jc-Thalamus-Derived-Molecules-Promote-Survival-and.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,Abstract,The mammalian neocortex is composed of various types of neurons that reflect its laminar and area structures.It has been suggested that,not only intrinsic but also afferent-derived extrinsic factors are involved in neuronal differentiation during development.,However,the,role and molecular mechanism of such extrinsic factors are almost unknown.Here,we attempted to identify molecules that are expressed,in the thalamus and affect cortical cell development.First,thalamus-specific molecules were sought by comparing gene expression,profiles of the developing rat thalamus and cortex using microarrays,and by constructing a thalamus-enriched subtraction cDNA library.,A systematic screening by in situ hybridization showed that several genes encoding extracellular molecules were strongly expressed in,sensory thalamic nuclei.,Exogenous and endogenous protein localization further demonstrated that two extracellular molecules,Neuritin-1(NRN1)and VGF,were transported to thalamic axon terminals.Application of,NRN1 and VGF to dissociated cell culture,promoted the dendritic growth.,An organotypic slice culture experiment further showed that,the number of primary dendrites in,multipolar stellate neurons increased in response to NRN1 and VGF,whereas dendritic growth of pyramidal neurons was not promoted.,These molecules also,increased neuronal survival,of multipolar neurons.,Abstract,Taken together,these results suggest that the thalamus-specific,molecules NRN1 and VGF play an important role,in the dendritic growth and survival,of cortical neurons in a cell type-specific manner.,Abstract,Identification of thalamus-specific genes,丘脑的特定基因的鉴定,Results,A,Flow chart for the identification of thalamus-specific genes.,B,Dissection of a,thalamic nucleus,the dLGN(left),and cortical plate(right)from,P3 rat,.,Asterisks,and dotted lines,mark the positions,of dissected tissues.,Figure1,Thalamus-specific genes identified by microarray analysis and screening of a subtraction cDNA library.,dLGN:dorsal lateral geniculate nucleus 外侧膝状体背侧核,dLGN,视觉的皮质下中枢,MG,C,Comparison of,gene expression,in the thalamus,and cortex,using microarrays.,The blue line represents,the average distribution of signals,and the,red line indicates,sevenfold,higher expression in the thalamus,than in the cortex.,A total of 137 transcripts,s,howed significantly higher expression in the,thalamus than in the cortex,D,Subtraction efficiency of,the constructed subtraction cDNA library.PCR amplification of,Gapdh,showsa reduced amount of,Gapdh,cDNA in subtracted cDNAs(bottom panel)compared with unsubtracted cDNAs(top panel).,Figure1,Thalamus-specific genes identified by microarray analysis and screening of a subtraction cDNA library.,obtained a total of 241 candidate thalamus-specific genes from both,analyses.,Figure1,Thalamus-specific genes identified by microarray analysis and screening of a subtraction cDNA library.,从两,种,分析，共获得了241个候选丘脑特定基因,A thalamic axon-derived influence is likely to be mediated by,extracellular molecules,including secretory or membranebound proteins.Based on the public database and previous reports,23 of 241 genes were found to encode these molecular,species,丘脑轴突,衍,生的影响很可能是,通过,细胞外分子介导,的,，包括分泌,蛋白,或膜结合蛋白。基于公共数据库和以前的,报道，,发现,241个基因里面有23个,基因编码这些分子,Table 1,In situ,hybridization demonstrated that,10 of these genes,were strongly expressed in the thalamus,and comprised of three,groups,Figure2,The thalamus-specific genes showed three different,expression patterns,A,In situhybridization shows dLGN-specific expression of,Tmem192.,B,Expression of,Vgf,is strong,in,sensory thalamic nuclei,including the dLGN,VB,and MG.,C,Kitl,is expressed in the entire thalamus,整个丘脑,.,D,Nissl staining of comparable sections.,NRN1,VGF,and WNT2B were chosen for thalamus-derived extrinsic factors,Figure2,The thalamus-specific genes showed three different,expression patterns,Accumulation of thalamus-specific proteins,in axon,terminals of thalamic neurons,丘脑神经元的轴突末梢,中,丘脑的特定蛋白的聚积,As thalamus-derived factors that affect cortical cell differentiation should be transported from cell bodies to axon terminals,transportation of the candidate proteins was tested in,dissociated thalamic cellsin vitro.,A,B,NRN1(A)and VGF(B)are distributed through,out a thalamic neuron,including the cell body,dendrites,and axon at 10 DIV.,A,B,NRN1(A,)and VGF(B,)localize strongly in the axon terminal of,thalamic neurons(arrows).,C,WNT2B is localized around the cell body,including the proximal parts of neurites.,C,Higher,magnification of the rectangular area in,C,shows,that WNT2B is,not detected in the axon(arrowheads).,Figure3,NRN1 and VGF are abundant in thalamic axon terminals,NRN1 and VGF were finally identified as molecules that are strongly,expressed in developing sensory thalamic nuclei,and,accumulate in the thalamic axon terminals,.,Figure3,NRN1 and VGF are abundant in thalamic axon terminals,Figure4,Nrn1 and,Vgf,are expressed in the thalamus during,development,A,In situhybridization revealed that,Nrn1,is strongly expressed in sensory thalamic,nuclei,dLGN,VB,and MG and,moderately expressed in other thalamic nuclei from E18 to P14.,B,Vgf,shows,more specific expression in sensory thalamic nuclei from E18 to P14.,C,Nissl staining of comparable sections,These results indicate that,Nrn1,and,Vgf,are strongly expressed,in sensory thalamic nuclei at those developmental stages when,thalamocortical axons interact with cortical neurons.,Figure4,Nrn1 and,Vgf,are expressed in the thalamus during,development,AD,Immunopositivity for NRN1 in the VB nucleus in the thalamus(B)and in barrels in layer 4 of the,primary somatosensory area in P7 mouse(D).,EL,VGF distribution in P7 mouse(EH)and P7 rat(IL)brains.Immunostaining signal of,VGF in the dLGN,VB,and reticular,nuclei(RT)in the thalamus,(F,J)and in layer 4 of the primary visual area(H,L).Nuclear DAPI staining is shown(A,C,E,G,I,K),Figure5,NRN1 and VGF are accumulated in thalamic axon terminals in cortical layer 4,These results indicate,that NRN1 and VGF,which are synthesized by thalamic,neurons,are transported to the developing cortex through thalamic axons,Figure5,NRN1 and VGF are accumulated in thalamic axon terminals in cortical layer 4,Figure6,NRN1 and VGF promote dendritic growth ofcortical neurons,(AC,A,-C,),Discrimination of,axons,and dendrites,in the cortical cell culture.Cortical neuronswere stained with antibodies,against,AcTub(A,A,)and MAP2(B,B,),after,7,days,in culture.,The longest,neurite is,AcTub-positive but,MAP2 negative,(arrowheads).The other,neurites,are double-positive.,DF,Purified candidate,proteins,were added to the culture medium of cortical cells,dissected from E18 rat,GJ,Quantitative analysis,of dendritic growth in the presence of NRN1(G,H),or VGF(I,J).,The number of primary,dendrites(G,I),and the total length,of,dendrites(H,J),were measured after,incubation with different,concentrations(conc.)of,these proteins.,Figure6,NRN1 and VGF promote dendritic growth ofcortical neurons,These results indicate,that,dendritic growth of cortical neurons is promoted by NRN1 and VGF.,Figure6,NRN1 and VGF promote dendritic growth ofcortical neurons,Whether NRN1 and VGF secreted from,thalamic,neurons promote dendritic,growth of cortical neurons,？,Figure 7,Block of endogenous NRN1 and VGF reduced dendritic growth of cortical neurons.,AF,Cortical neurons labeled with Cell,Tracker,(C,F,arrowheads)were cocultured with unlabeled,thalamic neurons,in the presence of control IgG(AC)or anti-NRN1 and anti-VGF antibodies,(DF).,G,H,Quantitative analysis,of the,number,of dendrites,(G),and the t,otal length,of dendrites,(H),.,This,result suggests that,NRN1 and VGF secreted from thalamic neurons can promote dendritic growth of cortical,neurons.,Figure 7,Block of endogenous NRN1 and VGF reduced dendritic growth of cortical neurons.,which cell type is affected in dendritic growth,？,Figure,8,NRN1 and VGF specifically affectdendritic growth of stellate neurons,A,Schematic representation of experimental design for cortical slice,culture.,B,C,Morphology of an EYFP-labeled,stellate(B)and pyramidal cell(C).,D,Expression of layer markers in particular cell types.CUX1 and ROR,are expressed in layer 4 stellate cells(arrowheads),whereas ER81 is expressed in a layer 5,pyramidal cell(arrow).,Table 2,Figure,8,NRN1 and VGF specifically affectdendritic growth of stellate neurons,EJ,Typical examples of individual layer 4 stellate cells(EG)and layer 5 pyramidal cells(HJ)at 7 DIV.,Figure,8,NRN1 and VGF specifically affectdendritic growth of stellate neurons,K,L,Quantitative analysis of the number and the total length of,primary dendritesof sampled stellate cells,was,performed afterculturing in the presence and absence of the thalamus-specific factors.,M,N,The same quantitative analysis,for,pyramidal cells.,These results suggest that,NRN1 and VGF promote dendritic growth of layer 4 stellate neurons,but not of layer 5 pyra-midal neurons,将两种蛋白加入,E13.5,转染过了脑组织培养，看是否是加强了突起的生长，结果显示明显促进了突起的长度和数量，但是和单独加没有区别。,Thus,it is,unlikely that NRN1 and VGF have different downstream signaling pathways,Figure,8,NRN1 and VGF specifically affectdendritic growth of stellate neurons,Figure,9,NRN1 and VGF enhance survival of cortical neurons,AC,Dissociated cortical cells were cultured for 7 days in the absence(A)and presence of either NRN1(B)or VGF(C)and were,visualized by immunostaining with anti-AcTub.D,Immunostaining for cleaved caspase-3(CC-3)together with AcTub.,AcTub-positive cellsare negative for CC-3,(arrows).Conversely,CC-3-positive,cells are negative for AcTub and exhibit pyknotic nucleus,(arrowheads).,E,F,Quantitative analysis showed that the application of NRN1(E)or VGF(F)significantly increased the density of,AcTub-positive survived neurons at 7 DIV,NRN1 and VGF promoted the,survival,of developing cortical neuronsin vitro.,Figure,9,NRN1 and VGF enhance survival of cortical neurons,which cell type is more affected in survival,？,background,PMI:pyramidal morphology index analysis,金字塔形态指标分析,defined as the ratio between the width of the largest,process,and the total number of processes emerging from the cell body,定义为最大,process,的宽度和processes emerging的总数的比例,Figure 1,0,NRN1 and VGF increase the population of multipolar neurons in cortical cell culture,A,B,A typical pyramidal,shaped cell(A)and a multipolar cell(B)were visualized by immunostaining with anti-MAP2 antibody.Each normalized PMI value(A,1.20;B,0.38)was calculated using a sampling circle(red).,C,Cumulative frequency distribution,of normalized PMI,in the control and in the presence of either NRN1 or VGF.,NRN1 and VGF application increased the population of,neurons that have smaller PMIs.,D,Average PMI values in the control,NRN1,and VGF,NRN1 and VGF increased the,proportion of,multipolar neurons,.,Figure 1,0,NRN1 and VGF increase the population of multipolar neurons in cortical cell culture,If,the thalamus-derived,factors may affect cell fate,？,Figure 11,NRN1 and VGF do notmarkedly affectcell fate ofcortical neurons,A,Time-lapse imaging oflabeled neuronsin slice culture at1 DIV and 7 DIV.Each number,represents,the same neuron,at the two time points.,B,C,Typical examples,of a neuron differentiating from,multipolar morphology(Mp),to a,stellate cell(St),(B),and from,unipolar morphology(Up),to a,pyramidal cell(Py),(C),.,Figure 11,NRN1 and VGF do notmarkedly affectcell fate ofcortical neurons,D,The histogramshowsthe proportionsof multipolar,cells,that,became stellate(black),pyramidal(white),and intermediate(gray),cell types,after,culturing.,E,The proportionsof unipolar,cells,that,became,stellate,(black),pyramidal(white),and intermediate(gray)cell types after culturing.,Taken together,these findings suggest that,the thalamus-derived,molecules NRN1 and VGF have,negligible effect,on the cell,fate of cortical neurons.,NRN1 and VGF may regulate the,development of postmitotic neurons but not progenitor,cells,as,is the case with BDNF and NT-3.,NRN1,和,VGF,可能调节的是神经细胞发育期的有丝分裂后期，而不是前体细胞，和,BDNF,、,NT-3,一样,Figure 11,NRN1 and VGF do notmarkedly affectcell fate ofcortical neurons,Discussion,In this study,we identified thalamus-specific genes using microarray analysis and a subtraction cDNA library.Throughout,further analyses we selected NRN1 and VGF as molecules that,may affect cortical cell development by being expressed specifically in sensory thalamic nuclei and transported to thalamic axon terminals.,In vitro analyses with dissociated cell,culture demonstrated that,NRN1 and VGF promoted dendritic growth of cortical neurons.,Organotypic slice cultures,further showed that,NRN1 and VGF specifically promote dendritic growth of stellate neurons.,survival of multipolar neurons was increased by these molecules,without,affecting cell fate,.,Taken together,these findings suggest that,thalamus-derived NRN1 and VGF promote the dendritic,growth and survival of a subset of cortical neurons.,All,results demonstrate that NRN1 and VGF,which are specifically expressed in sensory,thalamic nuclei,can be,transported to axon terminals and promote the survival and dendritic growth,of spiny stellate neurons.,These molecules may,contribute to the laminar configuration,of the neocortex as extrinsic,factors that are released from thalamic axons.,这些丘脑的轴突释放,的,分子,作为外源因素,可能,有助于新皮质 层状结构的形成。,Summary,