基于单细胞转录物组测序和加权基因共表达网络分析鉴定食蟹猴原始生殖细胞分化过程中的基因功能模块和分子网络

Identification of Functional Gene Modules and Molecular Networks During Differentiation of Primordial Germ Cells by Single Cell RNA-seq and Weighted Gene Co-expression Network Analysis

在哺乳动物中,作为能发育成精子或卵子的始祖细胞,原始生殖细胞(primordial germ cells,PGCs)在生命繁衍、遗传信息传递中发挥着关键作用。然而,目前人们对于灵长类PGCs分化过程中的基因功能模块和分子网络知之甚少。在本研究中,首先诱导食蟹猴胚胎干细胞向PGCs分化,在该过程的不同阶段(第0 d、第2 d与第4 d)分别进行单细胞转录物组测序(scRNA-seq),并基于加权基因共表达网络分析(WGCNA),鉴定其中的功能模块。结果共获得食蟹猴PGCs分化day 0、day 2、day 4三个时间点各91、55、66个单细胞样本。基于上述共计212个scRNA-seq数据,鉴定获得17个不同的基因功能模块。其中,与PGCs分化day 0显著正相关的模块为MEsalmon,对应的相关系数和显著性水平分别为0.89、2E-72。与day 2和day 4正相关性最高的模块均为MEblue,且随着分化时间的进行其相关系数从0.24(P-value=5E-04)增大到0.66(P-value=4E-28),并显著富集于BMP、Wnt信号通路以及上皮细胞分化、性腺发育等过程,推测该模块参与食蟹猴PGCs分化过程的起始和驱动。同时,对各个时间点的差异基因也进行了分析。最后,结合STRING数据库、Cytoscape和MCODE等工具,构建了食蟹猴PGCs分化过程中的PPI分子网络,鉴定到8个核心子网络及BMP4、WNT3、TFAP2C、SOX17等关键调控因子,并进行了人、食蟹猴和小鼠之间的比较分析。上述结果为深入理解非人灵长类胚胎干细胞早期发育规律,以及PGCs分化过程中的基因表达调控特征,提供了新的研究线索和理论参考。

In mammals,by functioning as the progenitor of matured gametes(sperms or oocytes),primordial germ cells(PGCs)are of remarkable importance in life reproduction and handing down genetic information from generation to generation.However,in the case of primates,very little is known on their gene modules and molecular networks underlying the specification of PGCs.In this study,firstly we carried out the single-cell RNA-seq(scRNA-seq)in PGC differentiation(at three time points of day 0,day 2,day 4)of Macaca fascicularis,and further identified the gene modules related to PGC development based on weighted gene co-expression network analysis(WGCNA).In total,we obtained 91,55 and 66 single cells at day 0,day 2 and day 4,respectively.Based on 212 scRNA-seq data at three different time points,we identified 17 different gene modules.Among which,the MEsalmon module was highly positively associated with undifferentiated day 0 with a Pearson correlation coefficient(PCC)of 0.89 and a P-value of 2 E-72.While the MEblue module was positively associated with both day 2 and day 4,and interestingly,its PCC remarkably increased from 0.24(P-value=5 E-04)to 0.66(P-value=4 E-28)with the PGC differentiation from day 2 to day 4.Enriched terms for the MEblue module were observed to be BMP and Wnt signaling pathways,and the biological processes of epithelial cell differentiation,male gonad development,etc.Thus,it is very likely that the co-expressed genes in this module are involved in driving the initiation of PGC differentiation in Macaca fascicularis.Furthermore,we identified the differentially expressed genes at each time points and performed GO/KEGG analysis.Finally,combined with the STRING database,we constructed the protein-protein interaction(PPI)networks for the development of PGCs in Macaca fascicularis using Cytoscape and MCODE.In total,we obtained 8 densely-connected core subnetworks closely related to PGC differentiation,identified the hub regulators such as BMP4,WNT3,TFAP2 C and SOX17 in the PPI networks,and performed comparative analysis among human,Macaca fascicularis and mouse.In summary,our findings provide the basis for deep understanding of the cell biology and gene regulation underlying the fate of PGCs at the single cell level and shed light on embryonic developmental characteristics at the level of systems biology in a highly evolved primate.