lmo4a基因在斑马鱼内耳发育中的功能研究
摘要
目的：世界范围内约有2.78亿人患有中度以上的听力损失,在中国听力语言残疾患者达2780万人。聋病防治主要包括传导性耳聋和感音神经性耳聋两个方面。半个世纪以来,传导性耳聋已经取得成熟的防治经验,感音神经性耳聋的预防和治疗是聋病基础研究的热点与难点。临床上,很多感音神经性耳聋为内耳发育异常所引起。内耳畸形和耳聋病程复杂,功能为不可逆缺失,治疗方法相对局限。寻找更多的内耳畸形、耳聋致病基因,探索致畸致聋机制,并筛选针对发病机制特异性治疗制剂,已成为耳科学基础与临床研究者的共同责任。LM04基因编码一种转录调节因子,小鼠耳泡中组织特异性敲除LM04,可见半规管发育异常、半规管嵴发育不良。因此LM04得到更多学者的关注。斑马鱼内耳发生机制以及前庭形态、功能保守,胚胎透明、易于操作,是研究内耳发育畸形和耳聋的优势模型。本实验主要研究lmo4基因对斑马鱼内耳发育的影响,并探索影响机制,为内耳畸形、感应神经性耳聋的防治奠定理论基础。
方法：本文主要采用斑马鱼胚胎内注射寡核苷酸Morpholino (MO)和mRNA的方法构建lmo4基因低表达和过表达模型；通过表型观察、原位杂交、免疫组化等方式研究lmo4基因对斑马鱼内耳发育的重要作用；通过筛选与内耳结构发生密切相关的基因表达变化研究lmo4基因影响内耳发育的机制；并针对机制筛选可治疗挽救表型的制剂。
结果：斑马鱼中lmo4的两个同源基因分别是lmo4a和lmo4b,两者均为母源性基因。10hpf,lmo4a主要在中胚叶及神经板中表达；14hpf开始表达于耳基板、耳泡及周围基质；随着耳泡发育,lmo4a表达范围与毛细胞区域部分重叠,并标记与半规管发生紧密相关的3个半规管嵴及半规管突起。lmo4b在耳泡、周围基质、及侧线中持续表达。通过Morpholino低表达lmo4b后未观察到内耳表型,我们后期的研究主要集中于lmo4a在内耳的功能。通过Morpholino低表达lmo4a后发现前基板发育受阻；耳泡大小、毛细胞数目、听平衡神经节内细胞数目呈等比减少；同时伴有特异的半规管发育异常(仅水平半规管突起形成)；与半规管发育紧密相关的bmp4,bmp2b明显上调,同时下调BMP信号可很大程度挽救低表达lmo4a诱导的半规管不发育表型。
结论：低表达lmo4a诱导的耳泡大小、内耳毛细胞和神经节的数量减小可归因于lmo4a对前基板早期发育的影响；而半规管畸形是由于BMP信号上调导致；耳泡内注射DM (AMP-activated protein kinase inhibitor, Dorsomorphin)小分子可下调BMP信号,促进半规管发育,挽救畸形。本实验探讨了lmo4a基因在斑马鱼内耳发育的重要作用和作用机制,耳泡内注射小分子DM可促进半规管发育,挽救半规管畸形。
Deafness effects the health of human seriously. Hearing loss affects approximately27million in the china alone and has a high incidence-about278million people suffer from moderate to severe hearing loss in the world. It can be safely assumed that all children with congenital sensory hearing loss would have detectable abnormalities in their inner ears if they could be examined histologically. Development of therapies to treat hearing loss and balance disorders iscomplicated by the diversity of disease processes leading to functional loss and the current approaches available for treatment. Despite the significant impact of hearing and balance disorders on the general population there are currently no dedicated pharmaceuticals that target the inner ear.
Incidence reduction, early detection, and early treatment of hearing loss have become a common interest of society. Prevention and treatment of Inner Ear Malformations and sensorineural deafness basic research is still hot and difficult.
The role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. In mouse, LMO4is initially expressed in the dorsolateral portion of the otic vesicle and its expression persists in the semicircular canals, macula, crista throughout embryogenesis indicating its important role in the mouse inner ear development. Targeted disruption of Lmo4resulted in the dysmorphogenesis of the vestibule and in the absence of three semicircular canals, anterior and posterior cristae.
Zebrafish has two homologous genes Imo4a and Imo4b. Transcripts of both genes were present in unfertilized eggs, suggesting a maternal origin. Imo4a expression is rather dynamic, mainly in the developing mesoderm and neural plate at late gastrula and segmentation stages. The onset of Imo4a in the otic organs was detected weakly in the otic placode at14hpf. From then on, Imo4a is expressed outside the developing otic vesicle, as well as at anterior-dorsal tip and the ventral part of the vesicle. At24hpf, Imo4a expression likely includes some ventral cells of the vesicle and perhaps newly emerged hair cell precursors. At36hpf, Imo4a expression is seen in the anterior, ventra-lateral and posterior positions, perhaps marking the3developing cristae. The epithelial protrusions, which form the future semicircular canal system, start to undergo morphological change at48hpf, and weak Imo4a expression is detected in the apical protrusion.No obvious otic phenotype was detected in Imo4b morphants,so further research was focus on the function of Imo4a in the inner ear.
Loss of Imo4a function leads to proportional reduction of otocyst size, hair cells statoacoustic ganglia. The markers that can highlight the preplacodal ectoderm (PPE. eyal and six4.1), or early sensory cells that derived from the PPE(i.e., ngnl, atoh1b and dlx3/4b) are down-regulated suggesting there is an overall decrease of gene expressions in the caudal PPE and PPE derived cells in the morphants and there is an early otic specification defect that leads to a general defect of inner ear functions.
However, a failure to grow the semicircular canals (SSCs) in Imo4a morphants suggests us a direct link between Imo4a and SSC epithelial budding event.Because bmp4, bmp2b,which, are normally expressed in sequence at the epithelial protrusion buds of the otic vesicle, are all elevated in lmo4aMO ears, we reason that up-regulation of Bmp signaling may prevent the outgrowth of the protrusions in the morphants. As our presumption,injection AMP-activated protein kinase (AMPK) inhibitor(DM, Dorsomorphin) to the otic vesicle can partically rescue the semicircular canals phenotype.
In summary, Imo4a in vivo may regulate Bmps, promoting semicircular morphogenesis. This is the first study exploring a significant opportunity for the development of local delivery of the small molecule DM as a totally new therapeutic approach for inner ear malformation and deafness.
方法：本文主要采用斑马鱼胚胎内注射寡核苷酸Morpholino (MO)和mRNA的方法构建lmo4基因低表达和过表达模型；通过表型观察、原位杂交、免疫组化等方式研究lmo4基因对斑马鱼内耳发育的重要作用；通过筛选与内耳结构发生密切相关的基因表达变化研究lmo4基因影响内耳发育的机制；并针对机制筛选可治疗挽救表型的制剂。
结果：斑马鱼中lmo4的两个同源基因分别是lmo4a和lmo4b,两者均为母源性基因。10hpf,lmo4a主要在中胚叶及神经板中表达；14hpf开始表达于耳基板、耳泡及周围基质；随着耳泡发育,lmo4a表达范围与毛细胞区域部分重叠,并标记与半规管发生紧密相关的3个半规管嵴及半规管突起。lmo4b在耳泡、周围基质、及侧线中持续表达。通过Morpholino低表达lmo4b后未观察到内耳表型,我们后期的研究主要集中于lmo4a在内耳的功能。通过Morpholino低表达lmo4a后发现前基板发育受阻；耳泡大小、毛细胞数目、听平衡神经节内细胞数目呈等比减少；同时伴有特异的半规管发育异常(仅水平半规管突起形成)；与半规管发育紧密相关的bmp4,bmp2b明显上调,同时下调BMP信号可很大程度挽救低表达lmo4a诱导的半规管不发育表型。
结论：低表达lmo4a诱导的耳泡大小、内耳毛细胞和神经节的数量减小可归因于lmo4a对前基板早期发育的影响；而半规管畸形是由于BMP信号上调导致；耳泡内注射DM (AMP-activated protein kinase inhibitor, Dorsomorphin)小分子可下调BMP信号,促进半规管发育,挽救畸形。本实验探讨了lmo4a基因在斑马鱼内耳发育的重要作用和作用机制,耳泡内注射小分子DM可促进半规管发育,挽救半规管畸形。
Deafness effects the health of human seriously. Hearing loss affects approximately27million in the china alone and has a high incidence-about278million people suffer from moderate to severe hearing loss in the world. It can be safely assumed that all children with congenital sensory hearing loss would have detectable abnormalities in their inner ears if they could be examined histologically. Development of therapies to treat hearing loss and balance disorders iscomplicated by the diversity of disease processes leading to functional loss and the current approaches available for treatment. Despite the significant impact of hearing and balance disorders on the general population there are currently no dedicated pharmaceuticals that target the inner ear.
Incidence reduction, early detection, and early treatment of hearing loss have become a common interest of society. Prevention and treatment of Inner Ear Malformations and sensorineural deafness basic research is still hot and difficult.
The role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. In mouse, LMO4is initially expressed in the dorsolateral portion of the otic vesicle and its expression persists in the semicircular canals, macula, crista throughout embryogenesis indicating its important role in the mouse inner ear development. Targeted disruption of Lmo4resulted in the dysmorphogenesis of the vestibule and in the absence of three semicircular canals, anterior and posterior cristae.
Zebrafish has two homologous genes Imo4a and Imo4b. Transcripts of both genes were present in unfertilized eggs, suggesting a maternal origin. Imo4a expression is rather dynamic, mainly in the developing mesoderm and neural plate at late gastrula and segmentation stages. The onset of Imo4a in the otic organs was detected weakly in the otic placode at14hpf. From then on, Imo4a is expressed outside the developing otic vesicle, as well as at anterior-dorsal tip and the ventral part of the vesicle. At24hpf, Imo4a expression likely includes some ventral cells of the vesicle and perhaps newly emerged hair cell precursors. At36hpf, Imo4a expression is seen in the anterior, ventra-lateral and posterior positions, perhaps marking the3developing cristae. The epithelial protrusions, which form the future semicircular canal system, start to undergo morphological change at48hpf, and weak Imo4a expression is detected in the apical protrusion.No obvious otic phenotype was detected in Imo4b morphants,so further research was focus on the function of Imo4a in the inner ear.
Loss of Imo4a function leads to proportional reduction of otocyst size, hair cells statoacoustic ganglia. The markers that can highlight the preplacodal ectoderm (PPE. eyal and six4.1), or early sensory cells that derived from the PPE(i.e., ngnl, atoh1b and dlx3/4b) are down-regulated suggesting there is an overall decrease of gene expressions in the caudal PPE and PPE derived cells in the morphants and there is an early otic specification defect that leads to a general defect of inner ear functions.
However, a failure to grow the semicircular canals (SSCs) in Imo4a morphants suggests us a direct link between Imo4a and SSC epithelial budding event.Because bmp4, bmp2b,which, are normally expressed in sequence at the epithelial protrusion buds of the otic vesicle, are all elevated in lmo4aMO ears, we reason that up-regulation of Bmp signaling may prevent the outgrowth of the protrusions in the morphants. As our presumption,injection AMP-activated protein kinase (AMPK) inhibitor(DM, Dorsomorphin) to the otic vesicle can partically rescue the semicircular canals phenotype.
In summary, Imo4a in vivo may regulate Bmps, promoting semicircular morphogenesis. This is the first study exploring a significant opportunity for the development of local delivery of the small molecule DM as a totally new therapeutic approach for inner ear malformation and deafness.
引文
[1]陈晓巍.重视遗传性耳聋的基因诊断[J].中华医学杂志,2007,87(16)：1081-1083
[2]韩东一.中国聋病防治现状[J].中华耳科学杂志.2007,5(4)：345-348
[3]Jackler RK, Luxford WM, House WF. Congenital malformations of the inner ear:a classification based on embryogenesis[J]. Laryngoscope.1987 Mar;97(3 Pt 2 Suppl 40):2-14.
[4]Goldfarb A, Avraham KB. Genetics of deafness:recent advances and clinical implicationa[J]. J Basic Clin Physiol Pharmacol.2002;13(2):75-88.
[5]胡浩,邬玲仟,梁德生,夏家辉.非综合征型感音神经性聋相关基因研究进展[J].中华耳鼻咽喉头颈外科杂志,2005,40(8)：633-641
[6]李建忠,程静,卢宇,孙艺,康东洋,张听,陈艾婷,袁慧军,韩东一.常染色体显性遗传性聋家系的临床表型及候选致病基因分析[J].中华耳科学杂志2010,8(1)：29-34
[7]Kerber KA. The genetics of vertigo[J]. Semin Neurol.2006; 26:484-491.
[8]Jen JC. Recent advances in the genetics of recurrent vertigo and vestibulopathy[J]. Curr Opin Neurol.2008 Feb;21(1):3-7.
[9]Karis A, Pata I, van Doorninck JH, et al.Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear[J].J Comp Neurol,2001 Jan 22,429(4):615-630.
[10]Wallis D, Hamblen M, Zhou Y, et al.The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival[J].Development,2003 Jan,130(1):221-232.
[11]Radde-Gallwitz K, Pan L, Gan L,et al.Expression of Islet 1 marks the sensory and neuronal lineages in the mammalian inner ear[J].J Comp Neurol,2004 Sep 27,477(4):412-421.
[12]Bondurand N, Kobetz A, Pingault V, et al.Expression of the SOX10 gene during human development[J].FEBS Lett,1998 Aug 7,432(3):168-172.
[13]Zheng JL, Gao WQ.Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears[J].Nat Neurosci,2000 Jun,3(6):580-586.
[14]Deng M, Pan L, Xie X,et al. Differential expression of LIM domain-only (LMO) genes in the developing mouse inner ear[J].Gene Expr Patterns.2006 Oct;6(8):857-63.
[15]Deng M, Pan L, Xie X,et al. Requirement for Lmo4 in the vestibular morphogenesis of mouse inner ear[J]. Dev Biol.2010 Feb 1;338(1):38-49.
[16]Lane ME, Runko AP, Roy NM,et al. Dynamic expression and regulation by Fgf8 and Pou2 of the zebrafish LIM-only gene, lmo4[J]. Mech Dev.2002 Dec;119 Suppl 1:S185-9.
[17]McCollum CW, Amin SR, Pauerstein P,et al. A zebrafish LMO4 ortholog limits the size of the forebrain and eyes through negative regulation of six3b and rx3[J].Dev Biol. 2007 Sep 15;309(2):373-85. Epub 2007 Jul 12.
[18]Wang, W, Chan, E,K, Baron, S, Van de Water, T, Lufkin, T. Hmx2 homeobox gene control of murine vestibular morphogenesis[J]. Development 2001128,5017-5029.
[19]Wang, W, Grimmer, J,F, Van De Water, T,R, Lufkin, T. Hmx2 and Hmx3 homeobox genes direct development of the murine inner ear and hypothalamus and can be functionally replaced by Drosophila Hmx[J]. Dev Cell.20047,439-453.
[20]Nasevicius A, Ekker SC. Effective targeted gene'knockdown'in zebrafish[J]. Nat Genet.200026:216-20.
[21]Leung AY, Mendenhall EM, Kwan TT,et al. Characterization of expanded intermediate cell mass in zebrafish chordin morphant embryos[J]. Dev Biol.2005 Jan 1;277(1):235-54.
[22]Branam AM, Hoffman GG, Pelegri F, Greenspan DS. Zebrafish chordin-like and chordin are functionally redundant in regulating patterning of the dorsoventral axis[J]. Dev Biol.2010 May 15;341(2):444-58. Epub 2010 Mar 11.
[23]Miller-Bertoglio VE, Fisher S, Sanchez A,et al. Differential regulation of chordin expression domains in mutant zebrafish[J]. Dev Biol.1997 Dec 15;192(2):537-50.
[24]Pyati UJ, Webb AE, Kimelman D. Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development. Developmen[J]. 2005 May;132(10):2333-43.
[25]Yu PB, Hong CC, Sachidanandan C,et al.Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism[J]. Nat Chem Biol.2008 Jan;4(1):33-41. Epub 2007 Nov 18.
[26]Hao J, Ho JN, Lewis JA,et al. In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors[J]. ACS Chem Biol.2010 Feb 19;5(2):245-53.
[27]Lindsay JR. Profound childhood deafness. Inner ear pathology[J]. Ann Otol Rhinol Laryngol.1973 Mar-Apr;82:Suppl 5:1-121.
[28]Grande, T, and Young, B. The ontogeny and homology of the Weberian apparatus in the zebrafish Danio rerio (Ostariophysi:Cypriniformes) [J]. Zool. J. Linn. Soc,2004, 140:241-254.
[29]Bever MM, Fekete DM. Atlas of the developing inner ear in zebrafish[J]. Dev Dyn. 2002 Apr;223(4):536-43.
[30]Haddon C, Lewis J. Early ear development in the embryo of the zebrafish, Danio rerio[J].J Comp Neurol.1996 Jan 29;365(1):113-28.
[31]Tanimoto M, Ota Y, Horikawa K, et al.Auditory input to CNS is acquired coincidentally with development of inner ear after formation of functional afferent pathway in zebrafish[J].J Neurosci.2009 Mar 4;29(9):2762-7.
[32]Kozlowski DJ, Murakami T, Ho RK, Weinberg ES. Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein[J]. Biochem Cell Biol 1997.75:551-562
[33]Akimenko M-A, Ekker M, Wegner J, Lin W, Westerfield M. Combinatorial expression of three zebrafish genes related to Distal-Less:part of a homeobox gene code for the head[J]. J Neurosci 1994.14:3475-3486.
[34]Ellies DL, Stock DW, Hatch G, Giroux G, Weiss KM, Ekker M. Relationship between the genomic organisation and the overlapping embryonic expression patterns of the zebrafish dlx genes[J].Genomics 1997.45:580-590.
[35]Kobayashi M, Osanai H, Kawakami K, Yamamoto M. Expression of three zebrafish six4 genes in the cranial sensory placodes and the developing somites[J]. Mech Dev 2000.98:151-155.
[36]Sahly I, Andermann P, Petit C. The zebrafish eyal gene and its expression pattern during embryogenesis[J]. Dev Genes Evol 1999.209:399-410.
[37]Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish[J]. Dev Dyn 1995.203:253-310.
[38]Haddon C, Lewis J. Early ear development in the embryo of the zebrafish, Danio rerio[J].J Comp Neural 1996.365:113-123
[39]Whitfield TT, Riley BB, Chiang MY, Phillips B. Development of the zebrafish inner ear[J]. Dev Dyn.2002 Apr;223(4):427-58.
[40]Bricaud O, Collazo A.The transcription factor sixl inhibits neuronal and promotes hair cell fate in the developing zebrafish (Danio rerio) inner ear[J].J Neurosci.2006 Oct 11;26(41):10438-51.
[41]Omata Y, Nojima Y, Nakayama S,et al. Role of Bone morphogenetic protein 4 in zebrafish semicircular canal development[J]. Dev Growth Differ.2007 Dec;49(9):711-9. Epub 2007 Oct 1.
[42]Kenny, D. A., Jurata, L. W., Saga, Y, Gill, G. N. Identification and characterization of LMO4, an LMO gene with a novel pattern of expression during embryogenesis[J]. Proc. Nat. Acad. Sci.199895:11257-11262.
[43]Racevskis, J., Dill, A., Sparano, J. A., Ruan, H. Molecular cloning of LMO4, a new human LIM domain gene[J]. Biochim. Biophys. Acta 1999 1445:148-153.
[44]Sum, E. Y. M., Segara, D., Duscio, B., Bath, M. L., Field, A. S., Sutherland, R. L Lindeman, G. J., Visvader, J. E. Overexpression of LMO4 induces mammary hyperplasia, promotes cell invasion, and is a predictor of poor outcome in breast cancer[J]. Proc. Nat. Acad. Sci.2005102:7659-7664.
[45]Raft S, Nowotschin S, Liao J, Morrow BE. Suppression of neural fate and control of inner ear morphogenesis by Tbx1[J]. Development 2004131:1801-1812.
[46]Riccomagno MM, Takada S, Epstein DJ. Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh[J]. Genes Dev 2005 19:1612-1623.
[47]Robledo RF, Lufkin T. Dlx5 and Dlx6 homeobox genes are required for specification of the mammalian vestibular apparatus[J]. Genesis 2006 44:425-437.
[48]Thompson DL, Gerlach-Bank LM, Barald KF, Koenig RJ. Retinoic acid repression of bone morphogenetic protein 4 in inner ear development[J]. Mol Cell Biol 2003 23: 2277-2286.
[49]Wang W, Grimmer JF, Van De Water TR, Lufkin T. Hmx2 and Hmx3 homeobox genes direct development of the murine inner ear and hypothalamus and can be functionally replaced by Drosophila Hmx[J]. Dev Cell 2004 7:439-453.
[50]Mowbray C, Hammerschmidt M, Whitfield TT. Expression of BMP signalling pathway members in the developing zebrafish inner ear and lateral line[J]. Mech Dev. 2001 108(1-2):179-84.
[51]Hammond KL, Loynes HE, Mowbray C,et al. A late role for bmp2b in the morphogenesis of semicircular canal ducts in the zebrafish inner ear.PLoS One. 2009.4(2):e4368.
[52]Yu PB, Deng DY, Lai CS, Hong CC, Cuny GD, Bouxsein ML, Hong DW, McManus PM, Katagiri T,Sachidanandan C, Kamiya N, Fukuda T, Mishina Y, Peterson RT, Bloch KD. BMP type Ⅰ receptor inhibition reduces heterotopic ossification[J]. Nat Med 2008 14:1363-1369.
[1]Waterman RE, Bell DH. Epithelial fusion during early semicircular canal formation in the embryonic zebrafish,Brachydanio rerio[J]. Anat Rec 1984.210:101-114.
[2]Platt C. Zebrafish inner ear sensory surfaces are similar to those in goldfish[J]. Hear Res 1993.65:133-140.
[3]Haddon C, Lewis J. Early ear development in the embryo of the zebrafish, Danio rerio[J]. J Comp Neurol 1996.365:113-123.
[4]Bever MM, Fekete DM. Atlas of the developing inner ear in zebrafish[J]. Dev Dyn 2002.223:536-543.
[5]Allende ML, Amsterdam A, Becker T,et al.Insertion mutagenesis in zebrafish identifies two novel genes,pescadillo and dead eye,enssential for embryonic development[J]. Genes Dev.1996 Dec 15; 10(24):3141-55.
[6]Driever W, Solnica-Krezel L, Schier AF,et al.A genetic screen for mutations affecting embryogenesis in zebrafish[J]. Development.1996 Dec; 123:37-46.
[7]Haffter P, Niisslein-Volhard C. Large scale genetics in a small vertebrate, the zebrafish[J]. Int J Dev Biol.1996 Feb;40(1):221-7.
[8]Nicolson T, R"usch A, Friedrich RW, Granato M, Ruppersberg JP, N"usslein-Volhard C. Genetic analysis of vertebrate sensory hair cell mechanosensation:the zebrafish circler mutants[J]. Neuron 1998.20:271-283.
[9]Whitfield TT, Riley BB, Chiang MY,et al. Development of the zebrafish inner ear[J]. Dev Dyn.2002 Apr;223(4):427-58.
[10]Whitfield TT. Zebrafish as a model for hearing and deafness[J]. J Neurobiol. 2002 Nov 5;53(2):157-71.
[11]Gaiano N, Amsterdam A, Kawakami K,et al. Insertional mutagenesis and rapid cloning of essential genes in zebrafish[J]. Nature.1996 Oct 31;383(6603):829-32.
[12]Summerton J, Weller D. Morpholino antisense oligomers:design, preparation, and properties[J]. Antisense Nucleic Acid Drug Dev.1997 Jun;7(3):187-95.
[13]Dutton K, Dutton JR, Pauliny A, Kelsh R.N.. A morpholino phenocopy of the colourless mutant[J]. Genesis 2001a 30:188-189.
[14]Chen W, Burgess S, Golling G, Amsterdam A, Hopkins N. High-throughput selection of retrovirus producer cell lines leads to markedly improved efficiency of germline-transmissible insertions in zebrafish[J]. J Virol 2002.76:2192-2198.
[15]Zeddies D G, Fay R R. Development of the acoustically evokedbehavioral response in zebrafish to pure tones[J]. J Exper Biol,2005,208:1363-1372
[16]HiggsD M, Rollo A K, SouzaMJ, et al. Development of form andfunction in peripheral auditory structures of the zebrafish (Danio rerio) [J]. J Acoust Soc Amer, 2003,113(2):1145～1154
[17]Goodrich L V. Hear, hear for the zebrafish[J]. Neuron,2005,45(1):3-5
[18]Williams, J.A. and Holder, N.2000.Cell turnover in neuromasts of zebrafish larvae[J].Hear. Res.143,171-181
[19]Owens, K.N. et al.2007. Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response[J]. J.Comp. Neurol.502,522-543
[20]Sarrazin AF, Villablanca EJ, Nunez VA, et al.2006.Proneural gene requirement for hair cell differentiation in the zebrafish lateral line[J]. Dev Biol. 15;295(2):534-45.
[21]Dambly-Chaudiere C, Sapede D, Soubiran F,et al.2003.The lateral line of zebrafish:a model system for the analysis of morphogenesis and neural development in vertebrates[J]. Biol Cell.95(9):579-87.
[22]Moorman S J, Burress C, Cordova R, et al. Stimulus dependence of the development of the zebrafish (Danio rerio) vestibular system[J].J Neurobiol,1999, 38(2):247～258
[23]Bang P I, Yelick P C, Malicki J J, et al. High-throughput behavioral screening method for detecting auditory response defects in zebrafish[J]. J Neurosci Meth, 2002,118(2):177～187
[24]Lieschke GJ, Currie PD.Animal models of human disease:zebrafish swim into view[J]. Nat Rev Genet.2007 May;8(5):353-67
[25]Sarrazin AF, Villablanca EJ, Nunez VA, et al. Proneural gene requirement for hair cell differentiation in the zebrafish lateral line[J]. Dev Biol.2006 Jul 15;295(2):534-45. Epub 2006 Apr 4.
[26]Dambly-Chaudiere C, Sapede D, Soubiran F,et al. The lateral line of zebrafish:a model system for the analysis of morphogenesis and neural development in vertebrates[J]. Biol Cell.2003 Dec;95(9):579-87.
[27]Ou HC, Raible DW, Rubel EW. Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line[J]. Hear Res.2007 Nov;233(1-2):46-53. Epub 2007 Jul 19.
[28]Murakami SL, Cunningham LL, Werner LA, et al. Developmental differences in susceptibility to neomycin-induced hair cell death in the lateral line neuromasts of zebrafish (Danio rerio) [J]. Hear Res.2003 Dec;186(1-2):47-56
[29]Sha, S.H. et al. Aspirin to prevent gentamicin-induced hearing loss[J]. N. Engl. J.Med.2006 354,1856-1857
[30]Feldman, L. et al. Gentamicin-induced ototoxicity in hemodialysis patients is ameliorated by N-acetylcysteine[J]. Kidney Int.200772,359-363
[31]Sugahara, K. et al. JNK signaling in neomycin-induced vestibular hair cell death[J]. Hear. Res.2006 221,128-135
[32]Cheng, A.G. et al. Hair cell death in the avian basilar papilla:characterization of the in vitro model and caspase activation[J]. J. Assoc. Res. Otolaryngol.2003 4, 91-105
[33]Chiu LL, Cunningham LL, Raible DW, et al. Using the zebrafish lateral line to screen for ototoxicity[J]. J Assoc Res Otolaryngol.2008 Jun;9(2):178-90. Epub 2008 Apr 12.
[34]Owens, K.N. et al. (2008) Identification of genetic and chemical modulators of zebrafish mechanosensory hair cell death[J]. PLoS Genet.4, e1000020
[35]Ou, H.C. et al. Identification of FDA-approved drugs and bioactives that protect hair cells in the zebrafish (Danio rerio) lateral line and mouse (Mus musculus) utricle[J]. J. Assoc. Res. Otolaryngol. (2009) 10,191-203
[36]Ma, E.Y. et al. Notch signaling regulates the extent of hair cell regeneration in the zebrafish lateral line[J]. J. Neurosci. (2008) 28,2261-2273
[2]韩东一.中国聋病防治现状[J].中华耳科学杂志.2007,5(4)：345-348
[3]Jackler RK, Luxford WM, House WF. Congenital malformations of the inner ear:a classification based on embryogenesis[J]. Laryngoscope.1987 Mar;97(3 Pt 2 Suppl 40):2-14.
[4]Goldfarb A, Avraham KB. Genetics of deafness:recent advances and clinical implicationa[J]. J Basic Clin Physiol Pharmacol.2002;13(2):75-88.
[5]胡浩,邬玲仟,梁德生,夏家辉.非综合征型感音神经性聋相关基因研究进展[J].中华耳鼻咽喉头颈外科杂志,2005,40(8)：633-641
[6]李建忠,程静,卢宇,孙艺,康东洋,张听,陈艾婷,袁慧军,韩东一.常染色体显性遗传性聋家系的临床表型及候选致病基因分析[J].中华耳科学杂志2010,8(1)：29-34
[7]Kerber KA. The genetics of vertigo[J]. Semin Neurol.2006; 26:484-491.
[8]Jen JC. Recent advances in the genetics of recurrent vertigo and vestibulopathy[J]. Curr Opin Neurol.2008 Feb;21(1):3-7.
[9]Karis A, Pata I, van Doorninck JH, et al.Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear[J].J Comp Neurol,2001 Jan 22,429(4):615-630.
[10]Wallis D, Hamblen M, Zhou Y, et al.The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival[J].Development,2003 Jan,130(1):221-232.
[11]Radde-Gallwitz K, Pan L, Gan L,et al.Expression of Islet 1 marks the sensory and neuronal lineages in the mammalian inner ear[J].J Comp Neurol,2004 Sep 27,477(4):412-421.
[12]Bondurand N, Kobetz A, Pingault V, et al.Expression of the SOX10 gene during human development[J].FEBS Lett,1998 Aug 7,432(3):168-172.
[13]Zheng JL, Gao WQ.Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears[J].Nat Neurosci,2000 Jun,3(6):580-586.
[14]Deng M, Pan L, Xie X,et al. Differential expression of LIM domain-only (LMO) genes in the developing mouse inner ear[J].Gene Expr Patterns.2006 Oct;6(8):857-63.
[15]Deng M, Pan L, Xie X,et al. Requirement for Lmo4 in the vestibular morphogenesis of mouse inner ear[J]. Dev Biol.2010 Feb 1;338(1):38-49.
[16]Lane ME, Runko AP, Roy NM,et al. Dynamic expression and regulation by Fgf8 and Pou2 of the zebrafish LIM-only gene, lmo4[J]. Mech Dev.2002 Dec;119 Suppl 1:S185-9.
[17]McCollum CW, Amin SR, Pauerstein P,et al. A zebrafish LMO4 ortholog limits the size of the forebrain and eyes through negative regulation of six3b and rx3[J].Dev Biol. 2007 Sep 15;309(2):373-85. Epub 2007 Jul 12.
[18]Wang, W, Chan, E,K, Baron, S, Van de Water, T, Lufkin, T. Hmx2 homeobox gene control of murine vestibular morphogenesis[J]. Development 2001128,5017-5029.
[19]Wang, W, Grimmer, J,F, Van De Water, T,R, Lufkin, T. Hmx2 and Hmx3 homeobox genes direct development of the murine inner ear and hypothalamus and can be functionally replaced by Drosophila Hmx[J]. Dev Cell.20047,439-453.
[20]Nasevicius A, Ekker SC. Effective targeted gene'knockdown'in zebrafish[J]. Nat Genet.200026:216-20.
[21]Leung AY, Mendenhall EM, Kwan TT,et al. Characterization of expanded intermediate cell mass in zebrafish chordin morphant embryos[J]. Dev Biol.2005 Jan 1;277(1):235-54.
[22]Branam AM, Hoffman GG, Pelegri F, Greenspan DS. Zebrafish chordin-like and chordin are functionally redundant in regulating patterning of the dorsoventral axis[J]. Dev Biol.2010 May 15;341(2):444-58. Epub 2010 Mar 11.
[23]Miller-Bertoglio VE, Fisher S, Sanchez A,et al. Differential regulation of chordin expression domains in mutant zebrafish[J]. Dev Biol.1997 Dec 15;192(2):537-50.
[24]Pyati UJ, Webb AE, Kimelman D. Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development. Developmen[J]. 2005 May;132(10):2333-43.
[25]Yu PB, Hong CC, Sachidanandan C,et al.Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism[J]. Nat Chem Biol.2008 Jan;4(1):33-41. Epub 2007 Nov 18.
[26]Hao J, Ho JN, Lewis JA,et al. In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors[J]. ACS Chem Biol.2010 Feb 19;5(2):245-53.
[27]Lindsay JR. Profound childhood deafness. Inner ear pathology[J]. Ann Otol Rhinol Laryngol.1973 Mar-Apr;82:Suppl 5:1-121.
[28]Grande, T, and Young, B. The ontogeny and homology of the Weberian apparatus in the zebrafish Danio rerio (Ostariophysi:Cypriniformes) [J]. Zool. J. Linn. Soc,2004, 140:241-254.
[29]Bever MM, Fekete DM. Atlas of the developing inner ear in zebrafish[J]. Dev Dyn. 2002 Apr;223(4):536-43.
[30]Haddon C, Lewis J. Early ear development in the embryo of the zebrafish, Danio rerio[J].J Comp Neurol.1996 Jan 29;365(1):113-28.
[31]Tanimoto M, Ota Y, Horikawa K, et al.Auditory input to CNS is acquired coincidentally with development of inner ear after formation of functional afferent pathway in zebrafish[J].J Neurosci.2009 Mar 4;29(9):2762-7.
[32]Kozlowski DJ, Murakami T, Ho RK, Weinberg ES. Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein[J]. Biochem Cell Biol 1997.75:551-562
[33]Akimenko M-A, Ekker M, Wegner J, Lin W, Westerfield M. Combinatorial expression of three zebrafish genes related to Distal-Less:part of a homeobox gene code for the head[J]. J Neurosci 1994.14:3475-3486.
[34]Ellies DL, Stock DW, Hatch G, Giroux G, Weiss KM, Ekker M. Relationship between the genomic organisation and the overlapping embryonic expression patterns of the zebrafish dlx genes[J].Genomics 1997.45:580-590.
[35]Kobayashi M, Osanai H, Kawakami K, Yamamoto M. Expression of three zebrafish six4 genes in the cranial sensory placodes and the developing somites[J]. Mech Dev 2000.98:151-155.
[36]Sahly I, Andermann P, Petit C. The zebrafish eyal gene and its expression pattern during embryogenesis[J]. Dev Genes Evol 1999.209:399-410.
[37]Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish[J]. Dev Dyn 1995.203:253-310.
[38]Haddon C, Lewis J. Early ear development in the embryo of the zebrafish, Danio rerio[J].J Comp Neural 1996.365:113-123
[39]Whitfield TT, Riley BB, Chiang MY, Phillips B. Development of the zebrafish inner ear[J]. Dev Dyn.2002 Apr;223(4):427-58.
[40]Bricaud O, Collazo A.The transcription factor sixl inhibits neuronal and promotes hair cell fate in the developing zebrafish (Danio rerio) inner ear[J].J Neurosci.2006 Oct 11;26(41):10438-51.
[41]Omata Y, Nojima Y, Nakayama S,et al. Role of Bone morphogenetic protein 4 in zebrafish semicircular canal development[J]. Dev Growth Differ.2007 Dec;49(9):711-9. Epub 2007 Oct 1.
[42]Kenny, D. A., Jurata, L. W., Saga, Y, Gill, G. N. Identification and characterization of LMO4, an LMO gene with a novel pattern of expression during embryogenesis[J]. Proc. Nat. Acad. Sci.199895:11257-11262.
[43]Racevskis, J., Dill, A., Sparano, J. A., Ruan, H. Molecular cloning of LMO4, a new human LIM domain gene[J]. Biochim. Biophys. Acta 1999 1445:148-153.
[44]Sum, E. Y. M., Segara, D., Duscio, B., Bath, M. L., Field, A. S., Sutherland, R. L Lindeman, G. J., Visvader, J. E. Overexpression of LMO4 induces mammary hyperplasia, promotes cell invasion, and is a predictor of poor outcome in breast cancer[J]. Proc. Nat. Acad. Sci.2005102:7659-7664.
[45]Raft S, Nowotschin S, Liao J, Morrow BE. Suppression of neural fate and control of inner ear morphogenesis by Tbx1[J]. Development 2004131:1801-1812.
[46]Riccomagno MM, Takada S, Epstein DJ. Wnt-dependent regulation of inner ear morphogenesis is balanced by the opposing and supporting roles of Shh[J]. Genes Dev 2005 19:1612-1623.
[47]Robledo RF, Lufkin T. Dlx5 and Dlx6 homeobox genes are required for specification of the mammalian vestibular apparatus[J]. Genesis 2006 44:425-437.
[48]Thompson DL, Gerlach-Bank LM, Barald KF, Koenig RJ. Retinoic acid repression of bone morphogenetic protein 4 in inner ear development[J]. Mol Cell Biol 2003 23: 2277-2286.
[49]Wang W, Grimmer JF, Van De Water TR, Lufkin T. Hmx2 and Hmx3 homeobox genes direct development of the murine inner ear and hypothalamus and can be functionally replaced by Drosophila Hmx[J]. Dev Cell 2004 7:439-453.
[50]Mowbray C, Hammerschmidt M, Whitfield TT. Expression of BMP signalling pathway members in the developing zebrafish inner ear and lateral line[J]. Mech Dev. 2001 108(1-2):179-84.
[51]Hammond KL, Loynes HE, Mowbray C,et al. A late role for bmp2b in the morphogenesis of semicircular canal ducts in the zebrafish inner ear.PLoS One. 2009.4(2):e4368.
[52]Yu PB, Deng DY, Lai CS, Hong CC, Cuny GD, Bouxsein ML, Hong DW, McManus PM, Katagiri T,Sachidanandan C, Kamiya N, Fukuda T, Mishina Y, Peterson RT, Bloch KD. BMP type Ⅰ receptor inhibition reduces heterotopic ossification[J]. Nat Med 2008 14:1363-1369.
[1]Waterman RE, Bell DH. Epithelial fusion during early semicircular canal formation in the embryonic zebrafish,Brachydanio rerio[J]. Anat Rec 1984.210:101-114.
[2]Platt C. Zebrafish inner ear sensory surfaces are similar to those in goldfish[J]. Hear Res 1993.65:133-140.
[3]Haddon C, Lewis J. Early ear development in the embryo of the zebrafish, Danio rerio[J]. J Comp Neurol 1996.365:113-123.
[4]Bever MM, Fekete DM. Atlas of the developing inner ear in zebrafish[J]. Dev Dyn 2002.223:536-543.
[5]Allende ML, Amsterdam A, Becker T,et al.Insertion mutagenesis in zebrafish identifies two novel genes,pescadillo and dead eye,enssential for embryonic development[J]. Genes Dev.1996 Dec 15; 10(24):3141-55.
[6]Driever W, Solnica-Krezel L, Schier AF,et al.A genetic screen for mutations affecting embryogenesis in zebrafish[J]. Development.1996 Dec; 123:37-46.
[7]Haffter P, Niisslein-Volhard C. Large scale genetics in a small vertebrate, the zebrafish[J]. Int J Dev Biol.1996 Feb;40(1):221-7.
[8]Nicolson T, R"usch A, Friedrich RW, Granato M, Ruppersberg JP, N"usslein-Volhard C. Genetic analysis of vertebrate sensory hair cell mechanosensation:the zebrafish circler mutants[J]. Neuron 1998.20:271-283.
[9]Whitfield TT, Riley BB, Chiang MY,et al. Development of the zebrafish inner ear[J]. Dev Dyn.2002 Apr;223(4):427-58.
[10]Whitfield TT. Zebrafish as a model for hearing and deafness[J]. J Neurobiol. 2002 Nov 5;53(2):157-71.
[11]Gaiano N, Amsterdam A, Kawakami K,et al. Insertional mutagenesis and rapid cloning of essential genes in zebrafish[J]. Nature.1996 Oct 31;383(6603):829-32.
[12]Summerton J, Weller D. Morpholino antisense oligomers:design, preparation, and properties[J]. Antisense Nucleic Acid Drug Dev.1997 Jun;7(3):187-95.
[13]Dutton K, Dutton JR, Pauliny A, Kelsh R.N.. A morpholino phenocopy of the colourless mutant[J]. Genesis 2001a 30:188-189.
[14]Chen W, Burgess S, Golling G, Amsterdam A, Hopkins N. High-throughput selection of retrovirus producer cell lines leads to markedly improved efficiency of germline-transmissible insertions in zebrafish[J]. J Virol 2002.76:2192-2198.
[15]Zeddies D G, Fay R R. Development of the acoustically evokedbehavioral response in zebrafish to pure tones[J]. J Exper Biol,2005,208:1363-1372
[16]HiggsD M, Rollo A K, SouzaMJ, et al. Development of form andfunction in peripheral auditory structures of the zebrafish (Danio rerio) [J]. J Acoust Soc Amer, 2003,113(2):1145～1154
[17]Goodrich L V. Hear, hear for the zebrafish[J]. Neuron,2005,45(1):3-5
[18]Williams, J.A. and Holder, N.2000.Cell turnover in neuromasts of zebrafish larvae[J].Hear. Res.143,171-181
[19]Owens, K.N. et al.2007. Ultrastructural analysis of aminoglycoside-induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response[J]. J.Comp. Neurol.502,522-543
[20]Sarrazin AF, Villablanca EJ, Nunez VA, et al.2006.Proneural gene requirement for hair cell differentiation in the zebrafish lateral line[J]. Dev Biol. 15;295(2):534-45.
[21]Dambly-Chaudiere C, Sapede D, Soubiran F,et al.2003.The lateral line of zebrafish:a model system for the analysis of morphogenesis and neural development in vertebrates[J]. Biol Cell.95(9):579-87.
[22]Moorman S J, Burress C, Cordova R, et al. Stimulus dependence of the development of the zebrafish (Danio rerio) vestibular system[J].J Neurobiol,1999, 38(2):247～258
[23]Bang P I, Yelick P C, Malicki J J, et al. High-throughput behavioral screening method for detecting auditory response defects in zebrafish[J]. J Neurosci Meth, 2002,118(2):177～187
[24]Lieschke GJ, Currie PD.Animal models of human disease:zebrafish swim into view[J]. Nat Rev Genet.2007 May;8(5):353-67
[25]Sarrazin AF, Villablanca EJ, Nunez VA, et al. Proneural gene requirement for hair cell differentiation in the zebrafish lateral line[J]. Dev Biol.2006 Jul 15;295(2):534-45. Epub 2006 Apr 4.
[26]Dambly-Chaudiere C, Sapede D, Soubiran F,et al. The lateral line of zebrafish:a model system for the analysis of morphogenesis and neural development in vertebrates[J]. Biol Cell.2003 Dec;95(9):579-87.
[27]Ou HC, Raible DW, Rubel EW. Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line[J]. Hear Res.2007 Nov;233(1-2):46-53. Epub 2007 Jul 19.
[28]Murakami SL, Cunningham LL, Werner LA, et al. Developmental differences in susceptibility to neomycin-induced hair cell death in the lateral line neuromasts of zebrafish (Danio rerio) [J]. Hear Res.2003 Dec;186(1-2):47-56
[29]Sha, S.H. et al. Aspirin to prevent gentamicin-induced hearing loss[J]. N. Engl. J.Med.2006 354,1856-1857
[30]Feldman, L. et al. Gentamicin-induced ototoxicity in hemodialysis patients is ameliorated by N-acetylcysteine[J]. Kidney Int.200772,359-363
[31]Sugahara, K. et al. JNK signaling in neomycin-induced vestibular hair cell death[J]. Hear. Res.2006 221,128-135
[32]Cheng, A.G. et al. Hair cell death in the avian basilar papilla:characterization of the in vitro model and caspase activation[J]. J. Assoc. Res. Otolaryngol.2003 4, 91-105
[33]Chiu LL, Cunningham LL, Raible DW, et al. Using the zebrafish lateral line to screen for ototoxicity[J]. J Assoc Res Otolaryngol.2008 Jun;9(2):178-90. Epub 2008 Apr 12.
[34]Owens, K.N. et al. (2008) Identification of genetic and chemical modulators of zebrafish mechanosensory hair cell death[J]. PLoS Genet.4, e1000020
[35]Ou, H.C. et al. Identification of FDA-approved drugs and bioactives that protect hair cells in the zebrafish (Danio rerio) lateral line and mouse (Mus musculus) utricle[J]. J. Assoc. Res. Otolaryngol. (2009) 10,191-203
[36]Ma, E.Y. et al. Notch signaling regulates the extent of hair cell regeneration in the zebrafish lateral line[J]. J. Neurosci. (2008) 28,2261-2273