Sox10在感觉神经元发育中的作用_英文_

第 30 卷 � 第 10期 � � 中 南 林 业 科 技 大 学 学 报� � Vol. 30 � No. 10 � 2010 年 10 月 Journal of Central South University of Forestry & Technology � Oct. 2010 A genetic investigation of the role of sox10 in sensory neuron development ZHANG Zhuo1 , SU Jian�ming2a , XIAO T iao�yi2b ( 1. China Animal Husbandry & Veterinar y Cent re, Beijing , 100094; 2a. Co llege of Veter inary Medicine; 2b. Colleg e of Animal Science & Techno lo gy , H unan Agr iculture Univer sity , Chang sha 410128, H unan, China) Abstract: Neural crest is a transient g roup o f mult ipo tent cells that mig rates ex tensively g iv ing rise to a w ide var iety o f der ivatives, including pigment cells, neurons and g lia. The understanding of sox10 function in neural cr est develop� ment is essential because sox10 mutations under lie several neurocr istopathies. Whereas the ro le of transcr iption factor sox10 in g lial lineage has been w ell established, sox10 function in sensor y neurons is more debat able. In this study, whole mount in situ hybridizations of developing w ild�ty pe and sox10 mutant zebrafish embryo s w ere used to examine all kinds of markers expression in neural crest development. T he results reveal that sox10 promo tes g lia l develop� ment, and also functions in specification o f senso ry neurons and autonomic lineage, and the sox10 function deficit leads a secondar y consequence for supernumerar y neuromasts. In addition, zebrafish sox10 mutant s ox10m618 play s a more sever e ro le in phenotype defect than a relativ ely novel s ox10 mutant s ox10baz 1 . A ccording to the results of this st udy, a well�fo unded assumpt ion can be put fo rw ard that Notch�Delta signalling maybe involved in early specification of senso ry neurons in sox10baz1 mutant, but this inference requir es fur thermore examines to be conf irmed. Key words: sox10; neural cr est ; zebr afish; senso ry neuron; glia CLC number: � Q953; Q754; Q959. 4 Document code: � A Article code: � 1673- 923X( 2010) 10- 0094- 13 Sox10在感觉神经元发育中的作用 章 � 卓1 , 苏建明2a , 肖调义2b ( 1. 农业部种畜质检中心,北京 100094; 2. 湖南农业大学 a.动物医学院; b.动物科技学院, 湖南 长沙 410128) 摘 � 要: � 神经嵴是在神经管形成过程中分离出的一组多能干细胞群,随着细胞迁移,可分化成成体多种特定的细 胞和组织。神经嵴的 sox10 突变体与一系列神经疾病相关, 尽管 sox10 基因与神经胶质之间的关系现已得到一定 的研究,但对其与感觉神经元之间作用的研究还留有很大空白。在本研究中, 我们主要运用 RNA 探针原位杂交技 术来检测一系列神经嵴系统发育标记在斑马鱼胚胎中的 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 达。本研究结果表明, sox10 基因既能促进神经胶质发 育,也能影响感觉神经元和自主神经的分化。在本研究中发现, sox 10 的功能缺陷, 能导致产生冗余神经丘二级效 应。此外,本研究还发现运用较广的斑马鱼 m618 突变体比近年新发现的 baz 1 突变体有着更为严重的表型缺陷。 根据本研究结果分析,可提出一种较为有依据的假设, N otch�Delt a信号通路极有可能影响了 sox10baz1突变体的早期 感觉神经元分化,该假设的证实仍需进一步研究。 关键词: � sox 10; 神经嵴; 斑马鱼; 感觉神经元; 神经胶质 � � Received date: 2010�06�12 � � Biography: ZHANG Zhuo( 1981- ) , female, nat ive place: Changsha, H unan. M aster of anim al medicine � � 作者简介:章 � 卓( 1981- ) ,女,湖南长沙人,硕士。从事动物学研究和种畜质检工作 1 � Introduction The neural crest , a t ransient st ructure, is a populat ion of cells derived fr om the neural fo lds. The format ion of neural crest to w hich the pro� spective neur al plate and the pr ospect ive epidermis contribute appears in the dor sal part of the neural tube w hen it is closed. Soon the cells m igrate ex� tensively f rom the neur al tube into the surrounding tissues to gener ate a prodigious numbers of differ� entiated cell types. T he variety of differ ent cell ty pes include: ( 1) the neurons and g lia cells of the sensory and autonomic systems; ( 2) the adr enal medulla and calcitonin cells of thyr oid; ( 3) pig� ment cells excluding those of the pigmented ret i� na; ( 4) skeletal t issues of the head; ( 5) par t of cardiac out flow tract ( Slack, 2006) [ 1] . One of the most important featur es of neural crest cells is their mult ipotency , w hich is the fate or fates that a clone derived from a single cell could adopt given an appr opriate environment ( Kelsh, 2006) [ 2] . A single neural crest cell can different iate into any of several dif ferent cell types in precise embryonic lo� cations, including skeletog enic ( o r ectomesen� chymal ) fates such as craniofacial cart ilage and non�skeletog enic ( or non�ectomesenchymal) fates including senso ry and autonomic neur ons, periph� eral g lia and diverse pigment cells ( Kelsh, 2006) [ 2] . According to some experiments of t rans� planted different regions o f neural crest , it is known that the differ ent iat ion of a neural crest cell depends on its ev entual locat ion, not on its place of or ig in ( Le Douarin et al . , 1975) [ 3] . T he pluri� potency of some neural crest cells is such that even regions o f the neural crest that never give rise to nerves in normal embryo s can be made to do so un� der certain conditions ( Gilbert , 2006) [ 4] . As cells become specif ied, they undergo fate rest rict ion, so that they are unable to generate all the diverse de� r iv at ives of the neural crest , but only a subset of all possible types of progeny, at least in v it ro ( Raible et al . , 1994; Kelsh, 2006) [ 2, 5] . Specif i� cation, w hich is defined as a restr ict ion in fate ( or the cell type no rmally produced in vivo) is dif fer� ent f rom commitment , w hich is a rest riction in po� tential ( or the capacity to pr oduce different fates under different env ironmental condit ions) ( Dorsky R. I. , 2000) [ 6] . Although specified, the cell that underg oes commitment may retain mult ipotency and may eventually adopt a different fate ( Kelsh, 2006) [ 2] . Abnormalit ies o f neural crest develop� ment in humans lead to a number of g enetic disea� ses, know n as neuro cristopathies ( neur al crest dis� o rders) , w hich include Waardenbur g�Shah syn� drome and H irschsprung� s disease. Sr y�r elated HMG box ( S ox ) pro teins are character ized by possession o f a DNA�binding do� main w ith similarity to the high�mobility group ( HMG ) domain of the sex determ ining factor SRY. Sox pro teins function as cel l type�specific accessory proteins and of ten exhibit a highly re� st ricted tissue dist ribut ion. T hey also fr equent ly funct ion in concert w ith other tr anscript ion factors ( Kuhlbrodt et al . , 1998) [ 7] . Sox10 transcript ion factor gene is specif ically expr essed t ransient ly in the early neural crest and later remains act ivated only in neural and melano� cyt ic neural crest lineages w hile it is never detected in mesenchymal cells, w hether deriv ed f rom me� sectoderm o r fr om mesoderm ( Dupin et al . , 2006) [ 8] . Early expression of sox10 in premigratory neu� r al crest cells is prominent in human, mouse, chick, fro g and zebr af ish. In later migrat ing cells expr ession o f sox10 is observ ed in mouse, chick and zebr af ish, including on both medial and do rso� lateral mig ration pathw ay s in the t runk, and in en� teric nervous system precursors m igrat ing along the gut primordium ( Kelsh, 2006) [ 2] . T hen it is rapidly dow nregulated in many cel ls, even in very early phases of dif ferentiat ion, but except the g lial derivat iv es, w here st rong and per sistent sox10 ex� pression is seen even probably into adulthood ( Kelsh, 2006) [ 2] . S ox10 expr ession in olig oden� drocytes and in the o t ic epithelium is conserved in 95第 30 卷 � � � � � 中 南 林 业 科 技 大 学 学 报 all major vertebrate model systems ( Kelsh, 2006) [ 2] . Recent ly , sox10 expression is detected t ransi� ent ly in the sensory neuron lineage ( Carney et al . , 2006 ) [ 9] . Loss�of�funct ion mutat ions in the human sox10 lo cus cause dominant , cell�autono� mous defects in Schw ann cell and melanocyte dif� ferent iation, w hile homozygous mutants exhibit embryonic lethal defect including failure o f oligo� dendrocy te different iation and reduct ion or absence of many NC derivat ives, w hich contain melano� cytes, enteric ganglia, g raded defects in sympa� thetic and do rsal root ganglia af fect ing bo th neu� r ons and glial components, but no effects on craniofacial cart ilage ( Kim et al . , 2003; Kelsh, 2006) [ 2, 10] . Sox10 has a role in maintaining mult i� potency of neural cr est stem cells and inhibit ing dif ferent iation o f neuronal fates at a very early stage ( Kelsh, 2006 ) [ 2] . T here is g row ing ev idence that defects in fate specif ication may be general. An essent ial part o f the mechanism is a series o f master sw itch� t ranscript ion�facto r�en� coding genes, w hich act separ ately or in combina� t ion to promote development o f specific fates ( Kelsh, 2006) [ 2] . Ext racellular gr ow th factors, signalling through transmembrane receptor s, act instr uctively to regulate master sw itch t ranscrip� t ion, and the downstream transcript ional ef fects of these signalling factors act w ith sox 10, w hich is an endogenous t ranscription factor expressed in developing neural cr ests ( Kelsh, 2006 ) [ 2] . In homozygous sox10 mutant mice, sensory neurons fo rm in dorsal r oot gang lia, and in the zebraf ish mutant only a few of the sensory neur ons form in dor sal r oot g ang lia ( Carney et al . , 2006) [ 9] , but satel lite cells or Schw ann cells do not develop, demonst rat ing a key ro le of this t ranscript ion facto r in the development of peripheral glial cells, including both g lial fate specificat ion and dif feren� t iat ion ( Britsch, 2001; Kelsh, 2006 ) [ 2, 11] . T his demonst rat ion comes fr om the study of E rbB3 and N otch1. In sox10 mutants, expression of erbB3, w hich is a component o f the Neuregulin ( Nrg ) receptor that drive glial fate specif icat ion, is lost in glial prog enitors, so this due to the failure of g lial fate specif icat ion. H ow ever, the sox10 mutant glial phenotype is mor e sever e than that of erbB3 mutants in bo th mouse and zebrafish, suggest ing that additional mechanisms are invo lved. One of such mechanism may depend upon N otch1, w hich drives g lial fate specif ication and loss of neuronal potent ial ( Kelsh, 2006) [ 2] . Sox10 contributes to no t only glial, but also autonom ic neur onal po ten� t ial f rom ex tinct ion by lineage commitment signals ( Kim et al . , 2003) [ 2, 10] . T his maintenance of neuronal potent ial involves tw o neuro genic t ran� script ion factors, Mash1 and Phox2b. Simultane� ously, sox 10 inhibits or delay s overt neuronal dif� ferent iation, bo th in v it ro and in vivo , but this requires a higher sox10 gene dosage than does the maintenance of neurogenic potent ial ( Kim et al . , 2003) [ 10] . One of the propo sals about neural crest derived sensory neurons is that sox10 funct ions in specificat ion of sensory neur ons and there are some ev idence show that in zebraf ish, early dor sal ro ot gang lia sensor y neur on survival is independent of different iated g lia ( Carney et al . , 2006) [ 9] . The homozygous sox10 mutants display a sim ilar phenotype as mouse sox10 nul l homozygotes ( Dut� ton et al . , 2001; Carney et al . , 2006) [ 9, 12] . How ever, in both mouse and zebraf ish, the phenotype of sox10 mutant sensory neuron is w eaker than that of other derivat ives. In zebraf ish, sensory neuron number is sharply reduced in the tail, but less affected in the t runk ( Car ney et al . , 2006) [ 9] . In this study, w e used tw o differ ent sox10 mutant , sox10 m618 and sox10baz 1 . sox10baz 1 mutant is a relat ively new sox10 mutant . In this allele, ther e is a G to A subst itut ion at positio n at 724, w hich creates a Valine to Methionine subst itut ion w ithin the HMG domain o f the sox10 protein at am ino acid posit ion 117. T his domain is highly conserved betw een human, chick and mouse sox10 pro tein sequences and this is also within HMG box which is DN A 96 ZH ANG Zhuo , et al: A genetic investig ation o f the role of sox10 in sensor y neuron development � � 第 8 期 binding domain ( Carney et al . , 2006 ) [ 9] . In sox10 m618 allele, there is a T to A subst itut ion at posit io n 425, w hich induces a L to G subst itut ion w ithin the HMG domain of the sox10 protein at am ino acid posit ion 142 and this domain also is in HMG box ( Dutton et al . , 2001) [ 12] . N eur ogenin ( N g n ) gene fam ily encodes a series of neural�specif ic basic helix�loop�helix ( bH LH ) t ranscr ipt ion facto rs that are key regula� to ry genes for the sensory neuron lineage in both mouse and zebraf ish ( Carney et al . , 2006) [ 9] . ngn1 is required for act ivat ion of a cascade of dow nst ream bHLH factors, including N eur oD, MA TH 3, and N SCL 1. Mor eover, N gn1 posit ive� ly regulates the Delta homolog DLL1 and can be negat ively regulated by Notch signalling ( M a et al . , 1998 ) [ 13] . Sox10 specif ies senso ry neuron precursors by regulating the proneural gene ngn1 ( Car ney et al . , 2006) [ 9] . 2 � Materials & Methods 2. 1 � Fish Husbandry Wild type ( AB ) , mutant , t ransgenic zebraf ish w ere kept in the aquarium at the U niv er� sity o f Bath. Natur al crosses w er e set up betw een fish overnight and the embryos collected the next day. Embryos w ere placed in embryo medium and grown at 28. 5 ! . Embryos w ere staged by Kimmel et al . ( 1995) . Where appropriate, melanisat ion was inhibi� ted by using 1 ∀ PTU ( 1�phenyl�2�thiourea) from 24 hours post fertilisat ion ( hpf) . 2. 2 � Whole�mount in Situ Hybridization of Zebr� afish Embryos � � RNA in situ hybridizat ion w as performed largely. Probes used w ere sox10, ngn1, mbp , f oxd3, i sl1, p hox2b, crestin, neuroD. Antibody used w as ant i�DIG alkaline phosphatase ( Roche) diluted 1/ 3000 w ith blo cking solution. 2. 3 � Microscopy Stained fish w ere viewed whole mount by placing them in a drop of 80 % ( volume fraction) glycerol be� tween stacks of No. 1 coverslips on a slide, with a cov� erslip placed over the top. Embryos were view ed by u� sing a Nikon Eclipse E800 microscope using appropri� ate filters and photographed by Nikon sight DS�US camera together with NIS Elements F softw are. 3 � Results 3. 1 � Neural crest migration is aberrant in sox10baz1 mutant � � Probes sox10 and crestin w er e used to detect the prem igratory and migrat ing neural crest cells at 24 and 30 hpf stages in zebrafish embryos. Compared w ith w ild�type, in sox 10baz 1 mutant migrat ing neural crest cells expressing sox 10 on medial pathw ay were reduced in tr unk at 24 hpf ( Fig . 1B) and 30 hpf ( Fig. 1D) , as same as express� ing crestin in this region at 24 hpf ( Fig. 2E, F) . In contrast , there w ere obviously g reater numbers of sox 10+ cells remaining in a prem igrator y po sit io n in sox10baz 1 mutant than w ild�ty pe at 24 hpf and 30 hpf ( F ig. 1) , as same as crest in + cells in pr em i� g ratory po sit io n ( Fig . 2E, F ) . Neural crest mig ra� t ion on the media pathw ay w as abno rmal in sox10baz 1 mutant compared w ith w ild�type. 3. 2 � Glial development is failed in sox10baz1 mutants 3. 2. 1 � PNS gl ial development is defective in sox 10baz1 mutants � � The sox10baz 1 mutant glial phenotype w as ex� am ined, using the mRNA in situ hybridizat ion de� tect ion of sox10, f oxd3 and mbp ( F ig. 3) . Periph� eral glial prog enitors w er e no t normally different i� ated in sox10baz 1 mutant . F irst, the po sterior later� al line nerv e in sox 10baz 1 mutant lacked mbp+ Schw ann cells ( Fig . 3F) , sox10+ Schw ann cells ( Fig . 3B, H ) and f oxd3 + Schw ann cells ( Fig. 3D) , but in the w ild�type these cells ( F ig . 3A, C, E, G ) show ed a consistent posit ive phenotype. Secondly, sox 10 + Schw ann cells ( F ig . 3I, K ) , f oxd3 + Schw ann cells ( F ig . 3M ) and f oxd3 + sat� 97第 30 卷 � � � � � 中 南 林 业 科 技 大 学 学 报 ellite g lial cells ( F ig. 3M ) associated with the spi� nal nerves w er e observ ed in w ild�type, but only a few of them could be seen in sox 10baz 1 mutant. Thus, neur al crest cells failed to dif ferent iate PNS glial as normal. Fig. 1 � Medial pathway at 24 hpf ( A, B) and 30 hpf ( C, D) inWT (A, C) and baz1 mutant (B, D) Migratin g neural crest cell s ( arrow s) ex pres sing sox 10 w ere redu ced in baz 1 mutan t compared w ith WT sibling. Th e num� ber of neural cres t cells remained in prem igratory posit ion ( as terisk s) w as greater in baz 1 mutant than in WT . Fig. 2 � Whole mount in situ hybridization of 24 hpf wild�type ( A�C) and baz1 mutant ( D�F) embryos for crestin The cell s expressing crest in reduced in head (D) , medial pathway in t runk ( E) , and lateral pathw ay in t runk ( F) of baz 1 mutant. 98 ZH ANG Zhuo , et al: A genetic investig ation o f the role of sox10 in sensor y neuron development � � 第 8 期 3. 2. 2 � CNS oligodendrocyte development in head is disrupted lightly in sox10 baz1 mutants � � Using sox10 expression as a mar ker, in situ hybr idization of w ild�type and sox10baz 1 reveals that at 48 hpf and 72 hpf sox10 + o lig odendro cyte pre� cur sor s in t runk spinal cord w er e similar ( F ig . 4A, B, E, F) . How ever, oligodendrocyte progenito rs in head w ere obviously reduced in sox10baz 1 mutant compared w ith w ild�type. T his could be detected by who le mount in situ hybridizat ion of 72 hpf w ild�ty pe and sox10baz 1 mutant embr yos for sox10 ( Fig . 4C, D) and mbp ( Fig. 4G, H ) . CNS oligo� dendrocy te development is defect ive in sox10baz 1 mutants. 3. 2. 3 � Glial development was less severely affected at 48 hpf in sox10baz1 than in sox10m618 mutants � � Sox10 expression in glia is reduced bo th in sox10 baz 1 and sox10 m618 mutants compared with w ild� type, but the reduct ion of glia in sox10baz 1 is not as se� verely as in sox10m618 at all axial levels. sox10+ cells a� round ot ic veside in head w as abundant in wild�type ( Fig. 5A) , but there was few these cells in sox10m618 Fig. 3 � PNS glial development is severely disrupted in sox10baz1 mutants ( A , B) At 48 hpf , sox 10 w as st rongly express ed in S chw ann cel ls of PLLn ( arrow s) in WT ( A) , but ab sen t in baz 1 m utants ( B) . ( C, D) At 48 hpf , f ox d3 ex pres sion in S chw ann cells of PLLn was r eadily seen in WT ( C) , bu t abs ent in baz 1 ( D) . (E , F) At 72 hpf , mbp w as expressed in Schw ann cell s of PLLn in WT ( E) , b ut absent in baz1 mutants ( F) . ( G , H) At 72 hpf , sox10 w as express ed in S chw ann cel ls of PLLn in WT ( G) , b ut abs ent in ba z1 mu tants ( H ) . ( I�L) Schw ann cel ls of spinal nerves ( ar row� h eads) w ere prominen tly labelled w ith sox10 in WT at 48 hpf ( I, K) , bu t highly reduced in baz 1 mutants ( J, L) . ( I, J ) ar e close�ups of ( K, L) . ( M , N ) f oxd3� expr ess ing Schw ann and satellit e glial cel ls ( asterisks ) as sociated w ith spinal nerves w ere pr om inent in WT (M) , but reduced in number in baz1 mutants at 48 hpf ( N) . All views are lateral view s of posterior t runk unless stated otherw ise. 99第 30 卷 � � � � � 中 南 林 业 科 技 大 学 学 报 Fig. 4 � Oligodendrocyte development in sox10baz1 and wild�type siblings ( A , B) Oligoden drocyte precursors ( arrow s) at 48 hpf in t run k ex pres sed sox10 and w ere indis tin guishable in num ber and dist ribut ion in WT ( A) and baz 1 m utant ( B) . ( C, D) At 72 hpf , sox 10 w as expressed in disp ersin g oligodendrocytes of head in both WT ( C) and baz 1 mutant ( D) . Note th at th e dist ribut ion w as st rongly clustered in WT, but loose in baz 1 mutant . ( E, F) T he region s of s om ites 7�11 of WT ( E) and baz 1 mutant ( F) show that sox 10 ex pres sion in oligoden drocyte prog enitors ( arrow� heads) in the vent ralmost spinal cord w ere unaf fected at 72 hpf in baz 1 mu tant . ( G , H ) Oligodendr ocyte dif f erent iat ion is abnor� mal as show n by st rongly decreased mbp ex pres sion in h indbrain read of 72 hpf emb ryo. ( F ig. 5I) . Although the sox10 + cells w ere obv i� ously r educed in the same place in head of sox10 baz 1 ( Fig . 5E) , there w ere st ill a few of them could be observed. In wild�type, sox10 was st rongly ex� pressed in glia associated spinal nerves in t runk ( Fig. 5B) and tail ( Fig. 5D) , but this expression w as ex t remely reduced in the same place of sox10 baz 1 mutant ( F ig. 5