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

用活体胚胎或原代细胞培养的形态技术研究了Mammlian神经脊细胞。全胚胎培养(WEC)使以更多的实验方式研究细胞成为可能，在这种方法中，胚胎被从其母体内切开以供操纵。WEC可以为发育生物学提供合适的方法。实际上，由于存在一些突变的面部畸形，因此使用小鼠胚胎进行WEC更具优势。本年度，从妊娠第10天开始，利用小鼠WEC进行了以下实验：1)用糜烂素D(CD)C57BL/6小鼠胚胎暴露于150 ng/ml的Cd中，用WEC处理2小时。在培养结束24小时时，70.4%的镉处理胚胎出现面部畸形(唇裂)。治疗后即刻罗丹明-鬼臼乙素染色图像显示F-肌动蛋白在鼻黏膜上皮细胞中的分布部分断裂。分裂的F-肌动蛋白可能与唇裂的形成有关。2)早期开放的卵黄囊诱导唇裂。卵黄囊(OYS)的形成是WEC不可缺少的。在C57BL/6小鼠胚胎中，在8尾体节体节前制作的胚胎，唇裂的诱发率为100%。用扫描电子显微镜观察早期绵羊胚胎的融合区，未见正常发育时观察到的微绒毛消失，但上皮表面呈球形，可见大量碎屑。

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.