Dataset Information

Pluripotency-State-Dependent Role of Dax1 in Embryonic Stem Cells Self-Renewal.

ABSTRACT: Dax1(also known as Nr0b1) is regarded as an important component of the transcription factor network in mouse embryonic stem cells (ESCs). However, the role and the molecular mechanism of Dax1 in the maintenance of different pluripotency states are poorly understood. Here, we constructed a stable Dax1 knockout (KO) cell line using the CRISPR/Cas9 system to analyze the precise function of Dax1. We reported that 2i/LIF-ESCs had significantly lower Dax1 expression than LIF/serum-ESCs. Dax1KO ES cell lines could be established in 2i/LIF and their pluripotency was confirmed. In contrast, Dax1-null ESCs could not be continuously passaged in LIF/serum due to severe differentiation and apoptosis. In LIF/serum, the activities of the Core module and Myc module were significantly reduced, while the PRC2 module was activated after Dax1KO. The expression of most proapoptotic genes and lineage-commitment genes were drastically increased, while the downregulated expression of antiapoptotic genes and many pluripotency genes was observed. Our research on the pluripotent state-dependent role of Dax1 provides clues to understand the molecular regulation mechanism at different stages of early embryonic development.

SUBMITTER: He J

PROVIDER: S-EPMC8286181 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Pluripotency-State-Dependent Role of Dax1 in Embryonic Stem Cells Self-Renewal.

He Jianrong J Cheng Yuda Y Ruan Yan Y Wang Jiali J Tian Yanping Y Wang Jiaqi J Wang Fengsheng F Zhang Chen C Xu Yixiao Y Liu Lianlian L Yu Meng M Wang Jiangjun J Zhao Binyu B Zhang Yue Y Yang Yi Y Liu Gaoke G Wu Wei W He Ping P Xiong Jiaxiang J Huang He H Zhang Junlei J Jian Rui R

Stem cells international 20210710

Dax1(also known as Nr0b1) is regarded as an important component of the transcription factor network in mouse embryonic stem cells (ESCs). However, the role and the molecular mechanism of Dax1 in the maintenance of different pluripotency states are poorly understood. Here, we constructed a stable Dax1 knockout (KO) cell line using the CRISPR/Cas9 system to analyze the precise function of Dax1. We reported that 2i/LIF-ESCs had significantly lower Dax1 expression than LIF/serum-ESCs. Dax1KO ES cell ...[more]

PMID: 34335791

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Project description:BackgroundSelf-renewal and pluripotency are considered as unwavering features of embryonic stem cells (ESCs). How ESCs regulate the self-renewal and differentiation is a central question in development and regenerative medicine research. Epidermal growth factor receptor (EGFR) was identified as a critical regulator in embryonic development, but its role in the maintenance of ESCs is poorly understood.MethodsHere, EGFR was disrupted by its specific inhibitor AG1478 in mouse ESCs (mESCs), and its self-renewal and pluripotency were characterized according to their proliferation, expression of pluripotency markers, embryoid body (EB) formation, and mRNA expression patterns. We also used another EGFR inhibitor (gefitinib) and RNA interference assay to rule out the possibility of non-specific effects of AG1478.ResultsEGFR inhibition by AG1478 treatment in mESCs markedly reduced cell proliferation, caused cell cycle arrest at G0/G1 phase, and altered protein expressions of the cell cycle regulatory genes (CDK2 (decreased 11.3%) and proliferating cell nuclear antigen (decreased 25.2%)). The immunoreactivities and protein expression of pluripotency factors (OCT4 (decreased 26.9%)) also dramatically decreased, while the differentiation related genes (GATA4 (increased 1.6-fold)) were up-regulated in mESCs after EGFR inhibition. Meanwhile, EGFR inhibition in mESCs disrupted EB formation, indicating its impaired pluripotency. Additionally, the effects observed by EGFR inhibition with another inhibitor gefitinib and siRNA were consistent with those observed by AG1478 treatment in mESCs. These effects were manifested in the decreased expression of proliferative and pluripotency-related genes and the increased expression of genes involved in differentiation. Moreover, RNA-seq analysis displayed that transcript profiling was changed significantly after EGFR inhibition by AG1478. A large number of differentially expressed genes were involved in cell cycle, apoptotic process, epigenetic modification, and metabolic process, which were related to self-renewal and pluripotency, confirming that EGFR deficiency impaired self-renewal and pluripotency in mESCs.ConclusionsTaken together, our results demonstrated the importance of EGFR in guarding the stemness of mESCs.

Dataset Information

Pluripotency-State-Dependent Role of Dax1 in Embryonic Stem Cells Self-Renewal.

Publications

Pluripotency-State-Dependent Role of Dax1 in Embryonic Stem Cells Self-Renewal.

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