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

最近，在禽类胚胎上开展了旨在阐明神经脊细胞迁移和组织贡献的实验工作。然而，鸟类没有牙齿发育。将标记的神经脊细胞移植到哺乳动物胚胎中，对于研究神经脊细胞在牙板发育中的作用是必要的。哺乳动物胚胎不适用于这些移植技术，因此这种方法对头部有严重的限制。因此，通过抗神经脊细胞抗体的免疫组织化学染色获得有关小鼠神经脊细胞行为的直接信息引起了人们的浓厚兴趣。在本研究计划中，发展了新的AMEX组织固定方法。筛选抗体的组织用50%丙酮，0，1M PBS固定，37℃，360W，微波处理机(H-2500，Biorad)输出。用体外免疫的方法对Bald/c小鼠的脾细胞产生抗体A13、A14(分别针对C57B1/6 13日龄、14日龄胚胎)。A13和A14都具有强烈的阶段特异性，并对牙胚下的神经脊细胞有强烈的反应。13天、14天胚胎连续切片经A13、A14染色后，用ABC法进行金胶体修饰，在暗场照明下观察。在端脑、眼球周围和牙囊间充质上可观察到信号。推测这些结果很好地反映了头神经脊细胞的通路。神经脊细胞原位标记和抑制的新技术是进一步发展的必要手段。

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.