双同源盒诱导小鼠胚胎干细胞向胚外内胚层分化的机制研究

doi:10.3969/j.issn.1674-8115.2024.11.003

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (11): 1359-1369.doi: 10.3969/j.issn.1674-8115.2024.11.003

• 论著 · 基础研究 • 上一篇

双同源盒诱导小鼠胚胎干细胞向胚外内胚层分化的机制研究

洪磊¹(), 郭传亮¹(), 蔡勤¹, 李婉睿¹, 曾溢滔¹, 薛燕¹^,²(), 曾凡一¹^,²()

^1.上海市儿童医院，上海交通大学医学附属儿童医院上海医学遗传研究所，国家卫健委医学胚胎分子生物学重点实验室，上海市胚胎与生殖工程重点实验室，上海 200040
^2.上海交通大学基础医学院组织胚胎学与遗传发育学系，上海 200025

收稿日期:2024-04-18 接受日期:2024-05-13 出版日期:2024-11-28 发布日期:2024-11-28
通讯作者: 薛燕,曾凡一 E-mail:honglei_shnu@163.com;chuanliangguo@163.com;xueyan@shsmu.edu.cn;fzeng@vip.163.com
作者简介:洪　磊（1996—），男，硕士生；电子信箱：honglei_shnu@163.com。
郭传亮（1986—），男，博士，助理研究员；电子信箱：chuanliangguo@163.com。第一联系人：洪　磊、郭传亮为共同第一作者。
基金资助:
国家重点研发计划(2019YFA0801402);国家自然科学基金(82271890);上海市临床重点专科项目(shslczdzk05705);上海市重点学科项目(2017ZZ02019);上海市地方高水平大学创新研究团队(SHSMU-ZDCX20212200);澳门科学技术发展基金（FDCT）(0092/2022/A2)

Mechanism of DUX-induced differentiation of mESC into extraembryonic endoderm

HONG Lei¹(), GUO Chuanliang¹(), CAI Qin¹, LI Wanrui¹, ZENG Yitao¹, XUE Yan¹^,²(), ZENG Fanyi¹^,²()

^1.Shanghai Institute of Medical Genetics, Shanghai Children′s Hospital, Shanghai Jiao Tong University School of Medicine, NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology & Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
^2.Department of Histoembryology, Genetics & Development, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China

Received:2024-04-18 Accepted:2024-05-13 Online:2024-11-28 Published:2024-11-28
Contact: XUE Yan,ZENG Fanyi E-mail:honglei_shnu@163.com;chuanliangguo@163.com;xueyan@shsmu.edu.cn;fzeng@vip.163.com
Supported by:
National Key Research and Development Program of China(2019YFA0801402);National Natural Science Foundation of China(82271890);Shanghai Key Clinical Specialty Project(shslczdzk05705);Shanghai Top Priority Key Discipline Project(2017ZZ02019);Innovative Research Team of High-Level Local Universities in Shanghai(SHSMU-ZDCX20212200);Macau Science and Technology Development Fund (FDCT)(0092/2022/A2)

摘要/Abstract

摘要：

目的·探索双同源盒（double homeobox，DUX）蛋白对小鼠胚胎干细胞（mouse embryonic stem cell，mESC）向胚外内胚层（extraembryonic endoderm，XEN）分化潜能的影响及可能的作用机制。方法·使用慢病毒体系在mESC中构建过表达DUX细胞系，利用流式细胞术检测DUX过表达前后2细胞样细胞（2-cell-like cell，2CLC）的比例，并使用实时定量聚合酶链反应（real-time quantitative reverse transcription polymerase chain reaction，RT-qPCR）检测2细胞期特异性基因，如内源性Dux、锌指和SCAN结构域的蛋白质4c（zinc finger and SCAN domain containing 4c，Zscan4c）、锌指蛋白352（zinc finger protein 352，Zfp352）和鼠内源性反转录病毒-聚合酶（murine endogenous retrovirus-L polymerase，MERVL-pol）的表达。RT-qPCR检测过表达DUX的mESC多能性因子［nanog homeobox（Nanog）、kruppel-like transcription factor 4（Klf4）、性别决定区Y框蛋白2（sex determining region Y-box 2，Sox2）、八聚体结合转录因子4（octamer-binding transcription factor 4，Oct4）］和自然分化状态下各胚层标志性基因［内胚层（endodermal）：GATA结合蛋白4（GATA binding protein 4，Gata4）、Gata6、Sox17；外胚层（ectodermal）：微Ⅲ型β微管蛋白3（tubulin beta 3 class Ⅲ，Tubb3）、巢蛋白（Nestin）；中胚层（mesodermal）：心脏和神经嵴衍生物表达转录本1（heart and neural crest derivatives expressed 1，Hand1）、肌源性分化蛋白1（myogenic differentiation 1，Myod1）、激酶插入结构域受体（kinase insert domain protein receptor，Flk1）］的表达。挖掘公共转录组测序（RNA sequencing，RNA-seq）数据，通过分析胚外内胚层标志基因的表达水平，明确DUX对mESC向胚外内胚层分化的影响；通过对差异基因的功能及通路进行基因本体论（Gene Ontology，GO）富集分析、京都基因和基因组数据库（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析和基因集富集分析（gene set enrichment analysis，GSEA），找出DUX作用的信号通路；深入分析已有的染色质免疫共沉淀技术结合二代测序（chromatin immunoprecipitation sequencing，ChIP-seq）数据，探究DUX的潜在靶基因。结果·2CLC比例升高和2细胞期标志基因表达上调，证明已成功构建过表达DUX细胞系。分子生物学实验显示过表达DUX后可有效维持mESC的多能性，与公共RNA-seq数据分析结果一致；差异基因分析发现，内胚层基因出现特异性上调；诱导mESC自然分化后，RT-qPCR检测实验表明XEN标志基因（Gata4、Gata6、Sox17）的mRNA表达出现显著上调（P<0.001），而中胚层、外胚层基因没有特异性变化。GSEA结果提示DUX可能激活了视黄醇代谢信号通路，ChIP-seq数据解析进一步揭示在DUX结合的peaks中存在已知的视黄酸受体motif，可激活下游与XEN发育相关的靶基因。结论·DUX与视黄酸信号通路密切关联，预示其激活了视黄酸信号通路，促进mESC倾向XEN分化。

关键词: 小鼠胚胎干细胞, 胚外内胚层细胞, 双同源盒, 视黄酸信号通路

Abstract:

Objective ·To explore the effect of double homeobox (DUX) protein on the differentiation potential of mouse embryonic stem cells (mESCs) into extraembryonic endoderm (XEN) and the possible mechanism of its action. Methods ·Overexpression of DUX cell lines in mESCs was achieved by using a lentiviral system. The proportion of 2-cell-like cells (2CLCs) before and after DUX overexpression was detected by flow cytometry, and the expression of 2-cell stage-specific genes, Dux, zinc finger and SCAN domain containing 4c (Zscan4c), zinc finger protein 352 (Zfp352) and murine endogenous retrovirus-L polymerase (MERVL-pol), were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). RT-qPCR assay was used to detect the expression of pluripotency factors, nanog homeobox (Nanog), kruppel-like transcription factor 4 (Klf4), sex determining region Y-box 2 (Sox2), and octamer-binding transcription factor 4 (Oct4), in pluripotent state, as well as the expression of signature genes for different germ layers in the differentiated state [endodermal: GATA binding protein 4 (Gata4), GATA binding protein 6 (Gata6), and sex determining region Y-box 17 (Sox17); ectodermal: Nestin and tubulin beta 3 class Ⅲ (Tubb3); mesodermal: heart and neural crest derivatives expressed 1 (Hand1), myogenic differentiation 1 (Myod1), and kinase insert domain protein receptor (Flk1)]. Public RNA sequencing (RNA-seq) data were mined to further clarify the effect of DUX on the differentiation of mESCs into extraembryonic endoderm. Functional and pathway enrichment analyses of differentially expressed genes were performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) to identify the signaling pathways regulated by DUX. Additionally, an in-depth analysis of existing chromatin immunoprecipitation sequencing (ChIP-seq) data was conducted to explore the potential target genes of DUX. Results ·Molecular biology experiments showed that overexpression of DUX could effectively maintain the pluripotency of mESCs, which was consistent with the analysis of public RNA-seq data. Differential gene analysis revealed that endodermal genes were specifically upregulated. After differentiation assay of mESCs, RT-qPCR assay experiments showed that mRNA expression of the XEN marker genes (Gata4, Gata6, Sox17) was significantly upregulated (P<0.001). In contrast, there was no specific change in mesodermal and ectodermal genes. GSEA enrichment analysis indicated that DUX might activate the retinoid metabolism signaling pathway, and the analysis of the ChIP-seq data further revealed the presence of a large number of known retinoic acid receptor motif in DUX-bound peaks, which could activate downstream target genes related to the development of the XEN. Conclusion ·DUX has a strong correlation with the retinoic acid signaling pathway and it is predicted to activate the retinoic acid signaling pathway, which could promote the tendency of mESCs toward XEN differentiation.

Key words: mouse embryonic stem cell (mESC), extraembryonic endoderm (XEN), double homeobox (DUX), retinoic acid signaling pathway

中图分类号:

R394.1

洪磊, 郭传亮, 蔡勤, 李婉睿, 曾溢滔, 薛燕, 曾凡一. 双同源盒诱导小鼠胚胎干细胞向胚外内胚层分化的机制研究[J]. 上海交通大学学报（医学版）, 2024, 44(11): 1359-1369.

HONG Lei, GUO Chuanliang, CAI Qin, LI Wanrui, ZENG Yitao, XUE Yan, ZENG Fanyi. Mechanism of DUX-induced differentiation of mESC into extraembryonic endoderm[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(11): 1359-1369.

图/表 7

表1 用于RT-qPCR的引物序列

Tab 1 Primer sequences used for RT-qPCR

Gene	Forward primer (5′→3′)	Reverse primer (5′→3′)
Gapdh	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
Dux	ACTTCTAGCCCCAGCGACTC	CCATGCTGCCAGGATTTCTA
Zscan4c	GTCCTGACAGAGGCCTGCC	GAGATGTCTGAAGAGGCAAT
Zfp352	AAGTCCCACATCTGAAGAAACAC	GGGTATGAGGATTCACCCACA
MERVL-pol	ATCTCCTGGCACCTGGTATG	AGAAGAAGGCATTTGCCAGA
Nanog	TCTTCCTGGTCCCCACAGTTT	GCAAGAATAGTTCTCGGGATGAA
Sox2	GCGGAGTGGAAACTTTTGTCC	CGGGAAGCGTGTACTTATCCTT
Klf4	GTGCCCCGACTAACCGTTG	GTCGTTGAACTCCTCGGTCT
Oct4	GGCTTCAGACTTCGCCTCC	AACCTGAGGTCCACAGTATGC
Gata4	CCCTACCCAGCCTACATGG	ACATATCGAGATTGGGGTGTCT
Gata6	GAGCTGGTGCTACCAAGAGG	TGCAAAAGCCCATCTCTTCT
Sox17	GAATTTCCATCTCCACCTCCG	CGATTGGCACCTTTCACCC
Nestin	CTGCAGGCCACTGAAAAGTT	GACCCTGCTTCTCCTGCTC
Tubb3	CGGCAACTATGTAGGGGACT	CCAGCACCACTCTGACCAA
Hand1	AAGGATGCACAAGCAGGTGAC	TTTAATCCTCTTCTCGCCGGG
Myod1	CCACTCCGGGACATAGACTTG	AAAAGCGCAGGTCTGGTGAG
Flk1	CCTGGTCAAACAGCTCATCA	AAGCGTCTGCCTCAATCACT

表2 用于RNA-seq数据分析的样本信息

Tab 2 Sample information for RNA-seq data analysis

Group	Sample	Description
DUX	Unsorted-NoDox-rep1/2	Untreated with tetracycline and unsorted 2CLCs
DUX	Unsorted-plusDox-rep1/2	Treated with tetracycline and unsorted 2CLCs
RA	2iL-ESCs-rep1/2/3	Control
RA	RA-ESCs-rep1/2/3	Treated with RA

表3 用于ChIP-seq数据分析的样本信息

Tab 3 Sample information for ChIP-seq data analysis

Sample	Description
Unsorted-plusDox-Input	Input. Treated with tetracycline and unsorted 2CLCs
Unsorted-plusDox-HAChIP-12hr-rep1/2	Addition of tetracycline-treated 12hr, unsorted 2CLCs
Unsorted-plusDox-HAChIP-18hr-rep1/2	Addition of tetracycline-treated 18hr, unsorted 2CLCs

图1 2CLC的鉴定Note： A. Microscopy pictures and flow cytometry showing the percentage of 2CLCs. B. RT-qPCR validation of selected 2C marker genes and MERVL-polymerase gene (MERVL-pol) in noDox (control) and plusDox (DUX overexpression) ES cells. ①P<0.001, compared with the control group.

Fig 1 Identification of 2CLCs

图2 DUX诱导的2CLC的发育潜能的检测Note： A. RT-qPCR validation of selected pluripotent marker genes in noDox (control) and plusDox (DUX overexpression) ES cells. B/C. Heatmap representation of expression levels of genes involved in 2CLCs (B) or endoderm (C) specification in noDox (control) and plusDox (DUX overexpression) cells. D. RT-qPCR was employed to assess the RNA expression levels of marker factors for endoderm, mesoderm and ectoderm on the fourth day of embryoid body differentiation in noDox (control) and plusDox (DUX overexpression) cells. ①P>0.05, ②P<0.001, ③P=0.001 3, compared with the control group.

Fig 2 Detection of developmental potential of DUX-induced 2CLCs

图3 DUX过表达ESC的功能富集分析Note： A. GO analysis of upregulated genes in plusDox ES cells. B. GSEA analysis showing collective changes in the retinol metabolism signaling pathway gene set. C. Expression levels (log2FC) (FC, fold change) of selected RA-pathway-related genes in plusDox and noDox cells (ESCs) based on RNA-seq data. D. Comparison of enrichment results from multiple gene lists.

Fig 3 Functional enrichment analysis of DUX overexpressed ESCs

图4 DUX调控ESC分化潜能的可能机制Note： A. Intersection of DUX-upregulated genes with RARG and DUX co-localized genes and possible mechanism of regulating mESCs. B. IGV view of RNA-seq and ChIP-seq results. C. KEGG analysis of the intersecting genes.

Fig 4 Potential mechanism of DUX regulation on ESCs differentiation

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双同源盒诱导小鼠胚胎干细胞向胚外内胚层分化的机制研究

Mechanism of DUX-induced differentiation of mESC into extraembryonic endoderm

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