转录组测序分析BMP4调控鸡原始生殖细胞形成的机制

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (5): 1644-1657.DOI: 10.1016/j.jia.2023.09.003

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转录组测序分析BMP4调控鸡原始生殖细胞形成的机制

收稿日期:2022-07-28 接受日期:2023-06-26 出版日期:2024-05-20 发布日期:2024-04-23

Transcriptome-based analysis of key signaling pathways affecting the formation of primordial germ cell in chickens

Ying Ding^{1, 2}, Qiong Zhi^{1, 2}, Qisheng Zuo^{1, 2}, Kai Jin^{1, 2}, Wei Han³, Bichun Li^{1, 2#}

¹ Key Laboratory of Animal Genetics, Breeding and Molecular Design of Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
² Joint International Research Laboratory of Agriculture and Agri-Product Safety of the Ministry of Education of China, Yangzhou University, Yangzhou 225009, China
³Poultry Research Institute, Chinese Academy of Agricultural Sciences/Jiangsu Institute of Poultry Science, Yangzhou 225009, China

Received:2022-07-28 Accepted:2023-06-26 Online:2024-05-20 Published:2024-04-23
About author:Ying Ding, E-mail: 2449317617@qq.com; #Correspondence Bichun Li, Tel: +86-514-87977207, Fax: +86-514-87350440, E-mail: yubcli@yzu.edu.cn
Supported by:
This work was supported by the National Key R&D Program of China (2021YFD1200301), the National Natural Science Foundation of China (32172718), the Yangzhou University Graduate International Academic Exchange Special Fund Project, China (YZUF2022206) and the “JBGS” Project of Seed Industry Revitalization in Jiangsu Province, China (JBGS(2021)029).

摘要/Abstract

摘要：

骨形成蛋白4 (Bone morphogenetic protein 4, BMP4) 是调控鸡原始生殖细胞(Primordial germ cells, PGCs)发育的关键因子，并且可在体外诱导鸡原始生殖细胞(Induced primordial germ cells, iPGCs)形成。然而，其诱导效率较低，在禽类中BMP4的调控机制尚不清楚。本研究以胚胎干细胞(Embryonic stem cells, ESCs)为对照组，对鸡胚体内的PGCs及体外BMP4诱导形成的iPGCs进行转录组测序分析，旨在明确调控PGCs发育的关键因素，解析PGCs的形成机制，为进一步优化PGCs诱导体系奠定理论基础。因此，本研究收集了第0天受精种蛋中的ESCs，孵化至第4.5天鸡胚性腺中的PGCs，以及体外添加BMP4诱导至第6天的iPGCs，利用RNA-seq分析三种细胞的转录图谱。结果表明，共有6142个基因在PGCs和iPGCs之间差异表达，其中2728个基因在iPGCs中表达上调，3414个基因在iPGCs中表达下调。与ESCs相比，BMP4在PGCs和iPGCs中均显著上调。KEGG结果表明，TGF-β和Wnt信号通路在鸡胚体内和体外PGCs形成过程中均被激活，且在iPGC中效果更显著。TGF-β信号通路的抑制因子Nodal在PGCs和iPGCs中表达均显著降低，甚至在iPGCs中不表达，这与以上结果一致。此外，在PGCs和iPGCs中，PI3K-AKT和溶酶体等自噬相关的信号通路显著富集。透射电镜结果表明，添加BMP4后，自噬溶酶体的数量显著增加，这表明自噬被显著激活，这与RNA-seq结果一致。此外，进一步的石蜡切片、糖原染色、流式分析、间接免疫荧光和qRT-PCR等结果均表明分别在鸡胚体内和体外干扰自噬基因ATG14后，PGCs的标记基因Cvh和C-kit显著下调，鸡胚性腺中的PGCs显著减少，iPGCs的诱导效率显著降低。综上所述，本研究筛选出调控PGCs形成的关键信号通路，旨在丰富调控鸡PGCs形成的网络机制，并为进一步提高体外诱导效率奠定理论基础。

Abstract:

Bone morphogenetic protein 4 (BMP4) can induce the formation of chicken primordial germ cells (PGCs) in vitro; however, its regulatory mechanism in poultry remains unknown. This study aimed to use RNA-seq to analyze PGCs in chicken embryos and iPGCs induced by BMP4 in vitro, clarify the internal regulatory factors of PGCs, analyze the mechanism of the formation of PGCs, and lay a theoretical foundation for the further optimization of PGCs induction systems. Embryonic stem cells (ESCs), PGCs and iPGCs induced by BMP4 in vitro were collected. The transcriptional maps of the three cell types were studied using RNA-seq. The results showed 6,142 genes differentially expressed between PGCs and iPGCs, of which 2,728 were upregulated in iPGCs and 3,414 were downregulated in iPGCs. Compared to that in ESCs, BMP4 was significantly upregulated in PGCs and iPGCs. KEGG results showed that both the TGF-β and Wnt signaling pathways were activated during the formation of PGCs in vitro and in vivo, and the activation was more significant during iPGCs induced by BMP4. The expression of Nodal, an inhibitory factor of TGF-β signaling, was significantly decreased in PGCs and iPGCs, but was not expressed in iPGCs, which further supports our conclusion. Additionally, the Lysosome and PI3K-AKT signaling pathways were significantly enriched in PGCs and iPGCs, respectively. Further, transmission electron microscopy (TEM) results showed that the number of autolysosomes was significantly higher after the addition of BMP4, which is consistent with the KEGG results. Furthermore, the number of PGCs was significantly reduced after ATG14 was interfered in vivo and in vitro. In conclusion, this study screened out the key signaling pathways during the formation of PGCs, aiming to provide help for enriching the mechanism network regulating PGCs formation in chicken and laying a theoretical foundation for further improving the efficiency of inducing PGCs in vitro.

Key words: RNA-seq , BMP4 , PGCs , ATG14 , chicken

. 转录组测序分析BMP4调控鸡原始生殖细胞形成的机制[J]. Journal of Integrative Agriculture, 2024, 23(5): 1644-1657.

Ying Ding, Qiong Zhi, Qisheng Zuo, Kai Jin, Wei Han, Bichun Li.

Transcriptome-based analysis of key signaling pathways affecting the formation of primordial germ cell in chickens [J]. Journal of Integrative Agriculture, 2024, 23(5): 1644-1657.

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