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

神经嵴细胞和HoxB基因在咽弓形态发生中的相互关系

基本信息

批准号：
07838031
负责人：
YASUI Kinya
金额：
$ 1.47万
依托单位：
Kagoshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07838031/
关键词：
house shrew whole embryo culture pharyngeal arches trigeminal nerve Smtwist 三又神経スンクス神経堤細胞 twist HoxB

项目摘要

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.

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。