Tbx1基因在小鼠胚肾中的表达规律及其相互作用蛋白研究
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摘要
前言
TBX1 (GeneID:6899)基因定位于染色体22q11.21,胞内定位于细胞核,全称为T-box1,属于T-box转录因子家族,后者在种系发生和进化中趋于保守,因其成员均包含一个相同的DNA结合域,即T-box而得名。近期研究发现,TBX1基因可影响包括耳、心脏、甲状旁腺、胸腺、牙齿等多种组织和器官的发育,并在精神疾病的发病中扮演一定角色。
多项研究提示泌尿生殖系统发育缺陷与染色体22q11.2微缺失密切相关,具体的畸形可涉及肾脏、输尿管／膀胱以及尿道等,畸形发生率高达36%,显著高于自然人群。TBX1基因是定位于上述缺失关键区内的关键基因。围绕TBX1基因的研究提示,TBX1基因可能通过剂量依赖形式,影响SHH、FGF、WNT以及BMP等相关信号通路,进而调控包括肾脏、心脏、耳、胸腺、甲状旁腺在内的多种组织器官的发育。动物实验亦证实,Tbx1基因缺失或单倍剂量不足可导致类似于人类22q11.2微缺失综合征的表型。
迄今为止,围绕TBX1基因的研究主要集中于其与心脏畸形的关系,而该基因是否与泌尿生殖系统畸形的发生存在联系尚未见报道。在本研究中,我们以小鼠为对象,对Tbx1基因在肾脏发育过程中的表达进行了检测,之后,通过获取其相互作用蛋白和RNA干扰实验,对该蛋白以及其相互作用蛋白在发育中的角色进行进一步探索,以期初步揭示调控肾脏发育的分子网络。
材料与方法
一、实验材料
1、健康昆明小鼠
2、Western Blot相关试剂
3、RT-PCR实验相关试剂
4、免疫沉淀实验相关试剂
5、细胞培养及转染相关试剂
二、实验方法
1、取胚龄为E13.5d、E15.5d、E17.5d、E19.5d,新生鼠以及正常成年昆明小鼠的肾脏组织,抽提RNA,分析Tbx1基因在小鼠不同发育时间点的表达情况。
2、免疫沉淀获取Tbx1的相互作用蛋白并进行质谱分析,通过Western Blot对结果进行验证,半定量RT-PCR分析该基因的时间表达情况。
3、干扰Tbx1及其相互作用蛋白的表达以确认二者的相互影响。
实验结果
1、Tbx1基因的表达在胚鼠肾脏发育的第E15.5天达到第一个峰值,随后逐渐减弱,至出生时又剧烈增高,达到第二峰值。此外,该基因在小鼠成体肾脏中存在低水平表达。
2、在小鼠的胚肾组织中,Hoxd10与Tbx1存在蛋白之间的相互作用。
3、在小鼠成肌细胞系C2Cl2中,干扰Hoxd10可以使该基因及Tbx1的表达量下调；干扰Tbx1则可以使该基因及Hoxd10的表达下调。
结论
1、Tbx1基因的表达水平随小鼠肾脏的发育而剧烈变化,提示其可能在肾脏的发育过程中扮演重要角色,并可能在成体肾脏中发挥某种功能。
2、在小鼠的胚肾组织中,Tbx1蛋白可能与Hoxd10蛋白存在相互作用,共同调节肾脏的发育。
3、在蛋白水平,Tbx1与Hoxd10之间可能存在调节和反馈作用。
Abstract
TBX1 (GeneID:6899) gene has been mapped to chromosomal region 22q11.21. Formally known as T-boxl, it belongs to the T-box transcription factor family, which is conserved through phylogeny and evolution. These genes are named for their similar DNA binding motif, T-box. TBX1 can impact development of ear, heart, parathyroid, thymus, teeth and other tissues and organs, and plays an important role in the development of schizophrenia.
Recent research has indicated that microdeletion in 22q11.2 is strongly associated with genitourinary malformations Specific anomalies include renal malformations, metanephric duct/urinary bladder and hypospadia. Among the carriers, the incidence of above defects is as high as 36%, which is significant higher than that of the general population. TBX1 has been mapped to the critical region of the deletion and is known as an important transcription factor during the development. Research has suggested that there may be a common genetic basis for the development of various tissues and organs including the kidneys, heart, ear, thymus and parathyroid glands. TBX1 may serve in dose-dependent manner as one of the important genes in SHH, FGF, WNT and BMP signaling pathways in the early development of such tissues and organs via a common regulatory mechanism. Animal experiment also confirmed that, Tbxl knockout or haploinsufficiency can lead to phenotypes similar to those seen in human 22q11.2 deletion syndrome.
So far, research of the TBX1 gene has been focused on its relationship with heart malformations, and there has been no report with regard to its connection with the development of genitourinary system. In the present study, we have analyzed the expression of Tbxl gene during the kidney development. In addition, by identifying its interacting protein, we also explored their role in the kidney development. This may help with delineation of the regulatory genetics networks for the embryonic development of the kidneys
Material and Methods
1、To analyze the expression of Tbxl gene in kidney tissue from mouse embryos with RT-PCR;
2、To identify its interacting protein with immunoprecipitation (IP) and mass spectrometry (MALDI-TOF-MS). Subsequently, the protein was verified with Western Blotting. Expression of the interacting genes in mouse embryonic kidneys was also studied with RT-PCR;
3、To explore the relationship between Tbx1 and Hoxd10 proteins through RNA interference.
Results
1、The expression of Tbxl gene reaches the first peak at the E15.5d in embryonic mouse kidneys, then decreases gradually, and reaches the second peak just after birth. In addition, the gene also expresses in adult mouse kidney at a low level;
2、Hoxd10 and Tbxl proteins may interact during embryonic development of mouse kidneys;
3、In myoblasts C2C12 cell line, interference of either Hoxd10 or Tbxl gene can downregulates the expression of both genes
Conclusion
1、The expression of Tbxl gene changes along with the development of mouse kidney, indicating that it may play an important role. The gene may also play certain roles in the adult kidney;
2、Tbxl may interact with Hoxd10 in embryonic kidneys of mice to regulate its development and function;
3、Tbxl and Hoxd10 may interact the protein level, and feedback to each other by expression.
TBX1 (GeneID:6899)基因定位于染色体22q11.21,胞内定位于细胞核,全称为T-box1,属于T-box转录因子家族,后者在种系发生和进化中趋于保守,因其成员均包含一个相同的DNA结合域,即T-box而得名。近期研究发现,TBX1基因可影响包括耳、心脏、甲状旁腺、胸腺、牙齿等多种组织和器官的发育,并在精神疾病的发病中扮演一定角色。
多项研究提示泌尿生殖系统发育缺陷与染色体22q11.2微缺失密切相关,具体的畸形可涉及肾脏、输尿管／膀胱以及尿道等,畸形发生率高达36%,显著高于自然人群。TBX1基因是定位于上述缺失关键区内的关键基因。围绕TBX1基因的研究提示,TBX1基因可能通过剂量依赖形式,影响SHH、FGF、WNT以及BMP等相关信号通路,进而调控包括肾脏、心脏、耳、胸腺、甲状旁腺在内的多种组织器官的发育。动物实验亦证实,Tbx1基因缺失或单倍剂量不足可导致类似于人类22q11.2微缺失综合征的表型。
迄今为止,围绕TBX1基因的研究主要集中于其与心脏畸形的关系,而该基因是否与泌尿生殖系统畸形的发生存在联系尚未见报道。在本研究中,我们以小鼠为对象,对Tbx1基因在肾脏发育过程中的表达进行了检测,之后,通过获取其相互作用蛋白和RNA干扰实验,对该蛋白以及其相互作用蛋白在发育中的角色进行进一步探索,以期初步揭示调控肾脏发育的分子网络。
材料与方法
一、实验材料
1、健康昆明小鼠
2、Western Blot相关试剂
3、RT-PCR实验相关试剂
4、免疫沉淀实验相关试剂
5、细胞培养及转染相关试剂
二、实验方法
1、取胚龄为E13.5d、E15.5d、E17.5d、E19.5d,新生鼠以及正常成年昆明小鼠的肾脏组织,抽提RNA,分析Tbx1基因在小鼠不同发育时间点的表达情况。
2、免疫沉淀获取Tbx1的相互作用蛋白并进行质谱分析,通过Western Blot对结果进行验证,半定量RT-PCR分析该基因的时间表达情况。
3、干扰Tbx1及其相互作用蛋白的表达以确认二者的相互影响。
实验结果
1、Tbx1基因的表达在胚鼠肾脏发育的第E15.5天达到第一个峰值,随后逐渐减弱,至出生时又剧烈增高,达到第二峰值。此外,该基因在小鼠成体肾脏中存在低水平表达。
2、在小鼠的胚肾组织中,Hoxd10与Tbx1存在蛋白之间的相互作用。
3、在小鼠成肌细胞系C2Cl2中,干扰Hoxd10可以使该基因及Tbx1的表达量下调；干扰Tbx1则可以使该基因及Hoxd10的表达下调。
结论
1、Tbx1基因的表达水平随小鼠肾脏的发育而剧烈变化,提示其可能在肾脏的发育过程中扮演重要角色,并可能在成体肾脏中发挥某种功能。
2、在小鼠的胚肾组织中,Tbx1蛋白可能与Hoxd10蛋白存在相互作用,共同调节肾脏的发育。
3、在蛋白水平,Tbx1与Hoxd10之间可能存在调节和反馈作用。
Abstract
TBX1 (GeneID:6899) gene has been mapped to chromosomal region 22q11.21. Formally known as T-boxl, it belongs to the T-box transcription factor family, which is conserved through phylogeny and evolution. These genes are named for their similar DNA binding motif, T-box. TBX1 can impact development of ear, heart, parathyroid, thymus, teeth and other tissues and organs, and plays an important role in the development of schizophrenia.
Recent research has indicated that microdeletion in 22q11.2 is strongly associated with genitourinary malformations Specific anomalies include renal malformations, metanephric duct/urinary bladder and hypospadia. Among the carriers, the incidence of above defects is as high as 36%, which is significant higher than that of the general population. TBX1 has been mapped to the critical region of the deletion and is known as an important transcription factor during the development. Research has suggested that there may be a common genetic basis for the development of various tissues and organs including the kidneys, heart, ear, thymus and parathyroid glands. TBX1 may serve in dose-dependent manner as one of the important genes in SHH, FGF, WNT and BMP signaling pathways in the early development of such tissues and organs via a common regulatory mechanism. Animal experiment also confirmed that, Tbxl knockout or haploinsufficiency can lead to phenotypes similar to those seen in human 22q11.2 deletion syndrome.
So far, research of the TBX1 gene has been focused on its relationship with heart malformations, and there has been no report with regard to its connection with the development of genitourinary system. In the present study, we have analyzed the expression of Tbxl gene during the kidney development. In addition, by identifying its interacting protein, we also explored their role in the kidney development. This may help with delineation of the regulatory genetics networks for the embryonic development of the kidneys
Material and Methods
1、To analyze the expression of Tbxl gene in kidney tissue from mouse embryos with RT-PCR;
2、To identify its interacting protein with immunoprecipitation (IP) and mass spectrometry (MALDI-TOF-MS). Subsequently, the protein was verified with Western Blotting. Expression of the interacting genes in mouse embryonic kidneys was also studied with RT-PCR;
3、To explore the relationship between Tbx1 and Hoxd10 proteins through RNA interference.
Results
1、The expression of Tbxl gene reaches the first peak at the E15.5d in embryonic mouse kidneys, then decreases gradually, and reaches the second peak just after birth. In addition, the gene also expresses in adult mouse kidney at a low level;
2、Hoxd10 and Tbxl proteins may interact during embryonic development of mouse kidneys;
3、In myoblasts C2C12 cell line, interference of either Hoxd10 or Tbxl gene can downregulates the expression of both genes
Conclusion
1、The expression of Tbxl gene changes along with the development of mouse kidney, indicating that it may play an important role. The gene may also play certain roles in the adult kidney;
2、Tbxl may interact with Hoxd10 in embryonic kidneys of mice to regulate its development and function;
3、Tbxl and Hoxd10 may interact the protein level, and feedback to each other by expression.
引文
1 Himmetoglu O, Tiras MB, Gursoy R, et al. The incidence of congenital malformations in a Turkish population. Int J Gynaecol Obstet.1996; 55:117-121.
2 Minguillon C, Logan M. The comparative genomics of T-box genes. Brief Funct Genomic Proteomic.2003; 2(3):224-233.
3 Merscher S, Funke B, Epstein JA, et al. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell.2001; 104:619-629.
4 Lindsay, E. A. Chromosome microdeletions:dissecting del22q11 syndrome. Nat. Rev. Genet. 2001; 2:858-868.
5 Zweier C, Sticht H, Aydin-Yaylagiil I, et al. Human TBX1 missense mutations cause gain of function resulting in the same phenotype as 22q11.2 deletions. Am J Hum Genet.2007; 80(3): 510-517.
6 Zhang Z, Huynh T, Baldini A. Mesodermal expression of Tbxl is necessary and sufficient for pharyngeal arch and cardiac outflow tract development. Development.2006; 133(18): 3587-3595.
7 Torres-Juan L, Rosell J, Morla M, et al. Mutations in TBX1 genocopy the 22q11.2 deletion and duplication syndromes:a new susceptibility factor for mental retardation. Eur J Hum Genet. 2007; 15(6):658-663.
8 Kobrynski LJ, Sullivan KE. Velocardiofacial syndrome, DiGeorge syndrome:the chromosome 22q11.2 deletion syndromes. Lancet.2007;370(9596):1443-1452.
9 Wu HY, Rusnack SL, Bellah RD, et al. Genitourinary malformations in chromosome 22q11.2 deletion. J Urol.2002; 168(6):2564-2565.
10 Kujat A, Schulz MD, Strenge S, et al. Renal malformations in deletion 22q11.2 patients. Am J Med Genet.2006; 140(14):1601-1602.
11 Jerome LA, Papaioannou VE. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat Genet.2001; 27(3):286-291.
12 Kerstjens-Frederikse WS, kurahashi H, Driscoll DA, et al. Microdeletion 22q11.2:clinical data and deletion size. J Med Genet.1999; 36:721-723.
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15 Merscher S, Funke B, Epstein JA, et al. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell.2001; 104:619-629.
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21 Merscher S, Funke B, Epstein JA, et al. TBX1 is responsible for cardio vascular defects in velo-cardio-facial DiGeorge syndrome. Cell.2001; 104:619-629.
22 Baldini A. DiGeorge syndrome:the use of model organisms to dissect complex genetics. Hum Mol Genet.2002; 11 (20):2363-2369.
23 Funke B, Epstein JA, Kochilas LK, et al. Mice overexpressing genes from the 22q11 region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle and inner ear defects. Hum Mol Genet.2001; 10(22):2549-2556.
24 Liao J, Kochilas L, Nowotschin S, et al. Full spectrum of malformations in velo-cardio-facial syndrome/DiGeorge syndrome mouse models by altering Tbx1 dosage. Hum Mol Genet.2004; 13(15):1577-1585.
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1 Minguillon C, Logan M. The comparative genomics of T-box genes. Brief Funct Genomic Proteomic.2003; 2(3):224-233.
2 Chieffo C, Garvey N, Gong W, et al. Isolation and characterization of a gene from the DiGeorge chromosomal region homologous to the mouse Tbxl gene. Genomics.1997; 43 (3): 267-277.
3 张立凤,桂永浩.Tbxl基因和DiGeorge综合征.国外医学遗传学分册.2005；28(5)：299-301.
4 Merscher S, Funke B, Epstein JA, et al. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell.2001; 104:619-629.
5 Lindsay E. A. Chromosome microdeletions:dissecting del22q11 syndrome. Nat. Rev. Genet. 2001; 2:858-868.
6 Zweier C, Sticht H, Aydin-Yaylagiil I, et al. Human TBX1 missense mutations cause gain of function resulting in the same phenotype as 22q11.2 deletions. Am J Hum Genet.2007; 80(3): 510-517.
7 Zhang Z, Huynh T, Baldini A. Mesodermal expression of Tbxl is necessary and sufficient for pharyngeal arch and cardiac outflow tract development. Development.2006; 133(18): 3587-3595.
8 Torres-Juan L, Rosell J, Morla M, et al. Mutations in TBX1 genocopy the 22q11.2 deletion and duplication syndromes:a new susceptibility factor for mental retardation. Eur J Hum Genet. 2007; 15(6):658-663.
9 Lindsay EA, Vitelli F, Su H, et al. Tbxl haploinsufficiency in the DiGeorge syndrome region causes aortic arch defects in mice. Nature.2001; 410:97-101.
10 Paylor R, Glaser B, Mupo A, et al. Tbxl haploinsufficiency is linked to behavioral disorders in mice and humans:implications for 22q11 deletion syndrome. Proc Natl Acad Sci U S A.2006; 103:7729-7734.
11 Baldini A. DiGeorge syndrome:the use of model organisms to dissect complex genetics Hum Mol Genet.2002; 11(20):2363-2369.
12 Funke B, Epstein JA, Kochilas LK, et al. Mice overexpressing genes from the 22q11 region deleted in velo-cardio-facial syndrome/DiGeorge syndrome have middle and inner ear defects Hum Mol Genet.2001; 10(22):2549-2556.
13 Liao J, Kochilas L, Nowotschin S, et al. Full spectrum of malformations in velo-cardio-facial syndrome/DiGeorge syndrome mouse models by altering Tbx 1 dosage. Hum Mol Genet.2004; 13(15):1577-1585.
14 Jerome LA, Papaioannou VE. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx 1. Nat Genet.2001; 27(3):286-291.
15 Vitelli F, Morishima M, Taddei I, et al. Tbx 1 mutation causes multiple cardiovascular defects and disrupts neural crest and cranial nerve migratory pathways. Hum Mol Genet.2002; 11(8): 915-922.
16 Moraes F, Novoa A, Jerome-Majewska LA, et al. Tbxl is required for proper neural crest migration and to stabilize spatial patterns during middle and inner ear development. Mech Dev. 2005; 122(2):199-212.
17 Kochilas LK, Potluri V, Gitler A, et al. Cloning and characterization of zebrafish tbxl. Gene Expr Patterns.2003; 3(5):645-651.
18 Piotrowski T, Ahn DG, Schilling TF, et al. The zebrafish van gogh mutation disrupts tbxl, which is involved in the DiGeorge deletion syndrome in humans. Development.2003; 130(20): 5043-5052.
19 Chapman DL, Garvey N, Hancock S, et al. Expression of the T-box family genes, Tbxl-Tbx5, during early mouse development. Dev Dyn.1996; 206(4):379-390.
20 Sauka-Spengler T, Le Mentec C, Lepage M, et al. Embryonic expression of Tbx1, a DiGeorge syndrome candidate gene, in the lamprey Lampetra fluviatilis. Gene Expr Patterns.2002; 2(1-2):99-103.
21 Garg V, Yamagishi C, Hu T, et al. Tbxl, a DiGeorge syndrome candidate gene, is regulated by sonic hedgehog during pharyngeal arch development. Dev Biol.2001; 235(1):62-73.
22 Riccomagno MM, Martinu L, Mulheisen M, et al. Specification of the mammalian cochlea is dependent on Sonic hedgehog. Genes Dev.2002; 16(18):2365-2378.
23 张立凤,桂永浩,钟涛等.外源性视黄酸对斑马鱼Tbxl基因的影响.中华儿科杂志.2007；45(4)：267-271.
24 Liu W, Levi G, Shanske A, et al. Retinoic acid-induced inner ear teratogenesis caused by defective Fgf3/Fgf10-dependent Dlx5 signaling. Birth Defects Res B Dev Reprod Toxicol. 2008; 83(2):134-144.
25 Vitelli F, Viola A, Morishima M, et al. TBX1 is required for inner ear morphogenesis Hum Mol Genet.2003; 12(16):2041-2048.
26 Raft S, Nowotschin S, Liao J, et al. Suppression of neural fate and control of inner ear morphogenesis by Tbx1. Development.2004; 131(8):1801-1812.
27 Arnold JS, Braunstein EM, Ohyama T, et al. Tissue-specific roles of Tbx1 in the development of the outer, middle and inner ear, defective in 22q11DS patients. Hum Mol Genet.2006; 15(10):1629-1639.
28 Xu H, Viola A, Zhang Z, et al. Tbx1 regulates population, proliferation and cell fate determination of otic epithelial cells. Dev Biol.2007; 302(2):670-682.
29 Xu H, Chen L, Baldini A. In vivo genetic ablation of the periotic mesoderm affects cell proliferation survival and differentiation in the cochlea. Dev Biol.2007; 310(2):329-340.
30 Baldini A. Dissecting contiguous gene defects:TBX1. Curr Opin Genet Dev.2005; 15(3): 279-284.
2 Minguillon C, Logan M. The comparative genomics of T-box genes. Brief Funct Genomic Proteomic.2003; 2(3):224-233.
3 Merscher S, Funke B, Epstein JA, et al. TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome. Cell.2001; 104:619-629.
4 Lindsay, E. A. Chromosome microdeletions:dissecting del22q11 syndrome. Nat. Rev. Genet. 2001; 2:858-868.
5 Zweier C, Sticht H, Aydin-Yaylagiil I, et al. Human TBX1 missense mutations cause gain of function resulting in the same phenotype as 22q11.2 deletions. Am J Hum Genet.2007; 80(3): 510-517.
6 Zhang Z, Huynh T, Baldini A. Mesodermal expression of Tbxl is necessary and sufficient for pharyngeal arch and cardiac outflow tract development. Development.2006; 133(18): 3587-3595.
7 Torres-Juan L, Rosell J, Morla M, et al. Mutations in TBX1 genocopy the 22q11.2 deletion and duplication syndromes:a new susceptibility factor for mental retardation. Eur J Hum Genet. 2007; 15(6):658-663.
8 Kobrynski LJ, Sullivan KE. Velocardiofacial syndrome, DiGeorge syndrome:the chromosome 22q11.2 deletion syndromes. Lancet.2007;370(9596):1443-1452.
9 Wu HY, Rusnack SL, Bellah RD, et al. Genitourinary malformations in chromosome 22q11.2 deletion. J Urol.2002; 168(6):2564-2565.
10 Kujat A, Schulz MD, Strenge S, et al. Renal malformations in deletion 22q11.2 patients. Am J Med Genet.2006; 140(14):1601-1602.
11 Jerome LA, Papaioannou VE. DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1. Nat Genet.2001; 27(3):286-291.
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