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

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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.