Periodic Reporting for period 4 - EmbryoMethFunc (Cell-Type Specific DNA Methylation Changes During Mammalian Development: Beyond Mapping)
Période du rapport: 2024-04-01 au 2024-09-30
One of the most remarkable and fascinating processes in biology is the development of a single fertilized egg into an embryo, within a short period of rapid growth and cellular specialization. However, many aspects of how this cellular diversity arises from cells carrying the same genetic blueprint, remain unanswered. Recent technology to extract information from multitudes of individual cells, can now describe the developmental process at unprecedented resolution. But, real understanding of how and why cells decide to follow a specific path toward a certain developmental fate, cannot be based solely on static observations. In our work, we reconstructed the genetic relationships between the cells of the embryo over time, in the same manner that a movie can be created from a series of snapshots. Alongside additional tools, such as genetic perturbations, cell localization technology, embryo manipulations, and complex computer algorithms, we gain insight into how cells commit and diversify in the early mammalian embryo. Our findings include defining the impact of epigenetic modifications on gene expression and cellular differentiation during early development and refining the understanding of the communication that takes place between the embryo and its developing placenta in generating a correct balance of cell lineages. Besides contributing to the understanding of one of the most fundamental processes in biology, the broader impact of our work includes guiding stem cell research efforts for regenerative medicine and advancing the study of gene regulation.
During the period of the ERC starting grant, we set to provide functional insight into the dynamics and function of DNA methylation during the course of embryonic development. Results of our work have all been published in a freely accessible maner.
1. To create the first fully time-resolved model for mouse gastrulation, we generated single-cell transcriptome data from over 150 individually isolated and processed embryos (Mittnenzweig, Mayshar et al., Cell. 2021). Since each embryo independently progresses along the same trajectory, cells making up each embryo could be placed on a time continuum. This facilitated a unified flow model of mouse gastrulation. The main findings of this study include: (i) We established scRNA-seq as a precise method for timing embryo development. (ii) Our work provides a holistic model of development that considers continuous, parallel, and converged differentiation towards multiple lineages. (iii) The flows model provides answers to many open questions on fate acquisition in the embryo. For example, it explains the pluripotency gradient in the differentiating epiblast and the lack of a stable meso-endoderm progenitor state. (iv) We show that at the population level - development is not a series of “yes/no” decisions. Rather, our model emphasizes semi-stable progenitor states that continuously multi-furcate in a time-dependent manner. (v) The time resolved atlas is demonstrated to be extremely useful to characterize mutant phenotypes with time-matched controls. Similarly, this model can also serve to evaluate the fidelity of stem-cell-cased embryo models, as we demonstrated in Lau, Rubinstein et al. (Cell Stem Cell 2022).
2. Building on this model, we focused on studying the regulation of DNA methylation as a paradigm for the emergence of epigenetic programs during gastrulation. As genes of interest, we focused on the Tet family of DNA demethylation enzymes. Importantly, Tet-null embryos fail to gastrulate, limiting the possibility to analyze cell-autonomous gene function. By comparative analysis of normal development, whole-embryo knockouts, and chimeras, distinguishing direct cell-autonomous vs. indirect non-cell-autonomous effects we could work out the precise functions of the Tet machinery (Cheng et al., Cell. 2022).
3. Imprinted genes are widely considered dosage-sensitive and nearly all imprinting clusters contain an intricate interplay between paternally and maternally expressed genes. In Weinberg-Shukron et al. (Nat Communications, 2022), we provide a significant advance in understanding mechanisms controlling imprinted genes and their impact on embryonic development. Our data shows that correct parent-of-origin imprinting pattern is secondary to balanced gene dosage. More provocatively, it suggests that an epigenetic state is irrelevant as long as appropriate gene expression is maintained at imprinted loci.
4. To lay the foundation for using the rabbit as a comparative system for understanding genome and cell regulation, we established a time-resolved atlas of rabbit gastrulation. By directly aligning to our previous mouse model, we were able to identify conserved and diverged processes over a shared time axis. Thus, the time course of different cellular lineages alongside the dynamics of the key genes involved could be determined. Many trajectories were found to be highly conserved, whereas others, mainly of extraembryonic tissues, but also primordial germ cells, were found to be highly species-specific (Mayshar et al., Cell 2023).
5. Chromatin properties such as three dimensional organization and genome regulation are influenced significantly by histones and their post-translational modifications. By measuring histone turnover in steady state, we could measure the exchange of different histone variants and their functional association, such as with transcription, both in mouse stem cells and in animals (Dunjic et al., Nat Communications, 2023).
6. By generating a model that combines placental development alongside gastrulation we revealed previously uncharacterized early cell specification events. This further allowed us to probe the functional importance of extraembryonic gene perturbation, and determine the fidelity of placentation in ex-utero and synthetic embryo model systems. Using a variety of gene perturbation strategies we could refine the roles of the key signaling peptide BMP4 for germline specification, identifying that its time-dependent production from different sources is at the same time crucial for their initial differentiation, but also in restricting their relative numbers (Hadas et al., Nature 2024).
1. To create the first fully time-resolved model for mouse gastrulation, we generated single-cell transcriptome data from over 150 individually isolated and processed embryos (Mittnenzweig, Mayshar et al., Cell. 2021). Since each embryo independently progresses along the same trajectory, cells making up each embryo could be placed on a time continuum. This facilitated a unified flow model of mouse gastrulation. The main findings of this study include: (i) We established scRNA-seq as a precise method for timing embryo development. (ii) Our work provides a holistic model of development that considers continuous, parallel, and converged differentiation towards multiple lineages. (iii) The flows model provides answers to many open questions on fate acquisition in the embryo. For example, it explains the pluripotency gradient in the differentiating epiblast and the lack of a stable meso-endoderm progenitor state. (iv) We show that at the population level - development is not a series of “yes/no” decisions. Rather, our model emphasizes semi-stable progenitor states that continuously multi-furcate in a time-dependent manner. (v) The time resolved atlas is demonstrated to be extremely useful to characterize mutant phenotypes with time-matched controls. Similarly, this model can also serve to evaluate the fidelity of stem-cell-cased embryo models, as we demonstrated in Lau, Rubinstein et al. (Cell Stem Cell 2022).
2. Building on this model, we focused on studying the regulation of DNA methylation as a paradigm for the emergence of epigenetic programs during gastrulation. As genes of interest, we focused on the Tet family of DNA demethylation enzymes. Importantly, Tet-null embryos fail to gastrulate, limiting the possibility to analyze cell-autonomous gene function. By comparative analysis of normal development, whole-embryo knockouts, and chimeras, distinguishing direct cell-autonomous vs. indirect non-cell-autonomous effects we could work out the precise functions of the Tet machinery (Cheng et al., Cell. 2022).
3. Imprinted genes are widely considered dosage-sensitive and nearly all imprinting clusters contain an intricate interplay between paternally and maternally expressed genes. In Weinberg-Shukron et al. (Nat Communications, 2022), we provide a significant advance in understanding mechanisms controlling imprinted genes and their impact on embryonic development. Our data shows that correct parent-of-origin imprinting pattern is secondary to balanced gene dosage. More provocatively, it suggests that an epigenetic state is irrelevant as long as appropriate gene expression is maintained at imprinted loci.
4. To lay the foundation for using the rabbit as a comparative system for understanding genome and cell regulation, we established a time-resolved atlas of rabbit gastrulation. By directly aligning to our previous mouse model, we were able to identify conserved and diverged processes over a shared time axis. Thus, the time course of different cellular lineages alongside the dynamics of the key genes involved could be determined. Many trajectories were found to be highly conserved, whereas others, mainly of extraembryonic tissues, but also primordial germ cells, were found to be highly species-specific (Mayshar et al., Cell 2023).
5. Chromatin properties such as three dimensional organization and genome regulation are influenced significantly by histones and their post-translational modifications. By measuring histone turnover in steady state, we could measure the exchange of different histone variants and their functional association, such as with transcription, both in mouse stem cells and in animals (Dunjic et al., Nat Communications, 2023).
6. By generating a model that combines placental development alongside gastrulation we revealed previously uncharacterized early cell specification events. This further allowed us to probe the functional importance of extraembryonic gene perturbation, and determine the fidelity of placentation in ex-utero and synthetic embryo model systems. Using a variety of gene perturbation strategies we could refine the roles of the key signaling peptide BMP4 for germline specification, identifying that its time-dependent production from different sources is at the same time crucial for their initial differentiation, but also in restricting their relative numbers (Hadas et al., Nature 2024).
The aforementioned studies represent major breakthroughs in their respective fields, in part attested by their reception by the scientific community.
Looking forward, we expect our work will have an important impact in several domains: (i) Establishment of quantitative models that truly represent development as a concurrent and interacting collection of intra-cellular processes with progressively robust functional identities. (ii) Providing a much-needed relatable conceptual reference for evaluating the fidelity of synthetic and in-vitro derived models to emulate mouse embryonic development, with great potential for directing stem cell research efforts for regenerative medicine. (iii) Our work will provide novel experimental frameworks for indexing spatial epigenomics from single cells and separating cell-intrinsic from non-cell-autonomous effects during cell specification, which can be widely implemented in diverse biological systems. (iv) Importantly, using these experimental frameworks will allow refining and revisiting embryonic phenotypes and dissecting the effects of DNA methylation on enhancer specificity and overall cell state and function, with unprecedented resolution.
Looking forward, we expect our work will have an important impact in several domains: (i) Establishment of quantitative models that truly represent development as a concurrent and interacting collection of intra-cellular processes with progressively robust functional identities. (ii) Providing a much-needed relatable conceptual reference for evaluating the fidelity of synthetic and in-vitro derived models to emulate mouse embryonic development, with great potential for directing stem cell research efforts for regenerative medicine. (iii) Our work will provide novel experimental frameworks for indexing spatial epigenomics from single cells and separating cell-intrinsic from non-cell-autonomous effects during cell specification, which can be widely implemented in diverse biological systems. (iv) Importantly, using these experimental frameworks will allow refining and revisiting embryonic phenotypes and dissecting the effects of DNA methylation on enhancer specificity and overall cell state and function, with unprecedented resolution.