Periodic Reporting for period 2 - EmbryoMethFunc (Cell-Type Specific DNA Methylation Changes During Mammalian Development: Beyond Mapping)
Berichtszeitraum: 2021-04-01 bis 2022-09-30
One of the most remarkable and fascinating processes in biology is the development of a single fertilized egg into an embryo within a short time period of rapid growth and cellular specialization. However, many aspects of how this cellular diversity arises from cells carrying the same genetic blueprint, remain a fundamental open question in biology. Recent technology that enables extracting information from multitudes of individual cells, now facilitates profiling the developmental process at unprecedented resolution. But, to really understand how and why cells decide to follow a specific path toward a certain developmental fate, cannot be based solely on such static observations. In our work, we integrate analysis of single cells with additional layers of information, such as genetic perturbations, cell localization technology, embryo manipulations, and complex computer algorithms to understand how cells commit and diversify in the early mammalian embryo. Our research is already delivering many novelties and establishing new paradigms with much potential impact for several domains, including basic science, synthetic embryology, and directing stem cell research efforts for regenerative medicine.
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.
1. Building a time resolved single-cell transcriptome map of mouse gastrulation. To create the first fully time-resolved model for mouse gastrulation, we generated single-cell transcriptome data fromover 150 individually isolated and processed embryos (Mittnenzweig, Mayshar et al., Cell. 2021). Since each embryo independently progresses at its own pace, towards ultimate convergence at a common outcome, individual cells making up each embryo could be placed on a bona fide time continuum. This facilitated a unified flow model of mouse gastrulation. The main findings include of this study include: (i) We establishes scRNA-seq as the most precise method for timing embryo development. (ii) Our work provides a holistic flow 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 mesendoderm 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.
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 particularly challenging and important Tet family of DNA demethylation enzymes. Importantly, Tet-null embryos fail to gastrulate, limiting the possibility to analyze cell-autonomous gene function. We demonstrated that interpretation of Tet function strongly depends on separating temporal, lineage, and cell-autonomous/inter-cellular effects. Eliminating the three Tets (Tet-TKO) in the entire embryo gives rise to major gastrulation defects much as expected. But, when Tet-TKO cells are let to develop in a WT niche by injecting deficient cells into normal host embryos, they can differentiate to nearly all embryonic lineages. From this starting point, we could work out the precise functions of the Tet machinery by comparative analysis of normal development, whole-embryo knockouts, and chimeras, distinguishing direct cell-autonomous vs. indirect non-cell-autonomous effects (Cheng et al., Cell. In revisions).
3. In the third study, (Weinberg-Shukron et al., Nat Communications, In revisions), we provide a significant advance in understanding mechanisms controlling imprinted genes and their impact on embryonic development. Imprinted genes are widely considered dosage-sensitive and nearly all imprinting clusters contain an intricate interplay between paternally and maternally expressed genes. Thus, perturbation of imprinted control regions results in antagonistic and reciprocal effects, complicating phenotype interpretation. The Dlk1-Dio3 locus, represents a highly conserved imprinting domain with an intergenic differentially methylated region (IG-DMR) essential for maintaining parent-of-origin expression of multiple genes. We find that rare compensatory de novo methylation in germline mutant embryos improves maternal to paternal gene expression ratios in the locus, rescuing the deleterious perinatal phenotype. Crossing mice with complete and partial IG-DMR deletions generated a unique genotype in which parental origin expression was flipped while retaining balanced gene dosage. This resulted in the synthetic rescue of each deletion on its own. 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. In addition, we were able to proceed beyond expectation in generating a new model that combines placental development alongside gastrulation. In this manner, we could reveal previously uncharacterized early cell specification events, probe the functional importance of extraembryonic gene perturbation, and determine the fidelity of placentation in ex-utero and synthetic embryo model systems (Hadas et al., in preparation).
1. Building a time resolved single-cell transcriptome map of mouse gastrulation. To create the first fully time-resolved model for mouse gastrulation, we generated single-cell transcriptome data fromover 150 individually isolated and processed embryos (Mittnenzweig, Mayshar et al., Cell. 2021). Since each embryo independently progresses at its own pace, towards ultimate convergence at a common outcome, individual cells making up each embryo could be placed on a bona fide time continuum. This facilitated a unified flow model of mouse gastrulation. The main findings include of this study include: (i) We establishes scRNA-seq as the most precise method for timing embryo development. (ii) Our work provides a holistic flow 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 mesendoderm 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.
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 particularly challenging and important Tet family of DNA demethylation enzymes. Importantly, Tet-null embryos fail to gastrulate, limiting the possibility to analyze cell-autonomous gene function. We demonstrated that interpretation of Tet function strongly depends on separating temporal, lineage, and cell-autonomous/inter-cellular effects. Eliminating the three Tets (Tet-TKO) in the entire embryo gives rise to major gastrulation defects much as expected. But, when Tet-TKO cells are let to develop in a WT niche by injecting deficient cells into normal host embryos, they can differentiate to nearly all embryonic lineages. From this starting point, we could work out the precise functions of the Tet machinery by comparative analysis of normal development, whole-embryo knockouts, and chimeras, distinguishing direct cell-autonomous vs. indirect non-cell-autonomous effects (Cheng et al., Cell. In revisions).
3. In the third study, (Weinberg-Shukron et al., Nat Communications, In revisions), we provide a significant advance in understanding mechanisms controlling imprinted genes and their impact on embryonic development. Imprinted genes are widely considered dosage-sensitive and nearly all imprinting clusters contain an intricate interplay between paternally and maternally expressed genes. Thus, perturbation of imprinted control regions results in antagonistic and reciprocal effects, complicating phenotype interpretation. The Dlk1-Dio3 locus, represents a highly conserved imprinting domain with an intergenic differentially methylated region (IG-DMR) essential for maintaining parent-of-origin expression of multiple genes. We find that rare compensatory de novo methylation in germline mutant embryos improves maternal to paternal gene expression ratios in the locus, rescuing the deleterious perinatal phenotype. Crossing mice with complete and partial IG-DMR deletions generated a unique genotype in which parental origin expression was flipped while retaining balanced gene dosage. This resulted in the synthetic rescue of each deletion on its own. 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. In addition, we were able to proceed beyond expectation in generating a new model that combines placental development alongside gastrulation. In this manner, we could reveal previously uncharacterized early cell specification events, probe the functional importance of extraembryonic gene perturbation, and determine the fidelity of placentation in ex-utero and synthetic embryo model systems (Hadas et al., in preparation).
The aforementioned studies represent major breakthroughs in their respective fields, in part attested by their reception by the scientific community.
By the end of the grant period, we expect our work will have an important impact in several domains: (i) We will establish quantitative models that truly represent development as a concurrent and interacting collection of intra-cellular processes with progressively robust functional identities. (ii) This will provide a much-needed relatable 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.
By the end of the grant period, we expect our work will have an important impact in several domains: (i) We will establish quantitative models that truly represent development as a concurrent and interacting collection of intra-cellular processes with progressively robust functional identities. (ii) This will provide a much-needed relatable 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.