Interrelationships between neural crest cells and HoxB genes in the morphogenesis of pharyngeal arches

本研究利用全胚胎培养系统，建立家鼠胚胎咽弓畸形的诱导技术，并通过改变培养液和供气条件，对发育过程中的细胞行为，特别是神经脊细胞进行全胚胎培养。从含有1-5个体节的胚胎开始培养时，在以下条件下效果最好。培养液为新鲜制备的纯大鼠血清。第一种气体流量为20%O_2(50ml/min)，与CO_2和5%N_2平衡，直到凌晨0点，然后改为60%O_2(25-30 ml/min)。放置12h，改变气体流量至50ml/min。用全反式维甲酸(RA)诱导咽弓畸形，而不用费氏素(N，N‘-八亚甲基-双-2，2-二氯乙酰胺)诱导咽弓畸形。RA处理表现出剂量依赖性和发育阶段依赖性。当RA浓度为60 ng/ml处理含4~6个体节的胚胎时，可引起第一咽弓和第二咽弓畸形及融合。用钨细针在具有4-6个体节的胚胎的第一-第二咽弓区域进行划痕操作也得到了类似的结果。到目前为止，家鼠HoxB基因的同源基因没有被克隆，而是twist同源基因，Smtwist已经从一个cdna文库中克隆出来。我们正在从家栖鼠胚胎中构建新的cDNA文库，并准备分析Smtwist在正常胚胎和处理后胚胎中的表达模式。

In this research project, we subjected to establish techniques to induce malformations of the pharyngeal arches in house shrew embryos using whole-embryo culture system and to reveal cell behavior, especially neural crest cells during the morphogenesis of pharyngeal metamerism.Whole-embryo culture was performed changing the conditions of medium and supplying gas. When the culture was started from embryos with 1-5 somites, the best result was obtained in the following conditions. Culture medium was freshly prepared pure rat serum. The first gas flow was with 20% O_2 (50ml/min) balanced with CO_2 and 5% N_2 until 0.00 am and then changed to 60% O_2 (25-30 ml/min). It was kept for 12h, and changed gas flow to 50ml/min. The gas flow was further changed to 75ml/min at next 12h later.The malformation of pharyngeal arches were induced with all-trans retinoic acid (RA) but not with Fertilysin (N,N'-Octamethylene-bis-2,2-dichloroacetamide). RA treatment showed dose-dependent and developmental stage-dependent effects. When RA was treated at 60ng/ml for embryos with 4-6 somites, it induced the malformation of the 1st and 2nd pharyngeal arches and the fusion of them. Similar results were obtained by the operation of scratch at the presumptive 1st-2nd pharyngeal arch region of embryos heving 4-6 somites with fine tungsten needle.Until now, house shrew's orthologues of HoxB genes were not cloned but twist orthologue, Smtwist has been cloned from a cDNA library. We are constructing new cDNA library from house shrew embryos and preparing the analysis of Smtwist expression patterns in normal and treated embryos.