1 Interpretation of Experimental Data

The study used the degradation fusion tag (dTAG) system to create a new Eomes gene editing tool to quickly and specifically manipulate the expression of Eomes protein, revealing its key functions in various stages of mouse embryonic development. By applying the dTAG system in mouse embryonic stem cells and mice, the research team deeply explored the dynamic expression and function of Eomes in various cell lineages. The results showed that the dTAG system can quickly and reversibly degrade Eomes protein, providing a powerful tool for studying the spatiotemporal specific role of Eomes in embryonic development and cell lineage differentiation.

Figure 1 presents the gene-editing strategy using CRISPR-Cas9 technology to construct a degradable variant of the Eomes protein (Eomes^deg). Researchers used crRNA targeting the Eomes gene along with an ssDNA repair template containing a degradation tag (Deg), a self-cleaving 2A peptide, and an mCherry reporter gene, successfully creating the Eomes^deg allele. This modification allows for tracking of Eomes protein expression via the mCherry label. In the experimental protocol, embryoid bodies (EBs) were formed under the induction of 2i/LIF medium and further promoted to differentiate into mesodermal/HE markers by adding factors such as BMP4, ActA, and VEGF. Western blot results show that the Eomes protein can be degraded within one hour of treatment with dTAG-13. Flow cytometry results confirmed that Eomes^deg/deg EBs treated with dTAG-13 lost Eomes protein expression. These results validate the effectiveness of the dTAG-13 mediated rapid degradation of Eomes protein, providing a powerful tool for in-depth study of Eomes functions during embryonic development.

Figure 3 presents the experimental design and results of administering dTAG treatment to mouse embryos at 6.5 days post-coitum (dpc) via intraperitoneal injection. The experimental flow diagram (Figure 3A) illustrates the method of dTAG treatment, while Figure 3B uses immuno fluorescence staining to show the expression and degradation of Eomes (green) and Foxa2 (red) in the Eomes^deg/deg homozygous embryos after treatment. DAPI (blue) stains the nuclei, revealing heterogeneity in the degree of Eomes protein degradation across different regions. Notably, embryos treated with dTAG-13 show Eomes-positive cells in the posterior region of the primitive streak, whereas embryos treated with dTAGV-1 still express Eomes in the extraembryonic ectoderm, the extraembryonic visceral endoderm (VE), and the primitive streak. These results indicate that dTAG-mediated protein degradation exhibits regional differences in vivo, highlighting the potential of this technique in studying the function of Eomes during embryonic development and the technical challenges associated with its application.

Figure 5 evaluates the recovery process of Eomes protein in Eomes ^deg/deg embryos and embryoid bodies (EBs) after washing with dTAG-13 or dTAGV-1 and validates the expression of the mCherry reporter gene. Figure 5A illustrates the processing and cleaning steps for small molecule dTAG. Figure 5B shows Eomes (green) and Foxa2 (red) staining in E6.5 embryos treated with DMSO, dTAG-13, or dTAGV-1, showing Eomes expression in the proximal posterior region after two hours of washing and reculture. Ectodermal/primitive bar area restoration. Figure 5C shows the staining of Eomes (green) and Nanog (red) in HE EBs after dTAG-13 treatment on the third day, evaluating the dynamic process of Eomes recovery. After 1 hour and 2 hours of dTAG-13 treatment, Eomes staining was undetectable; 1 hour after washing, Eomes nuclear staining was obvious, revealing the rapid recovery of Eomes protein. Figure 5D shows Eomes (green) and RFP (red) staining in E7.5 Eomes^deg/+ embryos. RFP detects the expression of the mCherry reporter gene. Figure 5E analyzes the expression of mCherry in E7.5 dissociated Eomes^deg/deg embryos by flow cytometry. About 70% of the cells express mCherry.

2 Insights of Research Findings

This study successfully applied the dTAG system to a mouse embryo model, achieving rapid and reversible control of Eomes protein, thus demonstrating the potential of this technology for controlling gene expression at the whole-organism level. Eomes, a key T-box transcription factor, plays a crucial role at multiple stages of embryonic development. By combining in vitro and in vivo experiments, this study extensively explored the functions of Eomes across different cell types and its temporally and spatially specific expression during embryonic development. In vitro experiments involved the introduction of dTAG labels at the Eomes site, creating an embryonic stem cell line that could rapidly degrade Eomes protein upon the application of the small molecule dTAG-13. The experiments showed that Eomes protein could be almost completely lost within one hour and quickly restored after the removal of dTAG-13, providing a powerful tool for studying its role in cell development. In vivo experiments involved injecting pregnant mice with dTAG-13, attempting to degrade Eomes protein specifically at different stages of embryonic development. Results revealed that the efficiency of Eomes protein degradation varied greatly among tissues, particularly during early embryonic stages, where its degradation significantly affected implantation and further development. This underscores the critical role of Eomes in determining early cell fate and reveals its complex regulatory mechanisms in cell differentiation and tissue formation. These findings not only showcase the potential of the dTAG system for studying key developmental genes with strict temporal and spatial expression but also highlight the importance of precise genetic manipulation techniques in understanding complex biological processes, offering new possibilities for future disease treatment research. Through this method, researchers can delve deeper into the roles of key transcription factors like Eomes in various biological processes.

3 Evaluation of the Research

This study used dTAG technology to introduce an innovative tool that can rapidly and reversibly manipulate the expression of target proteins. Compared with traditional genetic knockout methods, dTAG operations are more flexible and faster, and are particularly suitable for studying genes that are transiently expressed during development. Studies have shown that the dTAG system can efficiently degrade specific proteins in an in vitro environment, but there are differences in the efficiency and consistency of protein degradation in in vivo experiments. This may be affected by multiple factors such as the expression level of the target protein in different tissues, differences in cell types, drug bioavailability, and tissue permeability. In addition, protective structures in the early embryo may hinder the penetration of small molecules, thereby affecting experimental results. Despite the challenges, the dTAG system provides a unique mechanism to deeply explore the dynamic regulation of proteins and their role in development. In the future, optimizing the design of dTAG molecules will further expand their potential for application in disease models and treatments.

4 Concluding Remarks

Through the application of the dTAG system in this study, the researchers successfully degraded the Eomes protein specifically at different developmental stages, revealing its key role in early embryonic development in mice. As a T-box transcription factor, Eomes is particularly important in embryonic gastrulation, cell lineage determination, and organ development. The use of the dTAG system allows for rapid degrading of Eomes at specific times and cell types, thereby observing the dynamic effects of its loss of function on embryonic development. This method is superior to traditional gene knockout technology because it can observe the loss of specific gene function without affecting the overall survival of the embryo. This study provides a new perspective for understanding how Eomes regulates cell fate at different developmental stages, and demonstrates the potential of dTAG technology in disease model construction and the development of new therapeutic strategies.

5 Access Original Paper

A.M. Bisia et al., A degron-based approach to manipulate Eomes functions in the context of the developing mouse embryo. PNAS. 120(44), e2311946120 (2023). DOI: https://doi.org/10.1073/pnas.2311946120

Acknowledgments

Thank you to Proceedings of National Academy of Sciences of the United States of America for its open access strategy, which allows readers to freely access this valuable research result. The research work of Alexandra M. Bisia, Ita Costello, Maria Eleni Xypolita, Luke T.G. Harland, and others from the University of Oxford involved the use of the dTAG system to control the application of Eomes protein in embryonic development, providing a very good research basis for the animal researcher community. Due to the different perspectives of the reviewers on the understanding of the paper, if there may be differences in the research data, results, and evaluations compared to the author's wishes, I deeply apologize.

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