Trace of migration pathways of neural crest cells during mouse tooth germ formation by labeling with gold colloid probe.

最近，在禽类胚胎上进行了旨在阐明神经嵴细胞的迁移和组织贡献的实验工作。然而，鸟类没有牙齿。将标记的神经嵴细胞移植到哺乳动物胚胎中，是研究神经嵴细胞在牙板发育中作用的必要手段。哺乳动物胚胎不适合这些移植技术，因此这种方法对头部有严重的限制。因此，通过免疫组织化学方法对小鼠神经嵴细胞进行免疫组化染色，获得神经嵴细胞行为的直接信息，是一个很有意义的研究课题。用于筛选制备的抗体的组织用50%丙酮、0.1M PBS在37 ℃、360 W输出的条件下通过微波处理器（H-2500，Biorad）固定。用体外免疫Bald/c小鼠脾细胞的方法制备了抗C57 B1/6小鼠13天、14天胚胎的抗体A13、A14。A13和A14均具有较强的阶段特异性，对牙胚下神经嵴细胞有较强的反应。取13、14日龄胚胎连续切片，A13、A14染色后，用ABC法胶体金修饰切片，暗视野观察。在端脑、眼球周围和牙囊间充质上观察到信号。推测这些结果很好地反映了头神经嵴细胞的通路。神经嵴细胞原位标记和抑制的新技术是进一步研究的必要条件。

Relatively recently, experimental work aimed at elucidating the migration and tissue contribution of neural crest cells was carried out on avian embryos. However, no tooth develops in birds. Transplantation works of marked neural crest cells to mammalian embryos is necessary for studies on the contribution of neural crest cells to the development of dental laminas. Mammalian embryos are not amenable to these transplantation techniques, so this approach has serious limitations for the head. Hence there is intense interest in acquiring direct information about mouse neural crest cell behavior by immunohistochemical staining with antibody against neural crest cells.In this research program, new AMeX method for the tissue fixation was developed. Tissues for screening of prepared antibodies were fixed with 50% acetone, 0,1M PBS under the condition of 37.C, 360W output by a microwave processor (H-2500,Biorad). Antibodies A13,A14 (against mouse C57B1/6 13-day, 14-day embryo respectively) were produced by in vitro immunization method applied to the splenocytes from Bald/c mice. Both A13 and A14 have intense stage specificity and were intensely responded to neural crest cells beneath tooth germs. After staining with A13, A14 on serial section of 13-day, 14-day embryos, sections were modified with gold colloid by ABC method and observed under a dark field illumination. Signals were observed on a mesenchyme over the telencephalon, surrounding of eye ball and a mesenchyme of dental follicles. It was speculated that the pathways of cephalic neural crest cells was well reflected by these results. The new techniques of in situ labeling and inhibition on neural crest cells are necessary for further progress.