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

在本研究项目中，我们建立了利用全胚胎培养系统诱导家鼩胚胎咽弓畸形的技术，并揭示了细胞行为，特别是神经嵴细胞在咽变态形态发生过程中的行为。改变培养基条件和供气条件进行全胚培养。从1 ~ 5体胚开始培养时，在以下条件下效果最好。培养基为新鲜配制的纯大鼠血清。第一次气体流量为20% O_2 (50ml/min)，与CO_2和5% N_2平衡至00 am，然后改为60% O_2 （25- 30ml /min）。保存12h，将气体流量改为50ml/min。12小时后，进一步将气体流量改为75ml/min。全反式维甲酸（RA）可诱导大鼠咽弓畸形，而肥素（N，N′-八亚甲基-双-2,2-二氯乙酰胺）不能诱导咽弓畸形。RA治疗表现出剂量依赖性和发育阶段依赖性。当RA浓度为60ng/ml时，4-6个体的胚胎可诱导第一、第二咽弓畸形并融合。用细钨丝针在4 ~ 6个胚胎推定的第1 ~ 2咽弓区划伤，也得到了类似的结果。到目前为止，家鼩的HoxB基因同源物尚未克隆，但twist同源物Smtwist已从cDNA文库中克隆出来。我们正在从家鼩胚胎中构建新的cDNA文库，并准备分析Smtwist在正常和处理过的胚胎中的表达模式。

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.