Fine Mapping Of The Limb Defects And Embryonic Lethality QTL And Detection Of The Gene Expression Profiles Related To The Hind Limbs Development

The development of the mammalian limbs involves the cellular proliferation,differentiation,migration and apoptosis.The genes of HOX,SHH(Sonic hedgehog),FGF(fibroblast growth factor)and WNT signaling pathways play an important role in development of the mammalian limbs.The limb buds are mainly composed of three parts: the apical ectodermal ridge(AER),the progressive zone(PZ),and the polar activation zone(ZPA).The region of ARE is the main signal center of the limbs growth,and expresses some members of the FGF genes family,such as Fgf4 and Fgf8.The region of PZ is a mesenchymal cell region with strong splitting ability in the inner part of AER.It is mainly controlled by Hoxa genes and Hoxd genes.The region of ZPA exists in the posterior part of limb buds,and the genes of SHH and BMPs play a regulatory role in it.It is reported in literature that the abnormal limbs development may also complicate the infertility.There is the incomplete penetrance in the hind limb traits,but the maternal lethal phenotype is very stable.And we used the embryonic lethal phenotypes for fine mapping.Firstly,the wild mice were hybridized with the C57BL/6 strains mice,and the F2 mice experimental animal models were established.In the relevant sections of the chromosome 8,the 4 SNPs loci were selected,and then the genotyping was carried out by using the PCR-LDR typing technique.By fine mapping of the related QTL,the experiment successfully shortened the 10 Mb to 2.5Mb on the chromosome 8.And 10 candidate genes were screened by comparing the genes information of QTL segments in the MGI databases with the sequencing information of the mice.In order to study the co-expression of related genes(Hox genes family)with the candidate genes(Pbx4)in the developmental stage of the mice embryonic hind limbs(E10.5,E11.5,E12.5,E13.5),the expression profiles and a q PCR array technique was established.These were used to detect and analyze a large number of candidate genes for subsequent experiments.First,the Hox genes family,Wnt5 a,Pitx1,Fgf8 and Shh were selected for the detection,and the beta-actin and Gapdh were selected as the housekeeping genes.At the same time,the negative gene controls and sample negative controls were also set up.The gene primers were fixed on the PCR plates by heating and drying,and then the samples and q PCR reactants were added to the real time quantitative PCR reactions.In order to evaluate the q PCR array,and the amplification efficiency was tested by the gradient dilution methods.The standard deviation of the Ct values of all expressed genes was counted,and the reproducibility of the scheme was evaluated.The advantages of this technique were proved by the dissolution curves of q PCR array and agarose gel electrophoresis of the PCR products.In this study,we established a real-time quantitative polymerasechain reaction array method to study the expression differences of the genes of Hox,Wnt5 a,Pitx1,Fgf8 and Shh in the development stages of the C57BL/6 mice embryonic hind limbs.Using E10.5 as a control,we detected two kinds of the genes co-expression patterns in the period of the development of the mice hind limbs.The expression of the Hoxb6,Hoxb8,Hoxc8,Hoxc9,Hoxc10,Hoxd9,Shh,Hoxc9,Hoxc10,Hoxc11,Hoxd9,Hoxd12,Fgf8 and Pitx1 genes were down-regulated after up-regulated.And the expression of Hoxa11,Hoxa13,Hoxc12,Hoxc13 and Hoxd13 were down-regulated,and the candidate gene of the Pbx4 had the similar expression patterns.In addition,a small number of the Hox genes did not change significantly during the development of the mice hind limbs.To sum up,in the first part of this experiment,the fine mapping of the chromosomal related QTL was carried out on mice with the limbs developmental defects and embryonic lethality,and the ranges of the candidate genes was shortened.The candidate genes were screened preliminarily.The second part of the experiment is to study the expression of the genes related to hind limbs development in mice embryos,and the experiment established a stable,highly specific and reproducible q PCR array gene detecting program.The detection results described the relative expression differences of the Hox genes family,Wnt5 a,Pitx1,Fgf8,Pbx4 and Shh in the C57BL/6 mice embryonic hind limbs development periods,and established the gene expression profiles of the hind limbs development.It opens up new ideas for the limbs development work,also provides a small database for the future research work.It is important to explore the regulation networks of the Hox genes expression during the limbs development.