外胚叶发育不全基因及其受体在斑马鱼牙齿早期发育中的表达  

Expression patterns of ectodysplasin and ectodysplasin receptor during early dental development in zebrafish

作　　者：郑雪丹 杨其芬 徐智云[1] 杨德琴[1] Zheng Xuedan;Tang Qifen;Xu Zhiyun;Yang Deqin(Dept,of Endodontics,Stomatological Hospital of Chongqing Medical University,Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences,Chongqing Municipal Key Laboratory of Oral Biomedical Engineering ofHigher Education,Chongqing 401147,China;School of Life Sciences Southwest University,Chongqing 401147,China)

机构地区：[1]重庆医科大学附属口腔医院牙体牙髓科,口腔疾病与生物医学重庆市重点实验室,重庆市高等教育口腔生物医学工程重点实验室 [2]西南大学生命科学学院,重庆401147

出　　处：《华西口腔医学杂志》2019年第4期355-360,共6页West China Journal of Stomatology

基　　金：国家自然科学基金(31571508,31371473)~~

摘　　要：目的探讨外胚叶发育不全(EDA)基因及其受体(EDAR)基因在斑马鱼早期咽齿发育过程中的表达模式,为进一步研究其在牙齿发育过程中的功能奠定基础。方法提取受精后48 h(48 hpf)的斑马鱼胚胎总RNA,之后逆转录合成cDNA,以该cDNA为模板,特异性扩增斑马鱼咽齿特异性标记基因dlx2b、Eda信号通路相关基因eda及edar基因片段,用TA克隆试剂盒将这3个基因片段分别连接到pGEMT载体上,将对应质粒用聚合酶链反应(PCR)线性化,以PCR线性化做模板,选择相应的RNA聚合酶体外转录,合成dlx2b、eda及edar基因的反义mRNA探针。收集36、48、56、60、72、84 hpf的斑马鱼胚胎,运用全胚原位杂交技术检测eda及edar基因在斑马鱼咽齿发育部位的表达分布。比较原位杂交结果eda及edar表达区域与斑马鱼咽齿特异性标记基因dlx2b所指示咽齿部位的关系。结果获得了dlx2b、eda及edar基因的反义m RNA探针。原位杂交结果显示,48~72 hpf时eda及edar在鳃弓牙齿相应部位可见强棕褐色阳性信号,与dlx2b所指示咽齿部位吻合。结论Eda信号通路配体与受体在斑马鱼咽齿早期发育过程中即牙齿发生至形态发生阶段于咽齿部位特异性强表达,这一结果显示该信号通路可能参与斑马鱼咽齿早期发育的调控。Objective This study aims to study the expression patterns of ectodysplasin(EDA)and ectodysplasin receptor(EDAR)during the early development of zebrafish and provide a foundation for further research of the Eda signaling pathway in tooth development.Methods Total RNA was extracted from zebrafish embryos at 48 hours postfertilization(hpf)and then reverse transcribed for cDNA library generation.The corresponding RNA polymerase was selected for the synthesis of the digoxin-labeled antisense mRNA probe of zebrafish pharyngeal tooth specific marker dlx2 b and Eda signaling-associated genes eda and edar in vitro.The three sequences were ligated into a pGEMT vector with a TA cloning kit,and polymerase chain reaction(PCR)was applied to linearize the plasmid.The resultant PCR sequences were used as templates for synthesizing Dig-labeled mRNA probe dlx2 b,eda,and edar.Zebrafish embryos were collected at 36,48,56,60,72,and 84 hpf,then whole mount in situ hybridization was performed for the detection of eda and edar expression patterns.Then,their expression patterns at 72 hpf were compared with the expression pattern of dlx2 b.Results The mRNA antisense probes of dlx2 b,eda,and edar were successfully obtained.The positive signals of eda and edar were observed in zebrafish pharyngeal tooth region at 48-72 hpf and thus conform to the signals of dlx2 b in the positive regions.Conclusion The ligand eda and edar,which are associated with the Eda signaling pathway,are strongly expressed only at the pharyngeal tooth region in zebrafish from tooth initiation to the morphogenesis stage.Thus,the Eda signaling pathway may be involved in the regulation of the early development of zebrafish pharyngeal teeth.

关 键 词：外胚叶发育不全 外胚叶发育不全受体 牙齿发育 斑马鱼 原位杂交 

分 类 号：Q786[生物学—分子生物学]