Rho Mediates Cytokinesis and Epiboly Via ROCK in Zebrafish

行政院國家科學委員會專題研究計畫成果報告

Small GTPases 對斑馬魚胚發育之影響

計畫類別：個別型計畫計畫編號： NSC93-2311-B-002-020- 執行期間： 93 年 08 月 01 日至 94 年 07 月 31 日執行單位：國立臺灣大學動物學研究所

計畫主持人：李士傑

計畫參與人員：賴時磊

報告類型：精簡報告

處理方式：本計畫可公開查詢

中華民國 94年8月1日

MOLECULAR REPRODUCTION AND DEVELOPMENT 71:186–196 (2005)

Rho Mediates Cytokinesis and Epiboly via ROCK in Zebrafish

1 1 2 1,3 SHIH-LEI LAI, CHING-NUNG CHANG, PEI-JEN WANG, AND SHYH-JYE LEE * 1Institute of Zoology, National Taiwan University, Taipei, Taiwan, R.O.C. 2Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan, R.O.C. 3Department of Life Sciences, National Taiwan University, Taipei, Taiwan, R.O.C.

ABSTRACT To study the regulation of embry- The molecular controls of cytokinesis are beginning to onic development by Rho, we microinjected Clostri- be understood (Robinson and Spudich, 2000, 2004; dium botulinum C3-exoenzyme (C3) into zebrafish Glotzer, 2003). Among the important molecules involved embryos. We found that C3 inhibited cytokinesis in cytokinesis are the Rho family of small GTPases during early cleavages. C3 inhibition appeared to be (Guertin et al., 2002; Manser, 2002). Rho GTPases specific on RhoA, since the constitutively active RhoA regulate cytoskeleton-related cellular processes, includ- could partially rescued the C3-induced defects. Dis- ing exocytosis, endocytosis, vesicle transport/secretion, tributions of actin and the cleavage furrow associated cell migration, and also cytokinesis (Hall, 1998; Takai b-catenin were disrupted by C3. Belbbistatin, a myosin et al., 2001; Etienne-Manneville and Hall, 2002). The II inhibitor, also caused blastomeres disintegration. It formation of contractile ring and cleavage furrow de- suggested that Rho mediates cytokinesis via cleavage pends on the normal assembly of contractile actin- furrow protein assembly and actomyosin ring cons- myosin filament. Blocking Rho activity by Clostridium triction. Furthermore, C3 blocked cellular movements botulinum C3-exoenzyme (C3), results in multinucleate during epiboly and gastrulation as evident by the cells in Xenopus (Kishi et al., 1993), sea urchin (Mabuchi impairment on no tail and goosecoid expression in et al., 1993), Drosophila (Crawford et al., 1998), and blastoderm front runner cells and the dorsal lip of C. elegans (Jantsch-Plunger et al., 2000) embryos. These blastopore, respectively. Y-27632, an antagonist of observations suggest that karyokinesis occurs without Rho-associated kinase (ROK/ROCK), had the similar accompanied cytokinesis and that C3 exerts its inhibi- inhibitory effects on zebrafish development as the C3 tion on cytokinesis by interfering with the function of treatments. Taken together, these results suggest that actin-myosin in the contractile ring (Mabuchi et al., Rho mediates cleavage furrow protein assembly during 1993). cytokinesis and cellular migration during epiboly and The downstream targets of Rho during cytokinesis in- gastrulation via a ROK/ROCK-dependent pathway. clude Rho-associated kinase (ROK/ROCK) (Yasui et al., Mol. Reprod. Dev. 71: 186–196, 2005. 1998; Kosako et al., 1999, 2000; Goto et al., 2000), citron ß 2005 Wiley-Liss, Inc. kinase (Madaule et al., 1998), and formin-homology proteins (Verheyen and Cooley, 1994; Narumiya et al., Key Words: cytokinesis; gastrulation; C3-exoen- 1997; Watanabe et al., 1997; Severson et al., 2002). The zyme; Y-27632; belbbistatin; no tail; goosecoid phosphorylation of myosin regulatory light chain can be inhibited by knocking down ROK/ROCK pharmacologi- cally in vivo (Kosako et al., 2000). Myosin regulatory INTRODUCTION light chain phosphorylation is critical for subsequent Cellular cleavage is accomplished by coordinated activation of myosin II, which is the major protein actions of karyokinesis and cytokinesis. Karyokinesis providing the contractile force in the actomyosin- is the mitotic segregation of a dividing nucleus and contractile ring (Glotzer, 2001, 2003). However, since cytokinesis is the splitting of cytoplasm components. Y-27632, a specific ROCK inhibitor (Uehata et al., 1997) The process of cytokinesis is highly conserved in eukar- failed to block Hela cell cytokinesis at a concentration yotic organisms (Guertin et al., 2002). A structure called lower than 100 mM, ROCK is probably not required, but actomyosin ring, which contains actin, myosin, and other proteins, assembles at the equator of a dividing cell in coordination with the mitotic spindle. The Grant sponsor: Council of Agriculture, China (92Agriculture-9.2.4- actomyosin-contractile ring then ingresses to form the Fisheries-F1(Z)-2; Grant sponsor: National Science Council, China; cleavage furrow. In somatic cells, the cleavage furrow Grant number: NSC-93-2311-B-002-020. further constricts the components of spindle midzone *Correspondence to: Dr. Shyh-Jye Lee, 1 Roosevelt Road, Section 4, Institute of Zoology, National Taiwan University, 209 Fisheries and the two daughter nuclei separate from each other Science Building, Taipei, Taiwan 106. E-mail: jeffl[email protected] along the mitotic spindle. In contrast, the cells of Received 2 April 2004; Accepted 17 November 2004 embryos divide at an extraordinary high rate that the Published online in Wiley InterScience (www.interscience.wiley.com). daughter blastomeres remain adhered to each other. DOI 10.1002/mrd.20290 ß 2005 WILEY-LISS, INC. Rho AND ROCK IN ZEBRAFISH DEVELOPMENT 187 instead plays a facilitating role in cytokinesis (Madaule following primer pairs: (1) rhoA: forward (50-ATGGCA- et al., 1998). In contrast, Marlow et al. (2002) examin- GCAATTCGCAAGA-30) and reversed (50-TCACAGCA- ed the role of ROK/ROCK in zebrafish embryo by GACAGCATTTG-30). (2) ef1a: forward (50-CAAGGA- knocking down Rho kinase 2 (Rok2) activities using AGTCAGCGCATACA-30) and reversed (50-TGATGAC- dominant-negative Rok2 (dnRok2). They showed that CTGAGCGTTGAAG-30). The total RNAs were pre- the convergent extension impairment results in a pared by using Trizol reagent (Invitrogen Corporation, shorten-embryonic axis in Rok2-knock down embryos Carlsbad, CA) from different stages of embryos. For and they also claimed that abnormal cell division and synthesizing single-stranded cDNAs, 3 mg of total RNA, lethality occurred at a higher dosage of dnRok2. One can oligo dT primers with M-MLV reverse transcriptase also use morpholino oligonucleotides to suppress the (Promega Corporation, Madison, WI) were applied in a translation of Rok2 and study its involvement during total reaction volume of 25 ml. RT-PCR was performed early cleavages, however, it could be difficult due to the with respective primers for 30 cycles at a thermal cycler abundance of maternal proteins. Therefore, we have (PTC-200, MJ Research) according to the following pro- taken another approach to study the effects of Rho and tocol: denatured at 948C for 30 sec, annealed at 558C for ROCK on the early cleavage stages by using pharmaco- 45 sec, and elongated at 728C for 1 min. logical inhibitors. In zebrafish, following the rapid cleavage period, the Preparation of Embryo Extract, blastula cells separate into underlying deep cells, which Immunoprecipitation, and Western Blotting migrate to the dorsal side to form the embryo, and into Zebrafish embryos at designated stages were collected the overlying the epithelial sheet that spreads as a unit, and the extraction procedures were adapted from the during the process called epiboly, to enclose the deep starfish oocyte extraction protocols as described by Lee cells and the yolk cell (Kimmel et al., 1995). Cell con- et al. (2000). An anti-RhoA polyclonal antibody (sc-179, tractility, microtubule and actin polymerization are all Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was important for these cellular movements (Ridley, 2001). used for immunoprecipitating (1:20 dilution) Rho from Rho and Rac act via ROK/ROCK to phosphorylate zebrafish extract as described by Stapleton et al. (1998). myosin light chain for the formation of actin cytoskeletal After immunoprecipitation, the Rho-coated protein A structures including the formation of stress fibers and beads were settled down by centrifugation. The super- lamellipodia (Kaibuchi et al., 1999; Amano et al., 2000). natants were collected for the Western analysis of actin Through mDia, a mammalian formin homology protein, using an anti-actin polyclonal antibody (Sigma A2066, Rho has been demonstrated to regulate microtubule 1:200 dilution) as a control. The resulting beads were and F-actin polymerization (Ishizaki et al., 2001). The washed several times and resuspended in an equal noncanonical Wnt signaling pathways have been sug- amount of 2 SDS sample buffer. Protein samples were gested to control morphogenesis by regulating polariz- resolved in 15% SDS–PAGE gels, transferred onto ed cellular movement during convergent extension in PVDF membranes (Amersham Biosciences, Piscat- Xenopus via a novel formin homology protein Daam1 away, NJ) in 15 mM sodium borate at 100 mA for 9 hr. (Habas et al., 2001). We examine here the effects of Rho After transfer, Western analysis was done by following signaling on the cellular migration during gastrulation general protocols using the same anti-RhoA antibody in zebrafish. (1:400 dilution) and subjected to ECL Western detection Using pharmacological inhibitors, we show here that (Amersham Biosciences). Rho mediates cytokinesis via regulating actin and b- catenin assembly at the cleavage furrows by a ROK/ Microinjection Procedures ROCK-dependent pathway, which may work through Thin-wall (1.0 0.75 mm, 400) glass capillaries with myosin II. We also demonstrate that Rho regulates filaments (A-M systems, Inc. Carlsborg, WA) were pul- cellular movement during epiboly and gastrulation led using a horizontal puller (P-97, Sutter Instrument, through ROK/ROCK. Navato, CA). Embryos at desired stages as judged according to Kimmel et al. (1995) were immobilized at MATERIALS AND METHODS an injection trough on a 100 mm 2% agar plate. C3- Fish Husbandry and Embryo Collection exoenzyme (BIOMOL, Plymouth Meeting, PA), Y-27632 (Calbiochem, La Jolla, CA), and constitutively active Zebrafish, Danio rerio, were raised on a 14-hr day/ RhoA GST fusion protein (Cytoskeleton, Denver, CO) 8 10-hr night cycle at 28.5 C. Eggs were collected at 15– were dissolved in injection buffer (68.5 mM NaCl, 20 min intervals after spawning, washed and incubat- 1.35 mM KCl, 5 mM Na2HPO4,and1mMKH2PO4 and ed in Ringer’s solution (116 mM NaCl, 2.9 mM KCl, 0.25% (w/v) phenol red) alone or in different combina- 8 1.8 mM CaCl2, and 5 mM HEPES) at 28.5 C until use. tions as indicated. An injection pipette was forced into All chemicals were from Sigma (St. Louis, MO) unless the chorion and the yolk cell to reach the junction otherwise stated. between yolk cell and blastodisc where the solution was ejected by using a pressure injector (IM-300, Narishige, RT-PCR Analysis Japan). We estimated the injection volume by the clear- The expression of mRNA in zebrafish embryos at ance of cytoplasm (0.5% of 1-cell yolk volume). After in- selected stages was determined by RT-PCR using the jection, embryos were recovered from injection troughs 188 S.-L. LAI ET AL. and cultured in Ringer’s solution at 28.58C until Statistical Analysis examined. Experimental values are expressed as mean standard error of mean and analyzed in Excel using Actin and b-catenin Staining on pair-sample t-test. Whole Mount Embryos RESULTS To label F-actin, zebrafish embryos at designated stages were fixed in 3% paraformaldehyde in PBS The regulation of cytokinesis and cellular migration (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and by the small GTPase, Rho has been extensively studied 2mMKH2PO4) overnight at room temperature, washed in a variety of cellular systems including embryonic in PBS and manually dechorionated. Twenty embryos cells. However, little information is available for Rho were incubated with rhodamine phalloidin (R-415, expression and its functions during early embryonic 6.6 nM/ml, Molecular Probes, Inc., Eugene, OR) at room development in zebrafish. To study Rho regulation in temperature for 4–6 hr and then washed intensively zebrafish, we first located two zebrafish rhoA (zrhoA) with 0.5% Triton X-100 (PBT). To label b-catenin, cDNA in GeneBank, which are 582 bp (NM_212749) zebrafish embryos at designated stages were fixed in and 1777 bp (BC075938), respectively. Comparing the 4% paraformaldehyde in PBS overnight at 48C , washed coding regions of those two rhoA cDNAs, we found a in PBS and manually dechorionated. Twenty embryos mismatched nucleotide at 229 from C to T, which results were incubated with a polyclonal anti-b-catenin anti- in a change of translated amino acid residue from Ala body (Sigma C2206, diluted at 1:500) with continuous 110 to Val 110. To validate the zrhoA sequence, we rotation at 48C overnight and washed intensively with isolated a zrhoA cDNA containing the coding region by 0.5% Triton X-100 (PBT). b-catenin-labeled embryos RT-PCR. It was confirmed that the amino acid 110 is Val were incubated in a 1:250 dilution of a FITC-conjugated instead of Ala, which is also conserved in other animal goat anti-rabbit antibody (#111–095-144, Jackson Im- species as well. Zebrafish rhoA is 95.9% identical (185 of munoResearch Laboratories, West Grove, PA) at 48C 193 amino acids) to its human homologue, which reveals overnight. Both actin and b-catenin labeled embryos that rhoA is also highly conserved in teleost (Fig. 1). were transferred to 30% glycerol first and then 50% Expression of RhoA During Early glycerol for confocal microscopic observations at the Embryonic Development Center of Two-Photon Laser Confocal Microscope, National Taiwan University. 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 Removing Embryos From Their Chorions zRhoA (582 bp) expressed in all stages of embryos ex- for Blebbistatin Treatments amined (Fig. 2A). Ef1a was used as an RT-PCR control. One-cell stage eggs were treated with 0.5 mg/ml The relative expression level of zRhoA was much lower protease (Sigma 6917) in embryo medium (Westerfield, from 1-cell to 256-cell compared to later stages embryos. 8 2000) at 28.5 C for 1–2 min. Protease was removed It implied that RhoA exists as maternal protein at early immediately by several changes of embryo medium. stages. Therefore, we analyzed the changes of RhoA Chorions were removed by gentle swirling of embryos. protein expression by Western analysis using a poly- Some embryos were damaged upon protease treatment. clonal antibody against human RhoA (hRhoA), which Therefore, only morphologically healthy and cleaved can recognize RhoA in sea urchin embryos (Manzo et al., embryos were selected for experiments. 2003). Direct detection of RhoA in the zebrafish embryo lysate failed, which might be due to the amount of RhoA In Situ Hybridization on Whole was below detection limit (data not shown). Therefore, 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 Fig. 1. Sequence alignment of zebrafish and human RhoA gene. The stereomicroscope. All photographs were taken by a amino acid sequences of zebrafish RhoA (AAH75938.1) is aligned to that Nikon digital camera and analyzed using Adobe Photo- of human RhoA (AAC33178.1). Among 193 amino acids, only 8 amino acids are different as indicated, which represents a 95.9% identity. Val shop 6.0 except for the confocal images, which were 110 (enclosed by an empty rectangle) is the site of mismatch between photographed and edited utilizing the Leica Confocal RhoA cDNAs NM_212749 and BC075938. Q63 (underlined) was Software. mutated to L to make constitutively active RhoA used in this study. Rho AND ROCK IN ZEBRAFISH DEVELOPMENT 189

Fig. 3. C3-exoenzyme causes gradual death of embryos. No signi- ﬁcant 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.

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 Fig. 2. RhoA is present as maternal transcripts and proteins in zebrafish embryos. Expression of RhoA mRNAs or proteins in early 112 embryos treated with 1.2 nM C3 died in 24 hpf. zebrafish embryos from 1-cell to 6 somite stages was determined by RT- Therefore, we only analyzed the effects of C3 on embryos PCR (A) or Western blotting (B), respectively. Ef1a and actin was during the early cleavages and epiboly stages (up to served as RT-PCR and Western blotting internal controls, respectively. 6 hpf) unless otherwise stated. A recombinant hRhoA (100 ng per well) was also used as a positive Figure 4 shows the effects of C3 treatment on 8-cell control for RhoA detection. and sphere stage embryos. Sham-injected embryos showed distinct cleavage furrow boundaries at the we concentrated RhoA by immunoprecipitation with 8-cell (Fig. 4A) and sphere (Fig. 4B) stages. In contrast, subsequent Western analysis for detection. To have a embryos injected with 120 pM C3 at the 1-cell stage had Western blotting loading control, a polyclonal anti-actin incomplete or no cleavage furrows at the 8-cell (Fig. 4C) antibody was used to detect actin (42 kDa) in the and sphere stages (Fig. 4D). Therefore, C3 inhibited supernatant of zebrafish lysate after RhoA immuno- embryonic cleavages as demonstrated in other animal precipitation. RhoA protein (24 kDa) was detected in species. To analyze the dose-dependent response of C3, all stages of embryos examined as well as a recombinant we injected embryos with different amounts of C3 into hRhoA (Fig. 2B). However, it appeared that at least two 2–8 cell stage embryos. Injecting embryos after the bands around the region were detected, which might 1-cell stage allowed us to remove easily unfertilized be RhoC or RhoB, since the anti-RhoA antibody used eggs from our statistics. We also observed that phenol can also detect RhoC, but to a less extent to RhoB. For red, a visual indicator contained in the injection buffer, simplicity, we will refer them as Rho proteins here. Rho quickly diffused into all blastomeres from the site of proteins were present at high amounts in 1-cell and injection to ensure that all blastomeres received the gradually decreased until 256-cell, but significantly in- same treatments. We then examined the morphological creased after 30% epiboly stages embryos. The changes changes in particular the formation of cleavage fur- in Rho proteins level coincided very nicely with the rows at 4 hr post fertilization. As shown in Figure 4E, changes in rhoA mRNA expression. The appearance of 25.9 13.7% (N ¼ 4) of 111 embryos injected to an in- Rho proteins at early stages suggested they are maternal tracellular concentration of 0.4 nM C3 showed defects in deposited. More importantly, the presence of both cleavage. As intracellular C3 concentration increased to maternal RhoA mRNA and protein and the later acti- 1.2 and 3.6 nM, the percentage of embryonic cleavage vation of zygotic expression imply that Rho is function- defects increased significantly (P < 0.05) to 52.4 5.5% ally important in regulating early development in (N ¼ 4) of 115 embryos and 70.7 16.9% (N ¼ 4) of 97 zebrafish. embryos, respectively. Thus, C3 blocked early embryonic cleavage in a dose-dependent manner. Inhibiting Rho Activity Blocks Cytokinesis To study Rho functions during early embryonic devel- Inhibition of C3 can be Partially Rescued opment, we microinjected C3, a specific Rho antagonist by Constitutively Active RhoA (Aktories and Hall, 1989) into developing zebrafish To clarify the specificity of C3 inhibition on embryonic embryos. Embryos injected with increasing amounts of cleavage, we tried to rescue the C3-treated embryos 190 S.-L. LAI ET AL.

TABLE 1. Rescue of C3-Induced Embryonic Cleavage Defects by Constitutively Active RhoA

Treatment % cleavage defects (n)

Sham-injected 0.0 0.0 (161)a 4.5 mg/ml L63RhoA 1.5 1.3 (157)a 400 pM C3 47.7 14.1 (190)b C3 þ L63RhoA 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.

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 aggre- gated into patches in the presence of 1.2 nM C3 (Fig. 5C). Fig. 4. C3-exoenzyme blocks embryonic cleavage in a dose-depen- b dant manner. Fertilized eggs were injected with buffer (A, B) or 120 pM The -catenin staining was also severely disrupted in (C, D) C3-exoenzyme then incubated in Ringer’s solution until the C3-treated embryos as shown in Figure 5D. designated stages. We examined embryos at 8-cell (A, C) and sphere (B, D) stages. For the control embryos (A, B), distinct cleavage furrows Deterioration of Embryo by exist between blastomeres, while incomplete or no cleavage furrows (C, D) are shown in the C3-injected embryos (E). To test dosage Inhibiting Myosin II response of C3, embryos at 2-8 cell stage were injected with indicated Myosin II is a contractile ring protein that is known for amounts of C3-exoenzyme and examined at 4 and 6 hpf, respectively. its role in cytoplasmic constriction during cytokinesis Each data point represents the mean of percentage cleavage defects standard error of mean for four independent experiments. (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 by co-injection of the constitutively active RhoA of the cleavage furrow without disrupting mitosis or (L63RhoA), a constitutively active GST fusion proteins contractile ring assembly (Straight et al., 2003). The (see Fig. 1 for the mutation site). L63RhoA used is of zebrafish chorion is known to be a tough barrier for human origin and commercially available (Cytoskele- many chemicals. Therefore, we used protease to dechor- ton, Inc.). Since there is only 4% difference in amino acid inate 1-cell stage embryos as described in the ‘‘Materials sequences between human and zebrafish Rho A (Fig. 1) and Methods’’ and then incubated embryos with bleb- and the sequences around the site of mutation are bistatin at the 2-cell stage. Although 50 mM blebbistatin the same, it would be relatively safe to use human and 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)orb-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), required to maintain the adhesion of the cells with each 80% embryos kept dividing up to 3 hpf (1,000-cell stage) other or with the yolk cells. without interfering karyokinesis (data not shown), but Taken together, these observations suggest that cyto- started to deteriorate afterward showing loose blasto- kinesis can be speciﬁcally blocked by inhibiting RhoA meres. Disruption of the cells by blebbistatin was dose- via the disruptions of cleavage furrow protein assembly dependent as shown in Figure 6C. Thus, myosin II is and actomyosin ring constriction in zebraﬁsh embryos. 192 S.-L. LAI ET AL.

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)50mM 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 Fig. 7. C3-exoenzyme dose-dependently inhibits cellular migration number of the C3-treated embryos survived to reach during epiboly. Fertilized eggs were injected with buffer or C3 and epiboly at lower dosages of C3. However, cellular move- photographed at 50% epiboly stage on the animal pole view (A–D)or ment during epiboly could be impaired by C3 as low as the side views (E, F). Note that the 120 pM C3 injected embryo formed 120 pM. Figure 7A shows a sham-injected embryo at 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 50% epiboly and the beginning of gastrulation, noted by blastoderm were revealed by the labeling of a regular no tail mRNA ring the ring structure (arrows) that results from the in- (arrow, C), but the ring was distorted in the presence of C3. The volution of the mesoderm cells at the equator of the yolk involuting dorsal lip of blastopore (arrow) was shown by the labeling of cell. During epiboly the cells at the involuting epithelial 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 layer appeared to migrate to the equator of the yolk in 250 pM and examined accordingly (G). Each data point represents the the C3-injected embryos (Fig. 7B). However, the ring mean of percentage epiboly defects standard error of mean for three structure (arrows) typical of the involution of the meso- independent experiments. derm at 50% epiboly formed not at the equator, but near to the animal pole of the embryo (Fig. 7B). These migration (Hall, 1998), but its role in the spreading of embryos maintained the movements associated with the underlying deep cells is not understood. So we the involution of the mesoderm, but the presumptive further examined the involution of gastulating embryos mesodermal cells had not migrated over the yolk to the by monitoring the expressions of no tail, an immediate equator. Rho is required for adhesion during cell early gene known to turn on in the future germ ring, and Rho AND ROCK IN ZEBRAFISH DEVELOPMENT 193 goosecoid, another immediate early gene known to be volvement of Rho-associated kinase (ROK/ROCK) by exclusively expressed in the involuting dorsal lip of injecting a ROCK specific inhibitor, Y-27632 (Uehata blastopore (Schulte-Merker et al., 1994). Using whole- et al., 1997; Kosako et al., 2000). Y-27632 at 45.5 mM mount in situ hybridization, we observed that no tail disrupted the assembly of actin (Fig. 5E) and b-catenin mRNA was clearly expressed in the germ ring (arrow) of (Fig. 5F) as the C3 treatments (Fig. 5C,D), which the sham-injected embryo (Fig. 7C). However, irregular resulted in defects in cytokinesis (data not shown). As no tail rings were seen in the C3-treated embryo, which shown in Figure 9A, a sham-injected embryo clearly indicated an aberrant and unsynchronized migration of started epiboly formation and reached 30% of yolk front runner cells in the future germ ring (arrow, sphere, but the blastoderm margin of the embryo treated Fig. 7D). The inhibition of involution by C3 was obvious with 45.5 mM Y-27632 failed to spread toward vegetal by observing the presence but absence of goosecoid pole at the 30% epiboly stage (Fig. 9B). The inhibition of mRNA staining at the dorsal lip of blastopore (arrows) in Y-27632 on front runner cells migration was demon- the sham- (Fig. 7E) and C3- (Fig. 7F) injected embryos, strated by no tail mRNA staining where the no tail ring respectively. C3 also caused defects in epiboly forma- (arrow) was irregular and significant lagged (Fig. 9D) tion and gastrulation in a dose-dependent manner. As compared to the control embryos (Fig. 9C) at near 50% shown in Figure 7G, at an intracellular concentration epiboly stage. As in the C3-treated embryos, Y-27632 of 120 pM, C3 caused 44.3 8.0% (N ¼ 3) defects in also blocked involution of the dorsal lip of blastopore as 120 embryos. As the intracellular C3 concentration evident by the occurrence of goosecoid expression in the increased to 180 and 240 pM, the epiboly defects were control (arrow, Fig. 9E), but not in the Y-27632-treated increased to 68.4 7.1% (N ¼ 3, 198 embryos) and (arrow, Fig. 9F) embryos. To test the dosage response of 88.8 4.8% (N ¼ 3, 168 embryos), respectively. Y-27632, we also injected 2-8 cell stage embryos with Some of these embryos with delayed epiboly continued different amounts of Y-27632 and found that Y-27632 to develop. One such embryo, which was injected to inhibited cleavage and epiboly formation in a dose- 1.2 nM C3 at the 2- to 8-cell stage, is shown in Figure 8A. dependent manner as the C3 treatments (Fig. 9G). At an Compared to a sham-injected embryo incubated simi- intracellular concentration of 4.5 mM, Y-27632 caused a larly for 52–54 hpf (Fig. 8B), the C3-injected embryo had 15.5 6.9% (N ¼ 3) cleavage defects in 195 embryos. As a shorten-embryonic axis, but almost normal head injected concentration increased, the Y-27632-induced structures (Fig. 8A). Thus, migration of the tail meso- cleavage defects increased to 46.0 23.8% and derm was aberrant in the C3-treated embryos. 74.7 9.5% (N ¼ 3) at 11.4 mM (207 embryos) and 22.7 mM (179 embryos), respectively. In 126 embryos treated Inhibiting Rho-Associated Kinase Activity with 45.5 mM Y-27632, almost all embryos showed Blocks Cytokinesis and Epiboly cleavage defects (97.4 4.6%, N ¼ 3). Most of those To determine downstream targets of Rho during cyto- defective embryos died the next morning. However, a kinesis and epiboly, we examined the possible in- 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; Jantsch- Plunger 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, Y- 27632. (3) C3 and Y-27632 also block epiboly formation Fig. 8. C3 induces the shortening of embryonic axis. Embryos were and gastrulation in zebrafish embryos. These results 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 clearly demonstrated that Rho mediates cytokinesis, shorten-embryonic axis and a bent tail. In contrast, the sham-injected epiboly and gastrulation via its downstream effector embryo (B) appears normal. 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 pre- sumably 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 exogen- ous GFP fusion proteins would not occur until epiboly stage (our personal observations), which is too late to study early events like cytokinesis in zebraﬁsh. Alterna- tively, 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-ribosy- late a 25 kDa RhoA through out early development in sea urchin eggs and embryos (Manzo et al., 2003). ADP- ribosylation 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.

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 Fig. 9. Y-27632 inhibits the embryonic cleavage and gastrulation in zebrafish cytokinesis is specifically mediated by Rho. 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 Rho-Mediated Cytokinesis Is Regulated showed aberrant blastoderm front runner cell (arrows) migration (B, D) Through a ROK/ROCK-Dependent Pathway 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. Several known rho effectors that signal cytokinesis To test the dosage responses, embryos were injected with Y-27632 up to are citron kinase, mDia and ROK/ROCK (Glotzer, 2001, 45.5 mM and examined accordingly (G). Each data point represents the 2003). ROK/ROCK accumulates at the cleavage furrow mean of percentage epiboly defects standard error of mean for three and phosphorylates intermediate filaments and fibril- independent experiments. lary 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 195 stream target of ROCK appears to be myosin regulatory sion of Wnt5a and Wnt4 has been shown to disrupt light chain (MRLC), since MRLC phosphorylation at the convergent extension without dramatically affecting cleavage furrow is abolished by ROCK antagonists cell fate in both frogs and fish (Moon et al., 1993; Ungar (Kosako et al., 2000). ROCK has also been shown to in- et al., 1995). RhoA and ROK/ROCK have also been de- activate myosin phosphatase through phosphorylation monstrated to be downstream effectors of the planar cell on MBS that aids in keeping MRLC phosphorylated and polarity signaling (Strutt et al., 1997; Wunnenberg- subsequent activation of myosin II (Amano et al., 1996; Stapleton et al., 1999; Winter et al., 2001; Marlow et al., Kimura et al., 1996; Chihara et al., 1997). In Drosophila, 2002). Our results further strengthen the involvements Drok, a ROCK homologue, works downstream of the of RhoA and ROCK during gastrulation in zebrafish. Frizzled PCP signaling for phosphorylation of MRLC In summary, using specific pharmacological inhibi- and subsequent activation of Myosin II (Winter et al., tors against two important players of the Rho signaling 2001). In contrast, by overexpressing ROCK mutants, pathway, Rho and ROK/ROCK, we have essentially Madaule et al. (1998), failed to observe the multinuc- come to the same conclusion that Rho work via ROK/ leate cells, which leads to the conclusion that ROCK is ROCK to regulate key developmental processes, includ- not essential for cytokinesis. However, at concentra- ing the assembly of cleavage furrow proteins and tions from 10 to 30 mM, which are similar to previous actomysin ring constriction during cytokinesis and studies (Niggli, 1999; Kosako et al., 2000), Y-27632 in- cellular movements leading to epiboly formation and hibited cytokinesis and epiboly in over 50% zebrafish gastrulation in zebrafish. embryos in our hands (Fig. 9). When injected Y-27632 was increased to 45.5 mM, almost all embryos show- ACKNOWLEDGMENTS ed abnormality in development. At 45.5 mM, Y-27632 We thank Dr. Merrill B. Hille for helpful discussion may be too high to have some nonspecific inhibition. In and comments on the manuscript, Dr. C-H Hu for contrast, Y-27632 has been shown to have no effects generous gifts of no tail and goosecoid plasmids, and on p21-activated protein kinase even at 100 mM Dr. Yu-Fen Huang for help in confocal microscopy. (Narumiya et al., 1997). 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