The Research On Analyzing Differentially Expression Genes Of Embryonic Mesenchyme In Mice

Objective:To analyse the differentially expression genes of the mesenchyme taken from embryonic mice during the development of the inner ear and determinate its expression,then select the key genes which could promot the proliferation and differentiation of the sensory progenitors.Methods:Use the TGF?/BMP Signaling Pathway gene chip to analyse the differentially expression genes of the mesenchyme adjacent to auditory epithelia which was taken from embryonic day 12.5(E12.5),E13.5 and neonatal mouse by anotomic microscope.Then,use RT-PCR technoloy to determinate the amount of expression of some differencially expression genes(Mecom,Cdkn2 b,Ltbp2,Tgfb2)between mesenchyme from E12.5 and E13.5.At last,to abserve the dynamical expresstion tentency of Mecom gene at the early dewelopment of the inner ear by immunofluorescence(frozen sections).Results:1.Gene Chip:Through microarray analysis,11 genes were differentially expresse-d between mesenchyme from E12.5 and E13.5,including 6 up-regulated(Cdkn2b,Fos,Gsc,Herpud1,Tgfb1,Tgfb2)and 5 down-regulated(Bmpr1b,Fst,Gdf3,Mecom,Tgfbr1).All of the fold difference is more than 2 times.Among these,the expression of Gdf3 is 2.13 times down-regulation,but p=0.44,not statistically significant.The other 10 genes p<0.05.35 genes were differentially expressed between E12.5 of mesenchyme and neonatal mouse,including 25 up-regulated and 10 down-regulated(the fold difference is more than 2 times).Among these,although the fold difference of Col1 al,Igfbp3,Il6,Gdf2,Gdf7,Nodal and Serpineis more than 2 times,but the threshold of p>0.05,not statistically significant.The other 28 genes p<0.05.2.RT-PCR:A threshold of p<0.05,there was a significant statistical difference among E12.5,E13.5 and neonatal mouse of Cdkn2 b,Ltbp2,Tgfb2 and Mecom,which is consisten t with the results of microarray screening.3.The Expression Patterns of Mecom protein in the early development of inner ear Mecom was initially distributed throughout the mesenchyme adjacent to otic vesicle,cochleovestibular ganglia,and weaker expression in otic vesicle at E10.5,Then,Mecom gradually decreased and disappeared in sensory epithelia,and limited in the mesenchyme and cochleovestibular ganglia at E13.5.However,Sox2 was initially distributed throughout the otic vesicle and cochleovestibular ganglia at E10.5.At E11.5,Sox2 still broadly expressed in the prosensory cells.Then,Sox2 gradually limited in sensory epithelia and cochleovestibular ganglia.Conclusion:There are differentially expression genes of the mesenchyme during the development of the inner ear,and the differences may be related to the different abilities of the proliferation and differentiation of the inner ear sensory precursor cells.Analysis of these differentially expression genes may help us to understand the development mechanism of the inner ear and provide clues to study the hair cells regeneration.