毛軻，孟子秋，張永彪,3

綜 述

神經(jīng)嵴發(fā)育調(diào)控及顱面部遺傳基礎(chǔ)研究進展

毛軻1，孟子秋2，張永彪2,3

1. 北京航空航天大學(xué)生物與醫(yī)學(xué)工程學(xué)院，北京 100191 2. 北京航空航天大學(xué)醫(yī)學(xué)科學(xué)與工程學(xué)院，北京 100191 3. 工信部大數(shù)據(jù)精準醫(yī)療重點實驗室，北京 100191

顱面部賦予脊椎動物無與倫比的進化優(yōu)勢，其由顱神經(jīng)嵴細胞發(fā)育而來的骨、軟骨、神經(jīng)、肌肉等組織組成，使脊椎動物具備了復(fù)雜的神經(jīng)和感官系統(tǒng)。神經(jīng)嵴細胞是脊椎動物特有的具備遷移性、多能性的細胞類群，它們在增殖、遷移、分化過程中受到多個基因網(wǎng)絡(luò)的時序調(diào)控，從而參與復(fù)雜顱面部的形成。同時，顱面部又是一組高度可遺傳的表型組合，并具有兩個特征：在親緣后代中的可遺傳性及在不同個體間的高度可變性，這兩個特征分別提示了顱神經(jīng)嵴細胞發(fā)育調(diào)控網(wǎng)絡(luò)的精準性和可塑性。調(diào)控網(wǎng)絡(luò)內(nèi)基因適度突變會改變顱神經(jīng)嵴細胞的增殖和分化從而產(chǎn)生表型可塑性，而有害的遺傳突變則將導(dǎo)致畸形產(chǎn)生。本文梳理了對顱面部發(fā)育起決定作用的神經(jīng)嵴細胞的發(fā)育過程及基因調(diào)控網(wǎng)絡(luò)，在遺傳層面總結(jié)了已知的顱面部表型多樣性的決定基礎(chǔ)和顱面畸形的致病機制，以期為了解顱面部發(fā)育過程以及為顱面疾病的防控提供全面認知。

顱面發(fā)育；神經(jīng)嵴細胞；基因調(diào)控網(wǎng)絡(luò)；遺傳變異

顱面器官賦予了脊椎動物獨特的面部外觀，其發(fā)育受到復(fù)雜且高度協(xié)調(diào)的基因網(wǎng)絡(luò)調(diào)控。顱面部發(fā)育始于原腸胚期，需要多個不同的信號通路及各個胚層協(xié)調(diào)控制顱面形態(tài)生成，其中神經(jīng)嵴細胞(neural crest cells, NCCs)對顱面發(fā)育具有重要貢獻[1]。

NCCs是脊椎動物特有的呈集群遷移、具有短暫多能性的細胞類群，它們起源于原腸胚期的神經(jīng)板邊界，從閉合的神經(jīng)管背側(cè)遷移至胚胎多個部位[2]。NCCs的出現(xiàn)在脊椎動物進化中具有重要意義，它們賦予脊椎動物全新的頭部，促進了脊椎動物由被動的濾食行為向主動捕食的生活方式改變，使其輻射和適應(yīng)地球上大多數(shù)生態(tài)系統(tǒng)[3]。NCCs從誘導(dǎo)產(chǎn)生到分化可視為由多個連續(xù)變化階段組成，且在每個發(fā)展階段都存在一個核心基因調(diào)控網(wǎng)絡(luò)(gene regulatory network, GRN)。GRN由包含轉(zhuǎn)錄因子及信號分子的發(fā)育模塊組成，它整合了環(huán)境信號和細胞內(nèi)在基因信號，使NCCs在特定的時間和空間受到精細調(diào)控，從而分化形成骨、軟骨、神經(jīng)、肌肉等組織結(jié)構(gòu)，產(chǎn)生了顱面部的大部分衍生物[4]。

顱面部是一組高度可遺傳的表型組合，面部特征在親緣后代中表現(xiàn)為可遺傳性，然而在不同人群或個體間卻呈現(xiàn)高度可變性，其主要歸因于遺傳因素[5]。通過全基因組關(guān)聯(lián)分析發(fā)現(xiàn)與面部形態(tài)相關(guān)的位點超過300個，它們多富集于神經(jīng)嵴細胞或胚胎頜面組織的呈激活狀態(tài)的增強子區(qū)。此外，顱面部是脊椎動物中易發(fā)生畸形的部位之一，已證實遺傳突變是導(dǎo)致先天性顱面畸形的主因[6]。目前已鑒定的遺傳變異包括染色體缺失、重復(fù)、基因突變等功能性缺失或異常，這些變異在胚胎發(fā)育時期影響了神經(jīng)嵴細胞增殖或遷移[7]。本文總結(jié)了參與顱面發(fā)育的重要細胞類群——NCCs及其基因調(diào)控網(wǎng)絡(luò)，并對顱面部遺傳多樣性及顱面致病基因進行了總結(jié)。

1 神經(jīng)嵴細胞

神經(jīng)嵴于1868年由Wilhelm His Sr.在雞胚中首次發(fā)現(xiàn)，是脊椎動物胚胎發(fā)育過程中過渡性結(jié)構(gòu)[8]。神經(jīng)嵴起源于原腸胚期，在神經(jīng)板和非神經(jīng)外胚層交界處形成了神經(jīng)板邊界(neural plate border, NPB)，其可隆起成神經(jīng)褶皺，神經(jīng)板雙側(cè)的神經(jīng)褶皺互相接近后逐漸融合，形成閉合的神經(jīng)管(圖1A)；之后源于神經(jīng)管背側(cè)的NCCs成群向腹側(cè)遷移[9]。

NCCs是脊椎動物特有的細胞類群，具有很強的遷移潛能和短暫的多能性，可分化形成多種不同類型的細胞，如骨細胞、平滑肌細胞、神經(jīng)細胞、黑色素細胞等，它們直接或以衍生物形式分布于脊椎動物組織器官中[10]。根據(jù)起源，NCCs按照頭尾軸的排列順序分為4個主要亞群：顱、迷走、軀干和骶神經(jīng)嵴亞群(圖1B)。各個亞群的NCCs表現(xiàn)出不同的遷移模式，且其分化能力在開始遷移時已被確定[11]。顱神經(jīng)嵴細胞(cranial neural crest cells, CNCCs)是參與顱面部發(fā)育的重要類群，也是NCCs中唯一參與骨形成的細胞群[12]。CNCCs起源于前中樞神經(jīng)系統(tǒng)，即前腦、中腦和后腦(圖1C)，最前部的CNCCs構(gòu)成額鼻骨，而更多后部的CNCCs遷移至咽弓處，形成頜骨、中耳和頸部的骨骼及軟骨(圖1D)[12]。CNCCs賦予脊椎動物超級感官系統(tǒng)、復(fù)雜神經(jīng)網(wǎng)絡(luò)和武裝了牙齒的頜骨，使其相對于無脊椎動物擁有了“嶄新頭部”，在進化中具有重要意義。

圖1 神經(jīng)嵴細胞形成、遷移、分化示意圖

A：神經(jīng)嵴發(fā)育模式圖。神經(jīng)嵴細胞起源于神經(jīng)板邊界(綠色)，該結(jié)構(gòu)在胚胎原腸胚期位于神經(jīng)板(藍色)和非神經(jīng)外胚層之間(灰色)，隨著神經(jīng)管的發(fā)育，神經(jīng)嵴細胞經(jīng)過上皮間充質(zhì)轉(zhuǎn)化后形成成熟的神經(jīng)嵴細胞(綠色)，從神經(jīng)管背側(cè)遷移出。B：神經(jīng)嵴細胞亞群及分化類型。神經(jīng)嵴細胞亞群根據(jù)其軸向分布依次分為顱、迷走、軀干、骶神經(jīng)嵴細胞。C：以小鼠E9.5的模式圖為例展示神經(jīng)嵴遷移路線。神經(jīng)嵴遷移時，胚胎后腦便形成8個菱腦原節(jié)(rhombomeres1–8, R1～8)；來自前腦、中腦的神經(jīng)嵴細胞遷入胚胎顱面突起部位，來自菱腦R1和R2的細胞定向遷入第一咽弓(pharyngeal arch 1, PA1)，而第二咽弓(PA2)主要是由菱腦R3和R4遷出的細胞形成。D：神經(jīng)嵴在顱面部分化的組織展示。來自PA1、PA2的神經(jīng)嵴細胞貢獻了頜面部大部分顱面骨骼。

2 神經(jīng)嵴細胞的基因調(diào)控網(wǎng)絡(luò)

2.1 基因調(diào)控網(wǎng)絡(luò)在神經(jīng)嵴發(fā)育過程的概述

NCCs形成到命運決定是一個時序變化的過程，包括：神經(jīng)嵴誘導(dǎo)、特征化(specification)、上皮間充質(zhì)轉(zhuǎn)化(epithelial to mesenchymal transition, EMT)、遷移和分化；每一個階段都存在一個核心基因調(diào)控網(wǎng)絡(luò)[13,14]。研究者使用轉(zhuǎn)錄組、表觀組學(xué)以及動物模型等手段，建立了每個階段的復(fù)雜基因信號網(wǎng)絡(luò)，由信號分子模塊和階段特異的轉(zhuǎn)錄因子組成(圖2A)[5,15]。

2.1.1 神經(jīng)嵴的誘導(dǎo)和特征化

最早的NCCs可追溯到NPB形成時期，對小鼠()、雞()、爪蟾()的研究表明，在此時期，神經(jīng)細胞由和標(biāo)記，非神經(jīng)外胚層細胞由、和標(biāo)記，神經(jīng)嵴邊界介于二者之間，在3種主要信號通路BMP、WNT和FGF共同作用驅(qū)動神經(jīng)嵴邊界特定基因的表達，包括、、及等轉(zhuǎn)錄因子[16]。這些特異的轉(zhuǎn)錄因子及信號通路組合成為誘導(dǎo)神經(jīng)嵴的GRN，并將NCCs與另一種在NPB誘導(dǎo)的具有多能性的前基板區(qū)細胞區(qū)別開來[4,17]。在神經(jīng)嵴誘導(dǎo)形成后，神經(jīng)板會逐漸隆起，最后形成神經(jīng)管，而NPB內(nèi)會激活神經(jīng)嵴特征化模塊從而產(chǎn)生NCCs。在神經(jīng)管開始隆起時，NCCs開始特異性表達一些轉(zhuǎn)錄因子，如、、以及等。在脊椎動物中，這種GRN的組成是大致保守的，且以正反饋的形式在基因之間構(gòu)建網(wǎng)絡(luò)，如在爪蟾中通過激活及促進NPB的誘導(dǎo)，隨后在神經(jīng)嵴的特征化過程中驅(qū)動、、的表達[18,19]，而這些基因又是激活下游模塊的重要參與者(圖2A)。

2.1.2 神經(jīng)嵴上皮間充質(zhì)轉(zhuǎn)化

隨著神經(jīng)管形成，神經(jīng)嵴經(jīng)歷上皮細胞向間充質(zhì)細胞的轉(zhuǎn)變及分層，然后在整個胚胎中廣泛遷移。EMT協(xié)調(diào)信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄調(diào)控以觸發(fā)神經(jīng)嵴發(fā)生大的結(jié)構(gòu)變化，包括脫粘、細胞骨架重排、運動性能獲得等，但目前的分子基礎(chǔ)認識很大程度反映在控制脫粘的效應(yīng)基因模塊。在EMT過程中，神經(jīng)嵴特征化GRN涉及的轉(zhuǎn)錄因子如、、等，它們參與細胞粘附分子鈣粘蛋白超家族多個成員的調(diào)控。如在雞胚中，與結(jié)合抑制內(nèi)皮鈣粘素的表達，而和促進了間充質(zhì)鈣粘素的表達[20]。此外，EMT過程中神經(jīng)嵴GRN模塊也受細胞外信號調(diào)控，在爪蟾中Wnt可直接激活的表達促進EMT[21]。

2.1.3 神經(jīng)嵴細胞遷移和分化

NCCs經(jīng)過EMT發(fā)育成熟后從神經(jīng)管背側(cè)遷移離開，遷移的NCCs表達，它作為最早的神經(jīng)嵴特征基因之一可直接調(diào)控眾多下游效應(yīng)因子[22,23]。NCCs在遷移過程中響應(yīng)接觸性抑制以及信號性驅(qū)動等信號[24]，但控制NCCs何時停止遷移的機制目前還不清楚。研究表明在雞、小鼠遷移的NCCs中表達，但也受多個基因調(diào)控，如為雞顱神經(jīng)嵴細胞的活性調(diào)節(jié)區(qū)域，而該區(qū)域又受神經(jīng)嵴表達的、及等轉(zhuǎn)錄因子調(diào)控[25]。NCCs在遷移過程中維持足夠的可塑性，當(dāng)其遷移至特定區(qū)域后在相關(guān)信號通路作用下發(fā)生分化。如在雞第一咽弓中，bmp4介導(dǎo)的信號通路有助于表達，進而促進頜骨關(guān)節(jié)的正確定位[26]。在小鼠胚胎發(fā)育中，F(xiàn)gf8促進NCCs增殖發(fā)育[27]，并在NCCs空間特性及咽弓前后軸、近遠軸極性的建立過程中發(fā)揮作用[28]。NCCs可以分化形成30多種細胞類型，在此我們概述了顱神經(jīng)嵴衍生物中軟骨、神經(jīng)元和黑色素細胞等最具特征的終末分化基因模塊。CNCCs具備分化為軟骨細胞的潛能，驅(qū)動軟骨細胞發(fā)育的核心調(diào)控網(wǎng)絡(luò)涉及和，直接激活軟骨分化標(biāo)志物和[29,30]，此外還發(fā)現(xiàn)，介導(dǎo)的TGF-β通路在調(diào)節(jié)軟骨細胞發(fā)育中起著至關(guān)重要的作用[31]。NCCs分化的自主神經(jīng)細胞遍布全身，在模式動物等研究基礎(chǔ)上構(gòu)建了簡化的分化模塊，促進了和的表達，反過來，與一起激活神經(jīng)元分化基因[32]。此外，直接激活黑色素細胞發(fā)育的主要調(diào)控因子。與、因子共同作用并促進黑色素合成酶和的表達[33,34](圖2A)。

2.2 Hox/Dlx基因家族參與CNCCs位置識別

神經(jīng)嵴的遷移模式以及在特定位置的身份認定很大程度取決于它們的軸向起源，這賦予了NCCs在后期發(fā)育過程中對環(huán)境信號的適應(yīng)性，并最終導(dǎo)致細胞命運的差異。和基因家族在決定CNCCs的前后及近遠端模式中發(fā)揮著特別重要的作用[35,36]。

圖2 神經(jīng)嵴發(fā)育的基因調(diào)控網(wǎng)絡(luò)及位置識別的轉(zhuǎn)錄程序：Hox和Dlx

A：神經(jīng)嵴發(fā)育的基因調(diào)控網(wǎng)絡(luò)。簡化描繪了脊椎動物神經(jīng)嵴細胞的GRN，由不同層次組織的信號分子模塊和每個階段的轉(zhuǎn)錄因子組成，神經(jīng)嵴發(fā)育包括神經(jīng)嵴誘導(dǎo)(induction)、特征化(specification)、遷移(migration)以及分化(differentiation)過程，分別對應(yīng)不同顏色的模塊表示，箭頭代表調(diào)控激活。B：基因在小鼠胚胎咽弓的表達模式。沿胚胎前后軸方向，基因為神經(jīng)嵴細胞提供了在咽弓內(nèi)的空間識別信息，小鼠胚胎每個咽弓的不同顏色表示其特定的表達模式。C：基因在小鼠胚胎咽弓的表達模式。沿咽弓背腹側(cè)，基因為顱神經(jīng)嵴細胞提供空間識別信息，基因在咽弓中由近到遠端呈嵌套區(qū)域式表達。

基因在染色體上串聯(lián)成簇排列，并沿胚胎體軸表達，從后腦開始一直延續(xù)到脊髓，基因參與建立NCCs體軸前后位置的同一性[37,38]。NCCs根據(jù)基因的表達與否可分為Hox+NCCs和Hox–NCCs。Hox–NCCs從前腦、中腦及后腦前端遷至面部突起和第一咽弓，分化形成大部分的顱骨、內(nèi)耳骨、顴骨復(fù)合體，以及上下頜[12]。而從后腦遷出的Hox+NCCs可能預(yù)先形成對基因的識別，它們分別遷移至胚胎第二、三、四咽弓，對應(yīng)表達和(圖2B)。這些Hox+NCCs可分化成為構(gòu)成顱頜面的Reichert軟骨、顳骨及部分舌骨等[12]。有研究表明，在Hox–NCCs中強制表達基因會破壞顱面骨骼發(fā)育，同樣，Hox+NCCs也無法取代Hox–NCCs在胚胎發(fā)育中的作用[37]，如小鼠中Hoxa2功能缺失導(dǎo)致第二咽弓衍生物同源異形轉(zhuǎn)化為第一咽弓衍生的骨骼元素[39]。

基因是包含同源盒結(jié)構(gòu)的轉(zhuǎn)錄因子，該基因家族由6個成員組成，編號為Dlx1-6，它們彼此形成雙基因簇，在脊椎動物中具體存在形式為、、。該基因家族分布在與基因相同的染色體上，在建立第一二咽弓遠近端的NCC分布特征中發(fā)揮重要調(diào)控作用[40]。在胚胎發(fā)生過程中，基因沿第一、二咽弓的近遠端呈區(qū)域嵌套模式表達，和在大部分咽弓中表達，而和、和表達受限于咽弓遠端區(qū)域[41,42](圖2C)。研究表明，基因在顱頜面發(fā)育中調(diào)控NCCs的正確遷移和器官形態(tài)發(fā)生?；蚋弑磉_會導(dǎo)致CNCCs黏附成細胞團，在神經(jīng)管基側(cè)聚集，只有少部分CNCCs遷移至咽弓中[43]；在–/–敲除的小鼠模型中發(fā)現(xiàn)，基因表達的缺失會使小鼠表現(xiàn)出顱面部缺陷，包括Meckel軟骨、下頜骨和顱蓋骨等骨骼畸形[44]。

2.3 單細胞層面對CNCCs發(fā)育的研究

CNCCs是參與顱面部發(fā)育的重要細胞類群，然而從CNCCs到各種細胞類型的命運決定過程仍是該領(lǐng)域亟待解決的熱點問題。單細胞技術(shù)能夠依據(jù)細胞內(nèi)基因表達特征追溯分析細胞身份和細胞命運，在小鼠、人、雞、斑馬魚()等物種中完成了早期胚胎發(fā)育細胞圖譜繪制[45～50]。對NCCs的譜系追蹤和組學(xué)特征的研究有助于人們深入了解該類多能干細胞的命運決定過程以及該類細胞如何賦予脊椎動物強大的進化優(yōu)勢。

基于NCC的基因表達特征有助于解析細胞命運決定過程。Lignel等[51]采用多重單分子熒光原位雜交在雞胚發(fā)育早期檢測了35個基因在單細胞水平的表達特征，發(fā)現(xiàn)在雞胚神經(jīng)管中，早期遷移的NCC可以分為5個亞群。隨后由Williams等[52]采用單細胞染色質(zhì)可及性及轉(zhuǎn)錄組學(xué)手段在雞模型中全面揭示了遷移前NCCs的基因表達異質(zhì)性特征。他們發(fā)現(xiàn)在神經(jīng)嵴遷移前，CNCCs已經(jīng)形成獨立的亞群，并在表觀調(diào)控水平證實了順式動態(tài)調(diào)控過程；這些早期具有異質(zhì)性的NCCs在隨后的發(fā)育過程中形成了不同功能的細胞譜系[52]。

單細胞多組學(xué)除了揭示NCCs異質(zhì)性，還是當(dāng)前研究NCCs命運決定的優(yōu)勢策略。Soldatov等[53]結(jié)合單細胞測序及空間轉(zhuǎn)錄組技術(shù)，對小鼠胚胎期軀干及顱神經(jīng)嵴細胞比較分析后提出：NCCs命運決定是通過一系列的二元選擇達成的。也就是NCCs發(fā)育過程中，細胞內(nèi)存在兩個競爭性程序的激活，不同NCCs表現(xiàn)出傾向某一個程序表達，并最終完成命運決定。舉例來說，在小鼠中，NCCs從神經(jīng)管分層后其命運面臨初級分叉選擇，也就是將感官發(fā)育的細胞譜系與其他譜系分離出來，隨后又面臨自主神經(jīng)系統(tǒng)與間充質(zhì)譜系的命運抉擇。近期，F(xiàn)abian等[48]利用單細胞技術(shù)通過整合斑馬魚整個生命周期中CNCCs的轉(zhuǎn)錄組及染色質(zhì)可及性數(shù)據(jù)研究其細胞多樣性及譜系進展，發(fā)現(xiàn)CNCCs多能性的建立是通過漸進式的空間區(qū)域特異性調(diào)控來獲得的，并揭示了譜系啟動的候選轉(zhuǎn)錄因子，如在斑馬魚顱面軟骨譜系發(fā)育中發(fā)揮作用。單細胞技術(shù)幫助我們在轉(zhuǎn)錄組及染色質(zhì)可及性層面推測NCCs分化狀態(tài)及基因調(diào)控網(wǎng)絡(luò)，但CNCCs發(fā)育過程中產(chǎn)生細胞異質(zhì)性的過程和外界信號如何影響NCCs命運決定過程尚不清楚。

3 人顱面部的遺傳基礎(chǔ)及致病基因

3.1 人顱面部的遺傳基礎(chǔ)

顱面部是一組高度可遺傳的表型組合。在個體層面，人類同卵雙胞胎以及親屬之間面部相似而非親屬之間呈現(xiàn)較大差異；在群體層面，相同人種內(nèi)部的頜面部相似度遠大于不同人種的相似度，這主要歸因于遺傳因素[54,55]，但目前人類顱面遺傳差異的遺傳基礎(chǔ)認識有限。全基因組關(guān)聯(lián)分析(genome- wide association studies，GWAS)對不同個體的全基因組遺傳變異集進行觀測性研究，以確定是否有變異與某一性狀相關(guān)聯(lián)[56]。目前已有大量研究揭示了和面部特征相關(guān)聯(lián)的基因(表1)[57,58]。

已發(fā)表的十多項面部特征GWAS文章主要針對牙齒、耳朵、頭發(fā)等表型[75～77]，但這些研究大多局限于歐洲人群。采用顱面特征點(facial landmarks)獲取表型的GWAS研究中，有3個顯著位點在2個及以上獨立研究中重現(xiàn)：rs11093404()與眥間寬度[61,69]、rs2045323()與鼻子性狀[62,73]、rs3827760()與下頜前突，眼角到耳垂長度[62,66]。其他研究還鑒定到的強關(guān)聯(lián)包括：與鼻眼距離[59,60,65,66]，與鼻寬[61,62,65]，與唇形[57,74]。采用人工智能的面部圖像分割獲得面部表型的GWAS研究中，鑒定出更多全基因組水平顯著關(guān)聯(lián)的位點?；蚬δ茏⑨尠l(fā)現(xiàn)，90%的位點位于基因間區(qū)或內(nèi)含子區(qū)，且相關(guān)基因多與軟骨、第一二咽弓間充質(zhì)、面部骨骼和顎骨的發(fā)育等相關(guān)。ChIP-seq結(jié)果發(fā)現(xiàn)這些顯著關(guān)聯(lián)的位點富集于特定細胞或組織(如NCCs、胚胎頜面組織)的呈激活狀態(tài)的增強子區(qū)，特別是在人NCC細胞系中[5,57]。有趣的是，分析發(fā)現(xiàn)同源位點也落入黑猩猩的NCC細胞的高活性增強子區(qū)，從而推論這些變異位點可能影響人類的物種特異性和個體面部形態(tài)。

顱面部形態(tài)具有多維性和復(fù)雜性，對已發(fā)現(xiàn)的顱面部相關(guān)基因進行功能富集分析(https://maa-yanlab.cloud/Enrichr/)，篩選到-value 0.01的36個WikiPathway。對顯著富集的通路內(nèi)的蛋白進行蛋白互作分析(https://string-db.org/)(圖3)，結(jié)果顯示蛋白互作顯著富集于神經(jīng)嵴分化(=7.22E-13)。綜上所述，影響顱面形態(tài)的遺傳因素和神經(jīng)嵴細胞發(fā)育密切相關(guān)，提示顱面疾病也與神經(jīng)嵴細胞發(fā)育密不可分[78]。

表1 與顱面特征關(guān)聯(lián)的基因

3.2 先天顱面畸形及致病基因

先天性顱面畸形是一類出生缺陷疾患，該類疾病由遺傳突變或胚胎發(fā)育異常導(dǎo)致先天性顱骨、眼眶、顴骨、上下頜骨畸形及面部軟組織缺損，常見的有先天性唇/腭裂、半側(cè)顏面短小畸形、顱縫早閉、Treacher Collins綜合征等[79,80]。本文以先天顱面畸形中最高發(fā)的唇/腭裂、半側(cè)顏面短小畸形為例，梳理其相關(guān)風(fēng)險致病基因及可能的致病機制。

唇/腭裂(OMIM: 225060)是新生兒中較常見的先天頜面部畸形，發(fā)病率接近1/700[81]。唇裂可發(fā)生于單側(cè)、雙側(cè)或中間，當(dāng)上腭包含一個通向鼻子的裂隙便形成了腭裂。該疾病影響患者外觀、語言，甚至造成阻塞性呼吸等問題，嚴重影響患者身心健康。研究發(fā)現(xiàn)，染色體異常、單基因突變，遺傳和環(huán)境因素交互均可導(dǎo)致該畸形的產(chǎn)生。較為明確的致病基因為、、，他們分別與X連鎖的腭裂、唇裂/腭外胚層發(fā)育不良綜合征、范德沃爾綜合征相關(guān)。此外，揭示的風(fēng)險基因包括、、、、、、、和[82]。上述基因?qū)е录膊“l(fā)生的致病機制仍然未知，因此對唇和腭的發(fā)育基礎(chǔ)及調(diào)控網(wǎng)絡(luò)的研究成為基礎(chǔ)研究熱點。人胚胎期的第一咽弓和額鼻突參與了上唇、腭頂和下頜的發(fā)育，這些組織被外胚層上皮細胞覆蓋，其核心為NCCs來源的間充質(zhì)。當(dāng)NCCs或外胚層上皮細胞調(diào)控機制被擾亂即可導(dǎo)致唇/腭裂，如參與NCCs調(diào)控的轉(zhuǎn)錄因子、、發(fā)生突變，在人、小鼠和斑馬魚中會產(chǎn)生腭裂[83,84]。

圖3 人顱面部關(guān)聯(lián)基因的蛋白互作網(wǎng)絡(luò)

將目前研究發(fā)現(xiàn)的397個基因在enrichR上進行功能分析，以-value為0.01進行篩選，發(fā)現(xiàn)有76個基因在36個WikiPathway上顯著富集。將這些篩選基因通過STRING數(shù)據(jù)庫比對分析，以combined score > 0.7作為篩選蛋白互作的條件，將篩選到的蛋白互作網(wǎng)絡(luò)數(shù)據(jù)導(dǎo)入Cytoscape軟件，通過CytoNCA中介數(shù)中心性(betweenness centrality，BC)分析蛋白相互作用網(wǎng)絡(luò)中的核心基因，BC數(shù)值通過表示蛋白的圓框大小呈現(xiàn)，BC值越高，其在蛋白互作中越重要。神經(jīng)嵴分化相關(guān)的蛋白用橙色圓框表示。

半側(cè)顏面短小畸形(OMIM:164210)為發(fā)病率僅次于唇/腭裂的顱面畸形，也稱為第一二鰓弓綜合征，臨床表型主要為頜面部不對稱發(fā)育，伴有外耳和中耳表型異常、上頜和/或下頜發(fā)育畸形、頜面部軟組織發(fā)育不全等，其發(fā)病率介于1/3500～1/6500之間[85]。半側(cè)顏面短小畸形多為散發(fā)病例，可能與母體孕期狀態(tài)、海拔低氧環(huán)境、致畸劑等相關(guān)[86～88]，但越來越多的證據(jù)提示遺傳因素為發(fā)病的主因。Zhang等[89]通過對來自中國的939位患者的GWAS分析發(fā)現(xiàn)，與半側(cè)顏面短小畸形顯著相關(guān)的潛在致病基因多與顱神經(jīng)嵴發(fā)育相關(guān)，包括、、、、、、、、、、。另外，染色體缺失或重復(fù)也會導(dǎo)致該疾病發(fā)生，包括1p22.2-p31.2缺失、5p15缺失、14q23.1重復(fù)等[90-92]。14q為一重要的致病區(qū)域，其包含一個與神經(jīng)嵴發(fā)生密切相關(guān)的轉(zhuǎn)錄因子，其參與調(diào)控前腦、眼、耳的形成[93]，且敲除小鼠表現(xiàn)為嚴重的頜面畸形[94]。類似在染色體缺失或重復(fù)區(qū)間內(nèi)還發(fā)現(xiàn)、、和等基因[95]，多與NCCs的發(fā)育調(diào)控相關(guān)。此外，、、、基因中發(fā)生錯義突變或無義突變時，也會導(dǎo)致該疾病發(fā)生，這些基因功能缺失會破壞NCCs的增殖或遷移，并導(dǎo)致第一、二咽弓的發(fā)育異常[96～99]。綜上所述，先天性顱面畸形的產(chǎn)生與NCCs密切相關(guān)，在發(fā)育過程中當(dāng)調(diào)控網(wǎng)絡(luò)異?；蛑車h(huán)境改變影響NCCs的正常功能時，則可能導(dǎo)致先天性顱面疾病的發(fā)生。

4 結(jié)語與展望

顱面發(fā)育精妙且復(fù)雜，需要各個胚層相互協(xié)作完成，其中神經(jīng)嵴細胞在顱面發(fā)育過程中發(fā)揮著重要作用，也是脊椎動物區(qū)別于無脊椎動物的一個關(guān)鍵特征。神經(jīng)嵴發(fā)育是個迅速、精準調(diào)控的過程，雖然通過模式動物及組學(xué)分析構(gòu)建了NCCs發(fā)育過程中的GRN，但在NCCs持續(xù)變化的過程中，GRN中關(guān)鍵轉(zhuǎn)錄因子和基因模塊是否存在新的組合，GRN在NCCs遷移不同位置的實時變化特征，GRN是否存在表觀修飾以及基因突變對于GRN的影響等問題仍有待研究。單細胞組學(xué)技術(shù)有利于NCCs的GRN趨于完善，它可以通過追蹤多個發(fā)育時間點的單細胞譜系，并結(jié)合單個細胞的胚胎時空轉(zhuǎn)錄特征，在時空上辨識各個細胞類群的譜系變化和組學(xué)特征，可用于對GRN的深入解析[100]。

顱面部的身份標(biāo)志特征，能夠折射出年齡、性格、身體健康狀態(tài)等，解析其遺傳基礎(chǔ)對于顱面復(fù)雜性狀的認知以及先天疾病的探索具有重要意義。遺傳變異會使顱面部結(jié)構(gòu)在正常范圍內(nèi)發(fā)生改變，但也會導(dǎo)致顱面異常的產(chǎn)生。顱面畸形多為新生兒先天疾患，了解顱面畸形產(chǎn)生的遺傳基礎(chǔ)，可將致病基因篩查應(yīng)用于產(chǎn)前診斷，有利于控制顱面畸形患病風(fēng)險，幫助醫(yī)生進行醫(yī)學(xué)診斷。隨著生命科學(xué)的發(fā)展，人們對顱面遺傳認知會更加深刻，可為顱面疾病防治提供更多策略，而對顱面部發(fā)育具有關(guān)鍵貢獻的神經(jīng)嵴細胞可以作為重點研究目標(biāo)。另一方面，對神經(jīng)嵴細胞可塑性及其命運調(diào)控基因網(wǎng)絡(luò)的研究為細胞重編程和干細胞操作策略提供了線索。

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Progress on the regulation of neural crest and the genetics in craniofacial development

Ke Mao1, Ziqiu Meng2, Yongbiao Zhang2,3

The craniofacial features endow vertebrates with unparalleled evolutionary advantages. The craniofacial is composed of bone, cartilage, nerves, and connective tissues mainly developed from cranial neural crest cells (cNCCs). These tissues form complex organs which enable vertebrates to have powerful neural and sensory systems. NCCs are groups of migratory and pluripotent cells that are specific to vertebrates. The specification, premigration and migration, proliferation, and fate determination of the NCCs are precisely and sequentially controlled by gene regulatory networks, to ensure the ordered and accurate development of the craniofacial region. The craniofacial region represents a combined set of highly heritable phenotypes, which could be illustrated by the inherited facial features between relatives but perceptible differences among non-relatives. Such phenomena are termed heredity and variation, which are in accordance with the precision and plasticity of cNCCs gene regulatory network, respectively. Evidence has shown that genetic variations within the regulatory network alter the proliferation and differentiation of NCCs within a tolerable range, while deleterious mutations will lead to craniofacial malformations. In this review, we first summarize the development procedure of NCCs and their gene regulatory networks and then provide an overview on the genetic basis of the facial morphology and malformations. This review will benefit the understanding of craniofacial development and the prevention of craniofacial diseases.

craniofacial development; cranial neural crest cells; gene regulatory network; genetic variation

2022-06-28;

2022-08-29;

2022-09-26

國家自然科學(xué)基金項目(編號：31671312，82171844，81970898)資助[Supported by the National Natural Science Foundation of China (Nos. 31671312, 82171844, 81970898)]

毛軻，在讀博士研究生，研究方向：生物與醫(yī)學(xué)工程。E-mail: maocyy@126.com

張永彪，博士，副研究員，研究方向：生物信息學(xué)。E-mail: zhangyongbiao@buaa.edu.cn

10.16288/j.yczz.22-221

(責(zé)任編委: 閻言)