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 2Â 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 Â 0.75 mm, 400) glass capillaries with myosin II. We also demonstrate