热带爪蟾Dmrt5下游靶基因的全基因组水平分析及其在嗅觉神经形成中的功能研究
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摘要
DMRT/Dmrt (Doublesex and Mab3 Related Transcription factor)是一类与果蝇性别发育调控因子Doublesex以及线虫性别发育调控因子Mab3相关的转录因子,它们的共同特征就是具有一个富含半胱氨酸、类似锌指的、能和特异DNA序列结合的结构基序——DM保守盒。Dmrt基因家族是一类非常重要的与发育相关的基因,目前发现的大多数成员可能与性别分化和决定相关,也有些成员则可能在胚胎发育过程中参与了其它器官的发育。研究表明Dmrt4在爪蟾嗅觉神经形成中起着关键的作用,在研究中我们同时克隆了Dmrt4的两个同源基因Dmrt3和Dmrt5。通过对Dmrt3、Dmrt4、Dmrt5时空表达模式的分析发现,它们在前脑、嗅觉原基中都有表达。因此我们认为其功能可能存在冗余或者某种调控关系,据此我们对Dmrt3、4、5进行了基因芯片的筛选分析并通过体外和体内的过表达和基因敲落实验验证,主要研究结果如下
基因芯片数据表明被Dmrt5上调超过10倍的基因有233个被Dmrt5下调超过10倍的基因有99个,同样被Dmrt4上调超过10倍的基因有337个被Dmrt4下调的基因有361个,对Dmrt3的表达谱研究发现,被上调的基因有201个被下调基因的有289个。从这个数据可以看出尽管Dmrt4、Dmrt5表达模式进化和某些功能上具有极大的相似性但它们对所调控下游基因还是有比较大的差异。通过比较它们之间的差异发现Dmrt4、Dmrt5共同上调的下游基因有917个,共同下调的下游基因有510个,Dmrt3、Dmrt5共同上调的下游基因有117个,共同下调的下游基因有105个,Dmrt3、Dmrt4发现它们共同上调的下游基因有124个,共同下调的下游基因有228个,这表明Dmrt3、4、5可能共同参与了某些下游基因的调控,但同时它们对下游基因的调控亦有区别。
细化Dmrt5基因芯片我们发现数据表明过表达Dmrt5可以诱导Dmrt4、Neurogenin2、Ebf2、Ncam的表达,而这些因子都是爪蟾嗅觉神经形成过程中重要的分子标记。通过体内和体外试验我们证明Dmrt5在热带爪蟾嗅觉神经形成过程中起着重要作用,并且是通过Dmrt4来实现这种调控机制。
此外,Dmrt5除参与爪蟾嗅觉神经形成外,基因芯片数据分析还显示许多非经典Wnt信号通路中的组分也发生了变化包括多种非经典的Wnt配体,根据之前有文献报道,过表达或是抑制非经典的Wnt信号通路都会影响胚胎的原肠运动,而我们在过表达Dmrt5后发现胚胎的原肠运动受到影响,因此我们认为过表达Dmrt5可能激活了非经典的Wnt信号通路。同时我们发现多个中脑多巴胺神经元前体形成有关的基因如En2、Msxl、Gli3等发生了变化,这也进一步在两栖动物中验证了Dmrt5对于中脑多巴胺神经元形成具有重要的调控作用。
本研究第一次发现在热带爪蟾中Dmrt5调控爪蟾嗅觉神经形成,并对其调控机制进行了研究,同时我们通过基因芯片的分析发现Dmrt5在胚胎发育过程中发挥着多种功能,这为以后深入研究Dmrt5的其它功能奠定了坚实的基础。
DMRT/Dmrt (Doublesex and Mab-3 Related Transcription factor) genes encode a family of transcription factors related to Drosophila melanogaster doublesex (Dsx) and Caenorhabditis elegans Mab3, which are characterized by a unique zinc finger like DNA-binding motif known as the DM domain. Dmrt family genes are important genes related to development. Currently, majority of the members are associated with sex differentiation and determination, while some of the members are reported to participate in the process of embryonic development of other organs.
Our previous studies show that Dmrt4 plays a key role in the process of the Xenopus olfactory neurogenesis. During the cloning process of Dmrt4, we also cloned two other homologous genes of Dmrt4---Dmrt3 and Dmrt5. And our primarily data also reveal that these three homologues may have overlapping expression regions on olfactory placodes and dorsal telencephalon. Thus, in this study, we focus on their similarities and differences on regulation of downstream genes during embryogenesis by genome wide microarray analysis. Combining with previous study on Xenopus Dmrt4, and through a series of in vitro/in vivo studies, we propose that Dmrt5 is also involving in olfactory neurogenesis. The main findings are as follows:
Data from the microarray screen show that 233 genes'expression are raised and 99 genes' expression are reduced more than 10 times by Dmrt5;337 genes'expression are raised and 361 genes'expression are reduced more than 10 times by Dmrt4; while 201 genes'expression are raised and 289 genes'expression are reduced more than 10 times by Dmrt3. Through profile comparing among Dmrt3,4,5 with each other, we find that they are regulating different profiles of downstream genes, although they are overlapping expressed in forebrain and olfactory placodes. Data from the microarray screen also show that 917 genes'expression are raised and 510 genes'expression are reduced both by Dmrt4 and DmrtS; 117 genes'expression are raised and 105 genes'expression are reduced both by Dmrt3 and Dmrt5\while 124 genes'expression are raised and 228 genes'expression are reduced both by Dmrt3 and Dmrt4. Furthermore 558 genes' expression are raised and 373 genes'expression are reduced by all of them.These data indicate that Dmrt3、4、5 probably control different downstream gene profiles during embryogenesis, while they may share common characteristics in regulation of olfactory neurogenesis.
Further detailed analysis of Dmrt5's microarray data reveal that Dmrt5 can strongly up-regulate the expression of Dmrt4. Ncam、Neurogenin2 and Ebf2. Since these genes are important genes controlling olfactory neurogenesis in Xenopus tropicalis, we perform a series of in vivo/vitro assays by knockdown, overexpression and rescue to demonstrate that Dmrt5 also paly an important role in olfactory neurogenesis. Most interestingly, our data clearly indicate that Dmrt5 may promote olfactory neurogenesis via inducing Dmrt4's expression.
In addition, we also find Dmrt5 may be a multiple function regulator which participates in different developmental processes. For example Dmrt5 may be involved in embryo gastrulation and anterior cell fate determination by regulating noncanonical Wnt signalling, because over-expression of Dmrt5 could up-regulate some noncanonical Wnt ligands'expression, such as Wnt7a/b, Wntll and Wnt9a/b etc. Furthermore we find some genes like En2、Msx1、Gli3, which are related to ventral mesencephalic neural fate specification, are also up regulated by Dmrt5.
In general, our investigation firstly demonstrates that Dmrt5 is essential and critical for olfactory neurogenesis in Xenopus tropicalis. Besides, Dmrt5 could play multiple roles during embryogenesis. And our global gene expression analysis provides a valuable platform for further study.
基因芯片数据表明被Dmrt5上调超过10倍的基因有233个被Dmrt5下调超过10倍的基因有99个,同样被Dmrt4上调超过10倍的基因有337个被Dmrt4下调的基因有361个,对Dmrt3的表达谱研究发现,被上调的基因有201个被下调基因的有289个。从这个数据可以看出尽管Dmrt4、Dmrt5表达模式进化和某些功能上具有极大的相似性但它们对所调控下游基因还是有比较大的差异。通过比较它们之间的差异发现Dmrt4、Dmrt5共同上调的下游基因有917个,共同下调的下游基因有510个,Dmrt3、Dmrt5共同上调的下游基因有117个,共同下调的下游基因有105个,Dmrt3、Dmrt4发现它们共同上调的下游基因有124个,共同下调的下游基因有228个,这表明Dmrt3、4、5可能共同参与了某些下游基因的调控,但同时它们对下游基因的调控亦有区别。
细化Dmrt5基因芯片我们发现数据表明过表达Dmrt5可以诱导Dmrt4、Neurogenin2、Ebf2、Ncam的表达,而这些因子都是爪蟾嗅觉神经形成过程中重要的分子标记。通过体内和体外试验我们证明Dmrt5在热带爪蟾嗅觉神经形成过程中起着重要作用,并且是通过Dmrt4来实现这种调控机制。
此外,Dmrt5除参与爪蟾嗅觉神经形成外,基因芯片数据分析还显示许多非经典Wnt信号通路中的组分也发生了变化包括多种非经典的Wnt配体,根据之前有文献报道,过表达或是抑制非经典的Wnt信号通路都会影响胚胎的原肠运动,而我们在过表达Dmrt5后发现胚胎的原肠运动受到影响,因此我们认为过表达Dmrt5可能激活了非经典的Wnt信号通路。同时我们发现多个中脑多巴胺神经元前体形成有关的基因如En2、Msxl、Gli3等发生了变化,这也进一步在两栖动物中验证了Dmrt5对于中脑多巴胺神经元形成具有重要的调控作用。
本研究第一次发现在热带爪蟾中Dmrt5调控爪蟾嗅觉神经形成,并对其调控机制进行了研究,同时我们通过基因芯片的分析发现Dmrt5在胚胎发育过程中发挥着多种功能,这为以后深入研究Dmrt5的其它功能奠定了坚实的基础。
DMRT/Dmrt (Doublesex and Mab-3 Related Transcription factor) genes encode a family of transcription factors related to Drosophila melanogaster doublesex (Dsx) and Caenorhabditis elegans Mab3, which are characterized by a unique zinc finger like DNA-binding motif known as the DM domain. Dmrt family genes are important genes related to development. Currently, majority of the members are associated with sex differentiation and determination, while some of the members are reported to participate in the process of embryonic development of other organs.
Our previous studies show that Dmrt4 plays a key role in the process of the Xenopus olfactory neurogenesis. During the cloning process of Dmrt4, we also cloned two other homologous genes of Dmrt4---Dmrt3 and Dmrt5. And our primarily data also reveal that these three homologues may have overlapping expression regions on olfactory placodes and dorsal telencephalon. Thus, in this study, we focus on their similarities and differences on regulation of downstream genes during embryogenesis by genome wide microarray analysis. Combining with previous study on Xenopus Dmrt4, and through a series of in vitro/in vivo studies, we propose that Dmrt5 is also involving in olfactory neurogenesis. The main findings are as follows:
Data from the microarray screen show that 233 genes'expression are raised and 99 genes' expression are reduced more than 10 times by Dmrt5;337 genes'expression are raised and 361 genes'expression are reduced more than 10 times by Dmrt4; while 201 genes'expression are raised and 289 genes'expression are reduced more than 10 times by Dmrt3. Through profile comparing among Dmrt3,4,5 with each other, we find that they are regulating different profiles of downstream genes, although they are overlapping expressed in forebrain and olfactory placodes. Data from the microarray screen also show that 917 genes'expression are raised and 510 genes'expression are reduced both by Dmrt4 and DmrtS; 117 genes'expression are raised and 105 genes'expression are reduced both by Dmrt3 and Dmrt5\while 124 genes'expression are raised and 228 genes'expression are reduced both by Dmrt3 and Dmrt4. Furthermore 558 genes' expression are raised and 373 genes'expression are reduced by all of them.These data indicate that Dmrt3、4、5 probably control different downstream gene profiles during embryogenesis, while they may share common characteristics in regulation of olfactory neurogenesis.
Further detailed analysis of Dmrt5's microarray data reveal that Dmrt5 can strongly up-regulate the expression of Dmrt4. Ncam、Neurogenin2 and Ebf2. Since these genes are important genes controlling olfactory neurogenesis in Xenopus tropicalis, we perform a series of in vivo/vitro assays by knockdown, overexpression and rescue to demonstrate that Dmrt5 also paly an important role in olfactory neurogenesis. Most interestingly, our data clearly indicate that Dmrt5 may promote olfactory neurogenesis via inducing Dmrt4's expression.
In addition, we also find Dmrt5 may be a multiple function regulator which participates in different developmental processes. For example Dmrt5 may be involved in embryo gastrulation and anterior cell fate determination by regulating noncanonical Wnt signalling, because over-expression of Dmrt5 could up-regulate some noncanonical Wnt ligands'expression, such as Wnt7a/b, Wntll and Wnt9a/b etc. Furthermore we find some genes like En2、Msx1、Gli3, which are related to ventral mesencephalic neural fate specification, are also up regulated by Dmrt5.
In general, our investigation firstly demonstrates that Dmrt5 is essential and critical for olfactory neurogenesis in Xenopus tropicalis. Besides, Dmrt5 could play multiple roles during embryogenesis. And our global gene expression analysis provides a valuable platform for further study.
引文
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2 Zarkower, D., Establishing sexual dimorphism:conservation amidst diversity? [J],Nat. Rev., Genet.2001.2,175-185.
3 Smith, C.A., Sinclair, A.H.,2004. Sex determination:insights from the chicken.BioEssays 26, 120-132
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