Analysis on gene expression involved in migration and differentiation of cranial neural crest cells

分子和细胞分析，以阐明基因表达参与哺乳动物颅神经嵴细胞的迁移和分化。本年度取得的主要成果如下：1.颅神经嵴细胞迁移过程中Pax-6基因的表达我之前曾报道过，在Pax-6基因突变的纯合突变大鼠胚胎中，中脑嵴细胞向额鼻块的迁移受到损害。为了详细研究Pax-6基因在颅神经嵴迁移过程中的表达，建立了使用非放射性同位素标记探针的切片和整体标本原位杂交。Pax-6基因在脑神经嵴细胞中不表达，提示Pax-6基因产物可能参与介导环境因素对中脑嵴迁移的调控.维甲酸对微团培养小鼠面部间充质细胞软骨形成的影响如前所述，维甲酸诱导颅面畸形具有阶段依赖性，并且维甲酸受体基因的不同亚型在颅神经嵴来源的面部间充质的软骨形成和非软骨形成区域中表达。为了阐明维甲酸是否通过其受体介导软骨形成，使用从第11天小鼠胚胎分离的面部间充质细胞进行微团培养，并在体外再现软骨分化。不同水平的软骨形成中观察到个别面部原基的文化，和视黄酸抑制软骨形成剂量依赖性。这些结果与维甲酸受体基因表达的区域特异性一起，表明维甲酸参与面部间充质细胞的软骨形成。

Molecular and cellular analyzes were performed to elucidate gene expression involved in migration and differentiation of mammalian cranial neural crest cells. The major achievement obtained in this year is as follows :1. Expression of Pax-6 gene during migration of cranial neural crest cellsI have previously reported that migration of midbrain crest cells toward the frontonasal mass is impaired in homozygous mutant rat embryos which have mutation in Pax-6 gene. In order to examine Pax-6 gene expression in detail during cranial neural crest migration, in situ hybridization on sections and whole mounts using non-radioisotope labeled probes was established. Pax-6 gene was not expressed in cranial neural crest cells themselves ; thereby suggeseting that Pax-6 gene product has role in mediating environmental factors that are involved in controling midbrain crest migration.2. Effects of retinoic acid on hondrogenesis of mouse facial mesenchmal cells under micromass cultureAs previously described, retinoic acid induce craniofacial malformations stage dependently and different subtypes of retinoic acid receptor gene are expressed in chondrogenic and non-chondrogenic regions of facial mesenchyme derived from the cranial neural crest. To elucidate whether retinoic acid madiates chondrogenesis via its receptors, micromass culture was carried out using facial mesenchymal cells isolated from day 11 mouse embryos, and cartilage differentiation was reproduced in vitro. Different levels of chondrogenesis were observed among cultures of individual facial primordia, and retinoic acid inhibited cartilage formation dose-dependently. These results together with region-specificity of retinoic acid receptor gene expression, retinoic acid is suggested to be involved in chondrogenesis of facial mesenchymal eclls.