pea3在肾脏发育中的作用及其机制研究
摘要
肾脏疾病是儿童时期的常见病之一,现代生物学研究表明肾脏损伤后的修复过程类似胚胎期后肾发育过程的重演,因此,从发育生物学角度研究胚胎肾脏形成机制、肾小球发生和成熟的调控将为揭示肾脏发育不良和肾脏疾病的病理过程提供良好的前景,近年来备受广大肾脏病学者的关注。
本课题小组自2003年起开展肾脏发育相关基因的筛选工作,采用抑制性差减杂交(suppression subtractive hybridization ,SSH)技术构建了新生大鼠肾和生后21天大鼠肾脏组织发育差减文库,通过生物信息学分析,在新生鼠肾文库中,发现了与胚胎发育相关的pea3(polyomavirus enhancer activator3)基因。
pea3基因属于Ets癌基因家族成员,目前认为Ets家族蛋白在不同的生物体内可调控生长、转录、T细胞活化和器官发育等。pea3主要表达于鼠类多个发育中的器官,尤其是那些需经历上皮-基质相互作用的组织,如肺脏及乳腺,表明其在这些器官的形成中起重要作用。目前pea3在肾脏发育中的作用及机制,国内外尚未见报导。研究显示,pea3与wnt及wt1(肾脏发育中的两个关键蛋白)关系密切。本研究首先观察了pea3在大鼠肾脏不同发育阶段的时空表达规律;分别予wnt3a诱导、转染pea3真核表达载体、转染pea3 siRNA等方法在整体和细胞水平,进一步探讨该基因表达改变对胚胎肾脏发育的影响,及其与wnt、wt1之间的关系;最后应用斑马鱼为模型,显微注射pea3 mRNA及morpholino,在体观察pea3对肾脏发育的作用及机制。对该基因的研究有助于了解先天性肾脏发育不良和后天性肾脏疾病的病理机制,为基因治疗提供新的靶标。
第一部分:pea3基因在大鼠肾脏发育中的时空表达规律
目的观察pea3在大鼠肾脏发育中的时空表达规律。
方法选择E13(embryo day 13,胚胎第13天)、E15、E17、E19胚胎鼠肾脏和P0(postnatal day 0,生后0天,即新生鼠)、P7、P14、P21及成年鼠肾。分别提取不同时期肾组织总RNA及总蛋白,用于Real-time PCR及western blot,并以不同时期的肾组织进行原位杂交,观察不同时期pea3在大鼠肾脏发育中的时空表达规律。
结果Real-time PCR及western blot结果显示:pea3在E13表达很低,从E15至P0显著高表达,至P7表达又显著下调,后逐渐减少,成年大鼠肾脏中几乎不表达;原位杂交结果显示:E15,pea3弥漫表达于输尿管芽分枝及间充质;E17,集中于输尿管芽分枝、其周围间充质及髓质区;E19,主要局限于输尿管芽分枝的顶端,髓质区表达下降;P0,则局限于皮质生肾区,髓质仍可见弱阳性;P7,表达明显下降,只在外皮质的间质区有弱表达;在P14、P21和成年未见明显表达。
结论随着大鼠肾脏组织的不断成熟,pea3基因表达逐渐下调,提示该基因可能参与肾脏的发生发育,其在输尿管芽-后肾间充质相互作用时期(E15~P0)呈高表达,且主要表达在输尿管芽分枝端和浓缩的后肾间充质,提示该基因可能参与肾脏发育中上皮-基质之间相互诱导的发育过程。
第二部分: pea3基因在大鼠肾脏发育中的作用及机制
目的本研究旨在前期研究基础上探讨pea3基因在肾脏发育中的作用及可能的机制。
方法(1)应用Real-time PCR技术检测pea3基因在新生大鼠不同组织中的表达;(2)应用双标免疫荧光技术观察pea3基因和wt1基因在大鼠肾脏不同发育时期的表达;(3)人重组wnt3a诱导RIMM-18细胞(大鼠胚胎肾脏间充质细胞株)0~2天,在不同时间点分别做以下观察:a.形态学观察;b.应用Real-time PCR及western blot技术检测pea3、wt1、E-cadherin及vimentin基因的表达;(4)构建pcDNA3.1/ETV4(pea3的人同源基因)真核表达载体,转染RIMM-18细胞0~2天,以wnt3a诱导RIMM-18细胞为阳性对照,空载为阴性对照,分别做以下检测:a.应用western blot技术检测转染效率;b. 0.3%台盼蓝染色观察转染后细胞活力;c.形态学观察;d.应用western blot技术检测wt1、E-cadherin及vimentin基因的表达;(5)设计构建pea3基因的siRNA干扰序列,转染RIMM-18细胞,分别在24小时、48小时做核酸及蛋白水平检测,观察以下内容:a.应用Real-time PCR验证干扰效率及干扰后wt1基因的表达;b. western blot检测pea3、β-catenin、wt1、E-cadherin及vimentin基因的表达;(6)为进一步探讨wnt3a调节pea3的信号通路,分别用人重组wnt3a诱导RIMM-18细胞,或分别予ERK特异性抑制剂U0126及wnt/β-catenin特异性抑制剂DKK-1预处理,western blot技术检测β-catenin、p-ERK、pea3及wt1基因的表达。
结果(1)Real-time PCR观察多组织表达情况显示:pea3基因在肾、肺、脑、胰腺等在发育过程中需经历基质-上皮间相互诱导转化的组织中高表达;(2)双标免疫荧光染色发现:在E15、E17,pea3、wt1均表达于密集的后肾间充质细胞,从E19到P7,pea3和wt1都表达于输尿管芽及其分枝的上皮细胞,P14后pea3未再有阳性信号,而wt1局限于成熟的足细胞,验证了先前原位杂交的结果,并可初步提示在肾脏发育过程中pea3和wt1关系密切;(3)wnt3a诱导RIMM-18细胞2天后,细胞可见上皮化改变,Real-time PCR及western blot显示wnt3a诱导后,pea3、wt1及E-cadherin上调,vimentin下调,诱导间充质细胞向上皮细胞转分化(Mesenchymal epithelial Transition, MET)过程;(4)pea3真核表达载体转染RIMM-18细胞2天,western blot显示1:3转染效率最高,台盼蓝染色显示转染后细胞活力达95%以上,倒置显微镜观察转染2天后细胞发生上皮化改变,western blot显示转染后2天,wt1、E-cadherin上调,vimentin下调,诱导MET;(5)Real-time PCR显示代号为885的siRNA干扰效率近50%,pea3下降可致wt1表达下降,β-catenin无明显变化,vimentin稍上调,E-cadherin下调,MET过程受抑制;(6)wnt3a诱导RIMM-18细胞后3~6小时,p-ERK活化,应用U0126后,pea3、wt1表达下降;而应用DKK-1后,pea3、wt1无显著改变。
结论(1)已知wt1在胚胎肾发育中起关键作用,而pea3则可通过wt1在肾脏发育间质-上皮转化过程中发挥重要作用;(2)在肾发育间质-上皮转分化过程中,wnt3a可通过ERK途径上调pea3,并进一步上调wt1,但不能排除也存在其他的交叉信号途径共同调节肾发育中的间质-上皮间的转分化过程。
第三部分: pea3基因在斑马鱼前肾发育中作用及机制的探讨
目的在体观察pea3基因在斑马鱼前肾不同发育阶段的时空表达,并探讨其在斑马鱼前肾发育中的作用及机制,进一步验证先前研究结果,从而为肾脏的发育及某些肾脏疾病的发生机制提供新的理论依据及治疗靶标。
方法(1)选择25hpf(hours post fertilization,受精后25小时)、36hpf、51hpf,双色原位杂交技术检测pea3、wt1a在斑马鱼前肾不同发育时期中的表达,RT-PCR方法检测pea3在成年斑马鱼中肾组织中的表达;(2)予显微注射pea3 mRNA及对照mRNA,原位杂交分别检测24hpf、48hpf时,斑马鱼前肾标记基因wt1a、podocin、pax2.1、cdh17的表达;(3)予显微注射吗啡啉RNA(morpholino, MO)的方法抑制pea3及erm的表达,原位杂交观察wt1a及cdh17在24hpf及48hpf时的表达,Real-time PCR检测wt1a、podocin在显微注射后7dpf斑马鱼中的表达,随后分三组:分别注射对照、pea3/erm morpholino及pea3/erm morpholino + pea3 mRNA,原位杂交观察上述标记基因的表达;(4)分四组:分别注射对照、wt1a mRNA、pea3/erm morpholino及pea3/erm morpholino + wt1a mRNA,原位杂交观察48hpf时podocin、cdh17的表达。
结果(1)双色原位杂交观察斑马鱼前肾不同发育时期pea3、wt1a的表达:25hpf,wt1a大量表达于前肾小球原基,pea3未见表达;36hpf,pea3、wt1a均表达于前肾小球原基,wt1a较pea3丰富;51hpf,二者均表达于前肾小球,pea3表达较前增加;RT-PCR显示斑马鱼中肾亦见pea3表达;(2)显微注射pea3 mRNA,原位杂交观察:斑马鱼前肾发育延迟,wt1a、podocin分布不规则,未能在中线正常融合,pax2.1、cdh17表达无显著异常;(3)反向应用morpholino降低目的基因表达部分,同时注射pea3/erm morpholino,可致部分胚胎发生心包水肿,原位杂交观察:wt1a、podocin的表达面积显著减少;两肾小球间距离异常增宽,未能正常融合;cdh17和pax2.1表达亦无明显改变,仅单独注射pea3或erm morpholino时,前肾小球发育无显著影响;同时注射pea3 mRNA及pea3/erm morpholino,原位杂交显示wt1a、podocin的表达恢复至与对照组类似;(4)同时注射pea3/erm morpholino及wt1 mRNA,原位杂交观察podocin的表达亦可恢复。
结论(1)pea3表达于斑马鱼前肾;(2)pea3及erm可能通过调控wt1a的表达在斑马鱼前肾发育中起重要作用,从而进一步验证了前期在大鼠中的研究结果。
Renal disease is one of the most common diseases in children. It has been demonstrated that the repair process of kidney after damage is similar to the developing bioresearch. So investigating the nephrogenesis from the developmental biology will be benifitial for studying the mechanism of renal dysplasia and disease, and recently the development of kidney attract more and more attention.
We start to study the development of kidney from 2003, applied the suppression subtractive hybridization to build the neonatal renal and post-natal day 21 renal subtractive library to screen the new developmental genes. We found that the expression of pea3 (polyomavirus enhancer activator3) was upregulated in the neonatal renal subtractive library by bioinformatics.
Transcription factor pea3 belongs to the PEA3 subgroup of the Ets family, and Ets family proteins have been shown to play an important role in different biosystems , including regulating the growth, transcription, the activation of T cell, and the development of organs and so on. The expression of pea3 mainly located in the murine developing organs, especially in epithelial–mesenchymal interaction events, for example, kidney, lung and beast, which indicates that it plays a key role in these organogenesis. However, the role of pea3 in kidney development has not yet been clarified. Recent study has showed the close relationship between pea3 and other two moleculars (wnt and wt1, both of them are important to the kidney development). Here we firstly observe the spatial and temporal expression of pea3 during the development of rat kidney, then study it’s role and the relationship among pea3, wnt and wt1 by stimulation with wnt3a, overexpression of pea3 expression vector, and downregulation of pea3 by siRNA, respectively. We also apply zebrafish as the model to study the action and mechanism of pea3 in kidney development in vivo by micro-injection of pea3 mRNA or downregulation of pea3.
Part I: The temporal and spatial expression pattern of pea3 in rat kidney development
Object To explore the temporal and spatial expression pattern of pea3 in rat kidney development.
Methods Kidneys were dissected from embryos at E13, E15, E17 and E19, and from postnatal days P0, P7, P14, P21 and adult rats. Expression of pea3 was evaluated by real-time RT-PCR (polymerase chain reaction), western blot and in situ hybridization analysis.
Results Both real-time RT- PCR and western blot analyses revealed that pea3 exhibited dynamic developmental regulation, with high levels of expression from embryonic day E15 until birth, and declining levels thereafter. By in situ hybridization, pea3 mRNA was detected in the ureteric bud (UB) and surrounding metanephric mesenchyme of the kidneys from E15 until birth, but was undetectable in mature kidneys.
Conclusion These studies suggest that the newly identified gene pea3 may be involved in rat kidney development and differentiation, specially during the epthelial-mesenchymal interaction.
Part II: Role and mechanisms of pea3 played in rat kidney development
Object To develop the role of pea3 played in rat kidney development and it’s mechanisms.
Methods (1) The expression of pea3 in the different tissues of neonatal rat was investigated by real-time RT-PCR; (2) Pea3 and wt1 expression patterns during the rat kidney development was detected by double-immunofluorescence analysis; (3) RIMM-18 cells were incubated with wnt3a (100ng/ml) for 0-2 days, then the change of morphology was detected and the expression of pea3, wt1, E-cadherin and vimentin was examined by real-time RT-PCR and western blot, respectively; (4) The pea3 expression vector was cloned, then transfected into RIMM-18 cells, the change of morphology was detected and the expression of pea3, wt1, E-cadherin and vimentin was examined by real-time RT-PCR and western blot, respectively. Cells treated with wnt3a were used as the positive control, then observe cells’morphology and detect the expression of wt1, E-cadherin and vimentin by western blot; (5) The pea3 siRNA was synthesized, then transfected into RIMM-18 cells in the presence or absence of wnt3a, and the change of morphology was detected and the expression of pea3,β-catenin, wt1, E-cadherin and vimentin was examined by real-time RT-PCR and western blot, respectively; (6) To develop the signal pathway of wnt3a regulating pea3, RIMM-18 cells were pretreated with U0126 or wnt/β-catenin specific inhibitor DKK-1, then wnt3a was added, the expression ofβ-catenin, p-ERK, pea3 and wt1 were analysized by immunoblotting for investigating the signaling pathway involved in wnt3a-regulated pea3.
Results (1) The expression of pea3 in various tissues of newborn rat by SYBR green real-time RT-PCR analysis showed that pea3 is highest in kidney, lower in lung, pancreas and brain, little in spleen and liver; (2) Double-immunofluorescence staining for pea3 and wt1 showed the expression of pea3 was similar to the result of ISH before, and the expression of wt1 has many overlaps with that of pea3. The concordant expression of pea3 and wt1 in the condensing metanephric mesenchyme and the ureteric branches was showed; (3) Real-time RT-PCR data indicated that, incubation with wnt3a for 2 days led to an increase of pea3 mRNA at 6h and a decrease tendency in vimentin mRNA. By western blot, both pea3 and wt1 were obviously upregulated after stimulated with wnt3a for 12 hours, and E-cadherin was significantly increased after 2 days treatment. The morphologic changes were assessed by phase contrast microscopy. RIMM-18 cells exhibited cobble-stone-like epithelial morphology after 2 days incubation with wnt3a; (4) pea3 had increased wt1 and E-cadherin, and downregulated vimentin expression in western blot analysis after the cells transfected with pea3 expression vector, and the cells showed cobble-stone-like epithelial morphology change, which were also similar to the results of treated with wnt3a; (5) Firstly, real-time PCR showed Pea3 siRNA no. 885 could decreased pea3 expression at 24h post-transfection, then western blot analyses revealed that wnt3a stimulation treatment increased pea3, wt1, and E-cadherin and reduced vimentin expression respectively, and these expressions were reduced in wnt3a-treated cells transfected with pea3 siRNA; (6) Western blot analyses revealed that ERK1/2 was activated after treated by incubation with wnt3a for 3h and 6h, when pretreated with U0126, the ERK1/2 specific inhibitor, blocked the increase of both pea3 and wt1 induced by wnt3a.β-catenin was also increased after treated with wnt3a. While pretreated pretreatment with Dkk-1 prior to the addition of wnt3a,β-catenin was reduced, neither p-ERK, pea3 nor wt1 expression had no obviously changed.
Conclusion The data identify that: (1) wt1 is critical for kidney development, and pea3 may be as a wt1 trans-acting factor, important for mesenchymal-epithelial transitions. (2) wnt3a can induces pea3 through activation of ERK1/2 signaling pathway, however, there are maybe another cross-talk pathways during the proceed.
Part III: Expression and function of the Ets transcription factor pea3 during the formation of the zebrafish pronephros
Object To observe the temporal and spatial expression pattern of pea3 during formation of the zebrafish pronephros, and explore the roles of pea3 in pronephrogenesis, which further verify the results before, and provide clues to pathogensis of congenital renal disease and targets for therapy.
Methods (1) zebrafish embryos at different stages (25hpf, 36hpf, 51hpf) (hpf: hour post fertilization) were collected for whole mount double in situ hybridization and mesonephros were isolated from muture zebrafish for RT-PCR; (2) zebrafish embryos were microinjected pea3 mRNA for overexpression of pea3, then the markers of zebrafish pronephros - wt1a, podocin, pax2.1 and cdh17 were detected at 24hpf and 48hpf by in situ hybridization; (3) pea3/erm morpholinos were applied to knockdown the expression of pea3 and erm, respectively, then the expression of zebrafish pronephros markers were examined at 24hpf and 48hpf by in situ hybridization. The expression of wt1a and podocin was also detected at 7dpf after microinjection of pea3/erm morpholinos. Furthermore, pea3 mRNA was microinjected in addition with pea3/erm morpholinos, then the expression of zebrafish pronephros marker genes were assessed at 24hpf and 48hpf by in situ hybridization; (4) wt1a mRNA was microinjected in addition with pea3/erm morpholinos, then the expression of podocin and cdh17 was detected at 48hpf by in situ hybridization.
Results (1) By double in situ hybridization, pea3 mRNA was undetectedable at 25 hpf in pronephron, while wt1a is abundant in pronephron primordia. Both pea3 and wt1a expressed in glomerular primordia at 36 hpf, the area of pea3 expression was smaller than wt1a. Pea3 was expressed in pronephric glomerular at 51 hpf and had overlappping expression with wt1a. Pea3 also expressed in mature zebrafish mesonephros by RT-PCR; (2) Overexpression of pea3 by pea3 mRNA, embryos delayed the development. The distribution of wt1a and podocin was abnormal by in situ hybridization. Glomerular primordia of both sides were abnormal fusion. In addition, AP staining showed the distance between the two anteriors of pronephric tubulars also became larger, while the expression of pax2.1 and cdh17 had no obviously change; (3) Simultaneous microinjection of MO-pea3 and MO-erm could lead to the delayed development of embryos, some embryos appared pericardial edema, and in situ hybridization analysis showed the area of wt1a and podocin expression was reduced notablely. Moreover, the width between the two glomerulars was enlarged, and could not fuse normally at 48 hpf. However, the expression of cdh17 and pax2.1 was not reduced strikingly. While injected with single MO-pea3 or MO-erm, the development of embyos had no signally affected. In addition, when simultaneous microinjected of pea3/erm co-morpholinos with pea3 mRNA, the defects caused by pea3/erm co-morpholino were rescued; (4) Microinjection of wt1a mRNA in addition with pea3/erm co-morpholinos, the expression of podocin almost returned to the normal by in situ hybridization.
Conclusion In summary, we firstly observed pea3 expressed in zebrafish pronephron, and the pea3 subgroup of Ets family, including pea3 and erm, were required for the pronephrogenesis maybe through wt1a.
本课题小组自2003年起开展肾脏发育相关基因的筛选工作,采用抑制性差减杂交(suppression subtractive hybridization ,SSH)技术构建了新生大鼠肾和生后21天大鼠肾脏组织发育差减文库,通过生物信息学分析,在新生鼠肾文库中,发现了与胚胎发育相关的pea3(polyomavirus enhancer activator3)基因。
pea3基因属于Ets癌基因家族成员,目前认为Ets家族蛋白在不同的生物体内可调控生长、转录、T细胞活化和器官发育等。pea3主要表达于鼠类多个发育中的器官,尤其是那些需经历上皮-基质相互作用的组织,如肺脏及乳腺,表明其在这些器官的形成中起重要作用。目前pea3在肾脏发育中的作用及机制,国内外尚未见报导。研究显示,pea3与wnt及wt1(肾脏发育中的两个关键蛋白)关系密切。本研究首先观察了pea3在大鼠肾脏不同发育阶段的时空表达规律;分别予wnt3a诱导、转染pea3真核表达载体、转染pea3 siRNA等方法在整体和细胞水平,进一步探讨该基因表达改变对胚胎肾脏发育的影响,及其与wnt、wt1之间的关系;最后应用斑马鱼为模型,显微注射pea3 mRNA及morpholino,在体观察pea3对肾脏发育的作用及机制。对该基因的研究有助于了解先天性肾脏发育不良和后天性肾脏疾病的病理机制,为基因治疗提供新的靶标。
第一部分:pea3基因在大鼠肾脏发育中的时空表达规律
目的观察pea3在大鼠肾脏发育中的时空表达规律。
方法选择E13(embryo day 13,胚胎第13天)、E15、E17、E19胚胎鼠肾脏和P0(postnatal day 0,生后0天,即新生鼠)、P7、P14、P21及成年鼠肾。分别提取不同时期肾组织总RNA及总蛋白,用于Real-time PCR及western blot,并以不同时期的肾组织进行原位杂交,观察不同时期pea3在大鼠肾脏发育中的时空表达规律。
结果Real-time PCR及western blot结果显示:pea3在E13表达很低,从E15至P0显著高表达,至P7表达又显著下调,后逐渐减少,成年大鼠肾脏中几乎不表达;原位杂交结果显示:E15,pea3弥漫表达于输尿管芽分枝及间充质;E17,集中于输尿管芽分枝、其周围间充质及髓质区;E19,主要局限于输尿管芽分枝的顶端,髓质区表达下降;P0,则局限于皮质生肾区,髓质仍可见弱阳性;P7,表达明显下降,只在外皮质的间质区有弱表达;在P14、P21和成年未见明显表达。
结论随着大鼠肾脏组织的不断成熟,pea3基因表达逐渐下调,提示该基因可能参与肾脏的发生发育,其在输尿管芽-后肾间充质相互作用时期(E15~P0)呈高表达,且主要表达在输尿管芽分枝端和浓缩的后肾间充质,提示该基因可能参与肾脏发育中上皮-基质之间相互诱导的发育过程。
第二部分: pea3基因在大鼠肾脏发育中的作用及机制
目的本研究旨在前期研究基础上探讨pea3基因在肾脏发育中的作用及可能的机制。
方法(1)应用Real-time PCR技术检测pea3基因在新生大鼠不同组织中的表达;(2)应用双标免疫荧光技术观察pea3基因和wt1基因在大鼠肾脏不同发育时期的表达;(3)人重组wnt3a诱导RIMM-18细胞(大鼠胚胎肾脏间充质细胞株)0~2天,在不同时间点分别做以下观察:a.形态学观察;b.应用Real-time PCR及western blot技术检测pea3、wt1、E-cadherin及vimentin基因的表达;(4)构建pcDNA3.1/ETV4(pea3的人同源基因)真核表达载体,转染RIMM-18细胞0~2天,以wnt3a诱导RIMM-18细胞为阳性对照,空载为阴性对照,分别做以下检测:a.应用western blot技术检测转染效率;b. 0.3%台盼蓝染色观察转染后细胞活力;c.形态学观察;d.应用western blot技术检测wt1、E-cadherin及vimentin基因的表达;(5)设计构建pea3基因的siRNA干扰序列,转染RIMM-18细胞,分别在24小时、48小时做核酸及蛋白水平检测,观察以下内容:a.应用Real-time PCR验证干扰效率及干扰后wt1基因的表达;b. western blot检测pea3、β-catenin、wt1、E-cadherin及vimentin基因的表达;(6)为进一步探讨wnt3a调节pea3的信号通路,分别用人重组wnt3a诱导RIMM-18细胞,或分别予ERK特异性抑制剂U0126及wnt/β-catenin特异性抑制剂DKK-1预处理,western blot技术检测β-catenin、p-ERK、pea3及wt1基因的表达。
结果(1)Real-time PCR观察多组织表达情况显示:pea3基因在肾、肺、脑、胰腺等在发育过程中需经历基质-上皮间相互诱导转化的组织中高表达;(2)双标免疫荧光染色发现:在E15、E17,pea3、wt1均表达于密集的后肾间充质细胞,从E19到P7,pea3和wt1都表达于输尿管芽及其分枝的上皮细胞,P14后pea3未再有阳性信号,而wt1局限于成熟的足细胞,验证了先前原位杂交的结果,并可初步提示在肾脏发育过程中pea3和wt1关系密切;(3)wnt3a诱导RIMM-18细胞2天后,细胞可见上皮化改变,Real-time PCR及western blot显示wnt3a诱导后,pea3、wt1及E-cadherin上调,vimentin下调,诱导间充质细胞向上皮细胞转分化(Mesenchymal epithelial Transition, MET)过程;(4)pea3真核表达载体转染RIMM-18细胞2天,western blot显示1:3转染效率最高,台盼蓝染色显示转染后细胞活力达95%以上,倒置显微镜观察转染2天后细胞发生上皮化改变,western blot显示转染后2天,wt1、E-cadherin上调,vimentin下调,诱导MET;(5)Real-time PCR显示代号为885的siRNA干扰效率近50%,pea3下降可致wt1表达下降,β-catenin无明显变化,vimentin稍上调,E-cadherin下调,MET过程受抑制;(6)wnt3a诱导RIMM-18细胞后3~6小时,p-ERK活化,应用U0126后,pea3、wt1表达下降;而应用DKK-1后,pea3、wt1无显著改变。
结论(1)已知wt1在胚胎肾发育中起关键作用,而pea3则可通过wt1在肾脏发育间质-上皮转化过程中发挥重要作用;(2)在肾发育间质-上皮转分化过程中,wnt3a可通过ERK途径上调pea3,并进一步上调wt1,但不能排除也存在其他的交叉信号途径共同调节肾发育中的间质-上皮间的转分化过程。
第三部分: pea3基因在斑马鱼前肾发育中作用及机制的探讨
目的在体观察pea3基因在斑马鱼前肾不同发育阶段的时空表达,并探讨其在斑马鱼前肾发育中的作用及机制,进一步验证先前研究结果,从而为肾脏的发育及某些肾脏疾病的发生机制提供新的理论依据及治疗靶标。
方法(1)选择25hpf(hours post fertilization,受精后25小时)、36hpf、51hpf,双色原位杂交技术检测pea3、wt1a在斑马鱼前肾不同发育时期中的表达,RT-PCR方法检测pea3在成年斑马鱼中肾组织中的表达;(2)予显微注射pea3 mRNA及对照mRNA,原位杂交分别检测24hpf、48hpf时,斑马鱼前肾标记基因wt1a、podocin、pax2.1、cdh17的表达;(3)予显微注射吗啡啉RNA(morpholino, MO)的方法抑制pea3及erm的表达,原位杂交观察wt1a及cdh17在24hpf及48hpf时的表达,Real-time PCR检测wt1a、podocin在显微注射后7dpf斑马鱼中的表达,随后分三组:分别注射对照、pea3/erm morpholino及pea3/erm morpholino + pea3 mRNA,原位杂交观察上述标记基因的表达;(4)分四组:分别注射对照、wt1a mRNA、pea3/erm morpholino及pea3/erm morpholino + wt1a mRNA,原位杂交观察48hpf时podocin、cdh17的表达。
结果(1)双色原位杂交观察斑马鱼前肾不同发育时期pea3、wt1a的表达:25hpf,wt1a大量表达于前肾小球原基,pea3未见表达;36hpf,pea3、wt1a均表达于前肾小球原基,wt1a较pea3丰富;51hpf,二者均表达于前肾小球,pea3表达较前增加;RT-PCR显示斑马鱼中肾亦见pea3表达;(2)显微注射pea3 mRNA,原位杂交观察:斑马鱼前肾发育延迟,wt1a、podocin分布不规则,未能在中线正常融合,pax2.1、cdh17表达无显著异常;(3)反向应用morpholino降低目的基因表达部分,同时注射pea3/erm morpholino,可致部分胚胎发生心包水肿,原位杂交观察:wt1a、podocin的表达面积显著减少;两肾小球间距离异常增宽,未能正常融合;cdh17和pax2.1表达亦无明显改变,仅单独注射pea3或erm morpholino时,前肾小球发育无显著影响;同时注射pea3 mRNA及pea3/erm morpholino,原位杂交显示wt1a、podocin的表达恢复至与对照组类似;(4)同时注射pea3/erm morpholino及wt1 mRNA,原位杂交观察podocin的表达亦可恢复。
结论(1)pea3表达于斑马鱼前肾;(2)pea3及erm可能通过调控wt1a的表达在斑马鱼前肾发育中起重要作用,从而进一步验证了前期在大鼠中的研究结果。
Renal disease is one of the most common diseases in children. It has been demonstrated that the repair process of kidney after damage is similar to the developing bioresearch. So investigating the nephrogenesis from the developmental biology will be benifitial for studying the mechanism of renal dysplasia and disease, and recently the development of kidney attract more and more attention.
We start to study the development of kidney from 2003, applied the suppression subtractive hybridization to build the neonatal renal and post-natal day 21 renal subtractive library to screen the new developmental genes. We found that the expression of pea3 (polyomavirus enhancer activator3) was upregulated in the neonatal renal subtractive library by bioinformatics.
Transcription factor pea3 belongs to the PEA3 subgroup of the Ets family, and Ets family proteins have been shown to play an important role in different biosystems , including regulating the growth, transcription, the activation of T cell, and the development of organs and so on. The expression of pea3 mainly located in the murine developing organs, especially in epithelial–mesenchymal interaction events, for example, kidney, lung and beast, which indicates that it plays a key role in these organogenesis. However, the role of pea3 in kidney development has not yet been clarified. Recent study has showed the close relationship between pea3 and other two moleculars (wnt and wt1, both of them are important to the kidney development). Here we firstly observe the spatial and temporal expression of pea3 during the development of rat kidney, then study it’s role and the relationship among pea3, wnt and wt1 by stimulation with wnt3a, overexpression of pea3 expression vector, and downregulation of pea3 by siRNA, respectively. We also apply zebrafish as the model to study the action and mechanism of pea3 in kidney development in vivo by micro-injection of pea3 mRNA or downregulation of pea3.
Part I: The temporal and spatial expression pattern of pea3 in rat kidney development
Object To explore the temporal and spatial expression pattern of pea3 in rat kidney development.
Methods Kidneys were dissected from embryos at E13, E15, E17 and E19, and from postnatal days P0, P7, P14, P21 and adult rats. Expression of pea3 was evaluated by real-time RT-PCR (polymerase chain reaction), western blot and in situ hybridization analysis.
Results Both real-time RT- PCR and western blot analyses revealed that pea3 exhibited dynamic developmental regulation, with high levels of expression from embryonic day E15 until birth, and declining levels thereafter. By in situ hybridization, pea3 mRNA was detected in the ureteric bud (UB) and surrounding metanephric mesenchyme of the kidneys from E15 until birth, but was undetectable in mature kidneys.
Conclusion These studies suggest that the newly identified gene pea3 may be involved in rat kidney development and differentiation, specially during the epthelial-mesenchymal interaction.
Part II: Role and mechanisms of pea3 played in rat kidney development
Object To develop the role of pea3 played in rat kidney development and it’s mechanisms.
Methods (1) The expression of pea3 in the different tissues of neonatal rat was investigated by real-time RT-PCR; (2) Pea3 and wt1 expression patterns during the rat kidney development was detected by double-immunofluorescence analysis; (3) RIMM-18 cells were incubated with wnt3a (100ng/ml) for 0-2 days, then the change of morphology was detected and the expression of pea3, wt1, E-cadherin and vimentin was examined by real-time RT-PCR and western blot, respectively; (4) The pea3 expression vector was cloned, then transfected into RIMM-18 cells, the change of morphology was detected and the expression of pea3, wt1, E-cadherin and vimentin was examined by real-time RT-PCR and western blot, respectively. Cells treated with wnt3a were used as the positive control, then observe cells’morphology and detect the expression of wt1, E-cadherin and vimentin by western blot; (5) The pea3 siRNA was synthesized, then transfected into RIMM-18 cells in the presence or absence of wnt3a, and the change of morphology was detected and the expression of pea3,β-catenin, wt1, E-cadherin and vimentin was examined by real-time RT-PCR and western blot, respectively; (6) To develop the signal pathway of wnt3a regulating pea3, RIMM-18 cells were pretreated with U0126 or wnt/β-catenin specific inhibitor DKK-1, then wnt3a was added, the expression ofβ-catenin, p-ERK, pea3 and wt1 were analysized by immunoblotting for investigating the signaling pathway involved in wnt3a-regulated pea3.
Results (1) The expression of pea3 in various tissues of newborn rat by SYBR green real-time RT-PCR analysis showed that pea3 is highest in kidney, lower in lung, pancreas and brain, little in spleen and liver; (2) Double-immunofluorescence staining for pea3 and wt1 showed the expression of pea3 was similar to the result of ISH before, and the expression of wt1 has many overlaps with that of pea3. The concordant expression of pea3 and wt1 in the condensing metanephric mesenchyme and the ureteric branches was showed; (3) Real-time RT-PCR data indicated that, incubation with wnt3a for 2 days led to an increase of pea3 mRNA at 6h and a decrease tendency in vimentin mRNA. By western blot, both pea3 and wt1 were obviously upregulated after stimulated with wnt3a for 12 hours, and E-cadherin was significantly increased after 2 days treatment. The morphologic changes were assessed by phase contrast microscopy. RIMM-18 cells exhibited cobble-stone-like epithelial morphology after 2 days incubation with wnt3a; (4) pea3 had increased wt1 and E-cadherin, and downregulated vimentin expression in western blot analysis after the cells transfected with pea3 expression vector, and the cells showed cobble-stone-like epithelial morphology change, which were also similar to the results of treated with wnt3a; (5) Firstly, real-time PCR showed Pea3 siRNA no. 885 could decreased pea3 expression at 24h post-transfection, then western blot analyses revealed that wnt3a stimulation treatment increased pea3, wt1, and E-cadherin and reduced vimentin expression respectively, and these expressions were reduced in wnt3a-treated cells transfected with pea3 siRNA; (6) Western blot analyses revealed that ERK1/2 was activated after treated by incubation with wnt3a for 3h and 6h, when pretreated with U0126, the ERK1/2 specific inhibitor, blocked the increase of both pea3 and wt1 induced by wnt3a.β-catenin was also increased after treated with wnt3a. While pretreated pretreatment with Dkk-1 prior to the addition of wnt3a,β-catenin was reduced, neither p-ERK, pea3 nor wt1 expression had no obviously changed.
Conclusion The data identify that: (1) wt1 is critical for kidney development, and pea3 may be as a wt1 trans-acting factor, important for mesenchymal-epithelial transitions. (2) wnt3a can induces pea3 through activation of ERK1/2 signaling pathway, however, there are maybe another cross-talk pathways during the proceed.
Part III: Expression and function of the Ets transcription factor pea3 during the formation of the zebrafish pronephros
Object To observe the temporal and spatial expression pattern of pea3 during formation of the zebrafish pronephros, and explore the roles of pea3 in pronephrogenesis, which further verify the results before, and provide clues to pathogensis of congenital renal disease and targets for therapy.
Methods (1) zebrafish embryos at different stages (25hpf, 36hpf, 51hpf) (hpf: hour post fertilization) were collected for whole mount double in situ hybridization and mesonephros were isolated from muture zebrafish for RT-PCR; (2) zebrafish embryos were microinjected pea3 mRNA for overexpression of pea3, then the markers of zebrafish pronephros - wt1a, podocin, pax2.1 and cdh17 were detected at 24hpf and 48hpf by in situ hybridization; (3) pea3/erm morpholinos were applied to knockdown the expression of pea3 and erm, respectively, then the expression of zebrafish pronephros markers were examined at 24hpf and 48hpf by in situ hybridization. The expression of wt1a and podocin was also detected at 7dpf after microinjection of pea3/erm morpholinos. Furthermore, pea3 mRNA was microinjected in addition with pea3/erm morpholinos, then the expression of zebrafish pronephros marker genes were assessed at 24hpf and 48hpf by in situ hybridization; (4) wt1a mRNA was microinjected in addition with pea3/erm morpholinos, then the expression of podocin and cdh17 was detected at 48hpf by in situ hybridization.
Results (1) By double in situ hybridization, pea3 mRNA was undetectedable at 25 hpf in pronephron, while wt1a is abundant in pronephron primordia. Both pea3 and wt1a expressed in glomerular primordia at 36 hpf, the area of pea3 expression was smaller than wt1a. Pea3 was expressed in pronephric glomerular at 51 hpf and had overlappping expression with wt1a. Pea3 also expressed in mature zebrafish mesonephros by RT-PCR; (2) Overexpression of pea3 by pea3 mRNA, embryos delayed the development. The distribution of wt1a and podocin was abnormal by in situ hybridization. Glomerular primordia of both sides were abnormal fusion. In addition, AP staining showed the distance between the two anteriors of pronephric tubulars also became larger, while the expression of pax2.1 and cdh17 had no obviously change; (3) Simultaneous microinjection of MO-pea3 and MO-erm could lead to the delayed development of embryos, some embryos appared pericardial edema, and in situ hybridization analysis showed the area of wt1a and podocin expression was reduced notablely. Moreover, the width between the two glomerulars was enlarged, and could not fuse normally at 48 hpf. However, the expression of cdh17 and pax2.1 was not reduced strikingly. While injected with single MO-pea3 or MO-erm, the development of embyos had no signally affected. In addition, when simultaneous microinjected of pea3/erm co-morpholinos with pea3 mRNA, the defects caused by pea3/erm co-morpholino were rescued; (4) Microinjection of wt1a mRNA in addition with pea3/erm co-morpholinos, the expression of podocin almost returned to the normal by in situ hybridization.
Conclusion In summary, we firstly observed pea3 expressed in zebrafish pronephron, and the pea3 subgroup of Ets family, including pea3 and erm, were required for the pronephrogenesis maybe through wt1a.
引文
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