The Expression Of Shh In Mouse Limb Bud

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:typeâ… (PPD-â… ) is the duplication of one or more of the skeletal components of a biphalangeal thumb;typeâ…¡(PPD-â…¡)is the polydactyly of a triphalangeal thumb;typeâ…¢(PPD-â…¢) is the polydactyly of an index finger;typeâ…£(PPD-â…£) 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.