Dynamic Transcriptional Response and Activation of Integrin-YAP/TAZ Survival Signaling Pathway Revealed by Single-Cell Resolution Analysis of Embryo Dormancy

Introduction

Embryo dormancy, or diapause, is a unique reproductive adaptation that allows some mammals to pause embryonic development without compromising their developmental potential. Diapause is typically activated at the blastocyst stage, where the embryo has the selective capability to either implant or enter dormancy. This mechanism is regulated by maternal hormones such as estrogen and progesterone, which influence the uterine receptivity state, thereby deciding whether the embryo implants into the maternal uterus to continue development or enters dormancy. Although embryo diapause is a widely studied phenomenon, the molecular and cellular mechanisms during the diapause process remain incompletely understood. Therefore, by utilizing single-cell RNA sequencing (scRNA-seq) on mouse embryos, Chen and his team have defined dormant state embryos at the molecular level, revealing the dynamic transcriptional landscape in dormant embryos and how the activation of the Integrin-YAP/TAZ signaling pathway maintains the developmental potential of the embryos during dormancy.

Summary and Background of the Paper

This study, conducted by researchers Rui Chen, Rui Fan, Fei Chen, and others from the Max Planck Institute for Molecular Biomedicine, was published on September 5, 2024, in the journal “Cell Stem Cell.” The research primarily focuses on using a mouse model to explore the molecular mechanisms in embryos during diapause through single-cell RNA sequencing. Through this study, the authors not only uncovered the transcriptional dynamics within embryonic cells during diapause but also discovered the crucial role of the Integrin-YAP/TAZ signaling pathway in maintaining the developmental potential of dormant embryos. This discovery provides significant insights for understanding the biological mechanisms of embryo diapause and potentially developing technologies related to embryo preservation and embryo transfer.

Research Process and Experimental Design

a) Experimental Process and Methods

The study is divided into three stages: entry into diapause, maintenance of diapause, and exit from diapause. The authors collected embryonic samples from mouse embryos at embryonic days 4.5 (e4.5), 7.5 (edg7.5), 9.5 (edg9.5), and 5.5 (e5.5) for single-cell RNA sequencing to capture the dynamic transcriptional changes in the dormant state. To ensure comprehensive data, the study included both dormant state embryos and embryos exiting diapause induced by estrogen, combined with Wnt signaling reporter mice to verify signaling activity during the diapause period.

b) Exploration of Cellular Transcriptional Landscape and YAP/TAZ Signaling

During the diapause state, the authors found that both extraembryonic and embryonic tissues of dormant embryos maintained their cellular identities, exhibiting specific dynamic transcriptional responses unique to the diapause period. Notably, the activation of Integrin receptors in collaboration with the YAP/TAZ signaling pathway plays a critical role in maintaining embryonic stem cell vitality. Through integrative analysis networks, multiple gene expression patterns were analyzed, and the distribution of related proteins in dormant embryos was visually confirmed via immunohistochemistry.

In the study of the trophectoderm (TE) and inner cell mass (ICM), the authors observed that during diapause, the polarity formation of TE did not show significant differentiation, while the ICM exhibited unique aggregation. Integrin β1 serves as the main signaling receptor, assisting in maintaining polarity and cell vitality of the ICM. The Integrin-YAP signaling pathway plays a crucial role during the polarity formation of the ICM. When Integrin β1 is lost, nuclear accumulation of YAP protein significantly decreases, and cell death rates in the ICM significantly increase, indicating the critical role of the Integrin-YAP signaling in diapause maintenance.

Research Results

Transcriptional Dynamics Revealed by Single-Cell Transcriptomic Data

Through single-cell analysis, the research uncovered the entire transcriptomic landscape from blastocyst to diapause. During diapause, differentiation of embryonic tissues is suppressed, and all cells remain in a specific undifferentiated state. Once diapause ends and the implantation stage begins, cells gradually differentiate into specific cell lineages. This process is regulated by dynamic changes in multiple signaling pathways, including Wnt signaling, FGF4 signaling, and YAP/TAZ signaling.

Integrin-YAP/TAZ Signaling Maintains Embryo Viability

The study found that during diapause, Integrin-YAP/TAZ signaling maintains vitality and prevents cell death of the ICM. Integrin β1 is the key receptor for interaction between the embryo and the extracellular matrix, with nuclear accumulation of YAP protein primarily dependent on Integrin signaling. In experiments with embryonic stem cells (ESCs) genetically engineered to knock out YAP and TAZ, it was found that these two transcription factors jointly suppress apoptosis and significantly enhance embryo survival capability during diapause. This mechanism not only maintains the vitality of embryonic stem cells but also enhances the embryo’s adaptability to the dormant state.

Research Conclusions and Significance

This study deeply analyzes the dynamic transcriptional responses of embryos in a dormant state, revealing the key role of the Integrin-YAP/TAZ signaling pathway in maintaining the vitality of embryonic stem cells. This research provides new insights into the biological mechanisms of embryo diapause, while also unveiling the crucial role of the YAP/TAZ signaling pathway in cell survival and reactivation, holding significant scientific and applied value. The findings on diapause could offer new strategies in the biomedical field to maintain or regulate the state of embryos both in vitro and in vivo, thereby providing a reference for embryo transfer and assisted reproductive technologies.

Research Highlights

1. The first transcriptomic dynamic atlas of embryo diapause revealed at a single-cell level: The study employs single-cell RNA sequencing technology to capture changes in transcriptional activity throughout the development of dormant embryos, detailing the molecular characteristics of the dormancy state.

2. First revelation of the survival function of the Integrin-YAP/TAZ signaling pathway: The research demonstrates that during diapause, the activation of Integrin receptors activates YAP/TAZ signaling, maintaining the vitality of ICM cells, which provides critical insights into the regulatory mechanisms of embryo diapause.

3. Pioneering potential for optimizing embryonic stem cell culture conditions using the diapause state: The study shows that through regulation of the Integrin-YAP/TAZ signaling pathway, it is possible to better maintain the undifferentiated state of embryonic stem cells in vitro, which may offer new opportunities to improve the cultivation efficiency of embryonic stem cells.

4. Showing the similarity between diapause and cancer cell dormancy: The role of the YAP/TAZ signaling pathway in embryo diapause has certain similarities with its function in tumor dormancy, offering a new perspective for future exploration of the biological mechanisms of tumor dormancy.

5. Clear experimental design and detailed gene co-expression network analysis: The study not only includes in-depth bioinformatics analyses but also validates the expression distribution and role of key proteins in dormant embryos through experiments, laying a foundation for future diapause research.

Limitations of the Study

Although this study reveals the transcriptional dynamics of dormant embryos, further exploration is needed on how to precisely regulate the expression of related genes. Additionally, collecting sufficient numbers of embryos under real physiological conditions, especially genetically modified mutant embryos, poses significant challenges. Future research should further elucidate the regulatory mechanisms of Oct4 expression during diapause and the overall impact of other signaling pathways on the dormant state.

This study provides valuable insights into the molecular mechanisms of embryo diapause. Future work may further uncover more yet-to-be-discovered regulatory mechanisms of embryo diapause and explore its potential applications in regenerative medicine and biotechnology.