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

用胶体金探针标记小鼠牙胚形成过程中神经嵴细胞的迁移途径。

• 批准号: 02670806
• 负责人: YAMAAI Tomoichiro
• 金额: $ 1.28万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670806/
• 关键词: Mouse; Tooth germ; Neural crest cell; Monoclonal antibody; In vitro immunization; Microwave fixation; 単クロ-ン抗体; マイクロウエ-ブ固定; マイクロウェ-ブ

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.

最近，研究人员在鸟类胚胎上进行了神经嵴细胞迁移和组织贡献的实验研究。然而，鸟类没有牙齿。有标记的神经嵴细胞移植到哺乳动物胚胎是研究神经嵴细胞对牙板发育贡献的必要条件。哺乳动物胚胎不适合这些移植技术，所以这种方法对头部有严重的限制。因此，对神经嵴细胞免疫组化染色获得小鼠神经嵴细胞行为的直接信息有浓厚的兴趣。在本研究项目中，我们开发了一种新的组织固定方法AMeX。用于筛选制备抗体的组织用50%丙酮，0,1m PBS固定，微波处理机（H-2500,Biorad）在37℃，360W输出条件下固定。采用体外免疫的方法，在Bald/c小鼠脾细胞中产生A13、A14抗体（分别针对小鼠C57B1/6胚胎13天和14天）。A13和A14均具有强烈的分期特异性，对牙胚下的神经嵴细胞反应强烈。13日、14日胚连续切片用A13、A14染色后，用ABC法对切片进行金胶体修饰，暗场照明下观察。在远脑上的间质、眼球周围的间质和牙滤泡间质上观察到信号。推测这些结果很好地反映了头神经嵴细胞的通路。神经嵴细胞的原位标记和抑制新技术是进一步发展的必要条件。