Shh蛋白在小鼠肢芽上的表达研究
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摘要
背景和目的先天性多指(趾)(preaxial polydactyly,PPD)是一种常见的肢体畸形病征,临床表现主要为超出正常数目的手指(脚趾)赘生,在新生儿中的发生率居于各类肢体畸形之首。多指畸形除了独立发生外,还可以作为一些综合征的部分症状而伴随出现。轴前多指(趾)为发生在肢体桡(胫)侧的多余指(趾),按照受累指(趾)发生部位,可划分为4个亚型(Ⅰ-Ⅳ),分别代表二指节拇指多指、三指节拇指多指、食指多指与多指并指。轴前多指(趾)家系大多为常染色体显性遗传(autosomal dominant,AD),从简单的三指节拇指单个指骨增加直至完整的额外指(趾)的产生,患病个体表型有明显差异。指(趾)的发育过程是一个图式发育过程,在这个过程中,Shh(Sonic hedgehog)作为ZPA(Zoneof Polarizing Activity)区的形态发生蛋白起着重要的作用。有报道表明Shh通过扩散影响第二指(趾)的发生,而三到五指(趾)的发育不需要通过蛋白的扩散,第一指(趾)的发生不需要Shh的参与。Shh异位表达在肢芽前部就会出现多指现象,表现为先天性多指(趾)。通过对轴前多指家系血清进行连锁分析和单倍型构建,将该家系的致病位点定位在染色体7q36上的1.7cM范围内,并且通过遗传分析排除了shh基因自身序列改变致病的可能。ZPA调控序列(ZPA regulationsequence,ZRS)位于染色体7q36的LMBR1内含子5上,其基因位点发生突变后,影响Shh在肢芽上的表达。正常情况下,Shh只表达在肢芽后部,PPD突变的个体中Shh在肢芽前后部都有表达。本项目以小鼠为研究对象,研究胚胎发育时Shh在正常肢芽上的表达状况。以此为基础,将突变后的ZRS基因导入小鼠胚胎干细胞中,研究突变后的小鼠Shh表达状况和突变后的小鼠肢芽生长情况。
实验方法将适龄的ICR小鼠进行交配,次日检查,将产生阴栓的母鼠标记为E0.5天,E8.0天开始进行解剖取胚,E8.0-E12.5天进行整胚免疫组化染色,E12.5-E16.5天进行切片免疫组化染色,染色后的切片在显微镜下进行拍片。对Shh质粒扩增后抽提,进行酶切鉴定和电泳鉴定。
结果E8-E12.5天的小鼠全胚免疫组化棕黑色的部分表示的是阳性,黑色部分越大,阳性率越高。结果E10天时Shh开始有表达,E10.5天时表达量逐渐增多,E11天时在肢芽后部大量表达,E12天时表达量减少,E12.5天时已检测不出Shh的表达。E12.5-E16.5天的胚胎为切片免疫组化,Shh在E12.5-E16.5天时免疫组化染色结果都是阴性。电泳鉴定结果获得了清晰的Shh质粒酶切片段电泳图。
结论本研究运用整胚免疫组化和切片免疫组化相结合的方法,得到了Shh在各个时期的胚胎肢芽上的表达图,Shh在小鼠胚胎发育的E10-E12天在肢芽后部有表达。
Background and Objective Congenital multiple digits is a common limb malformation symptoms,clinical manifestations are beyond the normal number of digits supernumerary,it lives the first place in various types of newborns limb deformity. Besides occurred independence,polydactyly also can appear as part of a number of syndromes.Preaxial polydactyly(PPD) refers to an excess of parts on the radial side of the limb.According to the physical radial(tibial) side of the extra digits,Preaxial polydactyly can be divided into four types:typeⅠ(PPD-Ⅰ) is the duplication of one or more of the skeletal components of a biphalangeal thumb;typeⅡ(PPD-Ⅱ)is the polydactyly of a triphalangeal thumb;typeⅢ(PPD-Ⅲ) is the polydactyly of an index finger;typeⅣ(PPD-Ⅳ) is the polysyndactyly.Preaxial polydactyly is usually caused by single gene mutation and shows an autosomal-dominant mode of inheritance.The phenotype shows varation within families and range from a simple addition of a phalanx in triphalangeal thumb to whole digit duplication.The limb bud developmental process is a patterning developmental process,in this process,Shh(Sonic hedgehog) protein plays an important role as a ZPA(Zone of Polarizing Activity) morphogen.The digit begin to growth at the late of the limb bud development,it has been reported that Shh protein affects the second digit happening by diffusion.But when it affects the third to fifth digit happening,it does not need Shh diffusion,and the first digit happens without the involvement of Shh protein.Shh ectopic expression in the anterior of limb bud appears the phenomenon of multi-fingered,manifested as congenital multiple digits. It is reported that people used linkage analysis and haplotype construction PPD family serum,the family disease locus located at chromosome 7q36 on 1.7cM context,and ruled out the shh gene sequence changes in their disease-causing potential by genetic analysis.ZPA regulatory sequence(ZRS) is located in chromosome 7q36 of the LMBR1 intron 5,after mutation in the gene locus,Shh expression in the limb bud was effected. Under normal circumstances,Shh only expressed in the limb bud,PPD mutant individuals have Shh expression in anterior and posterior limb bud.This project studied the expression of Shh in normal limb bud during embryonic development.On this basis, after inducing the mutant gene of ZRS into mouse embryonic stem cells,it can study the expression of Shh and the growth of limb buds in the mutant mice.
Methods It takes age-appropriate ICR mice to mate,the female mouse having a vaginal suppository is marked as E0.5 day,calculating the number of days of their pregnancy accurately,we dissected the mouse to take embryos from E8.0 day,did the entire embryo immunohistochemistry from E8.0 to E12.5 day,and did slice immunohistochemistry from E 12.5 to E 16.5 day,put sections under microscope to make films.Importing Shh recombinant plasmid into Topl 0 bacteria to amplificate,extracting plasmid,extraction of plasmid after digestion was did electrophoretic analysis,carried out Shh fragment recovery experiments.
Results The mouse whole embryos immunohistochemistry from E8 to El2 day which shows brown-black line is positive,the more of black part,the higher of positive rate.We can know from the figure that,Shh expression begins at E10 day,the expression is gradually increasing at E10.5 day,Shh abundantly expressed at the posterior limb bud at E11 day,at E12 day,Shh sharply declined in expression,it can not detect Shh expression at E12.5 day.The E12.5 to E16.5 day embryos are did slice immunohistochemistry,it uses DAB coloring,brown-yellow part represents the positive,from the diagram we can know that,the embryo slice immunohistochemistry shows negative staining from E12.5 to E16.5 day,and obtained a clear Shh plasmid fragments by electrophoresis.
Conclusion this study uses whole embryo immunohistochemistry and sections immunohistochemistry method,obtains the Shh expression in embryonic limb bud at various times,Shh only expresses in the posterior of mouse embryo limb bud at E10-E12 days.
实验方法将适龄的ICR小鼠进行交配,次日检查,将产生阴栓的母鼠标记为E0.5天,E8.0天开始进行解剖取胚,E8.0-E12.5天进行整胚免疫组化染色,E12.5-E16.5天进行切片免疫组化染色,染色后的切片在显微镜下进行拍片。对Shh质粒扩增后抽提,进行酶切鉴定和电泳鉴定。
结果E8-E12.5天的小鼠全胚免疫组化棕黑色的部分表示的是阳性,黑色部分越大,阳性率越高。结果E10天时Shh开始有表达,E10.5天时表达量逐渐增多,E11天时在肢芽后部大量表达,E12天时表达量减少,E12.5天时已检测不出Shh的表达。E12.5-E16.5天的胚胎为切片免疫组化,Shh在E12.5-E16.5天时免疫组化染色结果都是阴性。电泳鉴定结果获得了清晰的Shh质粒酶切片段电泳图。
结论本研究运用整胚免疫组化和切片免疫组化相结合的方法,得到了Shh在各个时期的胚胎肢芽上的表达图,Shh在小鼠胚胎发育的E10-E12天在肢芽后部有表达。
Background and Objective Congenital multiple digits is a common limb malformation symptoms,clinical manifestations are beyond the normal number of digits supernumerary,it lives the first place in various types of newborns limb deformity. Besides occurred independence,polydactyly also can appear as part of a number of syndromes.Preaxial polydactyly(PPD) refers to an excess of parts on the radial side of the limb.According to the physical radial(tibial) side of the extra digits,Preaxial polydactyly can be divided into four types:typeⅠ(PPD-Ⅰ) is the duplication of one or more of the skeletal components of a biphalangeal thumb;typeⅡ(PPD-Ⅱ)is the polydactyly of a triphalangeal thumb;typeⅢ(PPD-Ⅲ) is the polydactyly of an index finger;typeⅣ(PPD-Ⅳ) is the polysyndactyly.Preaxial polydactyly is usually caused by single gene mutation and shows an autosomal-dominant mode of inheritance.The phenotype shows varation within families and range from a simple addition of a phalanx in triphalangeal thumb to whole digit duplication.The limb bud developmental process is a patterning developmental process,in this process,Shh(Sonic hedgehog) protein plays an important role as a ZPA(Zone of Polarizing Activity) morphogen.The digit begin to growth at the late of the limb bud development,it has been reported that Shh protein affects the second digit happening by diffusion.But when it affects the third to fifth digit happening,it does not need Shh diffusion,and the first digit happens without the involvement of Shh protein.Shh ectopic expression in the anterior of limb bud appears the phenomenon of multi-fingered,manifested as congenital multiple digits. It is reported that people used linkage analysis and haplotype construction PPD family serum,the family disease locus located at chromosome 7q36 on 1.7cM context,and ruled out the shh gene sequence changes in their disease-causing potential by genetic analysis.ZPA regulatory sequence(ZRS) is located in chromosome 7q36 of the LMBR1 intron 5,after mutation in the gene locus,Shh expression in the limb bud was effected. Under normal circumstances,Shh only expressed in the limb bud,PPD mutant individuals have Shh expression in anterior and posterior limb bud.This project studied the expression of Shh in normal limb bud during embryonic development.On this basis, after inducing the mutant gene of ZRS into mouse embryonic stem cells,it can study the expression of Shh and the growth of limb buds in the mutant mice.
Methods It takes age-appropriate ICR mice to mate,the female mouse having a vaginal suppository is marked as E0.5 day,calculating the number of days of their pregnancy accurately,we dissected the mouse to take embryos from E8.0 day,did the entire embryo immunohistochemistry from E8.0 to E12.5 day,and did slice immunohistochemistry from E 12.5 to E 16.5 day,put sections under microscope to make films.Importing Shh recombinant plasmid into Topl 0 bacteria to amplificate,extracting plasmid,extraction of plasmid after digestion was did electrophoretic analysis,carried out Shh fragment recovery experiments.
Results The mouse whole embryos immunohistochemistry from E8 to El2 day which shows brown-black line is positive,the more of black part,the higher of positive rate.We can know from the figure that,Shh expression begins at E10 day,the expression is gradually increasing at E10.5 day,Shh abundantly expressed at the posterior limb bud at E11 day,at E12 day,Shh sharply declined in expression,it can not detect Shh expression at E12.5 day.The E12.5 to E16.5 day embryos are did slice immunohistochemistry,it uses DAB coloring,brown-yellow part represents the positive,from the diagram we can know that,the embryo slice immunohistochemistry shows negative staining from E12.5 to E16.5 day,and obtained a clear Shh plasmid fragments by electrophoresis.
Conclusion this study uses whole embryo immunohistochemistry and sections immunohistochemistry method,obtains the Shh expression in embryonic limb bud at various times,Shh only expresses in the posterior of mouse embryo limb bud at E10-E12 days.
引文
[1].陈佳鹏 张蕾 陈功 宋新明 郑晓瑛。中国1993-1998年出生缺陷监测能力分析[J]。中华流行病学杂志 2006 May;27(5),392-395
[2].Zhou GX,Dai L,Zhu J,et al.Epidemiological Analysis of Polydactylies in Chinese Perinatals[J].Journal of Sichuan University(Medical Sciences Edition),2004;35(5):708-710.
[3].Heus HC,Hing A,van Baren MJ,et al.A physical and transcriptional map of the preaxial polydactyly locus on chromosome 7q36[J].Genomics,1999May;57(3):342-351.
[4].Lettice LA,Horikoshi T,Heaney SJ,et al.Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly[J].Proc Natl Acad Sci U S A,2002May;99(11):7548-7553.
[5].Echelard Y,Epstein DJ,St-Jacques B,et al.Sonic hedgehog,a member of a family of putative signaling molecules,is implicated in the regulation of CNS polarity[J].Cell,1993,75(7):1417-1430
[6].Testaz S,Jarov A,Williams KP,et al.Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched-Smoothened-Gli signaling pathway[J].Proc Natl Acad Sci USA,2001,98(22):12521-12526.
[7].Park HL,Bai C,Platt KA,et al.Mouse Glil mutants are viable but have defects in SHH signaling in combination with a Gli2 mutation[J].Development,2000,127(8):1593-1605
[8].Riddle RD,Johnson RL,Laufer E,et al.Sonic hedgehog mediates the polarizing activity of the ZPA[J].Cell,1993,75(7):1401-1416
[9].Tickle C.Patterning systems-from one end of the limb to the other[J].Dev Cell,2003,4(4):449-458
[10].Hil RE,Heaney SJ,Lettice LA.Sonic hedgehog:restricted expression and limb dysmorphologies[J].Anat,2003,202(1):13-20.
[11].Lopez-Martinez A, Chang DT, Chiang C, et al. Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage[J]. Curr Biol,1995,5(7):791-796.
[12].Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, and Beachy PA. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function[J]. Nature, 1996, 353,407-413.
[13].Kraus P, Fraidenraich D, and Loomis CA. Some distal structures develop in mice lacking Sonic hedgehog signaling[J]. Mech. Dev. , 2001, 100, 45-48.
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[2].Zhou GX,Dai L,Zhu J,et al.Epidemiological Analysis of Polydactylies in Chinese Perinatals[J].Journal of Sichuan University(Medical Sciences Edition),2004;35(5):708-710.
[3].Heus HC,Hing A,van Baren MJ,et al.A physical and transcriptional map of the preaxial polydactyly locus on chromosome 7q36[J].Genomics,1999May;57(3):342-351.
[4].Lettice LA,Horikoshi T,Heaney SJ,et al.Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly[J].Proc Natl Acad Sci U S A,2002May;99(11):7548-7553.
[5].Echelard Y,Epstein DJ,St-Jacques B,et al.Sonic hedgehog,a member of a family of putative signaling molecules,is implicated in the regulation of CNS polarity[J].Cell,1993,75(7):1417-1430
[6].Testaz S,Jarov A,Williams KP,et al.Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched-Smoothened-Gli signaling pathway[J].Proc Natl Acad Sci USA,2001,98(22):12521-12526.
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[8].Riddle RD,Johnson RL,Laufer E,et al.Sonic hedgehog mediates the polarizing activity of the ZPA[J].Cell,1993,75(7):1401-1416
[9].Tickle C.Patterning systems-from one end of the limb to the other[J].Dev Cell,2003,4(4):449-458
[10].Hil RE,Heaney SJ,Lettice LA.Sonic hedgehog:restricted expression and limb dysmorphologies[J].Anat,2003,202(1):13-20.
[11].Lopez-Martinez A, Chang DT, Chiang C, et al. Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage[J]. Curr Biol,1995,5(7):791-796.
[12].Chiang C, Litingtung Y, Lee E, Young KE, Corden JL, Westphal H, and Beachy PA. Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function[J]. Nature, 1996, 353,407-413.
[13].Kraus P, Fraidenraich D, and Loomis CA. Some distal structures develop in mice lacking Sonic hedgehog signaling[J]. Mech. Dev. , 2001, 100, 45-48.
[1]Saunders JW,Gasseling MT.Ectodermal-mesenchymal interactions in the origin of limb symmetry[M].In:Fleischmeyer R,Billingham RE,eds.Epithelial-Mesenchymal Interactions,Baltimore USA:Williams and Wilkins,1968,78-97
[2]Riddle RD,Johnson RL,Laufer E,et al.Sonic hedgehog mediates the polarizing activity of the ZPA[J]. Cell,1993,75(7):1401-1416
[3] Echelard Y, Epstein DJ, St-Jacques B,et al.Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity[J].Cell,1993,75(7):1417-1430
[4] Chiang C, Litingtung Y, Harris MP,et al.Manifestation of the limb prepattern:limb development in the absence of sonic hedgehog function[J]. Dev Biol,2001,236(2):421-435
[5] Testaz S,Jarov A,Williams KP,et al.Sonic hedgehog restricts adhesion and migration of neural crest cells independently of the Patched- Smoothened-Gli signaling pathway[J]. Proc Natl Acad Sci USA,2001, 98(22):12521-12526
[6] McGlinn E,Tabin CJ.Mechanistic insight into how Shh patterns the vertebrate limb[J]. Curr Opin Genet Dev, 2006,16(4):426-432
[7] Lewis PM, Dunn MP, McMahon JA, et al.Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1[J]. Cell,2001,105(5):599-612
[8] Tickle C,Summerbell D,Wolpert L.Positional signalling and specification of digits in chick limb morphogenesis[J]. Nature,l975, 254(5497): 199-202
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