MOLECULAR REPRODUCTION AND DEVELOPMENT 71:186–196 (2005)
Rho Mediates Cytokinesis and Epiboly via
ROCK in Zebrafish
SHIH-LEI LAI,1 CHING-NUNG CHANG,1 PEI-JEN WANG,2 AND SHYH-JYE LEE1,3*
1
Institute of Zoology, National Taiwan University, Taipei, Taiwan, R.O.C.
2
Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan, R.O.C.
3
Department of Life Sciences, National Taiwan University, Taipei, Taiwan, R.O.C.
ABSTRACT
To study the regulation of embryonic development by Rho, we microinjected Clostridium botulinum C3-exoenzyme (C3) into zebrafish
embryos. We found that C3 inhibited cytokinesis
during early cleavages. C3 inhibition appeared to be
specific on RhoA, since the constitutively active RhoA
could partially rescued the C3-induced defects. Distributions of actin and the cleavage furrow associated
b-catenin were disrupted by C3. Belbbistatin, a myosin
II inhibitor, also caused blastomeres disintegration. It
suggested that Rho mediates cytokinesis via cleavage
furrow protein assembly and actomyosin ring constriction. Furthermore, C3 blocked cellular movements
during epiboly and gastrulation as evident by the
impairment on no tail and goosecoid expression in
blastoderm front runner cells and the dorsal lip of
blastopore, respectively. Y-27632, an antagonist of
Rho-associated kinase (ROK/ROCK), had the similar
inhibitory effects on zebrafish development as the C3
treatments. Taken together, these results suggest that
Rho mediates cleavage furrow protein assembly during
cytokinesis and cellular migration during epiboly and
gastrulation via a ROK/ROCK-dependent pathway.
Mol. Reprod. Dev. 71: 186–196, 2005.
ß 2005 Wiley-Liss, Inc.
Key Words: cytokinesis; gastrulation; C3-exoenzyme; Y-27632; belbbistatin; no tail; goosecoid
INTRODUCTION
Cellular cleavage is accomplished by coordinated
actions of karyokinesis and cytokinesis. Karyokinesis
is the mitotic segregation of a dividing nucleus and
cytokinesis is the splitting of cytoplasm components.
The process of cytokinesis is highly conserved in eukaryotic organisms (Guertin et al., 2002). A structure called
actomyosin ring, which contains actin, myosin, and
other proteins, assembles at the equator of a dividing
cell in coordination with the mitotic spindle. The
actomyosin-contractile ring then ingresses to form the
cleavage furrow. In somatic cells, the cleavage furrow
further constricts the components of spindle midzone
and the two daughter nuclei separate from each other
along the mitotic spindle. In contrast, the cells of
embryos divide at an extraordinary high rate that the
daughter blastomeres remain adhered to each other.
ß 2005 WILEY-LISS, INC.
The molecular controls of cytokinesis are beginning to
be understood (Robinson and Spudich, 2000, 2004;
Glotzer, 2003). Among the important molecules involved
in cytokinesis are the Rho family of small GTPases
(Guertin et al., 2002; Manser, 2002). Rho GTPases
regulate cytoskeleton-related cellular processes, including exocytosis, endocytosis, vesicle transport/secretion,
cell migration, and also cytokinesis (Hall, 1998; Takai
et al., 2001; Etienne-Manneville and Hall, 2002). The
formation of contractile ring and cleavage furrow depends on the normal assembly of contractile actinmyosin filament. Blocking Rho activity by Clostridium
botulinum C3-exoenzyme (C3), results in multinucleate
cells in Xenopus (Kishi et al., 1993), sea urchin (Mabuchi
et al., 1993), Drosophila (Crawford et al., 1998), and
C. elegans (Jantsch-Plunger et al., 2000) embryos. These
observations suggest that karyokinesis occurs without
accompanied cytokinesis and that C3 exerts its inhibition on cytokinesis by interfering with the function of
actin-myosin in the contractile ring (Mabuchi et al.,
1993).
The downstream targets of Rho during cytokinesis include Rho-associated kinase (ROK/ROCK) (Yasui et al.,
1998; Kosako et al., 1999, 2000; Goto et al., 2000), citron
kinase (Madaule et al., 1998), and formin-homology
proteins (Verheyen and Cooley, 1994; Narumiya et al.,
1997; Watanabe et al., 1997; Severson et al., 2002). The
phosphorylation of myosin regulatory light chain can be
inhibited by knocking down ROK/ROCK pharmacologically in vivo (Kosako et al., 2000). Myosin regulatory
light chain phosphorylation is critical for subsequent
activation of myosin II, which is the major protein
providing the contractile force in the actomyosincontractile ring (Glotzer, 2001, 2003). However, since
Y-27632, a specific ROCK inhibitor (Uehata et al., 1997)
failed to block Hela cell cytokinesis at a concentration
lower than 100 mM, ROCK is probably not required, but
Grant sponsor: Council of Agriculture, China (92Agriculture-9.2.4Fisheries-F1(Z)-2; Grant sponsor: National Science Council, China;
Grant number: NSC-93-2311-B-002-020.
*Correspondence to: Dr. Shyh-Jye Lee, 1 Roosevelt Road, Section 4,
Institute of Zoology, National Taiwan University, 209 Fisheries
Science Building, Taipei, Taiwan 106. E-mail: jeffl[email protected]
Received 2 April 2004; Accepted 17 November 2004
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/mrd.20290
Rho AND ROCK IN ZEBRAFISH DEVELOPMENT
instead plays a facilitating role in cytokinesis (Madaule
et al., 1998). In contrast, Marlow et al. (2002) examined the role of ROK/ROCK in zebrafish embryo by
knocking down Rho kinase 2 (Rok2) activities using
dominant-negative Rok2 (dnRok2). They showed that
the convergent extension impairment results in a
shorten-embryonic axis in Rok2-knock down embryos
and they also claimed that abnormal cell division and
lethality occurred at a higher dosage of dnRok2. One can
also use morpholino oligonucleotides to suppress the
translation of Rok2 and study its involvement during
early cleavages, however, it could be difficult due to the
abundance of maternal proteins. Therefore, we have
taken another approach to study the effects of Rho and
ROCK on the early cleavage stages by using pharmacological inhibitors.
In zebrafish, following the rapid cleavage period, the
blastula cells separate into underlying deep cells, which
migrate to the dorsal side to form the embryo, and into
the overlying the epithelial sheet that spreads as a unit,
during the process called epiboly, to enclose the deep
cells and the yolk cell (Kimmel et al., 1995). Cell contractility, microtubule and actin polymerization are all
important for these cellular movements (Ridley, 2001).
Rho and Rac act via ROK/ROCK to phosphorylate
myosin light chain for the formation of actin cytoskeletal
structures including the formation of stress fibers and
lamellipodia (Kaibuchi et al., 1999; Amano et al., 2000).
Through mDia, a mammalian formin homology protein,
Rho has been demonstrated to regulate microtubule
and F-actin polymerization (Ishizaki et al., 2001). The
noncanonical Wnt signaling pathways have been suggested to control morphogenesis by regulating polarized cellular movement during convergent extension in
Xenopus via a novel formin homology protein Daam1
(Habas et al., 2001). We examine here the effects of Rho
signaling on the cellular migration during gastrulation
in zebrafish.
Using pharmacological inhibitors, we show here that
Rho mediates cytokinesis via regulating actin and bcatenin assembly at the cleavage furrows by a ROK/
ROCK-dependent pathway, which may work through
myosin II. We also demonstrate that Rho regulates
cellular movement during epiboly and gastrulation
through ROK/ROCK.
MATERIALS AND METHODS
Fish Husbandry and Embryo Collection
Zebrafish, Danio rerio, were raised on a 14-hr day/
10-hr night cycle at 28.58C. Eggs were collected at 15–
20 min intervals after spawning, washed and incubated in Ringer’s solution (116 mM NaCl, 2.9 mM KCl,
1.8 mM CaCl2, and 5 mM HEPES) at 28.58C until use.
All chemicals were from Sigma (St. Louis, MO) unless
otherwise stated.
RT-PCR Analysis
The expression of mRNA in zebrafish embryos at
selected stages was determined by RT-PCR using the
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following primer pairs: (1) rhoA: forward (50 -ATGGCAGCAATTCGCAAGA-30 ) and reversed (50 -TCACAGCAGACAGCATTTG-30 ). (2) ef1a: forward (50 -CAAGGAAGTCAGCGCATACA-30 ) and reversed (50 -TGATGACCTGAGCGTTGAAG-30 ). The total RNAs were prepared by using Trizol reagent (Invitrogen Corporation,
Carlsbad, CA) from different stages of embryos. For
synthesizing single-stranded cDNAs, 3 mg of total RNA,
oligo dT primers with M-MLV reverse transcriptase
(Promega Corporation, Madison, WI) were applied in a
total reaction volume of 25 ml. RT-PCR was performed
with respective primers for 30 cycles at a thermal cycler
(PTC-200, MJ Research) according to the following protocol: denatured at 948C for 30 sec, annealed at 558C for
45 sec, and elongated at 728C for 1 min.
Preparation of Embryo Extract,
Immunoprecipitation, and Western Blotting
Zebrafish embryos at designated stages were collected
and the extraction procedures were adapted from the
starfish oocyte extraction protocols as described by Lee
et al. (2000). An anti-RhoA polyclonal antibody (sc-179,
Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was
used for immunoprecipitating (1:20 dilution) Rho from
zebrafish extract as described by Stapleton et al. (1998).
After immunoprecipitation, the Rho-coated protein A
beads were settled down by centrifugation. The supernatants were collected for the Western analysis of actin
using an anti-actin polyclonal antibody (Sigma A2066,
1:200 dilution) as a control. The resulting beads were
washed several times and resuspended in an equal
amount of 2 SDS sample buffer. Protein samples were
resolved in 15% SDS–PAGE gels, transferred onto
PVDF membranes (Amersham Biosciences, Piscataway, NJ) in 15 mM sodium borate at 100 mA for 9 hr.
After transfer, Western analysis was done by following
general protocols using the same anti-RhoA antibody
(1:400 dilution) and subjected to ECL Western detection
(Amersham Biosciences).
Microinjection Procedures
Thin-wall (1.0 0.75 mm, 400 ) glass capillaries with
filaments (A-M systems, Inc. Carlsborg, WA) were pulled using a horizontal puller (P-97, Sutter Instrument,
Navato, CA). Embryos at desired stages as judged according to Kimmel et al. (1995) were immobilized at
an injection trough on a 100 mm 2% agar plate. C3exoenzyme (BIOMOL, Plymouth Meeting, PA), Y-27632
(Calbiochem, La Jolla, CA), and constitutively active
RhoA GST fusion protein (Cytoskeleton, Denver, CO)
were dissolved in injection buffer (68.5 mM NaCl,
1.35 mM KCl, 5 mM Na2HPO4, and 1 mM KH2PO4 and
0.25% (w/v) phenol red) alone or in different combinations as indicated. An injection pipette was forced into
the chorion and the yolk cell to reach the junction
between yolk cell and blastodisc where the solution was
ejected by using a pressure injector (IM-300, Narishige,
Japan). We estimated the injection volume by the clearance of cytoplasm (0.5% of 1-cell yolk volume). After injection, embryos were recovered from injection troughs
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and cultured in Ringer’s solution at 28.58C until
examined.
Actin and b-catenin Staining on
Whole Mount Embryos
To label F-actin, zebrafish embryos at designated
stages were fixed in 3% paraformaldehyde in PBS
(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and
2 mM KH2PO4) overnight at room temperature, washed
in PBS and manually dechorionated. Twenty embryos
were incubated with rhodamine phalloidin (R-415,
6.6 nM/ml, Molecular Probes, Inc., Eugene, OR) at room
temperature for 4–6 hr and then washed intensively
with 0.5% Triton X-100 (PBT). To label b-catenin,
zebrafish embryos at designated stages were fixed in
4% paraformaldehyde in PBS overnight at 48C , washed
in PBS and manually dechorionated. Twenty embryos
were incubated with a polyclonal anti-b-catenin antibody (Sigma C2206, diluted at 1:500) with continuous
rotation at 48C overnight and washed intensively with
0.5% Triton X-100 (PBT). b-catenin-labeled embryos
were incubated in a 1:250 dilution of a FITC-conjugated
goat anti-rabbit antibody (#111–095-144, Jackson ImmunoResearch Laboratories, West Grove, PA) at 48C
overnight. Both actin and b-catenin labeled embryos
were transferred to 30% glycerol first and then 50%
glycerol for confocal microscopic observations at the
Center of Two-Photon Laser Confocal Microscope,
National Taiwan University.
Removing Embryos From Their Chorions
for Blebbistatin Treatments
One-cell stage eggs were treated with 0.5 mg/ml
protease (Sigma 6917) in embryo medium (Westerfield,
2000) at 28.58C for 1–2 min. Protease was removed
immediately by several changes of embryo medium.
Chorions were removed by gentle swirling of embryos.
Some embryos were damaged upon protease treatment.
Therefore, only morphologically healthy and cleaved
embryos were selected for experiments.
In Situ Hybridization on Whole
Mount Embryos
Embryos treated with or without C3 or Y27632 were
fixed in 4% paraformaldehyde. In situ hybridization
against goosecoid or no tail genes on the whole mount
embryos were performed as described by Thisse et al.
(1994).
Embryo Observations and Photography
Observation of embryonic development was made at
designated time under a Leica Mz75 stereomicroscope, a
Leica DMIRB fluorescent microscope, or a Leica Mz75
stereomicroscope. All photographs were taken by a
Nikon digital camera and analyzed using Adobe Photoshop 6.0 except for the confocal images, which were
photographed and edited utilizing the Leica Confocal
Software.
Statistical Analysis
Experimental values are expressed as mean standard error of mean and analyzed in Excel using
pair-sample t-test.
RESULTS
The regulation of cytokinesis and cellular migration
by the small GTPase, Rho has been extensively studied
in a variety of cellular systems including embryonic
cells. However, little information is available for Rho
expression and its functions during early embryonic
development in zebrafish. To study Rho regulation in
zebrafish, we first located two zebrafish rhoA (zrhoA)
cDNA in GeneBank, which are 582 bp (NM_212749)
and 1777 bp (BC075938), respectively. Comparing the
coding regions of those two rhoA cDNAs, we found a
mismatched nucleotide at 229 from C to T, which results
in a change of translated amino acid residue from Ala
110 to Val 110. To validate the zrhoA sequence, we
isolated a zrhoA cDNA containing the coding region by
RT-PCR. It was confirmed that the amino acid 110 is Val
instead of Ala, which is also conserved in other animal
species as well. Zebrafish rhoA is 95.9% identical (185 of
193 amino acids) to its human homologue, which reveals
that rhoA is also highly conserved in teleost (Fig. 1).
Expression of RhoA During Early
Embryonic Development
To examine the expression of rhoA during early development, we performed RT-PCR analysis in zebrafish
embryos from 1-cell to 6-somite stages and found that
zRhoA (582 bp) expressed in all stages of embryos examined (Fig. 2A). Ef1a was used as an RT-PCR control.
The relative expression level of zRhoA was much lower
from 1-cell to 256-cell compared to later stages embryos.
It implied that RhoA exists as maternal protein at early
stages. Therefore, we analyzed the changes of RhoA
protein expression by Western analysis using a polyclonal antibody against human RhoA (hRhoA), which
can recognize RhoA in sea urchin embryos (Manzo et al.,
2003). Direct detection of RhoA in the zebrafish embryo
lysate failed, which might be due to the amount of RhoA
was below detection limit (data not shown). Therefore,
Fig. 1. Sequence alignment of zebrafish and human RhoA gene. The
amino acid sequences of zebrafish RhoA (AAH75938.1) is aligned to that
of human RhoA (AAC33178.1). Among 193 amino acids, only 8 amino
acids are different as indicated, which represents a 95.9% identity. Val
110 (enclosed by an empty rectangle) is the site of mismatch between
RhoA cDNAs NM_212749 and BC075938. Q63 (underlined) was
mutated to L to make constitutively active RhoA used in this study.
Rho AND ROCK IN ZEBRAFISH DEVELOPMENT
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Fig. 3. C3-exoenzyme causes gradual death of embryos. No significant death occurred prior to 6 hr post fertilization (shield stage), but
survival rate continued to decrease for 24 hr. Fertilized eggs were
injected at the one-cell stage with indicated amounts of C3-exoenzyme
in pM and cultured in Ringer’s solution at 28.58C. Embryos were examined at the designated time and the white/opaque embryos scored as
dead. These results are representative of at least four independent
experiments.
Fig. 2. RhoA is present as maternal transcripts and proteins in
zebrafish embryos. Expression of RhoA mRNAs or proteins in early
zebrafish embryos from 1-cell to 6 somite stages was determined by RTPCR (A) or Western blotting (B), respectively. Ef1a and actin was
served as RT-PCR and Western blotting internal controls, respectively.
A recombinant hRhoA (100 ng per well) was also used as a positive
control for RhoA detection.
we concentrated RhoA by immunoprecipitation with
subsequent Western analysis for detection. To have a
Western blotting loading control, a polyclonal anti-actin
antibody was used to detect actin (42 kDa) in the
supernatant of zebrafish lysate after RhoA immunoprecipitation. RhoA protein (24 kDa) was detected in
all stages of embryos examined as well as a recombinant
hRhoA (Fig. 2B). However, it appeared that at least two
bands around the region were detected, which might
be RhoC or RhoB, since the anti-RhoA antibody used
can also detect RhoC, but to a less extent to RhoB. For
simplicity, we will refer them as Rho proteins here. Rho
proteins were present at high amounts in 1-cell and
gradually decreased until 256-cell, but significantly increased after 30% epiboly stages embryos. The changes
in Rho proteins level coincided very nicely with the
changes in rhoA mRNA expression. The appearance of
Rho proteins at early stages suggested they are maternal
deposited. More importantly, the presence of both
maternal RhoA mRNA and protein and the later activation of zygotic expression imply that Rho is functionally important in regulating early development in
zebrafish.
Inhibiting Rho Activity Blocks Cytokinesis
To study Rho functions during early embryonic development, we microinjected C3, a specific Rho antagonist
(Aktories and Hall, 1989) into developing zebrafish
embryos. Embryos injected with increasing amounts of
C3 to an intracellular concentration of 1.2 nM, showed
no significant death up to 6 hr post fertilization (6 hpf,
shield stage) (see Fig. 3). However, 58 22% (N ¼ 4) of
112 embryos treated with 1.2 nM C3 died in 24 hpf.
Therefore, we only analyzed the effects of C3 on embryos
during the early cleavages and epiboly stages (up to
6 hpf) unless otherwise stated.
Figure 4 shows the effects of C3 treatment on 8-cell
and sphere stage embryos. Sham-injected embryos
showed distinct cleavage furrow boundaries at the
8-cell (Fig. 4A) and sphere (Fig. 4B) stages. In contrast,
embryos injected with 120 pM C3 at the 1-cell stage had
incomplete or no cleavage furrows at the 8-cell (Fig. 4C)
and sphere stages (Fig. 4D). Therefore, C3 inhibited
embryonic cleavages as demonstrated in other animal
species. To analyze the dose-dependent response of C3,
we injected embryos with different amounts of C3 into
2–8 cell stage embryos. Injecting embryos after the
1-cell stage allowed us to remove easily unfertilized
eggs from our statistics. We also observed that phenol
red, a visual indicator contained in the injection buffer,
quickly diffused into all blastomeres from the site of
injection to ensure that all blastomeres received the
same treatments. We then examined the morphological
changes in particular the formation of cleavage furrows at 4 hr post fertilization. As shown in Figure 4E,
25.9 13.7% (N ¼ 4) of 111 embryos injected to an intracellular concentration of 0.4 nM C3 showed defects in
cleavage. As intracellular C3 concentration increased to
1.2 and 3.6 nM, the percentage of embryonic cleavage
defects increased significantly (P < 0.05) to 52.4 5.5%
(N ¼ 4) of 115 embryos and 70.7 16.9% (N ¼ 4) of 97
embryos, respectively. Thus, C3 blocked early embryonic cleavage in a dose-dependent manner.
Inhibition of C3 can be Partially Rescued
by Constitutively Active RhoA
To clarify the specificity of C3 inhibition on embryonic
cleavage, we tried to rescue the C3-treated embryos
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TABLE 1. Rescue of C3-Induced Embryonic Cleavage
Defects by Constitutively Active RhoA
Treatment
Sham-injected
4.5 mg/ml L63RhoA
400 pM C3
C3 þ L63RhoA
% cleavage defects (n)
0.0 0.0 (161)a
1.5 1.3 (157)a
47.7 14.1 (190)b
24.5 11.6 (182)c
Embryos at 2–8 cells were injected with reagents indicated and
examined as described in the ‘‘Materials and Methods.’’ Data
are presented as mean standard error of mean with the
numbers of embryos observed in parentheses. Experimental
values are compared to each other within the same group. The
significance of difference between mean is analyzed by Excel
using pair-sample t-test with different superscript letters
representing P < 0.05.
zebrafish constructs interchangeably. The injection of
L63RhoA alone has no effect on cleavage (Table 1). In
contrast, the inhibition of cleavage by C3 at 400 pM
was partially (about 50%) rescued by co-injection with
L63RhoA at an intracellular amount of 4.5 mg/ml. These
results suggested that the C3 inhibition is specifically
targeting on RhoA.
Fig. 4. C3-exoenzyme blocks embryonic cleavage in a dose-dependant manner. Fertilized eggs were injected with buffer (A, B) or 120 pM
(C, D) C3-exoenzyme then incubated in Ringer’s solution until
designated stages. We examined embryos at 8-cell (A, C) and sphere
(B, D) stages. For the control embryos (A, B), distinct cleavage furrows
exist between blastomeres, while incomplete or no cleavage furrows
(C, D) are shown in the C3-injected embryos (E). To test dosage
response of C3, embryos at 2-8 cell stage were injected with indicated
amounts of C3-exoenzyme and examined at 4 and 6 hpf, respectively.
Each data point represents the mean of percentage cleavage defects
standard error of mean for four independent experiments.
by co-injection of the constitutively active RhoA
(L63RhoA), a constitutively active GST fusion proteins
(see Fig. 1 for the mutation site). L63RhoA used is of
human origin and commercially available (Cytoskeleton, Inc.). Since there is only 4% difference in amino acid
sequences between human and zebrafish Rho A (Fig. 1)
and the sequences around the site of mutation are
the same, it would be relatively safe to use human and
Inhibiting Rho Activity Blocks Actin
and b-Catenin Distributions at
the Cleavage Furrows
The cleavage furrow is assembled by cytoskeleton and
associate proteins. To further characterize the effects
of C3 on cleavage furrow formation, we examined the
distributions of actin, a major cytoskeleton protein, and
b-catenin, which are found at the lateral membranes
between blastomeres (Jesuthasan, 1998), by rhodamine
phalloidin and anti-b-catenin antibody staining, respectively. In the sham-injected sphere-stage embryos, both
actin (Fig. 5A) and b-catenin (Fig. 5B) were clearly
stained at the boundaries of blastomeres. In contrast,
actin was not present between blastomeres, but aggregated into patches in the presence of 1.2 nM C3 (Fig. 5C).
The b-catenin staining was also severely disrupted in
the C3-treated embryos as shown in Figure 5D.
Deterioration of Embryo by
Inhibiting Myosin II
Myosin II is a contractile ring protein that is known for
its role in cytoplasmic constriction during cytokinesis
(Robinson and Spudich, 2000). To examine the role of
myosin II during zebrafish embryonic development, we
incubated embryos with blebbistatin, a specific myosin
II inhibitor, which has been shown to inhibit contraction
of the cleavage furrow without disrupting mitosis or
contractile ring assembly (Straight et al., 2003). The
zebrafish chorion is known to be a tough barrier for
many chemicals. Therefore, we used protease to dechorinate 1-cell stage embryos as described in the ‘‘Materials
and Methods’’ and then incubated embryos with blebbistatin at the 2-cell stage. Although 50 mM blebbistatin
disrupted cytokinesis in a small portion of embryos
Rho AND ROCK IN ZEBRAFISH DEVELOPMENT
191
Fig. 5. C3 and Y-27632 disrupt distributions of actin and b-catenin at the cleavage furrows in injected
embryos. Embryos were fixed at sphere stage and stained with rodamine phalloidin (A, C, E) or b-catenin
(B, D, F) as described in the ‘‘Materials and Methods.’’ Injection of C3-exoenzyme (1.2 nM, A–D) or Y-27632
(4.5 mM, E, F) distorted the accumulation of actin and b-catenin at the cleavage furrows. All views are from
the animal pole.
(Fig. 6B) as compared to the control embryos (Fig. 6A),
80% embryos kept dividing up to 3 hpf (1,000-cell stage)
without interfering karyokinesis (data not shown), but
started to deteriorate afterward showing loose blastomeres. Disruption of the cells by blebbistatin was dosedependent as shown in Figure 6C. Thus, myosin II is
required to maintain the adhesion of the cells with each
other or with the yolk cells.
Taken together, these observations suggest that cytokinesis can be specifically blocked by inhibiting RhoA
via the disruptions of cleavage furrow protein assembly
and actomyosin ring constriction in zebrafish embryos.
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Fig. 6. Blebbistatin induces the loss of blastomeres and the disintegration of the embryos. Embryos were dechorinated at 1-cell stage as
described in the ‘‘Materials and Methods.’’ Dechorinated 2-cell stage
embryos were treated with (B) or without (A) 50 mM blebbistatin and
examined at sphere stage (4 hpf). The effects of blebbistatin were
dose-dependent as shown in (C) where the percentage of normal
embryos at 5 hpf are presented. These results are representative of at
least five independent experiments.
Inhibiting Rho Activity Blocks Epiboly
and Gastrulation
Following the rapid cleavages, zebrafish embryos
undergo massive cellular movement during epiboly
formation and subsequent gastrulation. We examined
the Rho-dependent cellular migration by observing
the development of C3-treated embryos. A significant
number of the C3-treated embryos survived to reach
epiboly at lower dosages of C3. However, cellular movement during epiboly could be impaired by C3 as low as
120 pM. Figure 7A shows a sham-injected embryo at
50% epiboly and the beginning of gastrulation, noted by
the ring structure (arrows) that results from the involution of the mesoderm cells at the equator of the yolk
cell. During epiboly the cells at the involuting epithelial
layer appeared to migrate to the equator of the yolk in
the C3-injected embryos (Fig. 7B). However, the ring
structure (arrows) typical of the involution of the mesoderm at 50% epiboly formed not at the equator, but
near to the animal pole of the embryo (Fig. 7B). These
embryos maintained the movements associated with
the involution of the mesoderm, but the presumptive
mesodermal cells had not migrated over the yolk to the
equator. Rho is required for adhesion during cell
Fig. 7. C3-exoenzyme dose-dependently inhibits cellular migration
during epiboly. Fertilized eggs were injected with buffer or C3 and
photographed at 50% epiboly stage on the animal pole view (A–D) or
the side views (E, F). Note that the 120 pM C3 injected embryo formed
an irregular and small ring structure (arrows) at the 50% epiboly stage
(B) compared to the sham-injected embryo (A). The front runner cells of
blastoderm were revealed by the labeling of a regular no tail mRNA ring
(arrow, C), but the ring was distorted in the presence of C3. The
involuting dorsal lip of blastopore (arrow) was shown by the labeling of
goosecoid mRNA in the control (E), but not in the C3-treated embryo
(F). To test the dosage responses, embryos were injected with C3 up to
250 pM and examined accordingly (G). Each data point represents the
mean of percentage epiboly defects standard error of mean for three
independent experiments.
migration (Hall, 1998), but its role in the spreading of
the underlying deep cells is not understood. So we
further examined the involution of gastulating embryos
by monitoring the expressions of no tail, an immediate
early gene known to turn on in the future germ ring, and
Rho AND ROCK IN ZEBRAFISH DEVELOPMENT
goosecoid, another immediate early gene known to be
exclusively expressed in the involuting dorsal lip of
blastopore (Schulte-Merker et al., 1994). Using wholemount in situ hybridization, we observed that no tail
mRNA was clearly expressed in the germ ring (arrow) of
the sham-injected embryo (Fig. 7C). However, irregular
no tail rings were seen in the C3-treated embryo, which
indicated an aberrant and unsynchronized migration of
front runner cells in the future germ ring (arrow,
Fig. 7D). The inhibition of involution by C3 was obvious
by observing the presence but absence of goosecoid
mRNA staining at the dorsal lip of blastopore (arrows) in
the sham- (Fig. 7E) and C3- (Fig. 7F) injected embryos,
respectively. C3 also caused defects in epiboly formation and gastrulation in a dose-dependent manner. As
shown in Figure 7G, at an intracellular concentration
of 120 pM, C3 caused 44.3 8.0% (N ¼ 3) defects in
120 embryos. As the intracellular C3 concentration
increased to 180 and 240 pM, the epiboly defects were
increased to 68.4 7.1% (N ¼ 3, 198 embryos) and
88.8 4.8% (N ¼ 3, 168 embryos), respectively.
Some of these embryos with delayed epiboly continued
to develop. One such embryo, which was injected to
1.2 nM C3 at the 2- to 8-cell stage, is shown in Figure 8A.
Compared to a sham-injected embryo incubated similarly for 52–54 hpf (Fig. 8B), the C3-injected embryo had
a shorten-embryonic axis, but almost normal head
structures (Fig. 8A). Thus, migration of the tail mesoderm was aberrant in the C3-treated embryos.
Inhibiting Rho-Associated Kinase Activity
Blocks Cytokinesis and Epiboly
To determine downstream targets of Rho during cytokinesis and epiboly, we examined the possible in-
Fig. 8. C3 induces the shortening of embryonic axis. Embryos were
injected with buffer (B) or 1.2 nM C3 (A) at 2- to 8-cell stage and
photographed at 52–54 hpf. The C3-treated embryo (A) shows a
shorten-embryonic axis and a bent tail. In contrast, the sham-injected
embryo (B) appears normal.
193
volvement of Rho-associated kinase (ROK/ROCK) by
injecting a ROCK specific inhibitor, Y-27632 (Uehata
et al., 1997; Kosako et al., 2000). Y-27632 at 45.5 mM
disrupted the assembly of actin (Fig. 5E) and b-catenin
(Fig. 5F) as the C3 treatments (Fig. 5C,D), which
resulted in defects in cytokinesis (data not shown). As
shown in Figure 9A, a sham-injected embryo clearly
started epiboly formation and reached 30% of yolk
sphere, but the blastoderm margin of the embryo treated
with 45.5 mM Y-27632 failed to spread toward vegetal
pole at the 30% epiboly stage (Fig. 9B). The inhibition of
Y-27632 on front runner cells migration was demonstrated by no tail mRNA staining where the no tail ring
(arrow) was irregular and significant lagged (Fig. 9D)
compared to the control embryos (Fig. 9C) at near 50%
epiboly stage. As in the C3-treated embryos, Y-27632
also blocked involution of the dorsal lip of blastopore as
evident by the occurrence of goosecoid expression in the
control (arrow, Fig. 9E), but not in the Y-27632-treated
(arrow, Fig. 9F) embryos. To test the dosage response of
Y-27632, we also injected 2-8 cell stage embryos with
different amounts of Y-27632 and found that Y-27632
inhibited cleavage and epiboly formation in a dosedependent manner as the C3 treatments (Fig. 9G). At an
intracellular concentration of 4.5 mM, Y-27632 caused a
15.5 6.9% (N ¼ 3) cleavage defects in 195 embryos. As
injected concentration increased, the Y-27632-induced
cleavage defects increased to 46.0 23.8% and
74.7 9.5% (N ¼ 3) at 11.4 mM (207 embryos) and 22.7
mM (179 embryos), respectively. In 126 embryos treated
with 45.5 mM Y-27632, almost all embryos showed
cleavage defects (97.4 4.6%, N ¼ 3). Most of those
defective embryos died the next morning. However, a
small portion of them did survive, but showed shorten or
curved tails (data not shown), which are similar to those
embryos expressed dnRok2 as described by Marlow et al.
(2002) and as seen for C3 injected embryos in Figure 8A.
Thus, we can conclude, as Marlow et al. did, that the
migration of the dorsal cell along straight paths that
dependent on the medial lateral elongation is inhibited
by both blocking Rho and Rho kinase activities.
DISCUSSION
Rho has been demonstrated to be a key molecular
switch during early embryonic development in both
vertebrate and invertebrate embryos (Kishi et al., 1993;
Mabuchi et al., 1993; Crawford et al., 1998; JantschPlunger et al., 2000). However, little is known regarding
the rho-dependent regulation of cytokinesis and gastrulation in zebrafish, a recently evolving vertebrate
model system. In this study, we demonstrate that (1) Rho
exists in early zebrafish embryos as maternal mRNAs
and proteins, (2) C3 blocks cytokinesis by disrupting the
assembly of actin and b-catenin and similar effects can
be mimicked by applying ROK/ROCK antagonist, Y27632. (3) C3 and Y-27632 also block epiboly formation
and gastrulation in zebrafish embryos. These results
clearly demonstrated that Rho mediates cytokinesis,
epiboly and gastrulation via its downstream effector
ROK/ROCK in zebrafish.
194
S.-L. LAI ET AL.
In this study, we observed the abundance of Rho
mRNAs and proteins in early embryos, which are presumably from a maternal origin. A later surge of zygotic
Rho transcription occurs no later than 30% epiboly
embryos. These phenomena suggest a functional role
for Rho to mediate early embryonic development. To
study Rho functions, several tools may be used for
knocking Rho activity, including dominant negative
Rho, Rho morpholino oligonucleotides, and C3-exoenzyme. Firstly, dominant negative Rho (N19RhoA) has
been widely used to inhibit Rho functions by ectopic
expression. However, a noticeable expression of exogenous GFP fusion proteins would not occur until epiboly
stage (our personal observations), which is too late to
study early events like cytokinesis in zebrafish. Alternatively, one can inject recombinant GST fusion protein of
N19RhoA instead. Unfortunately, N19RhoA GST fusion
protein has been known to be very unstable and its
activity lost after purification (Self and Hall, 1995; Sah
et al., 2000). We were also not able to generate N19RhoA
GST fusion protein, either. Secondly, the presence of
abundant maternal Rho prohibits the use of morphlino
oligonucleotides to knock down Rho activity, since the
morpholins can only block the newly synthesis of proteins, but not inhibiting activities of existing ones.
Lastly, C3-exoenzyme has been shown to ADP-ribosylate a 25 kDa RhoA through out early development in
sea urchin eggs and embryos (Manzo et al., 2003). ADPribosylation of Rho proteins shuts down Rho activity by
interfering Rho GTPase activity (Aktories and Hall,
1989). Therefore, we used C3-exoenzyme to block Rho
activity in our experiments.
Fig. 9. Y-27632 inhibits the embryonic cleavage and gastrulation in
a dose-dependant manner. Fertilized eggs were injected with buffer
(A, C, E) or 45.5 mM Y27632 (B, D, F) and photographed at 30% or 50%
epiboly stage on the side views. Note that the Y-27632-injected embryo
showed aberrant blastoderm front runner cell (arrows) migration (B, D)
and involution of dorsal blastolip (F) compared to controls (A, C, E). The
detection of no tail and goosecoid mRNAs was as described in Figure 6.
To test the dosage responses, embryos were injected with Y-27632 up to
45.5 mM and examined accordingly (G). Each data point represents the
mean of percentage epiboly defects standard error of mean for three
independent experiments.
C3 Inhibition on Cytokinesis can
be Rescued by Active Rho
Rho activity has been well established to be essential
for cleavage furrow formation during cytokinesis in
eukaryotic cells (Hall, 1998). The requirement for Rho
in cytokinesis has been demonstrated by application or
injection of C3, RhoGDI, or RhoA RNAi in Xenopus
(Kishi et al., 1993; Drechsel et al., 1997), sandollar
(Mabuchi et al., 1993), Drosophila (Crawford et al.,
1998), and C. elegans (Jantsch-Plunger et al., 2000).
Here, we report that the Rho-mediated cytokinesis also
occurs in zebrafish. In addition, we showed that C3 inhibition on cleavage could be rescued by constitutively active Rho, L63RhoA. These results suggest that
zebrafish cytokinesis is specifically mediated by Rho.
Rho-Mediated Cytokinesis Is Regulated
Through a ROK/ROCK-Dependent Pathway
Several known rho effectors that signal cytokinesis
are citron kinase, mDia and ROK/ROCK (Glotzer, 2001,
2003). ROK/ROCK accumulates at the cleavage furrow
and phosphorylates intermediate filaments and fibrillary acidic protein (Kosako et al., 1999; Goto et al., 2000).
Injection of dominant-negative ROK, has suggested that
ROK is required for cytokinesis in both Xenopus and
mammalian EL cells (Yasui et al., 1998). The down-
Rho AND ROCK IN ZEBRAFISH DEVELOPMENT
stream target of ROCK appears to be myosin regulatory
light chain (MRLC), since MRLC phosphorylation at the
cleavage furrow is abolished by ROCK antagonists
(Kosako et al., 2000). ROCK has also been shown to inactivate myosin phosphatase through phosphorylation
on MBS that aids in keeping MRLC phosphorylated and
subsequent activation of myosin II (Amano et al., 1996;
Kimura et al., 1996; Chihara et al., 1997). In Drosophila,
Drok, a ROCK homologue, works downstream of the
Frizzled PCP signaling for phosphorylation of MRLC
and subsequent activation of Myosin II (Winter et al.,
2001). In contrast, by overexpressing ROCK mutants,
Madaule et al. (1998), failed to observe the multinucleate cells, which leads to the conclusion that ROCK is
not essential for cytokinesis. However, at concentrations from 10 to 30 mM, which are similar to previous
studies (Niggli, 1999; Kosako et al., 2000), Y-27632 inhibited cytokinesis and epiboly in over 50% zebrafish
embryos in our hands (Fig. 9). When injected Y-27632
was increased to 45.5 mM, almost all embryos showed abnormality in development. At 45.5 mM, Y-27632
may be too high to have some nonspecific inhibition. In
contrast, Y-27632 has been shown to have no effects
on p21-activated protein kinase even at 100 mM
(Narumiya et al., 1997). Y-27632 has also been reported
to be only highly specific to ROCK-II and protein kinase
C-related kinase 2 (PRK2) (Davies et al., 2000). Here,
we could not rule out the possible inhibition on PRK2,
which is also involved in the regulation of actin cytoskeleton (Vincent and Settleman, 1997), in our experiments. However, since PRK2 has no known function in
mediating cytokinesis, therefore, our observations suggest that Rho mediated cytokinesis via a ROCK/ROK
dependent pathway.
Regulation of Gastrulation by Small
GTPases Signaling
During gastrulation, which begins at 50% epiboly,
directed migration of head mesoderm leads to the
elongation of dorsal mesoderm. In addition, the uniform
radial cell intercalation results in the thinning and
spreading of the mesoderm and ectoderm called epiboly.
A typical ring structure can be seen from the animal pole
view. However, we observed that zebrafish embryos
made a smaller and asymmetrical form of the mesodermal involuting ring structure in the presence of C3
(Fig. 7). In addition, the loss of no tail and goosecoid
expression at blastoderm front runner cells and the
dorsal lip of blastopore, respectively (Figs. 7 and 9)
further strengthen the involvement of Rho and ROCK in
regulation of gastrulation. The molecular regulation of
cellular movement during gastrulation is beginning to
unravel. The intracellular signals are conserved among
chordates (Wallingford et al., 2002). It has been shown
that Wnt signaling is central to cellular movement
during gastrulation in both Xenopus and zebrafish
(Keller et al., 2000). A similar pathway is used in the
establishment of planar cell polarity in Drosophila.
Noncanoical Wnt signaling regulates planar cell polarity and convergent extension, respectively. Overexpres-
195
sion of Wnt5a and Wnt4 has been shown to disrupt
convergent extension without dramatically affecting
cell fate in both frogs and fish (Moon et al., 1993; Ungar
et al., 1995). RhoA and ROK/ROCK have also been demonstrated to be downstream effectors of the planar cell
polarity signaling (Strutt et al., 1997; WunnenbergStapleton et al., 1999; Winter et al., 2001; Marlow et al.,
2002). Our results further strengthen the involvements
of RhoA and ROCK during gastrulation in zebrafish.
In summary, using specific pharmacological inhibitors against two important players of the Rho signaling
pathway, Rho and ROK/ROCK, we have essentially
come to the same conclusion that Rho work via ROK/
ROCK to regulate key developmental processes, including the assembly of cleavage furrow proteins and
actomysin ring constriction during cytokinesis and
cellular movements leading to epiboly formation and
gastrulation in zebrafish.
ACKNOWLEDGMENTS
We thank Dr. Merrill B. Hille for helpful discussion
and comments on the manuscript, Dr. C-H Hu for
generous gifts of no tail and goosecoid plasmids, and
Dr. Yu-Fen Huang for help in confocal microscopy.
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