劉鐘穎，黃霞，李紫怡，楊子豪，袁白銀

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肌動(dòng)蛋白細(xì)胞骨架在小鼠第二生心區(qū)祖細(xì)胞部署發(fā)育中的作用

劉鐘穎，黃霞，李紫怡，楊子豪，袁白銀

武漢科技大學(xué)生命科學(xué)與健康學(xué)院，武漢 430065

脊椎動(dòng)物心管起源于早期胚胎的生心中胚層細(xì)胞，隨后從鄰近的咽中胚層和內(nèi)臟中胚層中逐漸添加第二生心區(qū)(second heart field, SHF)祖細(xì)胞而延長(zhǎng)。SHF細(xì)胞向心管貢獻(xiàn)受損，使心管不能最大限度地延長(zhǎng)，導(dǎo)致一系列的心臟發(fā)育缺陷，包括最常見(jiàn)的右心室發(fā)育不良與流出道分隔旋轉(zhuǎn)異常等先天性出生缺陷。SHF祖細(xì)胞構(gòu)成非經(jīng)典頂端-基底極性上皮，并具有頂部單纖毛、動(dòng)態(tài)肌動(dòng)蛋白(actin)富集基底端的絲狀偽足等特點(diǎn)。本文總結(jié)了actin細(xì)胞骨架在小鼠SHF祖細(xì)胞部署發(fā)育過(guò)程中的研究進(jìn)展，揭示actin細(xì)胞骨架在SHF祖細(xì)胞發(fā)育特別是SHF細(xì)胞向流出道部署過(guò)程中的重要性，以期為闡明和理解SHF祖細(xì)胞遷移、部署的細(xì)胞生物學(xué)特性提供一定的理論參考。

肌動(dòng)蛋白；第二生心區(qū)；細(xì)胞部署；小鼠

先天性心臟病是目前人類最常見(jiàn)的出生缺陷，并且一直是引起嬰幼兒死亡的首要原因，其中約30%的先天性心臟缺陷為流出道(outflow tract, OFT)發(fā)育缺陷[1,2]。OFT和右心室(right ventricle, RV)是由第二生心區(qū)(second heart field, SHF)祖細(xì)胞發(fā)育形成，SHF祖細(xì)胞發(fā)育缺陷導(dǎo)致OFT和RV發(fā)育異常。因此，闡明SHF的發(fā)育過(guò)程和深入探討其調(diào)控機(jī)制對(duì)于破譯OFT發(fā)育缺陷的起源、控制和降低先天性心臟病的發(fā)病率、尋找臨床相關(guān)致病基因?qū)ζ湓\斷、防治以及試管嬰兒的篩選具有重要意義。

哺乳動(dòng)物的心臟是由第一生心區(qū)(first heart field, FHF)和SHF兩種主要的心臟祖細(xì)胞發(fā)育而來(lái)。位于前部側(cè)板中胚層的FHF祖細(xì)胞貢獻(xiàn)于早期分化的心肌細(xì)胞，形成原始心管，將來(lái)發(fā)育成左心室(left ventricle, LV)、房室通道、極小部分的右心室和心房心肌細(xì)胞[3~5]。位于咽中胚層(pharyngeal mesoderm, PM)和內(nèi)臟中胚層(splanchnic mesoderm, SpM)的SHF祖細(xì)胞添加晚分化的心肌細(xì)胞于伸展的心管，貢獻(xiàn)于OFT、RV及心房心肌細(xì)胞[4~6]。SHF祖細(xì)胞向心管貢獻(xiàn)、部署的缺陷使OFT的長(zhǎng)度不足以在心臟間隔形成期間正確的旋轉(zhuǎn)，升主動(dòng)脈和肺動(dòng)脈干與左心室和右心室對(duì)齊，導(dǎo)致心室分隔和心室/動(dòng)脈排列缺陷，引起雙出口右室(double outlet right ventricle, DORV)、大動(dòng)脈轉(zhuǎn)位(transposition of the great arteries, TGA)、永存動(dòng)脈干(persistent truncus arteriosus, PTA)等畸形[6,7]。

研究發(fā)現(xiàn)，SHF細(xì)胞構(gòu)成非經(jīng)典頂端-基底極性上皮，并具有頂部單纖毛、動(dòng)態(tài)肌動(dòng)蛋白(actin)富集基底端的絲狀偽足等特點(diǎn)，這表明actin細(xì)胞骨架在SHF發(fā)育中具有重要作用[8,9]。本文對(duì)哺乳動(dòng)物SHF的發(fā)現(xiàn)及特性、actin細(xì)胞骨架的特性、actin與細(xì)胞運(yùn)動(dòng)、actin與橫紋肌肌纖維以及actin與哺乳動(dòng)物SHF發(fā)育等方面進(jìn)行了闡述，以期為科研人員更深入地解析actin細(xì)胞骨架與哺乳動(dòng)物SHF發(fā)育之間的聯(lián)系提供參考，為闡明和理解SHF細(xì)胞遷移、部署的細(xì)胞生物學(xué)行為及調(diào)控網(wǎng)絡(luò)提供思路和方向，為尋找臨床相關(guān)致病基因并對(duì)先天性心臟病的診斷、防治提供理論基礎(chǔ)。

1 小鼠心臟第二生心區(qū)

1.1 第一生心區(qū)與第二生心區(qū)

心臟發(fā)育和形成是一個(gè)復(fù)雜的過(guò)程，涉及不同空間、不同細(xì)胞群體的整合，并被復(fù)雜的級(jí)聯(lián)調(diào)控網(wǎng)絡(luò)調(diào)控。心臟是由兩種主要的心臟祖細(xì)胞發(fā)育而來(lái)，分別是FHF和SHF祖細(xì)胞[5]。研究表明，F(xiàn)HF和SHF祖細(xì)胞在心臟新月(cardiac crescent, CC)期之前與共同的心臟祖細(xì)胞分離；FHF首先與心臟共同的祖細(xì)胞分離出來(lái)，并貢獻(xiàn)于心臟新月期和早期心管心肌細(xì)胞；而SHF是另外一個(gè)祖細(xì)胞群，它隨后將新分化的心肌細(xì)胞貢獻(xiàn)于生長(zhǎng)的心管[4,6,10]。FHF來(lái)源的原始心管將來(lái)發(fā)育為左心室、小部分的右心室、房室管和大部分的心房[3,5]。SHF祖細(xì)胞通過(guò)心臟動(dòng)脈極貢獻(xiàn)于OFT和右心室，并通過(guò)靜脈極貢獻(xiàn)于心房心肌細(xì)胞[4~6]。

在小鼠()胚胎發(fā)育的E8.5~E10.5時(shí)期，位于心管后部咽中胚層和內(nèi)臟中胚層的SHF祖細(xì)胞向原始心管添加分化的心肌細(xì)胞，使心管延伸并向右環(huán)化[11]。SHF祖細(xì)胞對(duì)心管的貢獻(xiàn)使心管得以足夠的伸展和生長(zhǎng)，這是心臟形態(tài)發(fā)生所必 需的。SHF祖細(xì)胞向心管貢獻(xiàn)、部署失敗導(dǎo)致OFT縮短，使OFT的長(zhǎng)度不足以在心臟間隔形成期間 正確的旋轉(zhuǎn)以使升主動(dòng)脈和肺動(dòng)脈干與左心室和右心室對(duì)齊，導(dǎo)致心室分隔和心室/動(dòng)脈排列缺 陷，如雙出口右室、大動(dòng)脈轉(zhuǎn)位、永存動(dòng)脈干及其他異常[6,12,13]。

1.2 第二生心區(qū)的發(fā)現(xiàn)及性質(zhì)

20世紀(jì)70年代，de la Cruz及其同事利用體內(nèi)氧化鐵顆粒標(biāo)記法揭示雞()心臟遠(yuǎn)端OFT和RV實(shí)際是由后來(lái)添加的細(xì)胞形成，并且心臟的生長(zhǎng)是通過(guò)添加位于早期心臟之外的細(xì)胞而實(shí)現(xiàn)[14]。另一項(xiàng)在小鼠中的實(shí)驗(yàn)也暗示，除了心臟新月以外，還有另外一種細(xì)胞群貢獻(xiàn)于發(fā)育的心臟[15]。然而，這些早期心臟之外細(xì)胞的來(lái)源直到2001年才被確定。

2001年發(fā)表的3項(xiàng)研究確定了在咽中胚層中的祖細(xì)胞群貢獻(xiàn)于胚胎期延伸的OFT。Mjaatvedt等[16]確定了由de la Cruz等[14]發(fā)現(xiàn)的前部心臟形成區(qū)，該前部心臟形成區(qū)是由圍繞在緊鄰現(xiàn)有心管的主動(dòng)脈囊周圍的中胚層組成。此外，Waldo等[17]鑒定出在貢獻(xiàn)于OFT期間，位于心包背壁(SpM)中表達(dá)和以及的祖細(xì)胞。同時(shí)，Kelly等[18]構(gòu)建了受成纖維細(xì)胞生長(zhǎng)因子10 ()基因調(diào)控元件驅(qū)動(dòng)的轉(zhuǎn)基因小鼠，發(fā)現(xiàn)在胚胎心臟的右心室和OFT以及相鄰的PM和SpM中具有β-半乳糖苷酶活性；在轉(zhuǎn)基因小鼠中使用DiI標(biāo)記法確定RV和OFT中的心肌細(xì)胞是從咽弓核心和SpM中添加。這3項(xiàng)研究揭示了位于PM和SpM中的另外一個(gè)祖細(xì)胞群，它們?cè)谠夹墓苄纬梢院筘暙I(xiàn)于心臟。目前認(rèn)為心臟SHF是由位于PM和SpM中的祖細(xì)胞群共同組成[4,6]，研究SHF細(xì)胞成為心臟發(fā)育領(lǐng)域的焦點(diǎn)。

SHF又可分為兩個(gè)亞細(xì)胞群：前部-SHF (A-SHF)和后部SHF (P-SHF)，A-SHF細(xì)胞通過(guò)動(dòng)脈極貢獻(xiàn)于OFT和右心室，P-SHF在靜脈極貢獻(xiàn)于心房及心房的分隔[4,6,10]。到目前為止，尚未確定背側(cè)心包壁中A-SHF祖細(xì)胞和P-SHF祖細(xì)胞之間的界限。利用染料-標(biāo)記和譜系-追蹤實(shí)驗(yàn)表明，P-SHF中存在能夠同時(shí)貢獻(xiàn)于OFT和心房心肌細(xì)胞的祖細(xì)胞群[19,20]。持續(xù)增殖和分化延遲是SHF祖細(xì)胞區(qū)別于FHF祖細(xì)胞的兩個(gè)限定特性，這兩個(gè)特性使SHF祖細(xì)胞能夠被逐漸添加到伸展的心管中，并受動(dòng)態(tài)咽信號(hào)環(huán)境和PM轉(zhuǎn)錄程序的調(diào)節(jié)[21]。FGF10是由Kelly團(tuán)隊(duì)鑒定得到的鼠科動(dòng)物SHF細(xì)胞的第一個(gè)分子標(biāo)記物，隨后的研究發(fā)現(xiàn)SHF祖細(xì)胞具有表達(dá)、、、和等基因的特征[22~26]。

2 肌動(dòng)蛋白細(xì)胞骨架

2.1 Actin細(xì)胞骨架及其解聚和加聚動(dòng)態(tài)

Actin是真核生物中表達(dá)量最豐富的骨架蛋白之一[27]。動(dòng)物細(xì)胞在不同環(huán)境中具有改變細(xì)胞形態(tài)的能力，這種能力主要是依賴actin細(xì)胞骨架系統(tǒng)[28]。細(xì)胞中actin具有兩種存在形式，即游離的單體肌動(dòng) 蛋白(globular actin, G-actin)和絲狀肌動(dòng)蛋白(filame-ntous actin, F-actin)。G-actin自身能夠聚合形成F-actin。F-actin是肌纖維細(xì)肌絲系統(tǒng)的核心組分，并被組織成各種不同的結(jié)構(gòu)以參與行使不同的生物學(xué)過(guò)程，如細(xì)胞周質(zhì)、應(yīng)力纖維、絲狀偽足和板狀偽足等[27,28]。

Actin細(xì)胞骨架是高度動(dòng)態(tài)的骨架蛋白，在生理?xiàng)l件下F-actin能夠持續(xù)在一端(正極端)聚合，在另一端(負(fù)極端)解聚。在細(xì)胞內(nèi)，被組裝成不同結(jié)構(gòu)的F-actin不斷進(jìn)行動(dòng)態(tài)且強(qiáng)烈地重組，使細(xì)胞適應(yīng)環(huán)境。因此，actin細(xì)胞骨架的動(dòng)態(tài)聚合和解聚在各種生理過(guò)程中發(fā)揮重要的調(diào)控作用[27]，如細(xì)胞運(yùn)動(dòng)[29~32]、細(xì)胞分裂和胞質(zhì)分裂[33,34]、細(xì)胞形狀調(diào)節(jié)[35]及轉(zhuǎn)錄調(diào)控[36]等。在沒(méi)有調(diào)節(jié)蛋白存在的生理離子條件下，actin自身能夠經(jīng)歷自發(fā)的聚合、解聚和成核過(guò)程，但是其更新遠(yuǎn)比在生物體內(nèi)緩慢的多。因此，為了適應(yīng)體內(nèi)F-actin快速更新的需求，需要大量的actin調(diào)節(jié)蛋白來(lái)調(diào)控actin成核、斷裂、聚合和解聚等過(guò)程[37]。細(xì)胞內(nèi)存在60多類G-actin和F-actin結(jié)合蛋白，協(xié)同調(diào)節(jié)actin的組裝和拆卸[38,39]。例如，作為成核因子的Arp2/3復(fù)合體，具有促進(jìn)分支狀F-actin成核和交聯(lián)F-actin的功能[40,41]；WASP 家族(其中最有名的是WASP蛋白(Wiskott-Aldrich syndrome protein)和SCAR/WAVE (suppressor of cyclic AMP receptor/WASP-family verprolin-homologous protein))作為Arp2/3復(fù)合體的主要活化因子，能夠結(jié)合并激活A(yù)rp2/3復(fù)合體，促進(jìn)新的F-actin成核[42,43]；Formins是另外一類actin成核因子，主要通過(guò)穩(wěn)定actin二聚體，促進(jìn)非分支F-actin成核和G-actin聚合[44,45]；Profilin促進(jìn)formins介導(dǎo)的actin聚合作用[41,44]；ADF/cofilin是actin解聚的主要調(diào)控因子，通過(guò)促進(jìn)F-actin切割和負(fù)極端G-actin解聚，增強(qiáng)actin的周轉(zhuǎn)[46~48]；WDR1/AIP1是ADF/cofilin的主要輔助因子，它能促進(jìn)與ADF/cofilin結(jié)合的F-actin的解聚作用[49,50]。

2.2 Actin細(xì)胞骨架在細(xì)胞遷移、橫紋肌中的調(diào)控作用

2.2.1 Actin細(xì)胞骨架與細(xì)胞遷移

細(xì)胞運(yùn)動(dòng)作為動(dòng)物細(xì)胞的重要特征，是與器官發(fā)育、宿主防御功能以及疾病發(fā)展密切相關(guān)的基本生命過(guò)程[51]。細(xì)胞遷移在許多生理和病理過(guò)程中發(fā)揮重要作用，如在后生動(dòng)物胚胎發(fā)育過(guò)程中，原腸胚呈現(xiàn)出廣泛的細(xì)胞遷移[52]，且在器官和腺體形成期間，存在細(xì)胞群的協(xié)調(diào)遷移[53]；在正常生理?xiàng)l件下，成纖維細(xì)胞、血管內(nèi)皮細(xì)胞等細(xì)胞遷移是傷口愈合所必需的[54,55]；免疫細(xì)胞通過(guò)遷移穿過(guò)血管和淋巴管并尋找入侵物質(zhì)[56,57]等。

盡管在不同細(xì)胞、組織及研究系統(tǒng)中，細(xì)胞遷移會(huì)有不同的類型和機(jī)制，但它們可被有效的概念化為一個(gè)循環(huán)過(guò)程：首先細(xì)胞發(fā)生極化，隨后在其前部形成突起，建立、穩(wěn)定細(xì)胞-基質(zhì)粘附作為細(xì)胞遷移的牽引位點(diǎn)，最后在其后部細(xì)胞脫離和縮回。細(xì)胞遷移過(guò)程是actin細(xì)胞骨架發(fā)生重大重塑的極端情況，而actin細(xì)胞骨架重塑是調(diào)控細(xì)胞遷移過(guò)程的關(guān)鍵和必需事件[29,58,59]。在細(xì)胞向前運(yùn)動(dòng)過(guò)程中，actin在細(xì)胞前端聚合以形成突起，同時(shí)這種聚合與后端板狀偽足的解聚相匹配，為細(xì)胞隨后的actin聚合反應(yīng)提供所需要的再循環(huán)G-actin[28,37]；另外，肌球蛋白馬達(dá)拉動(dòng)F-actin在遷移細(xì)胞尾部產(chǎn)生的收縮活性，提供細(xì)胞向前移動(dòng)的動(dòng)力[29,60]。F-actin作為肌球蛋白Ⅱ馬達(dá)的支架，是肌動(dòng)球蛋白收縮活動(dòng)的先決條件[61]。目前，已有大量研究確定actin細(xì)胞骨架的動(dòng)態(tài)特性是細(xì)胞運(yùn)動(dòng)所必需，使用actin解聚或穩(wěn)定F-actin的藥物處理運(yùn)動(dòng)細(xì)胞，將會(huì)終止細(xì)胞運(yùn)動(dòng)[62,63]。

2.2.2 橫紋肌中的actin細(xì)胞骨架

脊椎動(dòng)物的骨骼肌和心肌是典型的橫紋肌，秀麗隱桿線蟲(chóng)()的體壁肌是斜橫紋肌[64]。在橫紋肌纖維中，基于actin的細(xì)肌絲、基于肌球蛋白的粗肌絲和其他相關(guān)蛋白被組織成收縮裝置的最小重復(fù)單元—肌小節(jié)(sarcomere)，以產(chǎn)生肌肉組織的收縮力[65,66]。肌小節(jié)是位于兩個(gè)相鄰Z盤之間的區(qū)域，粗肌絲位于肌小節(jié)的中部，并具有雙極方向的肌球蛋白頭部；被原肌球蛋白-肌鈣蛋白復(fù)合物包裹的細(xì)肌絲在每個(gè)肌小節(jié)單元的末端以相反的方向取向；actin細(xì)肌絲的正極端被a-actinin蛋白錨定并交聯(lián)在Z帶上，并被加帽蛋白CapZ所加帽，而被加帽蛋白Tropomodulin加帽的細(xì)肌絲負(fù)極端則未被錨定在特定的結(jié)構(gòu)上[67,68]。

雖然在遷移的成纖維細(xì)胞和腫瘤細(xì)胞中，F(xiàn)-actin形成的調(diào)控機(jī)制已經(jīng)被廣泛研究，但在發(fā)育的肌細(xì)胞中，肌小節(jié)細(xì)肌絲的形成及調(diào)控過(guò)程目前仍不清楚。針對(duì)肌纖維的組裝，Sanger等提出了前肌原纖維模型[69,70]。在肌肉發(fā)育過(guò)程中，肌原纖維組裝是actin細(xì)胞骨架發(fā)生的主要形態(tài)改變，此外G-actin和F-actin的比率也發(fā)生劇烈的變化[71]。在成熟的橫紋肌肌小節(jié)中，整個(gè)actin細(xì)肌絲經(jīng)歷了不同速率的周轉(zhuǎn)[72~78]。有趣的是，雖然成熟肌小節(jié)細(xì)肌絲經(jīng)歷actin解聚和加聚動(dòng)態(tài)，但細(xì)肌絲卻具有相似的長(zhǎng)度[79,80]。橫紋肌肌纖維的形成、成熟肌小節(jié)細(xì)肌絲的結(jié)構(gòu)及動(dòng)態(tài)等特點(diǎn)預(yù)示在肌小節(jié)組裝和功能維持過(guò)程中，必然存在一套精確的調(diào)控系統(tǒng)來(lái)調(diào)節(jié)actin細(xì)肌絲的組裝和分解(表1)。大量的研究已經(jīng)表明，細(xì)肌絲正極端和負(fù)極端的加帽蛋白CapZ[81,82]和Tropomodulin[76,83~86]能夠調(diào)節(jié)肌小節(jié)細(xì)肌絲的組織和actin解聚和加聚動(dòng)態(tài)；其他actin相關(guān)蛋白如Nubulin[87~90]、Tropomyosin[91,92]、ADF/cofilin[78,93,94]、WDR1[95,96]、Leiomodin[86,97~99]和FHOD3[100]等也參與了肌小節(jié)細(xì)肌絲的組織和actin動(dòng)態(tài)的調(diào)控。作為actin解聚因子ADF/cofilin的輔助因子，WDR1能夠促進(jìn)與ADF/cofilin結(jié)合的F-actin的解聚。本課題組的研究表明，分別在胚胎期、幼年期和成年期心肌細(xì)胞中敲除，均會(huì)導(dǎo)致F-actin堆積、肌纖維的組裝和功能維持受損等表型[101,102]。

表1 橫紋肌中重要的肌動(dòng)蛋白動(dòng)態(tài)調(diào)控因子

在遺傳和/或環(huán)境因素等影響的病理?xiàng)l件下，肌小節(jié)細(xì)肌絲會(huì)發(fā)生改變。先天性肌病是一種遺傳性的肌肉病變，其特征是骨骼肌無(wú)力，并存在含有actin或其他肌纖維蛋白的棒狀物或聚集體[103]。在桿狀體肌病(nemaline myopathy)、肌動(dòng)蛋白肌病和細(xì)胞核內(nèi)桿狀體肌病這3種主要的先天性肌病中，均涉及編碼ACTIN及相關(guān)蛋白的基因突變[68,103]，包括cofilin (CFL2)[104,105]、a-actin (ACTA1)[106~110]、a-和b-原肌球蛋白(TPM3和TPM2)[111,112]、肌鈣蛋白T (TNNT1)[113~115]和nubulin (NEB)[116~118]等。

3 Actin細(xì)胞骨架在第二生心區(qū)祖細(xì)胞部署發(fā)育中的調(diào)控作用

近年來(lái)，一些調(diào)控SHF祖細(xì)胞部署發(fā)育的轉(zhuǎn)錄因子和信號(hào)通路相繼被報(bào)道，這些轉(zhuǎn)錄因子及信號(hào)通路缺失或改變導(dǎo)致OFT縮短、心臟環(huán)化受損等SHF發(fā)育的缺陷，并伴隨SHF細(xì)胞內(nèi)actin細(xì)胞骨架的破壞。這些結(jié)果揭示了actin細(xì)胞骨架在SHF細(xì)胞發(fā)育特別是SHF祖細(xì)胞向OFT部署過(guò)程的重要性(表2)。

3.1 Wnt5a-Dvl-PCP通路與SHF祖細(xì)胞actin細(xì)胞骨架

2005年，Kirby研究團(tuán)隊(duì)發(fā)現(xiàn)雞()中與遠(yuǎn)端OFT相鄰的SpM SHF細(xì)胞具有假?gòu)?fù)層柱狀上皮細(xì)胞層的特點(diǎn)[121]。直到2012年，這種緊密結(jié)合、上皮樣細(xì)胞層的SHF細(xì)胞形態(tài)才在小鼠SpM 中被發(fā)現(xiàn)，而靠近尾部SpM處的SHF細(xì)胞具有板狀偽足狀突起的細(xì)胞形態(tài)。因此有研究者提出一種假說(shuō)：位于尾部SpM中松散堆積的間充質(zhì)樣SHF祖細(xì)胞經(jīng)歷間充質(zhì)細(xì)胞到上皮細(xì)胞的轉(zhuǎn)化過(guò)程，以形成SpM中上皮樣SHF細(xì)胞層，通過(guò)細(xì)胞插入(cell intercalation)的方式促進(jìn)SHF祖細(xì)胞對(duì)OFT的貢獻(xiàn)；此外，在PCP活性被抑制的突變體中，尾部SpM處SHF祖細(xì)胞中actin聚合和板狀偽足的活性被破壞；該研究推測(cè)Wnt5a-PCP信號(hào)可能通過(guò)調(diào)節(jié)尾部SpM處SHF祖細(xì)胞內(nèi)actin聚合作用促進(jìn)SHF祖細(xì)胞的遷移部署，影響OFT延伸和心臟環(huán)化過(guò)程[8]。

2014年，王建波研究團(tuán)隊(duì)的結(jié)果表明：位于小鼠SpM中的SHF祖細(xì)胞被組織成上皮樣細(xì)胞層，在細(xì)胞層中的單個(gè)細(xì)胞顯示出多邊形形態(tài)，并且在細(xì)胞頂端皮質(zhì)周圍富集F-actin 的細(xì)胞被緊密擠在一起；SHF祖細(xì)胞在SpM某處經(jīng)歷間充質(zhì)到上皮樣細(xì)胞的轉(zhuǎn)變，被組織形成上皮樣細(xì)胞層，并以緊密結(jié)合、上皮樣細(xì)胞層的形式而非單細(xì)胞的方式向OFT遷移[7]。該研究與Sinha等[8]在缺失突變體小鼠中的研究結(jié)果一致，即抑制Wnt5a導(dǎo)致SpM處SHF祖細(xì)胞表現(xiàn)出減少且紊亂的actin聚合、細(xì)胞的形態(tài)和細(xì)胞排列方向被破壞等表型，進(jìn)一步揭示了SpM處SHF祖細(xì)胞中actin細(xì)胞骨架、細(xì)胞形態(tài)及排列方向在SHF祖細(xì)胞向OFT部署中的重要性。

2016年，王建波研究團(tuán)隊(duì)報(bào)道了利用突變體小鼠研究SpM中SHF祖細(xì)胞向OFT部署的另一項(xiàng)研究成果，進(jìn)一步揭示W(wǎng)nt5a-PCP信號(hào)通路在SpM SHF祖細(xì)胞向OFT部署過(guò)程中的時(shí)空調(diào)控作用[119]。在缺失的突變體小鼠中，通過(guò)Islet1- Cre驅(qū)動(dòng)SHF細(xì)胞過(guò)表達(dá)，能夠挽救SpM處SHF細(xì)胞中actin聚合、細(xì)胞極性的缺陷。在Islet1- Cre驅(qū)動(dòng)SHF細(xì)胞過(guò)表達(dá)的突變體小鼠中，與遠(yuǎn)端OFT相鄰的SpM處上皮樣SHF細(xì)胞的細(xì)胞粘附連接被破壞，SHF細(xì)胞滯留在該處，形成突起而不向OFT部署。雖然該研究并未通過(guò)實(shí)驗(yàn)證明過(guò)表達(dá)影響SpM處SHF細(xì)胞粘附連接的具體機(jī)制，然而研究表明上皮細(xì)胞極性和細(xì)胞連接相互影響，兩者的建立和維持需要actin細(xì)胞骨架的協(xié)同調(diào)控作用[35,122]。該研究進(jìn)一步揭示actin細(xì)胞骨架及細(xì)胞排列和極性在SHF祖細(xì)胞部署中的必要作用。

表2 影響SHF祖細(xì)胞actin細(xì)胞骨架的調(diào)控因子及信號(hào)通路

圖1 SHF祖細(xì)胞特異性敲除Wdr1小鼠的表型

E9.5 (A~F)、E10.5 (G~L)對(duì)照組(CTL)和SHF祖細(xì)胞敲除組(Wdr1/F; Mef2c-AHF-Cre)胚胎的顯微和組織學(xué)分析。與對(duì)照組胚胎相比，敲除組胚胎具有RV縮小、近端OFT和RV心肌細(xì)胞排列紊亂(黑箭頭)的表型。

3.2 TBX1與SHF祖細(xì)胞actin細(xì)胞骨架

2014年，F(xiàn)rancou等[9]研究證明：位于SpM及遠(yuǎn)端OFT處的SHF細(xì)胞組成非經(jīng)典頂端-基底極性的上皮，并具有頂部單纖毛、動(dòng)態(tài)actin富集基底端的絲狀偽足等特點(diǎn)；該研究還發(fā)現(xiàn)，除了調(diào)節(jié)SHF祖細(xì)胞的增殖和分化外，還通過(guò)參與調(diào)節(jié)SHF細(xì)胞上皮細(xì)胞極性、SHF祖細(xì)胞基底端actin動(dòng)態(tài)和絲狀偽足活性，影響SHF祖細(xì)胞向OFT的部署。該研究推測(cè)，在心管伸展過(guò)程中，SHF祖細(xì)胞基底端動(dòng)態(tài)actin重塑驅(qū)動(dòng)的絲狀偽足運(yùn)動(dòng)可能在維持SpM處SHF祖細(xì)胞狀態(tài)所需的信號(hào)通路中發(fā)揮作用。

3.3 Arid3b與SHF祖細(xì)胞actin細(xì)胞骨架

2014年，Uribe等[120]研究發(fā)現(xiàn)，通過(guò)基因捕獲(gene trap)方法構(gòu)建的突變體胚胎具有OFT和流入道(inflow tract, IFT)長(zhǎng)度縮短、房室墊發(fā)育受損等一系列心臟發(fā)育缺陷的表型；在E9.5的突變體胚胎中觀察到Islet1陽(yáng)性的SHF祖細(xì)胞在OFT區(qū)域積累，且SpM處SHF祖細(xì)胞中actin細(xì)胞骨架和細(xì)胞結(jié)構(gòu)均發(fā)生改變；Dil標(biāo)記、追蹤實(shí)驗(yàn)證明在突變體胚胎中，SHF細(xì)胞向心管的貢獻(xiàn)受損。該研究推測(cè)Arid3b可能通過(guò)actin細(xì)胞骨架調(diào)節(jié)SHF祖細(xì)胞向心管的運(yùn)動(dòng)，表明actin細(xì)胞骨架在SHF祖細(xì)胞遷移部署中的重要作用。

3.4 WDR1與SHF祖細(xì)胞actin細(xì)胞骨架

本課題組最近的研究揭示actin解聚調(diào)控因子WDR1在哺乳動(dòng)物SHF發(fā)育中的必要作用[102]。通過(guò)Mef2c-SHF-Cre構(gòu)建SHF細(xì)胞特異性敲除小鼠，該突變體小鼠從E11.5開(kāi)始死亡，且在E10.5表現(xiàn)出近端OFT和RV縮小的表型；敲除并不影響SHF細(xì)胞的數(shù)量以及SHF祖細(xì)胞由SpM向遠(yuǎn)端OFT的部署過(guò)程，但近端OFT和右心室細(xì)胞的空間排列和心肌細(xì)胞肌纖維的組裝過(guò)程被嚴(yán)重破壞(圖1)。我們推測(cè)：WDR1介導(dǎo)的actin解聚和加聚動(dòng)態(tài)可能通過(guò)調(diào)節(jié)上皮樣SHF祖細(xì)胞向心肌細(xì)胞分化過(guò)程中心肌細(xì)胞空間排列的重塑過(guò)程，進(jìn)一步調(diào)控OFT和RV大小。關(guān)于Mef2c-SHF-Cre敲除并不影響SHF祖細(xì)胞由SpM向遠(yuǎn)端OFT的部署過(guò)程，推測(cè)可能有兩方面原因：首先，基因不完全刪除導(dǎo)致WDR1的少量存留；其次，前人的研究表明SpM和遠(yuǎn)端OFT處的SHF細(xì)胞是伸展、極化的上皮細(xì)胞，且在這種上皮樣細(xì)胞層中可能存在一種推動(dòng)力以驅(qū)動(dòng)SHF細(xì)胞向遠(yuǎn)端OFT部署[7,119]，暗示SHF祖細(xì)胞向遠(yuǎn)端OFT部署過(guò)程中可能對(duì)actin解聚和加聚動(dòng)態(tài)的需求較少。

4 結(jié)語(yǔ)與展望

SHF祖細(xì)胞整合、部署的精確、具體細(xì)胞機(jī)制尚不完全清楚，如是否通過(guò)主動(dòng)細(xì)胞遷移、細(xì)胞凝聚及細(xì)胞張力、細(xì)胞插入還是有方向的細(xì)胞分裂等機(jī)制，仍需要進(jìn)行深入的研究來(lái)闡明。然而，進(jìn)一步研究SHF祖細(xì)胞部署的細(xì)胞生物學(xué)過(guò)程及生物力學(xué)等特性，需要開(kāi)發(fā)高分辨率動(dòng)態(tài)成像技術(shù)。SHF細(xì)胞具有非典型上皮樣的頂端-基底細(xì)胞極性、細(xì)胞連接、排列和動(dòng)態(tài)actin在基底端細(xì)胞膜富集等特點(diǎn)，這些研究揭示actin細(xì)胞骨架在SHF祖細(xì)胞部署、發(fā)育中發(fā)揮關(guān)鍵的調(diào)控作用。因此，利用actin細(xì)胞骨架作為切入點(diǎn)，通過(guò)分子細(xì)胞生物學(xué)手段構(gòu)建SHF祖細(xì)胞標(biāo)記、追蹤體系，結(jié)合活細(xì)胞成像和高穿透性電子顯微成像系統(tǒng)，將會(huì)為深入闡明、理解SHF祖細(xì)胞遷移、部署的細(xì)胞生物學(xué)特征以及生物力學(xué)提供新的研究思路和方向。

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The role of actin cytoskeleton in regulating the deployment process of mouse cardiac second heart field progenitor cells

Zhongying Liu, Xia Huang, Ziyi Li, Zihao Yang, Baiyin Yuan

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The vertebrate heart tube originates from cardiogenic mesodermal cells in the early embryo, and subsequently elongates by progressive addition of second heart field (SHF) progenitor cells from adjacent pharyngeal mesoderm and splanchnic mesoderm. Insufficient addition of SHF cells to the heart tube causes the failure of maximal elongation of the heart tube, which results in a series of developmental defects including the most common congenital birth defects, such as right ventricular dysplasia and outflow tract septation, and alignment anomalies. SHF cells form an atypical, apicobasally polarized epithelium which is characterized by apical monocilia, and dynamic actin-rich basal filopodia. In this review, we summarize recent research progresses of actin cytoskeleton in the deployment process of mouse SHF progenitor cells, and reveal the significance of actin cytoskeleton in SHF development, especially in the deployment of SHF cells to the outflow tract, to provide theoretical reference for elucidating and understanding the biological characteristics of SHF deployment.

actin; second heart field; cell deployment; mouse

2018-10-29;

2019-01-18

國(guó)家自然科學(xué)基金項(xiàng)目(編號(hào)：31701266)資助[Supported by the National Natural Science Foundation of China (No. 31701266)]

劉鐘穎，碩士研究生，專業(yè)方向：遺傳及細(xì)胞生物學(xué)。E-mail: 757967423@qq.com

袁白銀，博士，講師，研究方向：遺傳及發(fā)育生物學(xué)。E-mail: yuanby@wust.edu.cn

10.16288/j.yczz.18-293

2019/1/29 16:14:30

URI: http://kns.cnki.net/kcms/detail/11.1913.R.20190129.1614.002.htm

(責(zé)任編委: 楊中州)