Ciona intestinalis as an emerging model organism: its regeneration under controlled conditions and methodology for egg dechorionation

Ciona intestinalis as an emerging model organism: its regeneration under controlled conditions and methodology for egg dechorionation 1 2 first-author †E-mail:patrick-01@sohu.comjhxiang@ms.qdio.ac.cn †E-mail:patrick-01@sohu.comjhxiang@ms.qdio.ac.cn patrick-01@sohu.com 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:patrick-01@sohu.comjhxiang@ms.qdio.ac.cn †E-mail:patrick-01@sohu.comjhxiang@ms.qdio.ac.cn patrick-01@sohu.com 1 3 1 1 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China College of Aqua-Life Science and Technology, Shanghai Fisheries University, Shanghai 200090, China College of Aqua-Life Science and Technology, Shanghai Fisheries University, Shanghai 200090, China Department of Aquatic Biology and Fisheries, University of Kerala, Trivandrum 695034, India Department of Aquatic Biology and Fisheries, University of Kerala, Trivandrum 695034, India The ascidian Ciona intestinalis is a model organism of developmental and evolutionary biology and may provide crucial clues concerning two fundamental matters, namely, how chordates originated from the putative deuterostome ancestor and how advanced chordates originated from the simplest chordates. In this paper, a whole-life-span culture of C . intestinalis was conducted. Fed with the diet combination of dry Spirulina , egg yolk, Dicrateria sp., edible yeast and weaning diet for shrimp, C . intestinalis grew up to average 59 mm and matured after 60 d cultivation. This culture process could be repeated using the artificially cultured mature ascidians as material. When the fertilized eggs were maintained under 10, 15, 20, 25 °C, they hatched within 30 h, 22 h, 16 h and 12 h 50 min respectively experiencing cleavage, blastulation, gastrulation, neurulation, tailbud stage and tadpole stage. The tadpole larvae were characterized as typical but simplified chordates because of their dorsal nerve cord, notochord and primordial brain. After 8~24 h freely swimming, the tadpole larvae settled on the substrates and metamorphosized within 1~2 d into filter feeding sessile juvenile ascidians. In addition, unfertilized eggs were successfully dechorionated in filtered seawater containing 1% Tripsin, 0.25% EDTA at pH of 10.5 within 40 min. After fertilization, the dechorionated eggs developed well and hatched at normal hatching rate. In conclusion, this paper presented feasible methodology for rearing the tadpole larvae of C . intestinalis into sexual maturity under controlled conditions and detailed observations on the embryogenesis of the laboratory cultured ascidians, which will facilitate developmental and genetic research using this model system. Ciona intestinalis Model organism Laboratory culture Embryogenesis INTRODUCTION 1 Ciona intestinalis (subphylum Urochordata, class Ascidiacea, family Ciona) is hermaphrodite, sessile and filter-feeding marine solitary invertebrate. Its tadpole larvae possess a prototypical chordate body plan that includes a dorsal neural tube, an axial notochord flanked by muscle cells, and a ventral endodermal strand (Satoh, 1994 1994 ). As a cosmopolitan species, they are ubiquitous throughout the world and also scatter along the Chinese coast (Zheng, 1995 1995 ). C . intestinalis was used as a popular model for embryo biology and was also one of the animals which were first introduced for cell lineage research (Conklin, 1905 1905 ; Satoh, 1994 1994 ; Xiang, 2003 2003 ); In the 1940s, Morgan ( 1940 1940 ; 1945 1945 ) studied self and cross fertilization of C . intestinalis ; Tung et al.( 1977 1977 ) investigated the interaction between oocyte plasm and nucleus taking ascidians as material. In the last decades, researchers showed a surge of interest in ascidians because of their crucial evolutionary position and representative early developmental mode (Satoh and Jeffery, 1995 1995 ; Simmen et al., 1995 1995 ; Di Gregorio and Levine, 1998 1998 ; Corbo et al., 2001 2001 ; Dehal et al., 2002 2002 ; Nishida, 2002 2002 ). The whole genome of C . intestinalis has now been sequenced, analyzed and annotated (Sordino et al., 2001 2001 ; Dehal et al., 2002 2002 ; Satoh et al., 2003 2003 ); Hundreds of thousands of ESTs (expressed sequence tags) were disclosed; a great number of development involved genes were cloned and hundreds of gene expression patterns were published (Nishikata et al., 2001 2001 ; Satou et al., 2001a 2001a ; 2002 2002 ; Kusakabe et al., 2002 2002 ; Inaba et al., 2002 2002 ; Fujiwara et al., 2002 2002 ; Shida et al., 2003 2003 ; Satoh et al., 2003 2003 ). As a potential model system of developmental and evolutionary biology, C . intestinalis gained so much attention that it was regarded as a “biology’s rising star” (Pennisi, 2002 2002 ). However, it is not always very convenient to obtain C . intestinalis as experimental materials because their sources vary among seasons and regions. Almost all the biological materials of the published articles were collected from the seaside (Corbo et al., 1997 1997 ; Dehal et al., 2002 2002 ; Inaba et al., 2002 2002 ; Tsuda et al., 2003 2003 ; Awazu et al., 2004 2004 ; Kawai et al., 2005 2005 ). On the other hand, there are always requirements on the population in regard to some special experimental designs, for example genetic mutation, inbred strain construction, micromanipulation and so on. With the rapid increasing interest in ascidian research, it is necessary to establish a laboratory culture mode as in the case of other model organisms, such as fruit fly, Caenorhabditis elegans , zebra fish and mouse. Therefore, it is essential to carry out the whole life span laboratory culture of C . intestinalis under controlled conditions (Kano et al., 2001 2001 ; Sordino et al., 2001 2001 ). Up to date, there are no informative references on this. This paper presents methodology for artificial propagation, removal of the egg chorion and laboratory cultivation of C . intestinalis . In addition, some new observations on the development of the laboratory cultured eggs and dechorionated eggs together with some details of the biological characteristics of the juvenile ascidians were also provided in order to better exploit this emerging model organism. MATERIALS AND METHODS 1 Source of experimental animals 2 Healthy adult ascidians, more than 6 cm in height, with tunica semitransparent and integral in appearance and both incurrent siphon and outcurrent siphon naturally spread, were collected from Jiaozhou Bay and maintained in seawater aquariums with constant illumination at 18 °C. They were fed with microalgae and egg yolk till sexual maturity. The eggs were fertilized and hatched in filtered seawater at 20 °C. Several batches of hundreds of hatched tadpole larvae were settled on plastic dishes for cultivation purpose. Laboratory culture method 2 1. Food 3 Dried powder of several microalgae species, namely Spirulina sp., fresh Nitzschia sp., Chlorella sp., Dicrateria sp., and egg yolk, nauplius of brine shrimp, a commercial micro pellet diet (Seafood™, Qingdao, China), a commercial weaning food for shrimp (HighSciTech™, Qingdao, China), soybean milk, egg yolk, edible yeast were selected as food for the juvenile ascidians. Among them, Nitzschia sp., Chlorella sp., Dicrateria sp., and soybean milk were fed directly. The dried powder of Spirulina sp. and edible yeast were rehydrated for 30 min before feeding. The solid diets were triturated to make the particles less than 50 μm. 2. Culture management 3 Different groups of juvenile ascidians were fed using the above diets with adjustments according to the fullness in the intestines. Food efficiency was evaluated and the diet combination was optimized based on the surviving rate and growth rate resulting from different diet. The ascidians were cultured at 15~18 °C with the optimized diet combinations as final food. When the juvenile ascidians grew up to about 1 cm, they were maintained in 200 L aquariums at a density of not more than 500 individuals/m 3 . The diet remnants were removed by plastic siphons and half of the seawater was changed every day. After a 60 d culture, the ascidians reached sexual maturity. 3. Collection and culture of fertilized eggs from the artificially cultivated ascidians 3 Mature artificially cultured ascidians with eggs and sperm visible in the gonoducts were used. The animals were dissected to expose gonoducts. Excess seawater and body fluid were removed with tissue paper. After their oviduct was cut open by sharp scissors, their eggs were collected by a Pasteur pipette and transferred immediately into a petri dish containing filtered seawater. The sperm duct was cut and sperms (from all individuals) were collected using a Pasteur pipette in a 1.5 ml micro centrifuge tube and stored at 4 °C. Only sperm, but not seawater or body fluid were collected as neatly as possible. With all things prepared, the sperms suspension was diluted with into about 1 ml (depending on the original volume of sperm suspension) filtered and sterile seawater. One to two drops of diluted sperm suspension were added into a petri dish containing eggs. The dish was allowed to stand for 10 min to complete fertilization and then the eggs were washed three times to prevent polyspermy. The fertilized eggs were cultured at 10, 15, 20, 25 °C respectively with 3 replicates. The time for different development stages and their different hatching rates at different temperature were recorded. 4. Removal of the follicle cells and test cells 3 Mature eggs are covered with follicle cells in the outer layer, and test cells in the inner layer, between them are the chorions. These two layers of cells should be removed for better observations. Unfertilized eggs were repeatedly pipetted to remove follicle cells. After two times of washing, the eggs were transferred to a 15 ml centrifuge tube containing 10 ml of dechorionation solution of filtered seawater containing 10 ml of 1% Tripsin (Sigma) and 0.25% EDTA (Gibco) at pH of 10.5, and incubated on a shaker at 25 °C. Normally, both the chorion and test cells would be removed within 40 min. Once the eggs were dechorionated, they were collected swiftly into petri dishes, with floor coated with 1% agarose, and washed several times with filtered seawater. After that, the dechorionated eggs were fertilized and cultured at 20 °C. 5. Embryogenesis observation and biological traits of juvenile ascidians 3 The eggs from the artificially cultured ascidians and the dechorionated eggs were fertilized with the methods mentioned above. The embryo development process and juvenile ascidians were observed and recorded using ZEISS Axioplan microscope and Kodak MDS digital camera. 6. Data analysis 3 Data were documented as mean±standard error. T -test was applied to verify the resulting differences. RESULTS AND DISCUSSION 1 Effect of diets on growth performance 2 Our priliminary test, revealed that soybean milk, nauplius and micropellet diet were not fit food for C . intestinalis because of shortcomings such as bad growth performance, trouble in preparation and difficulty for storage. If we fed C . intestinalis with egg yolk, weaning diet of only crustacea and yeast, the ascidians grew slowly at low surviving rate (data not shown). Feeding the C . intestinalis larvae with Spirulina powder, fresh Nitzschia sp., Chlorella sp., Dicrateria sp., could meet their need for growth in the early stage (body height less than 1 cm), but resulted great growth diversity and increasing mortality rate in the late culture period. At last, we optimized a diet combination of Dicrateria sp., dry Spirulina powder, egg yolk, weaning diet for shrimp, edible yeast, which yielded best results (Fig. 1 1 ). After 45 d culture with the optimized diet, white sperms appeared in the spermaduct; after 50 d, orange eggs were found in the oviduct. In about 2 months, the ascidians reached average body height of 59 mm, survival rate rose to 85%, and most of them matured (Fig. 1 1 ). *: Sperm emerged; **: Oocyte emerged Developmental timetable and hatching rate at different temperatures 2 Water temperature has obvious effect on the developmental process (Fig. 2 2 ). At 10, 15, 20, 25 °C, it required 3 h 10 min, 1 h 20 min, 50 min, 40 min for fertilized eggs to become two cells, and 30 h, 22 h, 16 h, 12 h 50 min to become tadpole larvae, respectively. At 10 °C, fertilized eggs cleaved one time at approximately an hour, while at 20 °C, it required 20~30 min. At 10 °C, eggs could be normally fertilized but the hatching rate was obvious lower than that at 15, 20 and 25 °C ( P < 0.01) (Fig. 3 3 ). In the aquariums, matured ascidians could spawn at 12 °C, but the hatching rate was high (average 93%) only at water temperature of 15~25 °C (Fig. 3 3 ). ** P < 0.01 Embryogenesis and characteristics of tadpole larvae 2 The eggs of C . intestinalis were floating. The first two blastomeres, destined to form the two sides of the tadpole larvae, were equal in size. The eggs ooplasm rearranged within 10 min post fertilization. The fertilized eggs developed underwent egg cleavage, blastula, gastrula, neurula stage, tailbud stage and then hatched as tadpole larvae (Figs. 4a~4i 4a~4i ). The 0.7 mm long tadpole larvae were composed of head and tail (Fig. 4j 4j ). The head had a cerebral ganglion with black ocellus and black otolith; the tail contained notochord, muscles and a hollow dorsal neural tube connecting with the anterior cerebral ganglion. Tunica was formed in the tadpole larva stage and enwrapped the larva. The tadpole larvae were phototactic and could swim for a short distance by intermittent swinging of the tail. During this period, the larva did not feed. The tadpole larvae then attached to substrates by the attachment villi. In the following days, notochord and muscles of the tail disappeared, neural tube and sensory organs transformed into cerebral ganglion; With the appearance of the incurrent and outcurrent siphons, the freely swimming tadpole turned into filter feeding and sessile juveniles. Scale bar=100 μm After dechorionation, the fertilized eggs could develop into tadpole larvae (Fig. 5 5 ). The dechorionated eggs were sinkable and developed at the same speed at that of the normal fertilized eggs (Mita-Miyazawa et al., 1985 1985 ), while the hatching rate decreased and a few embryos developed abnormally, especially after neurulation. Scale bar=100 μm Observations on the juvenile ascidians 2 Microscopic observations showed that after metamorphosis, some adult organs such as incurrent siphon, outcurrent siphon, branchial sac, intestine, heart appeared, while other advanced characteristics of the tadpole such as notochord and dorsal neural tube disappeared, with only neural complex left positioned between the incurrent siphon and outcurrent siphon. Chiba et al.( 2004 2004 ) presented detailed observations of development of the tissues and organs in Ciona intestinali s larva and juveniles. The blood cells of the juvenile ascidians are distinguishable flowing in the body and are countable in number. The circulation mode is unique because the circulation direction is reversible with a specific frequency. Under our laboratory culture conditions, the ascidians showed evident growth diversity (Fig. 1 1 ); A few of the cultivated asicidians grew slowly, with the tunica broken, viscera became whitish and the incurrent siphon and outcurrent siphon not extending freely, and finally died. Prospect of Ciona intestinalis as a future model organism 2 Ascidians are some of the prevailing marine sessile organisms, among which are many ascidians with bioactivities (He and Cheng, 2002 2002 ). As far as evolutionary theory and developmental biology and genetic domain are concerned, C . intestinalis is considered as an attractive simple experimental system. For evolutionists, phylogenetics inferred by molecular evidences and cladistic analysis suggested (Wada and Satoh, 1994 1994 ) that among the three subphyla of chordata, subphylum urochordata (including ascidians) are the basal group; the other two subphyla, namely subphylum cephalochordate (the amphioxus are the most famous representative species) and vertebrata are sister groups. Furthermore, it is now widely believed that all the chordates originate from the same deuterostome ancestor, thus, ascidians facilitate understanding of the origins and evolutionary diversification of the chordates (Satoh and Jeffery, 1995 1995 ; Satoh et al., 2003 2003 ). For developmental biologists and geneticists, the fertilized egg develops rapidly into a tadpole larva, with a small number of organs including epidermis, central nervous system with two sensory organs, endoderm and mesenchyme in the trunk, and notochord and muscle in the tail. This configuration of the ascidian tadpole is thought to represent the most simplified and primitive chordate body plan. Disclosure of the molecular mechanisms underlying ascidian embryogenesis is essential for understanding the ancestral developmental program of human beings and other chordates (Corbo et al., 2001 2001 ; Satoh et al., 2003 2003 ). There are several potential advantages in taking ascidians as material. (1) Voluminous data including hundreds of thousands of ESTs and the whole genome sequences were published, which are free for use in academic research (Dehal et al., 2002 2002 ; Satoh et al., 2003 2003 ); (2) The body of C . intestinalis is transparent regardless of its size and the embryos develop quickly; the whole life span is only 2~3 months; therefore it is convenient to conduct mutation and genetic screening (Nakatani et al., 1999 1999 ; Moody et al., 1999 1999 ; Sordino et al., 2001 2001 ); (3) Every blastomere of the early developmental stages is distinguishable in appearance and is named based on the nomenclature of Conklin ( 1905 1905 ), and the fate of the blastomeres before gastrulation was documented (Conklin, 1905 1905 ; Nishida, 1987 1987 ); also each lineage leading to the formation of epidermis, central nervous system, mesenchyme, tail muscle and notochord was well characterized (Nishida, 1987 1987 ); (4) Exogenous DNA can be introduced into fertilized eggs using simple electroporation methods (Corbo et al., 1997 1997 ; Di Gregorio and Levine, 1998 1998 ; Bertrand et al., 2003 2003 ) enabling transforming enough synchronously developing embryos. By these means, the upstream regulatory sequences can be quickly identified. Besides, whole-mount in-situ hybridization techniques were successfully applied to embryos, even to juvenile ascidians, thus temporal and spatial expression patterns of specific genes could be obtained (Satoh et al., 2003 2003 ; Tsuda et al., 2003 2003 ; Tokuoka et al., 2004 2004 ); (5) The only 2500~2600 cells of the tadpole larvae made up of epidermis, nervous system (including cerebral neural complex and spinal cord with two sense organs), muscle, mesenchyme, notochord and endodermal strand, which represent the main traits of the chordates (Satoh and Jeffery, 1995 1995 ; Corbo et al., 2001 2001 ; Satoh et al., 2003 2003 ); (6) A newly emerging technique-gene knockdown, which includes morphlinos (Satou et al., 2001b 2001b ) to identify gene functions could be applied in ascidian embryos because they are large enough and easy to be handled (Nishida, 2002 2002 ; Tsuda et al., 2003 2003 ; Inada et al., 2003 2003 ; Tokuoka et al., 2004 2004 ); (7) C . intestinalis has a small genome (1/17 of that of human) of about 1.55×10 8 bp/haploid containing approximately 15800 genes, which share 62% similar genes with human and fruit fly. In addition, 17% of the genes are exclusively similar to human (Dehal et al., 2002 2002 ). The compact genome is easy for gene regulatory network assays (Awazu et al., 2004 2004 ; Yagi et al., 2004 2004 ). In conclusion, this paper presents methodology for artificial propagation, removal of egg chorion, and laboratory cultivation of C . intestinalis . Moreover, some new observations on the development of normal and dechorionated eggs together with some details of biological characteristics of the juvenile ascidians were also provided in order to better understand and exploit this emerging model organism. In addition, the application of various molecular techniques to the ascidian system highlights its advantages as a future experimental system for exploring the molecular mechanisms underlying the expression and function of developmental genes as well as genetic circuitry responsible for the establishment of the basic chordate body plan. References S Awazu , A Sasaki , T Matsuoka , N Satoh , Y Sasakura . An enhancer trap in the ascidian Ciona intestinalis identifies enhancers of its Musashi orthologous gene . Dev Biol , 2004 . 275 ( 2 ): 459 - 472 . 15501231 DOI: 10.1016/j.ydbio.2004.08.029 http://doi.org/10.1016/j.ydbio.2004.08.029 . An enhancer trap in the ascidian Ciona intestinalis identifies enhancers of its Musashi orthologous gene V Bertrand , C Hudson , D Caillol , C Popocici , P Lemaire . Neural tissue in ascidian embryosis induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors . Cell , 2003 . 115 ( 5 ): 615 - 627 . 14651852 DOI: 10.1016/S0092-8674(03)00928-0 http://doi.org/10.1016/S0092-8674(03)00928-0 . Neural tissue in ascidian embryosis induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors S Chiba , A Sasaki , A Nakayama , K Takamura , N Satoh . Development of Ciona intestinalis juveniles (through 2nd ascidian stage) . Zoolog Sci , 2004 . 21 ( 3 ): 285 - 298 . 15056923 DOI: 10.2108/zsj.21.285 http://doi.org/10.2108/zsj.21.285 . Development of Ciona intestinalis juveniles (through 2nd ascidian stage) EG Conklin . The organization and cell-lineage of the ascidian egg . J Acad Nat Sci Philadelphia , 1905 . 2 ( 13 ): 1 - 19 . . The organization and cell-lineage of the ascidian egg JC Corbo , M Levine , RW Zeller . Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis . Development , 1997 . 124 589 - 602 . 9043074 . Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis JC Corbo , A Di Gregorio , M Levine . The ascidian as a model organism in developmental and evolutionary biology . Cell , 2001 . 106 ( 5 ): 535 - 538 . 11551501 DOI: 10.1016/S0092-8674(01)00481-0 http://doi.org/10.1016/S0092-8674(01)00481-0 . The ascidian as a model organism in developmental and evolutionary biology P Dehal , Y Satou , RK Campbell , J Chapman , B Degnan , A de Tomaso , B Davidson , A Di Gregorio , M Gelpke , DM Goodstein , 等 . The draft genome of Ciona intestinalis : insights into chordate and vertebrate origins . Science , 2002 . 298 ( 5601 ): 2157 - 2167 . 12481130 DOI: 10.1126/science.1080049 http://doi.org/10.1126/science.1080049 . The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins A Di Gregorio , M Levine . Ascidian embryogenesis and the origins of the chordate body plan . Curr Opin Genet Dev , 1998 . 8 ( 4 ): 457 - 463 . 9729723 DOI: 10.1016/S0959-437X(98)80118-4 http://doi.org/10.1016/S0959-437X(98)80118-4 . Ascidian embryogenesis and the origins of the chordate body plan S Fujiwara , Y Maeda , T Shin-I , Y Kohara , N Takatori , Y Satou , N Satoh . Gene expression profiles in Ciona intestinalis cleavage-stage embryos . Mech Develop , 2002 . 112 ( 1-2 ): 115 - 127 . DOI: 10.1016/S0925-4773(01)00651-7 http://doi.org/10.1016/S0925-4773(01)00651-7 . Gene expression profiles in Ciona intestinalis cleavage-stage embryos SS He , YX Cheng . The drug value and progress in study of ascidian . J Shanghai Fish Univ , 2002 . 11 ( 2 ): 167 - 170 . (in Chinese) . The drug value and progress in study of ascidian K Inaba , P Padma , Y Satouh , T Shin-I , Y Kohara , N Satoh , Y Satou . EST analysis of gene expression in testis of the ascidian Ciona intestinalis . Mol Reprod Dev , 2002 . 62 ( 4 ): 431 - 445 . 12112576 DOI: 10.1002/mrd.10131 http://doi.org/10.1002/mrd.10131 . EST analysis of gene expression in testis of the ascidian Ciona intestinalis K Inada , T Horie , T Kusakabe , M Tsuda . Targeted knockdown of an opsin gene inhibits the swimming behaviour photoresponse of ascidian larvae . Neurosci Lett , 2003 . 347 ( 3 ): 167 - 170 . 12875912 DOI: 10.1016/S0304-3940(03)00689-X http://doi.org/10.1016/S0304-3940(03)00689-X . Targeted knockdown of an opsin gene inhibits the swimming behaviour photoresponse of ascidian larvae S Kano , S Chiba , N Satoh . Genetic relatedness and variability in inbred and wild populations of the solitary ascidian Ciona intestinalis revealed by arbitrarily primed polymerase chain reaction . Mar Biotechnol , 2001 . 3 ( 1 ): 58 - 67 . 14961391 DOI: 10.1007/s101260000048 http://doi.org/10.1007/s101260000048 . Genetic relatedness and variability in inbred and wild populations of the solitary ascidian Ciona intestinalis revealed by arbitrarily primed polymerase chain reaction N Kawai , H Takahashi , H Nishida , H Yokosawa . Regulation of NF-κB/Rel by IκB is essential for ascidian notochord formation . Dev Biol , 2005 . 277 ( 1 ): 80 - 91 . 15572141 DOI: 10.1016/j.ydbio.2004.09.007 http://doi.org/10.1016/j.ydbio.2004.09.007 . Regulation of NF-κB/Rel by IκB is essential for ascidian notochord formation T Kusakabe , R Yoshida , I Isao Kawakami , R Kusakabe , Y Mochizuki , L Lixy Yamada , T Shin-I , Y Kohara , N Satoh , M Tsuda , 等 . Gene expression profiles in tadpole larvae of Ciona intestinalis . Dev Biol , 2002 . 242 ( 2 ): 188 - 203 . 11820815 DOI: 10.1006/dbio.2002.0538 http://doi.org/10.1006/dbio.2002.0538 . Gene expression profiles in tadpole larvae of Ciona intestinalis I Mita-Miyazawa , S Ikegami , N Satoh . Histospecific acetylcholinesterase development in the presumptive muscle cells isolated from 16-cell-stage ascidian embryos with respect to the number of DNA replications . J Embryol Exp Morphol , 1985 . 87 1 - 12 . 3928795 . Histospecific acetylcholinesterase development in the presumptive muscle cells isolated from 16-cell-stage ascidian embryos with respect to the number of DNA replications R Moody , SW Davis , F Cubas , WC Smith . Isolation of developmental mutants of the ascidian Ciona intestinalis . Molecular and General Genetics MGG , 1999 . 262 ( 1 ): 199 - 206 . DOI: 10.1007/s004380051075 http://doi.org/10.1007/s004380051075 . Isolation of developmental mutants of the ascidian Ciona intestinalis TH Morgan . An interim report on cross and self fertilization in Ciona . J Exp Zool , 1940 . 85 ( 1 ): 1 - 32 . DOI: 10.1002/jez.1400850102 http://doi.org/10.1002/jez.1400850102 . An interim report on cross and self fertilization in Ciona TH Morgan . The conditions that lead to normal or abnormal development of Ciona . Biol Bull , 1945 . 88 50 - 62 . . The conditions that lead to normal or abnormal development of Ciona Y Nakatani , R Moody , WC Smith . Mutation affecting tail and notochord development in the ascidian Ciona intestinalis . Development , 1999 . 126 3293 - 3301 . 10393109 . Mutation affecting tail and notochord development in the ascidian Ciona intestinalis H Nishida . Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage . Dev Biol , 1987 . 121 ( 2 ): 526 - 541 . 3582738 DOI: 10.1016/0012-1606(87)90188-6 http://doi.org/10.1016/0012-1606(87)90188-6 . Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage H Nishida . Patterning the marginal zone of early ascidian embryos: localized maternal mRNA and inductive interactions . Bio Essays , 2002 . 24 613 - 624 . . Patterning the marginal zone of early ascidian embryos: localized maternal mRNA and inductive interactions T Nishikata , L Yamada , Y Mochizuki , Y Satou , T Shin-I , Y Kohara , N Satoh . Profiles of maternally expressed genes in fertilized eggs of Ciona intestinalis . Dev Biol , 2001 . 238 ( 2 ): 315 - 331 . 11784013 DOI: 10.1006/dbio.2001.0370 http://doi.org/10.1006/dbio.2001.0370 . Profiles of maternally expressed genes in fertilized eggs of Ciona intestinalis E Pennisi . Comparative biology joins the molecular age . Science , 2002 . 296 ( 5574 ): 1792 - 1795 . 12052937 DOI: 10.1126/science.296.5574.1792 http://doi.org/10.1126/science.296.5574.1792 . Comparative biology joins the molecular age N Satoh . Developmental Biology of Ascidians , 1994 . : Cambridge, UK Cambridge University Press , . Developmental Biology of Ascidians N Satoh , WR Jeffery . Chasing tails in ascidians: developmental insights into the origin and evolution of chordates . Trends in Genetics , 1995 . 11 ( 9 ): 354 - 359 . 7482787 DOI: 10.1016/S0168-9525(00)89106-4 http://doi.org/10.1016/S0168-9525(00)89106-4 . Chasing tails in ascidians: developmental insights into the origin and evolution of chordates N Satoh , Y Satou , B Davidson , M Levine . Ciona intestinalis : an emerging model for whole-genome analyses . Trends in Genetics , 2003 . 19 ( 7 ): 376 - 381 . 12850442 DOI: 10.1016/S0168-9525(03)00144-6 http://doi.org/10.1016/S0168-9525(03)00144-6 . Ciona intestinalis: an emerging model for whole-genome analyses Y Satou , N Takatori , L Yamada , Y Mochizuki , M Hamaguchi , H Ishikawa , S Chiba , K Imai , S Kano , SD Murakami , 等 . Gene expression profiles in Ciona intestinalis tailbud embryos . Development , 2001 . 128 2893 - 2904 . 11532913 . Gene expression profiles in Ciona intestinalis tailbud embryos Y Satou , KS Imai , N Satoh . Action of morpholinos in Ciona embryos . Genesis , 2001 . 30 ( 3 ): 103 - 106 . 11477683 DOI: 10.1002/gene.1040 http://doi.org/10.1002/gene.1040 . Action of morpholinos in Ciona embryos Y Satou , L Yamada , Y Mochizuki , N Takatori , T Kawashima , A Sasaki , M Hamaguchi , S Awazu , K Yagi , Y Sasakura , 等 . A cDNA resource from the basal chordate Ciona intestinalis . Genesis , 2002 . 33 ( 4 ): 153 - 154 . 12203911 DOI: 10.1002/gene.10119 http://doi.org/10.1002/gene.10119 . A cDNA resource from the basal chordate Ciona intestinalis K Shida , D Terajima , R Uchino , S Ikawa , M Ikeda , K Asano , T Watanabe , K Azumi , M Nonaka , Y Satou , 等 . Hemocytes of Ciona intestinalis express multiple genes involved in innate immune host defense . Biochem Bioph Res Commun , 2003 . 302 ( 2 ): 207 - 218 . DOI: 10.1016/S0006-291X(03)00113-X http://doi.org/10.1016/S0006-291X(03)00113-X . Hemocytes of Ciona intestinalis express multiple genes involved in innate immune host defense MW Simmen , S Leitgeb , VH Clark , SJM Jones , A Bird . Gene number in an invertebrate chordate, Ciona intestinalis . Proc Natl Acad Sci USA , 1995 . 95 4437 - 4440 . DOI: 10.1073/pnas.95.8.4437 http://doi.org/10.1073/pnas.95.8.4437 . Gene number in an invertebrate chordate, Ciona intestinalis P Sordino , L Belluzzi , R De Santis , WC Smith . Developmental genetics in primitive chordates . Philos Trans R Soc Lond B , 2001 . 356 ( 1414 ): 1573 - 1582 . DOI: 10.1098/rstb.2001.0919 http://doi.org/10.1098/rstb.2001.0919 . Developmental genetics in primitive chordates M Tokuoka , KS Imai , Y Satou , N Satoh . Three distinct lineages of mesenchymal cells in Ciona intestinalis embryos demonstrated by specific gene expression . Dev Biol , 2004 . 274 ( 1 ): 211 - 224 . 15355799 DOI: 10.1016/j.ydbio.2004.07.007 http://doi.org/10.1016/j.ydbio.2004.07.007 . Three distinct lineages of mesenchymal cells in Ciona intestinalis embryos demonstrated by specific gene expression M Tsuda , T Kusakabe , H Iwamoto , T Horie , Y Nakashima , M Nakagawa , K Okunou . Origin of the vertebrate visual cycle: II. Visual cycle proteins are localized in whole brain including photoreceptor cells of a primitive chordate . Vision Res , 2003 . 43 ( 28 ): 3045 - 3053 . 14611940 DOI: 10.1016/j.visres.2003.09.012 http://doi.org/10.1016/j.visres.2003.09.012 . Origin of the vertebrate visual cycle: II. Visual cycle proteins are localized in whole brain including photoreceptor cells of a primitive chordate TC Tung , SC Wu , YF Yeh , KS Li , MC Hsu . Cell differentiation in ascidian studied by nuclear transplantation . Sci Sinica , 1977 . 22 222 - 233 . . Cell differentiation in ascidian studied by nuclear transplantation H Wada , N Satoh . Details of the evolutionary history from invertebrates to vertebrates as deduced from the sequences of 18srDNA . Proc Natl Acad Sci USA , 1994 . 91 1801 - 1804 . 8127885 . Details of the evolutionary history from invertebrates to vertebrates as deduced from the sequences of 18srDNA JH Xiang . Marine Biology , 2003 . : Beijing Science Press , (in Chinese) . Marine Biology K Yagi , N Satoh , Y Satou . Identification of downstream genes of the ascidian muscle determinant gene Ci-macho1 . Dev Biol , 2004 . 274 ( 2 ): 478 - 489 . 15385173 DOI: 10.1016/j.ydbio.2004.07.013 http://doi.org/10.1016/j.ydbio.2004.07.013 . Identification of downstream genes of the ascidian muscle determinant gene Ci-macho1 CX Zheng . Species diversity of ascidians in the coastal China seas . Chinese Biodivers , 1995 . 3 ( 4 ): 201 - 205 . (in Chinese) . Species diversity of ascidians in the coastal China seas The authors profoundly thank Prof. Hidetoshi Saiga and his assistant Tetsurou from Tokyo Metropolitan University for good advice on techniques. 10.1631/jzus.2006.B0467 Project supported by the Hi-Tech Research and Development Program (863) of China (No. 2003AA603440) and the Knowledge Innovation Program from the Chinese Academy of Sciences (No. KZCX2-211) 2005-11-01 2006-03-01 2006-05-12 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 07