration
of the yolk cell from an embryo at 60% epiboly. As in the
external YSL, microtubules of the internal YSL form a dense
network surrounding the YSN. Such an organization of the
internal YSL microtubules was observed from the dome stage,
until the completion of epiboly.
Microtubule arrays of the zebrafish yolk cell 2449
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 cells
is 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 the
embryonic shield, (60% epiboly; 6.5 hours), deep cells approach 60% of the yolk sphere latitude, and the EVL rim is ahead of the deep cells
margin (A,C). The YSN and their associated microtubules are again visible in front of the blastoderm (B,C). At this stage some YSN are
extended along the animal-vegetal axis (A,B). The YCL microtubules appear to be continuous with the YSN microtubules (B,C). Bars, 100 µm.
Microtubule drugs reveal functions of the yolk cell
microtubules during epiboly
To investigate a potential role of microtubules in epibolic
movements, we studied the effects of the microtubule depolymerizing agent nocodazole (Lee et al., 1980), and the microtubule stabilizing agent taxol (Schiff et al., 1979), on epiboly
of the YSN, YSL and blastoderm. Embryos were cultured
in the presence of drugs starting at the late blastula (3.7-4 h,
Fig. 3D), or 40% epiboly stages. The progress of development was monitored in treated and control embryos for
the next several hours until the completion of epiboly in
control embryos. First, at the time when control embryos
reached 30 or 40% epiboly, treated embryos were inspected
with Nomarski optics to determine whether the thick
external YSL became narrow and the YSN densely packed
(Trinkaus, 1993). Second, the epibolic movements during the
later stages of epiboly were monitored by determining the
vegetal extent of the YSN, the rim of EVL and of deep cells
in live and fixed embryos. The distribution of YSN and the
organization of microtubules were also assessed in embryos
Fig. 6. Organization of microtubules of the internal-YSL in embryos at 60% epiboly. To expose the internal YSL, the blastoderm was removed
manually 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 was
not completely removed (arrow). The YSN are present in both internal YSL, and also in front of the EVL rim in the external YSL. (B) Brightfield 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) Confocal
image of microtubules of the internal YSL at the animal pole. A dense network of microtubules surrounds the YSN (n).
2450 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 the
top. 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/ml
nocodazole. Microtubule arrays of the blastoderm and yolk cells are completely disorganized (compare with Fig. 3B). (B,C) Effect of different
concentrations of nocodazole on the initial phase of epiboly. In embryos treated at the sphere stage with 0.5 µg/ml nocodazole crowding of the
YSN 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-stage
embryos, no YSN are visible in front of the blastoderm rim, indicating that the crowding of the YSN took place. A very dense microtubule
network is visible vegetal to the blastoderm. (E) Effect of taxol on the organization of yolk cell microtubules, 8 hours after beginning of
treatment (12h of development). Big gaps and bundles of microtubules (black arrow) are apparent in the microtubule array of the YCL.
fixed and stained with anti-tubulin antibodies and a DNA
dye.
(i) Nocodazole inhibits contraction of the YSL and
crowding of the YSN
Treatment of the late blastula stage embryos with 5-20 µg/ml
nocodazole dramatically affected the organization of microtubules, cell divisions, epiboly and gastrulation. In embryos
fixed 30 minutes after addition of 10 µg/ml nocodazole, microtubule arrays of blastoderm and yolk cell were completely disorganized (Fig. 7A). After one hour of incubation with nocodazole, microtubules were no longer detected in the blastoderm
cells, while only few short microtubules still persisted in the
yolk cell (data not shown). Blastoderm cells remained large
throughout the experiment, indicating that cell divisions were
inhibited (Fig. 8). Neither germ ring nor embryonic shield were
detected, indicating that both involution and convergence
towards the dorsal side (Warga and Kimmel, 1990) were either
greatly repressed or completely stopped.
Distinct aspects of epiboly were variably impaired in the
absence of microtubules. The external YSL remained a wide
belt below the blastoderm ring and no crowding of the YSN
was observed (Fig. 7C). Embryos treated at the sphere stage
(4h) for 1h with 0.5 µg/ml nocodazole exhibited numerous
long microtubules of the yolk cell (data not shown). In these
embryos, in contrast to the embryos treated with higher concentrations of nocodazole, the YSN concentrated close to the
blastoderm rim (Fig. 7B versus C). These observations are con-
Microtubule arrays of the zebrafish yolk cell 2451
Fig. 8. Effects of nocodazole on vegetal movements of the YSN, EVL and deep cells, and on the process of localized endocytosis. Nomarski
micrographs (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 of
deep 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 yolk
sphere (75% epiboly). The magnified view of the vegetal portion of the same embryo (D), shows that the YSN are already reaching 80% of the
yolk 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) Embryos
treated 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 at
55% 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 that
deep cells almost completely cover the YSN. (G-L) Visualization of the process of endocytosis at the 50% of epiboly (5h) in an untreated
embryo (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% Lucifer
Yellow. Endocytic vesicles form a ring positioned vegetally to the margin of blastoderm (large arrowheads) in both control (H,I) and
nocodazole-treated embryos (K,L).
sistent with a function of the yolk cell microtubules in the compaction of YSN and contraction of the YSL at the beginning
of epiboly.
(ii) Nocodazole blocks vegetal migration of the YSN but
only partially inhibits epiboly of the EVL and deep cells
In the later stages of epiboly, the movements of the YSN
towards the vegetal pole appeared blocked in the nocodazoletreated embryos, while the vegetal expansion of EVL and deep
blastoderm cells were only partially inhibited. At 75% epiboly
in control embryos, YSN covered up to 80% of the yolk sphere
(Fig. 8A,D). At the same time in treated embryos, the YSN
reached only 40% of the yolk sphere latitude and did not
exhibit elongated shapes, characteristic to this stage of epiboly
(Fig. 8F). The border between the YSL and the YCL was not
easily visible in the nocodazole-treated embryos. Therefore,
we could not determine whether movements of other components of the YSL were also inhibited. The margin of deep cells
2452 L. Solnica-Krezel and W. Driever
was uneven, and reached only to 40-50% of the yolk sphere
latitude, almost completely covering the YSN (Fig. 8B,F). The
EVL rim arrived, however, at the 55-60% of the yolk sphere
latitude, and was vegetal to both the deep cells and the YSN
(Figs 8B,E, 1E). The EVL and deep cells of treated embryos
moved very slowly toward the vegetal pole. When control
embryos were at the tail bud stage (9.5h), the EVL rim of the
treated embryos was about 70% of the yolk sphere latitude, and
treated embryos started to degenerate (data not shown).
(iii) Localized endocytosis can occur in the absence of
microtubules
Thus, in the nocodazole-treated embryos all epibolic
movements of the YSN were either severely inhibited or completely blocked. In contrast, epiboly of EVL was only partially
repressed. It has been proposed that epiboly of the EVL is
passive and is driven by epiboly of the surface of the YSL to
which the EVL is tightly linked (Betchaku and Trinkaus,
1978). Epiboly of the surface of the YSL has been explained
in part by the process of endocytosis localized during epiboly
to the narrow ring of the external YSL, vegetally to the EVL
rim (Betchaku and Trinkaus, 1986). To test whether the
process of endocytosis is still
occurring in embryos in which
microtubules
have
been
disrupted, embryos treated for 1
or 2 hours with 5 or 10 µg/ml
nocodazole and control embryos
were incubated in a solution of
Lucifer Yellow (Betchaku and
Trinkaus, 1986). Fluorescent
microscopy revealed a ring of
endocytic vesicles localized
vegetally to the blastoderm
margin in control embryos (Fig.
8G-I), as well as in nocodazoletreated embryos (Fig. 8J-L). This
observation suggests that the disruption of microtubules does not
equally affect all the aspects of
epiboly in zebrafish embryos,
but rather primarily impairs
movements of the YSN.
(iv) Taxol delays all epibolic
movements toward the
vegetal pole
30 minutes after incubation of
sphere-stage embryos (4h) in
100 µM taxol, the YSN were
covered by the blastoderm, and
only a belt of a dense network of
the YSL microtubules was
visible vegetal to the blastoderm
rim (Fig. 7D). Thus the contraction of the YSL was not
inhibited.
In
taxol-treated
embryos both the YSL, and the
YCL microtubule arrays had a
denser appearance than in
control embryos (Fig. 7D versus
Fig. 4D), covered the vegetal pole completely and were more
resistant to nocodazole (data not shown). The movements of
the YSN, EVL and deep cells towards the vegetal pole in the
later stages of epiboly were delayed. In contrast to nocodazole
treatment, in embryos treated with taxol, epibolic movements
of YSN, EVL and deep cells were affected to a similar extent.
When control embryos reached 60% epiboly (s.d.±6%, 48
embryos examined), treated embryos exhibited only 48%
epiboly (s.d.±6%, 46 embryos examined). At the completion
of epiboly in control embryos (Fig. 9A, 9h; 96±5%, 23
embryos examined), the blastoderm of treated embryos
arrived at 82±7% of the yolk sphere latitude (35 embryos
examined; Fig. 9B,C). However, when control embryos were
at the 2-somite stage (10.5h), the embryos treated with taxol
also exhibited two somites and a forming notochord (Fig.
9D,E). This indicated that taxol treatment affected only
epiboly, but not gastrulation or morphogenesis. However,
most of the taxol-treated embryos have not completed epiboly
at this stage. Instead, they formed abnormally shaped
gastrulae, with a portion of the yolk cell protruding from contracted blastopore lips (Fig. 9D). Embryos continued development in the taxol solution for the next several days,
Fig. 9. Effect of 100 µM taxol on later stages of epiboly. Nomarski images of live embryos, animal
pole is towards the top. In A,B,D, dorsal is to the right. The margin of deep cells (del) is indicated by
large arrowheads, the margin of EVL by arrows, and the vegetal extent of the external YSL by small
arrowheads. Bars, 100 µm. (A-C) 5 hours after addition of taxol, 9 hours of development. In control
embryos yolk plug closure takes place (A), whereas the blastoderm of an embryo treated with taxol
remains at 60% of the yolk sphere latitude (B,C). (B,C) Micrographs show that the cortical cytoplasm
of the yolk cell exhibits gaps or thinnings (g). The YSN are in front of the blastoderm (C). The EVL
rim is visible between the YSN and the margin of deep cells (C). (D-F) Taxol-treated embryo 6 hours
after addition of the drug, 10 hours of development. Similar to control embryos (not shown) the
embryonic body is formed (D), and notochord (nt) and the first somites (s) are apparent in the dorsal
view (E). The blastoderm still does not cover the whole yolk sphere. (F) shows a magnified view of
the yolk cell surface which exhibits distortions (small black arrowheads), and gaps or thinnings in the
cortical cytoplasm (g).
Microtubule arrays of the zebrafish yolk cell 2453
although up to 30% of them exhibited tail defects (data not
shown).
Examination of the tubulin staining pattern of taxol-treated
embryos delayed in epiboly revealed bundles of microtubules
and large gaps in the microtubule arrays of the YCL and YSL
(Fig. 7E). Examination of live taxol-treated embryos with
Nomarski optics indicated that the yolk cortical cytoplasm
exhibited apparent discontinuities or thinner regions between
thicker cytoplasmic regions (Fig. 9C,F). The apparent discontinuities of the YCL observed in live embryos most likely
corresponded to gaps in the microtubule array, with thick
cytoplasmic regions corresponding to bundles of microtubules.
DISCUSSION
Two distinct microtubule arrays of the yolk cell
This work demonstrates that the yolk cell of the zebrafish
embryo is equipped with two distinct microtubule arrays. The
YSL exhibits a network of mitotic or interphase microtubules,
while an array of microtubules aligned along the animalvegetal axis exists in the YCL (Fig. 1A). The developmental
changes in the organization of these microtubule arrays
correlate with epibolic movements (Fig. 1A-E). We provide
evidence that these microtubule arrays are actively involved in
the epibolic movements of the YSN, and contribute to other
aspects of epiboly.
Yolk cell microtubules during cleavage and blastula
stages
During early cleavage stages the YCL microtubules appear to
emerge from the marginal blastomeres. Most likely microtubules extend through large cytoplasmic bridges connecting
these blastomeres with the YCL (Kimmel and Law, 1986a).
After formation of the YSL, microtubules of the YCL appear
to emanate from the centers associated with the syncytial
nuclei. Since these centers also radiate mitotic spindle microtubules during the mitotic divisions of the YSN, they probably
correspond to true microtubule organizing centers. We
presume that when the marginal blastomeres completely fuse
with the yolk cell, generating the YSL (Kimmel and Law,
1985b), both nuclei and microtubule organizing centers are
introduced into the yolk cell. Additionally, the animal-vegetal
YCL microtubules are likely to exhibit uniform polarity: the
minus ends in the blastomeres or in the YSL with plus ends
pointing toward the vegetal pole.
During the period of mitotic divisions of the YSN, the
external YSL expands and the YSN spread towards the vegetal
pole while maintaining a very regular distribution (Trinkaus,
1993 and this work). We found that the astral microtubules of
neighboring spindles in the YSL overlap and interdigitate. A
similar configuration of interdigitating astral microtubules has
been reported for syncytial nuclei undergoing cortical
migration during the 8th and 9th division cycles in the
Drosophila embryo (Baker et al., 1993). These authors propose
that microtubule-dependent forces generated by plus-end
directed microtubule motors act between anti-parallel astral
microtubules of adjacent spindles to push nuclei apart. These
putative repulsive forces drive nuclei toward the surface (Baker
et al., 1993). A similar array of forces could explain both the
spreading of the YSN and their regular distribution during
mitotic cycles.
Yolk cell microtubules during epiboly
Our analysis revealed that the changes in the organization of
the yolk cell microtubules correlate with both the process of
crowding of the YSN at the beginning of epiboly, and with
their subsequent vegetal movements (Fig. 1). These correlations could result from the action of a yet different cellular
component, such as a microfilament network (Betchaku and
Trinkaus, 1978), acting on both the YSN and the microtubules,
or on the entire YSL. Alternatively, the correlations could
indicate that microtubule activity is required for epibolic
movements. This alternative is supported by analysis of
embryos treated with nocodazole. In the absence of microtubules, the YSL did not contract and the YSN did not become
densely packed. Further, YSN movement toward the vegetal
pole in the later stages of epiboly was greatly inhibited. Additionally, the YSN of the nocodazole-treated embryos did not
exhibit elongated shapes observed during epiboly in control
embryos (Fig. 1D,E). This indicated that the tension that
stretches the nuclei along the animal-vegetal axis during
epiboly is reduced or absent from the nocodazole-treated
embryos.
Several observations argue that the inhibition of nuclear
movements in the nocodazole-treated embryos results specifically from the loss of microtubule function. First, some treatments with nocodazole that led to inhibition of the nuclear
movements were initiated at the sphere or at later stages of
epiboly, after the cessation of mitotic divisions of the syncytial
nuclei (Kane et al., 1992). Thus, observed effects are rather
unlikely to be an indirect consequence of interference with proliferation of the YSN. Second, when sphere-stage embryos
were treated with a low concentration of nocodazole such that
disruption of microtubules was delayed, then the YSN became
densely packed near the blastoderm rim. Finally, epiboly of
blastoderm proceeded farther than epiboly of the YSN. We
cannot exclude, however, that some aspects of inhibition of
epiboly in nocodazole-treated embryos were secondary to the
disruption of microtubules.
Microtubule depolymerizing drugs are known to inhibit
directional movements of treated cells (Vasiliev, 1991). Loss
of directional movements of deep cells could both explain the
impairment of gastrulation movements (Winklbauer and
Nagel, 1991), and account for the observed slow vegetal
expansion of deep cells in the embryos treated with nocodazole. Epiboly of the EVL was the least affected in the absence
of microtubules. It has been proposed that epiboly of the EVL
is accompanied and driven in part by a process of endocytosis
localized vegetally to the blastoderm rim (Betchaku and
Trinkaus, 1986). We have demonstrated that localized endocytosis does occur in embryos treated with nocodazole and,
thus, it could account for the observed expansion of EVL. This
observation underscores the requirement for microtubule
function specifically in YSN movements.
Taxol treatment experiments also support the involvement
of microtubules in epiboly. In embryos treated with taxol, the
vegetal movements of both the YSN and blastoderm were
delayed to the same degree. The inhibition of epiboly was
specific, since gastrulation and morphogenesis seemed to
progress normally. In treated embryos, microtubules were
2454 L. Solnica-Krezel and W. Driever
longer than those of untreated embryos, more resistant to nocodazole, and often microtubule bundles and gaps in microtubule
arrays were observed. Thus, inhibition of epiboly is most likely
mediated by the effect of taxol on microtubules of the yolk cell.
Interphase PtK2 cells treated with taxol form long microtubule
bundles. This is accompanied by the loss of centrosomenucleated microtubules (DeBrabander et al., 1981). Therefore,
the changes in the microtubule organization in the yolk cell of
taxol-treated embryos are consistent with effects of taxol on
interphase cells.
How might taxol delay epiboly? Stabilization of microtubules by taxol has been proposed to explain the inhibition of
pronuclear movement in sea urchin embryos (Schatten et al.,
1982). Since YCL microtubules become progressively shorter
during epiboly, taxol stabilization could interfere with this
process. Alternatively, impairment of epiboly could be only a
secondary consequence of the increased microtubule stability.
Toward a model of epiboly
The YSL provides the major force in the vegetal spreading of
the overlaying blastoderm (Trinkaus, 1951; Betchaku and
Trinkaus, 1978). The following mechanisms were proposed to
be involved in the epiboly of the YSL by Trinkaus and his colleagues (Trinkaus, 1984). Microfilaments forming a concentric
ring in the external YSL were proposed to generate a constrictive force that would drive expansion of the YSL. Epiboly
of the surface of the yolk cell involves expansion of the surface
of the YSL with simultaneous diminishing of the surface of the
YCL. The expanding surface of the YSL would tag along the
EVL attached to it. The diminishing of the surface of the YCL
is explained by a process of programmed endocytosis taking
place in the area of the external YSL in front of the EVL. Concurrent expansion of the YSL surface would be achieved by
the release of the excess of membrane stored in the internal
YSL in a form of membranous microvilli.
Recently, it has been proposed that an array of YCL microtubules would aid epiboly by pulling the blastoderm toward the
vegetal pole (Strähle and Jesuthasan, 1993). The taxol-induced
general delay in epiboly that we observed is reminiscent of the
phenotype described for embryos treated with UV or short
pulses of nocodazole, when the YCL microtubules are present
but somewhat disorganized (Strähle and Jesuthasan, 1993).
The above treatments indicate that microtubules contribute to
general epibolic movements. However, the observation of a
block of YSN movements, with only partial inhibition of
epiboly of the blastoderm and an unaffected localized endocytosis in the absence of microtubules, points to a more complex
role of microtubules in epiboly. Therefore, we propose that the
epibolic movements of the yolk syncytial nuclei are not
coupled to the process of endocytosis and to epiboly of the
blastoderm. Further, epibolic movements of the YSN but not
of the blastoderm are absolutely dependent on microtubules.
We hypothesize that both the YSL network and the YCL
microtubules contribute differently to various aspects of
epiboly, and that they are primarily involved in movements of
the YSN. Microtubules have been implicated in nuclear
movements in a variety of organisms, based on either morphological criteria (Picket-Heaps, 1991; Lloyd et al., 1987),
correlation between the organization of microtubules and
nuclear movements (Baker et al., 1993), sensitivity of nuclear
movements to anti-microtubule drugs (Picket-Heaps, 1991;
Lutz and Inouye, 1982; Oakley and Morris, 1981), or on a
genetic evidence (Huffaker et al., 1988; Morris, 1976). One
possible mechanism for microtubule-mediated nuclear
migration involves molecules that interact between the microtubules and a nucleus generating force to move it along microtubules (e.g. Osmani et al., 1990). Such a mechanism,
involving the YSL and YCL microtubules and a plus enddirected microtubule motor, could explain epibolic movements
of the YSN. Alternatively, motor molecules working between
interdigitating microtubules in the YSL network would
generate a pushing force similar to that used in anaphase B of
mitosis, 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 the
expansion of the margin of the YSL and possibly in transport
of cellular components localized in the external YSL. Since the
cellular components of the external YSL, like microfilaments,
may also be involved in epiboly; interference with the YCL
microtubules could affect all epibolic movements. Motor
molecules, similar to those involved in pulling the spindle pole
by astral microtubules (Saunders, 1993), would pull their organizing centers located in the YSL toward the vegetal pole.
Coordinate action of the YSL and YCL arrays would result in
the expansion of the whole YSL, whereas action of the YSL
network alone at the beginning of epiboly could account for
crowding of the YSN.
Distinguishing between these models will require identification of the involved motor molecules and their functional
analysis during epiboly. Additionally, it is important to understand the relationship between the microtubule and microfilament systems in this process. Since the large syncytial yolk cell
proves very accessible to experimental analysis, further studies
of microtubule arrays of the yolk cell in zebrafish can contribute to our understanding of the role of the cytoskeleton in
vertebrate development.
We are thankful to Elisabeth Vogelsang for sectioning embryos
stained with anti-tubulin antibodies, and to Dr Rocco Rotello for help
with western blotting. Special thanks to Dr Robert M. Ezzell for
giving us access to the confocal microscope, and Dr Yimin Ge for
instructions on collection and processing of confocal images. We
would like to thank our colleagues Derek Stemple, Timothy Burland,
Alexander Schier, Eliza Shah, Brant Weinstein, Fried Zwartkruis and
Dr John Trinkaus for the constructive comments on the manuscript.
Part of this work has been presented in an abstract form at the
Northeast Regional Developmental Biology Conference in Woods
Hole, March 1993. This work has been supported by National Institute
of Health grant HD29761 and by Bristol-Myers Squibb.
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