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遗传谱系示踪技术揭示小鼠胚胎前体心外膜向心外膜转化过程

Analysis of the Process of Proepicardium-epicardium Transition in Mouse Embryos by Genetic Fate-mapping Approaches
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摘要 心外膜的形成是胚胎心脏发育的关键生理过程之一。利用遗传谱系示踪技术示踪观察前体心外膜向心外膜细胞转化过程,具有重要的科学研究价值。本研究拟利用Tbx18+前体/心外膜祖细胞遗传谱系示踪模型,揭示胚胎心外膜的起源及前体心外膜向心外膜转化的过程。利用整胚和切片原位杂交技术揭示,Tbx18 mRNA特异性表达于胚龄(E)9.5 d小鼠胚胎前体心外膜;故Tbx18是前体心外膜的特异性标记基因。利用整胚X-Gal染色,揭示报告基因Lacz在E9.5 d遗传谱系示踪模型鼠胚前体心外膜中大量表达,此时报告基因从前体心外膜逐渐迁移并开始少量表达于心外膜。Lacz在E10~E10.5 d双杂合鼠胚前体心外膜中表达逐渐减少,而在心外膜组织中逐渐增多;在E11.5 d,报告基因在前体心外膜中表达基本消失,而在心外膜组织中大量表达。切片进行XGal染色也揭示,报告基因Lacz定位于早期胚胎前体心外膜及心外膜。免疫荧光染色证实,早期胚胎心外膜细胞呈现未分化的祖细胞状态。通过报告基因的表达变化模式揭示,胚胎心外膜的形成经历了启动、转化、完成3个阶段;E9.5~11.5 d左右这个时间段发生的前体心外膜向心外膜转化,可能是心外膜形成的主要来源和形式。 The formation of epicardium is one of the key physiological processes during embryonic heart development.Therefore,it has important scientific value by using genetic lineage tracing technique to trace and observe the process of proepicardium-epicardium transition.This study is to reveal the origins of proepicardium and the processes of the proepicardium-epicardium transition by using genetic fate-mapping models of Tbx18+pro/epicardial progenitor cells.The results of whole-mount and tissue sections in situ hybridization revealed that the Tbx18 mRNA specifically expressed in the mouse proepicardium at embryonic(E) 9.5 day.Therefore,Tbx18 is a specific marker gene for proepicardium.The Lacz reporter genes were highly expressed in the embryonic proepicardium in genetic lineage tracing model atE9.5 day and then were gradually migrated to epicardium and slightly expressed in epicardium.The expression of Lacz gradually reduced in the proepicardium,meanwhile its expression gradually increased in epicardial tissues at E10 ~ E10.5 day.The expression of Lacz disappeared in proepicardium and became abundantly expressed in epicardium at E11.5 day.X-Gal staining of tissue sections showed that Lacz reporter genes were detected in the proepicardium and epicardium of embryonic heart.Immunofluorescence staining further confirmed that embryonic epicardial cells showed an undifferentiated state of progenitor cells.The expression patterns of Lacz reporter genes revealed that the formation of the embryonic epicardium went through three phases:initiation,transition and completion.The proepicardium-epicardium transition,which happened during the period of E9.5 ~ 11.5 day,may be the main origins and forms of epicardium.
出处 《中国生物化学与分子生物学报》 CAS CSCD 北大核心 2016年第8期962-966,共5页 Chinese Journal of Biochemistry and Molecular Biology
基金 国家自然科学基金(No.NSFC81270211) 重庆市科委前沿与应用基础研究计划项目(No.cstc2015jcyj A10070) 重庆教委科学技术研究项目(No.KJ130324)资助~~
关键词 T-box转录因子18 前体心外膜 心外膜 遗传谱系示踪 T-box transcription factor 18(Tbx18) proepicardium epicardium genetic fate-mapping
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参考文献18

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