TRIM32 Deficiency Leads To Decreasing Generation Of Neurons During Cortex Development In Mice

BackgroundThere is no doubt that the cortex development is the most sophisticated process during the embryo stage.In the rodent animals,neurons are produced from E12 to E18,and the peak is at E15,then finish after birth.Neurogliocytes are produced from E18.5,and the peak is neonatal period.RGSs are located in the VZ zone and could self-renewal,and then migrate and differentiate to mature neurons,finally make the six layer cortex.TRIM32(tripartite motif containing protein 32)is an E3 ubiquitin ligase.This prorein include a particular six repetitive NHL domains except for Zinc-binding domains,B-box domains and coiled-coil region in its protein structure.TRIM32 has an important role in muscular system.According to previous reports,TRIM32 is involved in regulation of muscular stem cell differentiation and is usually lead to limb-girdle muscular dystrophy type 2H(LGMD2H)when the loss of TRIM32.In our lab,we have found that TRIM32 is high express in the cortex neuron of adult mice and in the neural progenitor cells from MGE in the embryonic brain,and we found that TRIM32 deficiency leads to decreased number of GABA-ergic interneurons in the cortex.In this study,we focus on the effect of TRIM32 during the cortex development.Purpose1.To explore the espression patterns of TRIM32 in the cortex of embryonic mice brain during the embryonic cortex development.2.To investigate the effects of TRIM32 deficiency in the genereation of mature neurons during embryonic cortex development.3.To investigate the effects of TRIM32 deficicency in the generation of NPCs in the VZ/SVZ zone during embryonic cortex development.4.To investigate the mechanism of TRIM32 deficiency impacting the generation of NPCs during the embryonic cortex development.Methods1.TRIM32 expression patterns in the mice cortex.Using immunofluorescent staining of TRIM32 in the slide of E13.5,E16.5 and E18.5.2.The effect of cortex size and mature neuron production caused by TRIM32 deficency.Evaluate the NCx and CP size by DAPI staining.Using immunofluorescent staining of layer specific marker to label the neurons at E14.5,E16.5,E18.5 and P30,and then estimate the result.3.The effect of neural precursor cells produce in the VZ/SVZ zone caused by TRIM32 deficiency.Using immunofluorescent staining of specific markers to label NPC at E14.5 and E16.5 and then estimate the result.4.The effect of TRIM32 in the proliferation and mitosis of NPCs,and in the apoptosis of cortex cells during embryo stage.Using staining of Edu and PH3 to label proliferation and mitosis cells at E14.5 and E16.5 and then estimate the result.5.The effect of TRIM32 in the cortex cells apoptosis during embryo stage.Using staining of Caspase3 to label apoptotic cells at E14.5 and E16.5 and then estimate the result.6.Statistical analysisAll measurements were performed in triplicate and all values are expressed as the mean ± the standard error.P value<0.05 was considered statistically significant.Results1.The expression pattern of TRIM32 in embryo mouse cortex.At E13.5,TRIM32 is high expressed in the VZ/SVZ zone cells,and located in thecytoplasm,then at E16.5,TRIM32 is high expressed in the IZ zone cells,soon afterwards E18.5,TRIM32 is high expressed in the TBR1 positive neurons,located in the cell nucleus.2.TRIM32 deficiency leads to decreased CNx and CP size and decreased number of mature neurons in the embryo mouse cortex.At E18.5,the CNx and CP size are decreased in TRIM32-/? mouse,and the difference is significant(NCx,t=-4.709,P=0.026;CP,t=-7.634,P=0.O01);At E14,5,E16.5 and E18.5,TBR1 positive neurons are decreased in TRIM32-/? mouse,and the diffenence are all significant(E14.5,t=-4.433,P=0.032;E16.5,t=-5.549,P=0.022;E18.5,t=-7.793,P=0.021).At E16.5 and E18.5,BCL11b positive neurons are decreased in TRIM32-/-mouse,the differenceare all significant(E16.5,t=-6.691,P=0.037;E18.5,t=-12.047,P=0.036).At E18.5 and P30,CUX1 positive neurons are decreased in TRIM32-/-A mouse,and the difference are all significant(E18.5,t=-7.622,P=0.047;P30,t=33.956,P=0.014).3.TRIM32 deficiency leads to decreased neural precursor cells.At E14.5 and E16.5,the number of PAX6 and TBR2 positive cells are both decreased in TRIM32-/-mouse cortex compare to wild type mouse,and the differency is significant.(PAX6,E14.5,t=9.125,P=0.018,E16.5,t=13.230,P=0.018;TBR2,E14.5,t=7.143,P=0.028,E16.5,t=12.548,P=0.024).4.TRIM32 deficiency leads to decreased proliferation and mitosis in neural precursor cells.The proliferation of NPCs:at E14.5 and E16.5,the Edu labeled cells are decreased in TRIM32-/-mouse,and the difference is significant(E14.5,t=14.061,P=0.000;E16.5,t=8.715,P=0.038)The mitosis of NPCs:at E14.5,both basal and apical area,the PH3 positive cells are decreased in TRIM32 deficiency mouse cortex compare to wild type mouse,the differency is significant(basal zone:t=16.234,P=0.000;apical zone:t=26.415,P=0.000).At E16.5,the differency are still exist,but it is not significant in basal area,but it's still significant in apical area(basal zone,t=1.784,P=0.231),apical zone,t=4.051,P=0.022).5.TRIM32 deficiency do not effect the cell apoptosis status in the embryo cortex.The number of Caspase3 positive cells do not have differency both at E14.5 and E18.5 in the TRIM32 deficiency mouse cortex compare to wild type mouse(E14.5,t=0.083,P=0.938;E18.5,t=-1.350,P=0.283).