Analysis of teratogenic mechanisms of retinoic acid on mammalian craniofacial morphogenesis---as models for human congenital anomalies

维甲酸(RA)是维生素A的天然代谢物，对哺乳动物的正常发育至关重要。然而，过量的维生素A和其他天然和合成的维甲酸(包括RA)也会导致胚胎异常。实验动物用维甲酸诱导的颅面畸形被认为是人类先天畸形的模型，如Treacher-Collins和Di George综合征。鉴于颅神经脊细胞在头面部发育中的重要作用，我们建立了可重复诱导典型头面部畸形表型的体外培养条件，并对其行为进行了检测。用全反式维甲酸体外处理9.0d和9.5d大鼠胚胎6h，进一步培养至11.5d胚胎。前者导致中枢神经系统和咽弓的后部化，后者使第一、第二弓以及三叉神经节和听面神经节融合。有趣的是，DiI标记研究表明，来自前脑和先天后脑的CREST细胞不能在融合的弓内混合。它支持这样一种观点，即脊细胞群体的差异身份仍然保持着。相反，在9.0d培养的大鼠胚胎中，来自后脑前部的CREST细胞异位迁移到第二弓，这表明RA不仅影响后脑神经外胚层的识别，也影响来自后脑的CREST细胞的识别。因此，RA以一种阶段依赖的方式影响脊细胞的迁移和脑神经的特征。这种RA效应的阶段依赖性也在分子水平上观察到，因为CRABP-I蛋白的分布模式在晚期治疗中没有变化，但在早期治疗中发生了显著变化。

Retinoic acid (RA), a natural metabolite of vitamin A,is essential for normal mammalian development. However, embryos exhibit anomalies also by excess vitamin A and other natural and synthesized retinoids including RA.Craniofacial malformations experimentally induced with retinoids in laboratory animals are regarded as models for human congenital anomalies such as Treacher-Collins and Di George syndromes. Since cranial neural crest cells play important roles in development of head and face, here we have established in vitro conditions which reproducibly induce typical phenotypes of craniofacial abnormalities and examined behavior of the crest cells.9.0 day and 9.5 day rat embryos were treated in vitro with all-trans-RA for 6 hours and further cultured up to the stage corresponding to 11.5 day in vivo embryos. The former treatment induced posteriorization in the CNS and pharyngeal arches, while the latter treatment resulted in fusion of the first and second arches as well as that of the trigeminal and acousticofacial ganglia. Interestingly, DiI labeling study revealed that crest cells derived from the anterior and preotic hindbrain do not mix within the fused arch. It supports the idea that differential identities of the crest cell populations are still maintained. In contrast, crest cells from the anterior hindbrain ectopically migrate into the second arch in cultured rat embryos treated at 9.0day, suggesting that RA affected not only the identity of the hindbrain neuroectoderm but also that of the crest cells derived from the hindbrain. Thus, RA affected migration of crest cells and features of cranial nerves in a stage-dependent manner. Such stage dependency in RA's effects was also observed at the molecular level since the distribution pattern of CRABP-I protein was unchanged in the late-stage treatment but drastically altered in the early-stage treatment.