Liu Li-ping, Xiang Jian-hai, Dong Bo, Natarajan Pavanasam, Yu Kui-jie, Cai Nan-er
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
J Zhejiang Univ Sci B. 2006 Jun;7(6):467-74. doi: 10.1631/jzus.2006.B0467.
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 degrees 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.
海鞘Ciona intestinalis是发育生物学和进化生物学的模式生物，可能为两个基本问题提供关键线索，即脊索动物如何从假定的后口动物祖先起源，以及高等脊索动物如何从最简单的脊索动物起源。本文进行了Ciona intestinalis的全生命周期培养。以干螺旋藻、蛋黄、叉鞭金藻、食用酵母和虾用断奶饲料的组合为食，Ciona intestinalis长到平均59毫米，并在培养60天后成熟。这个培养过程可以用人工培养的成熟海鞘作为材料重复进行。当受精卵分别在10、15、20、25摄氏度下培养时，它们分别在30小时、22小时、16小时和12小时50分钟内孵化，经历卵裂、囊胚形成、原肠胚形成、神经胚形成、尾芽期和蝌蚪期。蝌蚪幼虫因其背神经索、脊索和原脑而被表征为典型但简化的脊索动物。在自由游动8 - 24小时后，蝌蚪幼虫附着在基质上，并在1 - 2天内变态为滤食性固着幼体海鞘。此外，未受精卵在含有1%胰蛋白酶、0.25%乙二胺四乙酸（EDTA）、pH值为10.5的过滤海水中40分钟内成功去壳。受精后，去壳卵发育良好并以正常孵化率孵化。总之，本文提出了在可控条件下将Ciona intestinalis的蝌蚪幼虫饲养至性成熟的可行方法，以及对实验室培养海鞘胚胎发生的详细观察，这将有助于利用该模式系统进行发育和遗传研究。
