在Sox2陽性細(xì)胞敲除β-catenin對小鼠內(nèi)耳發(fā)育和極性的影響

錢曉青　任冬冬　叢　寧　趙　萌　遲放魯

(1 復(fù)旦大學(xué)附屬眼耳鼻喉科醫(yī)院耳鼻喉科研究院,2耳鼻喉科,5實(shí)驗(yàn)中心　上?！?00031;3上海市聽覺醫(yī)學(xué)臨床中心　上?！?00031; 4衛(wèi)生部聽覺醫(yī)學(xué)重點(diǎn)實(shí)驗(yàn)室　上?！?00031)

耳蝸和前庭分別是重要的聽覺和位覺平衡覺器官,它們具有感知機(jī)械作用的毛細(xì)胞,通過其表面的靜纖毛束來探測聲音和位置信號。在此過程中,規(guī)律的內(nèi)耳毛細(xì)胞極性排列對于聽力的傳導(dǎo)和平衡的維持必不可少。在內(nèi)耳發(fā)育過程中,經(jīng)典的Wnt/β-catenin信號通路參與調(diào)控細(xì)胞增殖、分化等重要環(huán)節(jié)[1],對誘導(dǎo)Pax2陽性的耳基板細(xì)胞形成聽囊至關(guān)重要[2],并可介導(dǎo)感覺區(qū)和非感覺區(qū)前庭器的形成[3]。有研究發(fā)現(xiàn),Atoh1是Wnt信號通路的直接靶點(diǎn)[4],而Atoh1是已知的在毛細(xì)胞發(fā)育過程中不可或缺的基因,其缺失將造成耳蝸和前庭無法形成[5]。Lgr5是Wnt信號增強(qiáng)正反饋調(diào)節(jié)回路上的一員,標(biāo)記內(nèi)耳干細(xì)胞,研究發(fā)現(xiàn)Lgr5在調(diào)控毛細(xì)胞增殖和分化過程中具有重要作用[6-8]。

耳蝸Corti’s器包含1排內(nèi)毛細(xì)胞和3排外毛細(xì)胞,毛細(xì)胞頂端具有呈V型階梯狀排列的靜纖毛束和一根居于一側(cè)的動纖毛,V型的頂點(diǎn)指向動纖毛側(cè),從而形成亞細(xì)胞平面極性,而極化細(xì)胞的規(guī)律排列則構(gòu)成平面細(xì)胞極性(planar cell polarity,PCP)[9]。Wnt5a通過非經(jīng)典Wnt/PCP信號通路調(diào)節(jié)耳蝸感覺上皮的PCP及匯聚伸展作用[10]。前庭除了具有PCP之外,還具有整體組織極性,表現(xiàn)為具有一條極性分界線(line of polarity reverse,LPR)穿囊斑而過,將囊斑分為動纖毛方向相反的兩個區(qū),橢圓囊斑的動纖毛朝向LPR,球囊斑的動纖毛背離LPR[11]。在毛細(xì)胞成熟過程中伴隨LPR的形成,此過程可被Wnt抑制劑干擾[10,12],從而推斷該過程受到Wnt信號通路調(diào)節(jié)。

β-catenin作為經(jīng)典Wnt/β-catenin信號通路的重要組成蛋白,對內(nèi)耳毛細(xì)胞的排列和極性發(fā)育具體作用目前尚不知曉。本實(shí)驗(yàn)中,我們構(gòu)造了在內(nèi)耳感覺上皮的前體細(xì)胞(Sox2陽性細(xì)胞)條件敲除β-catenin的小鼠動物模型,在體研究β-catenin缺失對內(nèi)耳發(fā)育,尤其是對毛細(xì)胞的排列和極性的影響。

材 料 和 方 法

實(shí)驗(yàn)動物β-cateninflox(exon2-6)小鼠[13]由李華偉教授(復(fù)旦大學(xué)附屬眼耳鼻喉科醫(yī)院)惠贈,Sox2CreERT小鼠[14]由柴人杰教授(東南大學(xué))惠贈,兩種小鼠均為C57BL/6小鼠。將以上兩種小鼠雜交得Sox2CreERTβ-cateninf/flox(exon2-6)雙陽性小鼠[15],再將β-cateninf/flox(exon2-6)雌鼠與Sox2CreERTβ-cateninf/flox(exon2-6)雄鼠雜交,對孕鼠行他莫昔芬(40 mg/mL溶解于玉米油,美國Sigma公司)腹腔注射(0.1 mg/g),7天后麻醉孕鼠,取出胚胎,解剖得聽泡、耳蝸和前庭組織,解剖方法已有詳述[16-18]?；蛐蜑镾ox2CreERTβ-cateninf/flox(exon2-6)的小鼠作為實(shí)驗(yàn)組,無Cre表達(dá)的同窩小鼠作為對照組。本實(shí)驗(yàn)通過了復(fù)旦大學(xué)倫理審查委員會的倫理審查。

基因型鑒定取5 mm鼠尾,溶于150 μL的Direct PCR試劑、Proteinase K試劑(美國Viagen Biotech公司)混合液(50∶1),55 ℃ 金屬浴(北京天根公司)加熱過夜,次日85 ℃ 金屬浴加熱45 min后,12 500×g快速離心2 min,吸出上層清亮液體100 μL,即為提取的DNA。取2.5 μL DNA,行PCR擴(kuò)增DNA,通過電泳凝膠成像檢測DNA片段長度。β-cateninflox(exon2-6)引物為5’-AAGGTAGAG-TGATGAAAGTTGTT-3’,5’-CACCATGTCCT-CTGTCTATTC-3’;Cre引物為5’-TGCCACGAC-CAAGTGACAGCAATG-3’;5’-ACCAGAGACG-GAAATCCATCGCTC-3’。檢測β-cateninflox(exon2-6)片段長度為300 bp,β-catenin為223 bp,Cre為300～400 bp(圖1)。

免疫熒光染色Corti’s器、橢圓囊斑、球囊斑從小鼠取出后,立即用4%多聚甲醛(美國Sigma-Aldrich公司)室溫固定30 min,0.1%Triton X-100(溶于PBS,美國Sigma-Aldrich公司)通透30 min后,用10%的驢血清(溶于0.1% Triton X-100,美國Merkmilli Pore公司)封閉1 h,然后加一抗4 ℃過夜。第2天用0.1%Triton X-100漂洗3～5 h后,加二抗,置于室溫2 h后用0.1% Triton X-100漂洗1 h,最后封片。本實(shí)驗(yàn)中用到的一抗有:山羊來源的抗β-catenin抗體(1∶200,美國Sigma公司),Arl13B (1∶200,美國Emory大學(xué)陳平教授惠贈),鼠來源的抗β-spectrin抗體(1∶200,美國BD Biosciences公司)。用到的二抗有:驢抗山羊-647 (1∶1 000),驢抗兔-647 (1∶1 000),驢抗鼠-555 (1∶1 000)。綠色熒光偶聯(lián)phalloidin (1∶1 000)用于肌動蛋白染色,以上試劑均購自美國Jackson ImmunoResearch公司。共聚焦掃描顯微鏡(Leica SP8)用于免疫熒光染色標(biāo)本的掃描。

WT:Wild type;H:Heterozygous;MT:Mutant.

圖1β-cateninflox(exon2-6)和CreDNA凝膠電泳圖
Fig1β-cateninflox(exon2-6)andCreDNAgelelectrophoresis

結(jié)　　　果

β-catenin敲除影響內(nèi)耳形態(tài)發(fā)育Atoh1作為經(jīng)典Wnt/β-catenin通路的下游目的基因[4],在內(nèi)耳毛細(xì)胞發(fā)育中不可或缺[18 - 22]。我們猜測經(jīng)典Wnt/β-catenin信號通路對毛細(xì)胞發(fā)育極為關(guān)鍵。對懷有Sox2CreERTβ-cateninf/flox(exon2-6)胚胎的孕鼠行雌激素類似物他莫昔芬腹腔注射,至少2天后,激活的Cre重組酶切除2個LoxP位點(diǎn)之間的β-catenin外顯子2-6,導(dǎo)致雙陽性胚胎小鼠的Sox2陽性細(xì)胞的β-catenin基因被敲除[13]。取已激活Cre重組酶的Sox2CreERTβ-cateninf/flox(exon2-6)突變小鼠Corti’s器及前庭斑行β-catenin免疫熒光染色,在相同光強(qiáng)下行共聚焦顯微鏡掃描,因Sox2廣泛分布,可觀察到與對照組相比,β-catenin在Corti’s器和前庭的整個感覺上皮表達(dá)均明顯下降,與對照組相同部位的細(xì)胞-細(xì)胞連接β-catenin染色在實(shí)驗(yàn)組中明顯減弱(圖2、3),驗(yàn)證了β-catenin基因在Sox2陽性細(xì)胞中被敲除。

The arrows in the boxes indicats the β-catenin-staining cell-cell connections.

圖2對照組和β-catenin敲除組在Corti’s器的β-catenin免疫熒光染色
Fig2Theimmunofluorescencestainingofβ-cateninintheorganofCortiinthecontrolandtheβ-cateninknockoutgroup

前期研究發(fā)現(xiàn),小鼠內(nèi)耳的極性發(fā)育從胚胎13.5天(E13.5)開始[23]。我們在E11.5進(jìn)行他莫昔芬誘導(dǎo),48 h后即E13.5 Sox2陽性細(xì)胞的β-catenin被敲除,E18.5解剖胚胎小鼠,獲取聽泡。與對照組相比,實(shí)驗(yàn)組小鼠的聽泡體積明顯變小(圖4)。為量化聽泡體積,我們用Photoshop軟件分別測量了對照組及實(shí)驗(yàn)組的聽泡長徑的相對長度(聽泡頂端與底端連線,圖4左圖黃線),兩者長徑相對長度分別為732.16±3.31 (n=6)和675.09±7.78 (n=6),差異有統(tǒng)計(jì)學(xué)意義(圖4,P<0.001)。進(jìn)一步解剖聽泡,得到耳蝸和前庭組織。從大體結(jié)構(gòu)觀察,在Sox2CreERTβ-cateninf/flox(exon2-6)小鼠中,Corti’s器明顯變短、變小,前庭組織也整體變小(圖5)。我們進(jìn)一步定量測量解剖得到的Corti’s器相對長度以及橢圓囊斑、球囊斑相對面積,并將對照組與實(shí)驗(yàn)組數(shù)據(jù)進(jìn)行了比較。測量得Corti’s器相對長度分別為1.79±0.04 (n=3)和1.57±0.08 (n=4),橢圓囊斑相對面積分別為39 615.67±1 808.66 (n=3)和33 371.72±679.64 (n=4),球囊斑相對面積分別為50 293.67±1 710.82 (n=3)和36 581.76±2 607.93 (n=4),差異均有統(tǒng)計(jì)學(xué)意義(圖6,P<0.01、P<0.01、P<0.001),與定性觀察結(jié)果一致。由此可見,在Sox2陽性細(xì)胞中敲除β-catenin對小鼠內(nèi)耳的形態(tài)發(fā)育影響較大,β-catenin在內(nèi)耳發(fā)育中具有重要作用。

SA:Saccule.The figures below are the amplified views of correspondent boxes above.

圖3對照組和β-catenin敲除組球囊斑
β-catenin免疫熒光染色
Fig3TheImmunofluorescencestainingofβ-catenininthesaccularmaculaeinthecontrolandβ-cateninknockoutgroup

β-catenin敲除影響Corti’s器毛細(xì)胞的排列和極性E11.5注射他莫昔芬激活Cre重組酶,經(jīng)極性蛋白染色我們發(fā)現(xiàn),此時毛細(xì)胞發(fā)育尚在早期,許多極性蛋白尚未表達(dá),無法觀察極性情況。故我們推遲他莫昔芬注射時間至E15.5,48 h后即E17.5 Sox2陽性細(xì)胞中的β-catenin被敲除,此時細(xì)胞分化和極性建立尚未完全形成[23],以此進(jìn)一步探究β-catenin敲除對Corti’s器以及前庭毛細(xì)胞極性形成的影響。我們?nèi)15.5激活Cre重組酶、出生后2天(P2)解剖的小鼠Corti’s器,行Phalloidin染色。Phalloidin可以標(biāo)記細(xì)胞骨架,同時可以染出毛細(xì)胞的表皮板和呈V型排列的靜纖毛束。β-catenin突變小鼠Corti’s器Phalloidin染色顯示出紊亂的毛細(xì)胞排列(圖7),出現(xiàn)連續(xù)多個外毛細(xì)胞缺失(圖7白色Δ標(biāo)示),伴有或不伴有內(nèi)毛細(xì)胞的插入(圖7*標(biāo)示)。中底圈毛細(xì)胞缺失或插入情況較多,但仍有清晰的內(nèi)外毛細(xì)胞形態(tài)差異和兩者的分界線。越接近頂圈,毛細(xì)胞排列越紊亂,甚至出現(xiàn)內(nèi)外毛細(xì)胞混雜無法區(qū)分界限的情況(圖7F)。值得一提的是,仍有部分區(qū)域,毛細(xì)胞排列較為規(guī)整,無毛細(xì)胞的缺失和插入(圖7B)。毛細(xì)胞的極性方向通過標(biāo)記靜纖毛束的Phalloidin聯(lián)合標(biāo)記動纖毛的Arl13B來顯示(圖8白色箭頭標(biāo)示)。對照組毛細(xì)胞呈現(xiàn)出規(guī)律一致的V型靜纖毛束和居于一側(cè)的動纖毛(圖8左),與此不同,β-catenin敲除小鼠的耳蝸毛細(xì)胞靜纖毛束雖仍保持V型,但V型頂端朝向不再整齊劃一。從底圈到頂圈,紊亂程度加強(qiáng)。在頂圈,我們觀察到動纖毛出現(xiàn)在細(xì)胞中間(圖8藍(lán)色箭頭指示居于表皮板中間位置的動纖毛);動纖毛數(shù)量也發(fā)生了變化,有些毛細(xì)胞具有2～3根動纖毛(圖8黃色箭頭指示單個毛細(xì)胞具有多根動纖毛)。由此可以發(fā)現(xiàn)β-catenin缺失干擾了Corti’s器毛細(xì)胞的排列和動、靜纖毛的排布。

圖4對照組和β-catenin敲除組的聽泡長徑比較
Fig4Thecomparisonofnormalizedlongdiameterofoticvesiclebetweenthecontrolandβ-cateninknockoutgroup

SA:Saccule;UT:Utricle;AC:Anterior cristae;LC:Lateral cristae;PC:Posterior cristae.

圖5對照組和β-catenin敲除組的Corti’s器與前庭
Fig5ThevestibulesandCorti’softhecontrolandβ-cateninknockoutgroup

β-catenin敲除影響前庭毛細(xì)胞的數(shù)量和極性為了探討β-catenin敲除對前庭毛細(xì)胞的數(shù)量和極性的影響,我們對E15.5行他莫昔芬誘導(dǎo)、P2解剖固定的小鼠橢圓囊斑進(jìn)行了染色。Phalloidin染色后定性觀察,可見橢圓囊毛細(xì)胞明顯變稀疏(圖9)。選取橢圓囊、球囊感覺上皮區(qū)100μm×100μm面積,統(tǒng)計(jì)其內(nèi)毛細(xì)胞數(shù)。結(jié)果顯示,對照組和實(shí)驗(yàn)組橢圓囊斑內(nèi)毛細(xì)胞數(shù)分別為117.7±10.5(n=9)和101.4±10.3(n=14),球囊斑內(nèi)毛細(xì)胞數(shù)分別為100.8±16.3 (n=6)和72.9±8.6 (n=9),Sox2CreERTβ-cateninf/flox (exon2-6)小鼠的橢圓囊和球囊毛細(xì)胞數(shù)均明顯減少(圖10,P<0.01、P<0.001)。毛細(xì)胞的靜纖毛附著于表皮板上,動纖毛根部為基體,其所在處表皮板缺如?？贵wβ-spectrin可以染出毛細(xì)胞的表皮板,故在動纖毛根部染色呈空洞,我們用β-spectrin染出動纖毛所在的一側(cè)來指示毛細(xì)胞的極性方向。已知橢圓囊斑和球囊斑各有1條LPR,其兩側(cè)毛細(xì)胞極性方向相反,橢圓囊斑毛細(xì)胞方向均指向LPR,球囊斑毛細(xì)胞則均背離LPR[11]。本實(shí)驗(yàn)中,與對照組一樣,β-catenin敲除小鼠的橢圓囊斑和球囊斑仍均具有相對清晰的極性分界線,其兩側(cè)毛細(xì)胞指示方向大致正常(圖11)。但值得注意的是,在橢圓囊,分界線兩側(cè)存在散在的毛細(xì)胞極性方向與其附近大多數(shù)毛細(xì)胞的極性方向相反。這種情況在突變小鼠的球囊也有出現(xiàn)。

圖6　對照組和β-catenin敲除組的Corti’s器長度、橢圓囊斑及球囊斑面積比較Fig 6　The comparison of normalized length of Corti’s organs area of utricular maculea and saccular maculeabetween the control and β-catenin knockout group

經(jīng)典Wnt/β-catenin信號通路在調(diào)控細(xì)胞增生、定向、分化等步驟中具有重要作用[1]。Wnts是一種短距分泌糖肽,與frizzled受體相結(jié)合,通過激活細(xì)胞間的連鎖瀑布反應(yīng)來調(diào)控細(xì)胞生長、增殖以及細(xì)胞命運(yùn)。β-catenin是經(jīng)典Wnt信號通路的中介者,控制下游靶基因的表達(dá)。同時它也是一種結(jié)構(gòu)蛋白,參與構(gòu)成鈣黏蛋白介導(dǎo)的細(xì)胞黏附連接及有絲分裂期的中心體[24]。

Δ:The missing outer HCs;*:The inserted inner HCs.

圖7對照組和β-catenin敲除組的Corti’s器毛細(xì)胞排列
Fig7TheHCarrangementoftheorgansofCorti’sinthecontrolandβ-cateninknockoutgroup

White arrow heads indicate HC polarity orientation.White dashed line outlines the hair cells with more than one kinocilium indicated by yellow arrows.The hair cells with three kinoclia were circled by the yellow dashed line and amplified in the upper right.The hair cells with a kinocilium in the center of cell surface indicated by a blue arrowhead were circled by the blue dashed line and amplified in the lower right.

圖8對照組和β-catenin敲除組的Corti’s器毛細(xì)胞極性
Fig8TheHCpolarityoftheorgansofCortiinthecontrolandβ-cateninknockoutgroup

UT:Utricle.

圖9對照組和β-catenin敲除組的橢圓囊Phalloidin染色
Fig9Thephalloidinstainingofutriclesinthecontrolandβ-cateninknockoutgroup

本實(shí)驗(yàn)結(jié)果顯示,Sox2CreERTβ-cateninf/flox (exon2-6)小鼠的Corti’s器變短變小,前庭器變小,前庭斑感覺上皮區(qū)毛細(xì)胞數(shù)減少。對毛細(xì)胞發(fā)育過程中經(jīng)典Wnt活性的一系列研究表明,Wnt活性增加可以促進(jìn)毛細(xì)胞的正常發(fā)育[8,25-26]。此外,Atoh1在啟動毛細(xì)胞分化、促進(jìn)毛細(xì)胞成熟等方面都有關(guān)鍵作用[5,27-28],并且已證實(shí)Atoh1是Wnt/β-catenin通路的下游直接靶基因[4]。本實(shí)驗(yàn)結(jié)果與上述實(shí)驗(yàn)結(jié)果相一致,我們推測β-catenin敲除能降低Wnt活性,抑制細(xì)胞增殖、分化,同時抑制Atoh1表達(dá),進(jìn)而抑制毛細(xì)胞產(chǎn)生,導(dǎo)致Corti’s器和前庭器變小,毛細(xì)胞減少。有研究發(fā)現(xiàn),耳蝸感覺上皮的匯聚伸展作用受非經(jīng)典Wnt/PCP信號通路調(diào)節(jié)[10]。本實(shí)驗(yàn)中,β-catenin敲除后Corti’s器變短,推測β-catenin依賴的經(jīng)典Wnt信號通路共同參與調(diào)節(jié)耳蝸匯聚伸展,但其機(jī)制有待探究。

圖10　對照組和β-catenin敲除組的橢圓囊、球囊　毛細(xì)胞計(jì)數(shù)比較Fig 10　　　　The comparison of HC numbers in utricle and saccule

The white line indicates the line of polarity reverse (LPR).The white arrows and yellow arrows indicate the normal and abnormal HC orientation,respectively.

圖11對照組和β-catenin敲除組的橢圓囊及球囊
β-spectrin染色和極性分界線
Fig11Theβ-spectrinstainingandLPRofutriclesandsacculesinthecontrolandβ-cateninknockoutgroup

我們發(fā)現(xiàn),在Sox2陽性細(xì)胞條件性敲除β-catenin導(dǎo)致耳蝸毛細(xì)胞排列紊亂、極性改變,同時導(dǎo)致前庭斑毛細(xì)胞散在極性改變。Sox2主要在前體細(xì)胞中表達(dá),β-catenin是細(xì)胞連接的重要組成部分。我們推測,在Sox2陽性細(xì)胞敲除β-catenin導(dǎo)致細(xì)胞之間連接作用減弱,從而毛細(xì)胞排布發(fā)生紊亂。發(fā)育中的Corti’s器由毛細(xì)胞的前體細(xì)胞(支持細(xì)胞)向毛細(xì)胞的分化開始于底圈,并向頂圈方向延伸進(jìn)行[29-31]。因此,越接近頂圈,毛細(xì)胞的分化越不成熟,尚未分化的支持細(xì)胞越多。在接近頂圈的過程中,隨著支持細(xì)胞數(shù)增多,β-catenin缺失導(dǎo)致的細(xì)胞連接減弱的作用便表現(xiàn)得越明顯,細(xì)胞排列越加紊亂,甚至內(nèi)外毛細(xì)胞界限無法區(qū)分。Vangl2是一種平面極性核心蛋白,受不依賴β-catenin的非經(jīng)典Wnt/PCP通路調(diào)節(jié),實(shí)驗(yàn)發(fā)現(xiàn)Vangl2條件敲除的小鼠耳蝸毛細(xì)胞的靜纖毛束不再沿中間-外側(cè)軸統(tǒng)一朝向[32]。本實(shí)驗(yàn)中,敲除β-catenin同樣可以引起耳蝸毛細(xì)胞靜纖毛束V型朝向紊亂,還導(dǎo)致前庭斑散在毛細(xì)胞極性改變,提示經(jīng)典Wnt/β-catenin通路可能在毛細(xì)胞PCP建立過程中也起到作用。核心PCP基因突變并不影響單個靜纖毛束的極化結(jié)構(gòu),PCP突變的小鼠毛細(xì)胞靜纖毛束仍保持V形,動纖毛仍然在其頂端[33-37]。本實(shí)驗(yàn)中Sox2CreERTβ-cateninf/flox(exon2-6)小鼠的耳蝸毛細(xì)胞靜纖毛束仍呈V型,但動纖毛位置發(fā)生改變,出現(xiàn)在細(xì)胞表皮板中央,以及出現(xiàn)一個毛細(xì)胞具有2～3根動纖毛的情況,表明單個毛細(xì)胞的內(nèi)在極性受到影響,提示經(jīng)典Wnt/β-catenin通路對亞細(xì)胞極性也有作用。

我們認(rèn)為,經(jīng)典Wnt/β-catenin信號通路是介導(dǎo)內(nèi)耳感覺上皮發(fā)育的重要通路。同時,β-catenin對于穩(wěn)定的細(xì)胞-細(xì)胞連接、規(guī)律的細(xì)胞排列很重要。我們還發(fā)現(xiàn),經(jīng)典Wnt/β-catenin信號通路可能與非經(jīng)典PCP通路相似,不僅參與毛細(xì)胞PCP建立和耳蝸延伸過程,還可能在亞細(xì)胞層面上調(diào)控毛細(xì)胞的極性。

[1]AMERONGEN RV,NUSSE R.Towards an integrated view of Wnt signaling in development[J].Development,2009(19),136:3205-3214.

[2]OHYAMA T,MOHAMED OA,TAKETO MM,etal.Wnt signals mediate a fate decision between otic placode and epidermis[J].Development,2006,133(5):865-875.

[3]RAKOWIECKI S,EPSTEIN DJ.Divergent roles for Wnt/β-catenin signaling in epithelial maintenance and breakdown during semicircular canal formation[J].Development,2013,140(8):1730-1739.

[4]SHI F,CHENG Y,WANG X,etal.β-catenin up-regulatesAtoh1expression in neural progenitor cells by interaction with anAtoh1 3’enhancer[J].JBiolChem,2010,285(1):392-400.

[5]BERMINGHAM NA,HASSAN BA,PRICE SD,etal.Math1:an essential gene for the generation of inner ear hair cells[J].Science,1999,284(5421):1837-1841.

[6]CHAI R,KUO B,WANG T,etal.Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea[J].ProcNatlAcadSciUSA,2012,109(21):8167-8172.

[7]SHI F,KEMPFLE J,EDGE AS.Wnt-responsive Lgr5-expressing stem cells are hair cell progenitors in the cochlea[J].JNeurosci,2012,32(28):9639-9648.

[8]JACQUES BE,PULIGILLA C,WEICHERT RM,etal.A dual function for canonical Wnt/β-catenin signaling in the developing mammalian cochlea[J].Development,2012,139(23):4395-4404.

[9]GUBB D,GARCIA-BELLIDO A.A genetic analysis of the determination of cuticular polarity during development inDrosophilamelanogaster[J].JEmbryolExpMorph,1982,68(5):37-57.

[10]QIAN D,JONES C,RZADZINSKA A,etal.Wnt5a functions in planar cell polarity regulation in mice[J].DevBiol,2007,306(1):121-133.

[11]DEANS MR.A balance of form and function:planar polarity and development of the vestibular maculae[J],SeminCellDevBiol,2013,24(5):490-498.

[12]DABDOUB A,DONOHUE MJ,BRENNAN A,etal.Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea[J].Development,2003,130(11):2375-2384.

[13]BRAULT V,MOORE R,KUTSCH S,etal.Inactivation of the β-catenin gene byWnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development[J].Development,2001,128(8):1253-1264.

[14]ARNOLD K,SARKAR A,YRAM MA,etal.Sox2+ adult stem/progenitor cells are important for tissue regeneration and survival of mice[J].CellStemCell,2011,9(4):317-329.

[15]SHI F,HU L,JACQUES BE,etal.β-catenin is required for hair-cell differentiation in the cochlea[J].JNeurosci,2014,34(19):6470-6479.

[16]DABDOUB A,PULIGILLA C,JONES JM,etal.Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea[J].ProcNatlAcadSciUSA,2008,105(47):18396-18401.

[17]HAN Z,YANG J,CHI F,etal.Survival and fate of transplanted embryonic neural stem cells byAtoh1 gene transfer in guinea pigs cochlea[J].Neuroreport,2010,21(7):490-496.

[18]HUANG Y,CHI F,HAN Z,etal. New ectopic vestibular hair cell-like cells induced byMath1 gene transfer in postnatal rats[J].BrainRes,2009,1276:31-38.

[19]SHOU J,ZHENG JL,GAO W.Robust generation of new hair cells in the mature mammalian inner ear by adenoviral expression ofHath1[J].MolCellNeurosci,2003,23(2):169-179.

[20]WOODS C,MONTCOUQUIOL M,KELLEY MW.Math1 regulates development of the sensory epithelium in the mammalian cochlea[J].NatNeurosci,2004,7(12):1310-1318.

[21]YANG J,BOUVRON S,LV P,etal.Functional features of trans-differentiated hair cells mediated byAtoh1 reveals a primordial mechanism[J].JNeurosci,2012,32(11):3712-3725.

[22]ZHENG JL,GAO W.Overexpression ofMath1 induces robust production of extra hair cells in postnatal rat inner ears[J].NatNeurosci,2000,3(6):580-586.

[23]YANG X,QIAN X ,MA R,etal.Establishment of planar cell polarity is coupled to regional cell cycle exit and cell differentiation in the mouse utricle[J].SciRep,2017,7:43021.

[24]VALENTA T,HAUSMANN G,BASLER K.The many faces and functions of β-catenin[J].EMBOJ,2012,31(12):2714-2736.

[25]DASGUPTA R,FUCHS E.Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation[J].Development,1999,126(20):4557-4568.

[26]SHI F,HU L,EDGE AS.Generation of hair cells in neonatal mice by β-catenin overexpression in Lgr5-positive cochlear progenitors[J].ProcNatlAcadSciUSA,2013,110(34):13851-13856.

[27]CAI T,SEYMOUR ML,ZHANG H,etal. Conditional deletion ofAtoh1 reveals distinct critical periods for survival and function of hair cells in the organ of Corti [J].JNeurosci,2013,33(24):10110-10122.

[28]CHONKO KT,JAHAN I,STONE J,etal.Atoh1 directs hair cell differentiation and survival in the late embryonic mouse inner ear[J].DevBiol,2013,381(2):401-410.

[29]LEE YS,LIU F,SEGIL N.A morphogenetic wave ofp27Kip1transcription directs cell cycle exit during organ of Corti development[J].Development,2006,133(15):2817-2826.

[30]CHEN P,SEGIL N.p27Kip1links cell proliferation to morphogenesis in the developing organ of Corti[J].Development,1999,126(8):1581-1590.

[31]CHEN P,JOHNSON JE,ZOGHBI HY,etal.The role ofMath1 in inner ear development:uncoupling the establishment of the sensory primordium from hair cell fate determination[J].Development,2002,129(10):2495-2505.

[32]COPLEY CO,DUNCAN JS,LIU C,etal.Postnatal refinement of auditory hair cell planar polarity deficits occurs in the absence ofVangl2[J].JNeurosci,2013,33(35):14001-14016.

[33]YIN H,COPLEY CO,GOODRICH LV,etal.Comparison of phenotypes between differentVangl2 mutants demonstrates dominant effects of theLooptailmutation during hair cell development[J].PLoSOne,2012,7(2):e31988.

[34]MONTCOUQUIOL M,RACHEL RA,LANFORD PJ,etal. Identification ofVangl2 andScrb1 as planar polarity genes in mammals[J].Nature,2003,423(6936):173-177.

[35]CURTIN JA,QUINT E,TSIPOURI V,etal. Mutation ofCelsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse[J].CurrBiol,2003,13(13):1129-1133.

[36]WANG J,HAMBLET NS,MARK S,etal.Dishevelledgenes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation[J].Development,2006,133(9):1767-1778.

[37]WANG Y,GUO N,NATHANS J.The role ofFrizzled3 andFrizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells[J].JNeurosci,2006,26(8):2147-2156.