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The study of neural crest cells by the mammalian whole embryo culture

哺乳动物全胚胎培养神经嵴细胞的研究

基本信息

批准号：
61870073
负责人：
ETO Kazuhiro
金额：
$ 1.92万
依托单位：
Tokyo Medical & Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1988
项目状态：
已结题

项目摘要

Mammlian neural crest cells have been investigated using morphological techniques with in vivo embryos or primary cell culture. Whole embryo culture (WEC) has made it possible to study the cells in more experimental way in which embryos are dossected out from their mother for manipulation. WEC can provide with appropriate method for developmental biology. In praticular, it has more advantages to use mouse embryos for wec since there are some mutants which have facial abmormality. In this current year, the following experiments were made to perturb the facial formation by chemical or operative treatment using mouse WEC from day 10 of gestation.1) Perturbation of facial formation with chytochalasin D (CD)C57BL/6 mouse embryos were exposed to 150ng/ml of CD for 2 hours using WEC. Facial mal-formation (cleft lip) was observed in 70.4% of the CD-treated embryos at the end point of the culture for 24 hours. The distribution of F-actin in nasal epithelium was observed to be partially broken in rhodamine-phalloidin staining image just after the treatment. Distrubed F-actin might be related to the cleft lip formation.2) Induction of cleft lip by early open yolk sacOrening of yolk sac (OYS) is indispensable to WEC. In C57BL/6 mouse embryos, when it has been made before 8-tail somite stage, cleft lip was induced to 100% of the embryos. Scanning electron microscopy of the presumptive fusion area in early OYS embryos showed that the disappearance of microvilli which were observed in mormal development was not occured, but that the surface of epithelium was spherical, and many debrises were observed.

哺乳动物的神经嵴细胞已经研究了在体内胚胎或原代细胞培养的形态学技术。全胚胎培养（WEC）使得以更实验性的方式研究细胞成为可能，其中胚胎从母体中分离出来进行操作。WEC可为发育生物学研究提供合适的方法。尤其是，由于存在一些具有面部畸形的突变体，因此使用小鼠胚胎进行wec更具优势。在本年度中，进行了以下实验，以从妊娠第10天起使用小鼠WEC通过化学或手术处理来扰乱面部形成。1）用chytochalasin D（CD）扰乱面部形成使用WEC将C57 BL/6小鼠胚胎暴露于150 ng/ml CD 2小时。在培养24小时的终点时，在70.4%的CD处理的胚胎中观察到面部畸形（唇裂）。结果：治疗后即刻，若丹明-鬼笔环肽染色显示，F-actin在鼻粘膜上皮的分布被部分破坏。F-actin的紊乱可能与唇裂的形成有关。2）早期开放的卵黄囊诱发唇裂卵黄囊的形成是WEC形成的必要条件。在C57 BL/6小鼠胚胎中，在8尾体节期之前制作的胚胎，唇裂诱导率为100%。对早期OYS胚胎融合区的扫描电镜观察表明，未出现正常发育时所见的微绒毛消失，但上皮表面呈球形，并有大量碎屑。