Ubc9對雞胚神經(jīng)嵴細胞發(fā)育的影響

李明洋，劉乙蒙，劉 超，李淑玲，欒治東*

(遼寧醫(yī)學院 基礎醫(yī)學院 1.發(fā)育生物學教研室；2.組胚教研室；3.生物化學教研室, 遼寧 錦州 121000)

研究論文

Ubc9對雞胚神經(jīng)嵴細胞發(fā)育的影響

李明洋1，劉乙蒙3，劉 超1，李淑玲2，欒治東1*

(遼寧醫(yī)學院 基礎醫(yī)學院 1.發(fā)育生物學教研室；2.組胚教研室；3.生物化學教研室, 遼寧 錦州 121000)

目的探索Ubc9和SUMO通路對雞胚神經(jīng)嵴細胞發(fā)育的影響。方法1)采集雞胚，通過免疫熒光實驗和原位雜交實驗來檢測SUMO結(jié)合酶Ubc9及神經(jīng)嵴細胞標志基因的表達情況。2)設置對照組(非特異性嗎啉代處理)和Ubc9-嗎啉代組來處理各組8期雞胚，隨后通過原位雜交實驗檢測Snail2，Sox9和FoxD3等神經(jīng)嵴細胞晚期標志基因的表達情況。結(jié)果1)Ubc9與標志基因Pax7共表達于4、6和8期雞胚神經(jīng)嵴細胞前體。2)與對照組相比，Ubc9-嗎啉代組雞胚單側(cè)上Snail2(P<0.05)，Sox9(P<0.01)和FoxD3(P<0.05)的mRNA水平均有下降。結(jié)論Ubc9表達于4、6和8期雞胚的神經(jīng)脊細胞前體，雞胚神經(jīng)嵴細胞的形成需要SUMO通路參與。

雞胚；神經(jīng)嵴細胞；Ubc9；SUMO通路

神經(jīng)嵴細胞(neural crest cell, NCC)起源于脊椎動物背部神經(jīng)管，為多潛能的干細胞并具有高度遷移性，能夠分化產(chǎn)生多種組織，包括頭部的大部分骨骼和肌肉、 外周神經(jīng)系統(tǒng)的神經(jīng)元和膠質(zhì)細胞及皮膚黑色素細胞等[1]。NCC發(fā)育異常會導致缺陷病。研究表明，許多蛋白參與NCC形成和遷移，如NCC早期標志基因Pax7[2]以及晚期調(diào)控因子如Snail2、Sox9和FoxD3[3]等。

小泛素相關修飾蛋白(small ubiquitin-related modifier，SUMO)化是一種廣泛存在的蛋白質(zhì)翻譯后修飾形式[4]，參與核質(zhì)轉(zhuǎn)運，細胞周期調(diào)控，信號傳導和轉(zhuǎn)錄活性調(diào)控等[5]。本研究將觀察SUMO化中唯一的E2結(jié)合酶-Ubc9[6]在早期雞胚中的表達情況，隨后檢測Ubc9敲除后雞胚的NCC晚期標志基因的表達水平，探索SUMO通路對雞胚NCC發(fā)育的影響。

1 材料與方法

1.1 試劑

受精雞蛋(遼寧醫(yī)學院動物實驗中心)；嗎啉代 (Gene Tools)；抗體:anti-DIG, anti-rabbit alexa fluor 448(invtrogen)。

1.2 雞胚的制備

受精雞蛋于37 ℃孵育箱中孵育到4(18 h)、6(24 h)和8(28 h)期[7]。用無菌注射器抽取部分蛋清后敲開蛋殼，濾紙覆蓋雞胚，再沿濾紙邊剪下雞胚，于4%多聚甲醛中固定2 h。

1.3 原位雜交

雞胚先經(jīng)4%多聚甲醛固定，然后經(jīng)過梯度濃度的甲醛處理，再用100%甲醇洗兩次。65 ℃預雜交1 h，80 ℃預熱探針5 min,再65 ℃孵育過夜，用含有2% 勃林格試劑(Boehringer)和滅活的胎牛血清的三羥甲基氨基甲烷-鹽酸緩沖液室溫封閉2 h，anti-DIG經(jīng)1∶1 000稀釋，4 ℃搖床過夜，堿性磷酸酶顯色[8]。

1.4 免疫熒光及成像

免疫熒光實驗按照標準步驟[9]。簡要概括，將胚胎固定在4 %多聚甲醛中，用磷酸鹽緩沖液 (含有2 g/L牛血清白蛋白,0.1% Tween)潤洗(3×60 min)。一抗孵育，4 ℃過夜。特異性熒光二抗經(jīng)1∶2 000稀釋，顯色。用Nikon體視顯微鏡(Spot SE鏡頭和Nikon Eclipse 80i顯微鏡的軟件)成像。

1.5 電穿孔法敲除Ubc9

將嗎啉代(Mo)置于3%蔗糖溶液使其濃度為0.48 μmol/L，添加1 μg DNA (pCIG)作為載體和提供綠熒光，單側(cè)電穿孔法[10]轉(zhuǎn)染入固定在濾紙環(huán)中的4期雞胚。雞胚孵育[11]過夜，隨后用于原位雜交和免疫熒光分析。

1.6 統(tǒng)計學分析

所得實驗數(shù)據(jù)采用四格表的Fisher確切概率法分析，統(tǒng)計學處理由SPSS 17.0統(tǒng)計軟件完成。

2 結(jié)果

2.1 Ubc9在不同發(fā)育時期雞胚的表達

4～6期雞胚中Ubc9 mRNA廣泛而分散地表達(圖1A，B)，有逐漸向神經(jīng)褶匯聚的趨勢，在8期可見Ubc9在神經(jīng)褶表達明顯增強(圖1C)，并且8期3個切片處的原位雜交檢測也顯示Ubc9在此處表達增強(圖1D)。

A～B.Ubc9 expression in chick embryos at st 4(18 hours)and 6(24 hours)；C.Ubc9 expression in chick embryos at st 8(28 hours)；D.sections at the indicated level of the st 8 embryo

圖1原位雜交檢測Ubc9在早期(4,6,8期)雞胚中的表達
Fig1Ubc9expression(insituhybridization)inchickembryosatearlystages(4,6,8st)

2.2 Ubc9與 Pax7在雞胚神經(jīng)褶上的共定位情況

NCC早期標志基因Pax7(圖2A) 與Ubc9(圖2B)共表達于神經(jīng)褶(圖2C，D)，即神經(jīng)脊細胞前體。

2.3敲除8期雞胚的Ubc9后，神經(jīng)嵴細胞無法形成

對照組(圖3A,B,C)經(jīng)5堿基錯配的對照-Mo(非特異性)處理后， 9個雞胚出現(xiàn)了1個Snail2(A)的異常表達(1/9),Sox9(B,1/10),FoxD3(C,1/8)，近似無影響。而實驗組(圖3 D,E,F)經(jīng)Ubc9-Mo處理后，與對照組相比，Snail2(D,7/9),Sox9(E,5/6)和FoxD3(F,7/10)的mRNA表達明顯減少(P<0.05,P<0.01和P<0.05) (表 1)。

3 討論

本研究首先用原位雜交法檢測了Ubc9的mRNA的表達情況，發(fā)現(xiàn)Ubc9在8期雞胚的神經(jīng)褶即NCC的前體表達，同時，免疫熒光實驗發(fā)現(xiàn)NCC早期標志基因Pax7[2]與Ubc9共表達于8期雞胚神經(jīng)褶，即神經(jīng)嵴前體細胞所在位置。這些結(jié)果提示，Ubc9可能在NCC形成過程中起作用。

A.Pax7 expression at NC precursors of the neural fold；B.Ubc9 expression at NC precursors of the neural fold；C.Ubc9 are coexpressed with Pax7 in NC precursors of the neural fold of st 8 embryos；D.Framed regions of merged images at higher magnification;Ubc9(green) and Pax7(red) in C,D

圖2免疫熒光顯示Ubc9和Pax7共定位于雞胚8期的神經(jīng)嵴細胞前體(神經(jīng)褶)

Fig2Ubc9proteinsarecoexpressed(Immunofluorescence)withPax7inNCprecursorsoftheneuralfoldofst8embryos

A～C.unilateral electroporation of 5 mismatch control morpholinos have no effect on the expression of NC markersSnail2,Sox9 andFoxD3； D～F.Ubc9-morpholino downregulates the expression ofSnail2,Sox9 andFoxD3; right panels show higher-magnification of framed regions, arrows point to untreated internal control signal and brackets demarcate treated areas; insets show the localization of morpholinos (green) beforeinsituhybridization

圖3 嗎啉代法敲除Ubc9導致了Snail2(D), Sox9(E),和FoxD3(F)表達的下降Fig 3 Unilateral Ubc9-morpholino downregulates the expression of Snail2(D), Sox9(E), and FoxD3(F)

*P<0.05,**P<0.01 compared with control group.

Ubc9是SUMO化唯一的E2結(jié)合酶。SUMO化反應包括3個步驟：活化，結(jié)合和連接，涉及多個酶的級聯(lián)反應：E1活化酶、E2結(jié)合酶以及E3連接酶，其中Ubc9，參與SUMO化中結(jié)合和連接兩步[12]，阻斷了Ubc9的表達，即阻斷了SUMO通路[4]。

敲除小鼠SUMO1會導致唇裂等缺陷病[13]的發(fā)生，說明NCC形成或遷移需要SUMO通路的參與，并且，一些NCC相關轉(zhuǎn)錄因子如Sox9等可被SUMO化修飾[14- 15]。但是NCC形成和遷移是否需要Ubc9或需要SUMO通路的參與，目前沒有直接證據(jù)。本研究通過嗎啉代敲除法降低了雞胚Ubc9的mRNA表達水平，發(fā)現(xiàn)NCC晚期標志基因Snail2,Sox9和FoxD3的表達明顯下降或不表達，說明NCC形成需要Ubc9 和SUMO通路的參與。

綜上所述，Ubc9表達于早期雞胚神經(jīng)嵴細胞前體，NCC形成需要SUMO通路的參與。本研究為神經(jīng)嵴細胞的進一步探索提供了新的思路，而Ubc9和SUMO通路如何影響神經(jīng)嵴細胞形成及機制仍有待研究。

[1] Achilleos A, Trainor PA. Neural crest stem cells: discovery, properties and potential for therapy[J]. Cell Res, 2012,22:288- 304.

[2] Basch ML, Bronner-Fraser M, García-Castro MI. Specification of the neural crest occurs during gastrulation and requires Pax7[J]. Nature, 2006,441: 218- 222.

[3] Stuhlmiller TJ, García- Castro MI. Current perspectives of the signaling pathways directingneural crest induction[J]. Cell Mol Life Sci, 2012,69:3715- 3737.

[4] Wadosky KM, Willis MS. The story so far: post-translational regulation of peroxisome proliferator-activated receptors by ubiquitination and SUMOylation[J]. Am J Physiol Heart Circ Physiol, 2012,302:H515- 526.

[5] 陳泉,施蘊渝.小泛素相關修飾物SUMO研究進展[J].生命科學, 2004,16:1- 6.

[6] Jentsch S, Psakhye I.Control of nuclear activities by substrate-selective and protein-group SUMOylation[J].Annu Rev Genet,2013,47:167- 186.

[7] Hamburger V, Hamilton HL.A series of normal stages in the development of the chick embryo[J]. J Morphol, 1951, 88:49- 92.

[8] Henrique D,Adam J, Myat A,etal.Expression of a Delta homologue inprospective neurons in the chick[J]. Nature, 1995,375:787- 790.

[9] Betters E, Liu Y, Kjaeldgaard A,etal.Analysis of early human neural crest development[J]. Dev Biol,2010,344:578- 592.

[10] Muramatsu T, Mizutani Y, Ohmori Y,etal.Comparison of three nonviral transfection methods for foreign gene expression in early chicken embryos in ovo[J]. Biochem Biophys Res Commun, 1997,230:376- 380.

[11]Chapman SC, Collignon J, Schoenwolf GC,etal.Improved method for chick whole-embryo culture using a filter paper carrier[J]. Dev Dyn, 2001,220:284- 289.

[12] 韋瑋，張浩.蛋白質(zhì)SUMO化修飾研究進展[J].中國生物工程雜志,2008，28:122- 126.

[13] Alkuraya FS, Saadi I, Lund JJ,etal. SUMO1 haploin sufficiency leads to cleft lip and palate[J]. Science, 2006, 313: 1751.

[14] Song T,Li G,Jing G,etal. SUMO1 polymorphisms are associated with non-syndromic cleft lip with or without cleft palate[J].Biochem Biophys Res Commun, 2008,377:1265- 1268.

[15] Taylor KM, Labonne C.SoxE factors function equivalently during neural crest and inner ear development and their activity is regulated by SUMOylation[J]. DevCell, 2005,9:593- 603.

The effect of Ubc9 on neural crest cell development of chicken embryo

LI Ming-yang1, LIU Yi-meng3, LIU Chao1, Li Shu-ling2, LUAN Zhi-dong1*

(1.Dept. of Developmental Biology; 2.Dept. of Histology and Embryology; 3.Dept. of Biochemical andMolecular Biology，Liaoning Medical College.Jinzhou 121001，China)

ObjectiveTo examine the effect of Ubc9 and SUMO pathway on neural crest cell development of chicken embryo.Methods1)Chicken embryos were collected,Insituhybridization and Immunofluorescence were used to observe the expression of SUMO-conjugating enzyme-Ubc9 and early markers of neural crest. 2)In the control group, 5-mismatch Ubc9-control morpholino (5MM-Mo) was used to treat chick embryos; In the Ubc9-Mo group, sequence-specific Morpholinos(Mo) was used to reduce Ubc9 level in chicken embryos, then the mRNA of neural crest markers(Snail2，Sox9andFoxD3) was examined.Results1)Ubc9 was coexpressed with Pax7 in neural crest precursors of the neural fold at 4,6 and 8 stage of early chicken embryos. 2)Compared to the control group,Ubc9-Mo treatment led to a reduce of mRNA level of neural crest markers,Snail2(P<0.05)，Sox9 (P<0.01)andFoxD3(P<0.05).ConclusionsSUMO-conjugating enzyme, Ubc9 expression start at neural fold at 4,6 and 8 stage of early chicken embryo, Ubc9 and SUMO pathway are required for neural crest cell development of chicken embryo.

chicken embryo；neural crest cell; Ubc9；SUMO pathway

2014- 04- 10

2014- 06- 20

*通信作者(correspondingauthor)：zhidongluan@lnmu.edu.cn

1001-6325(2014)08-1079-04

R 392.11

A