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The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamics of the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and for allantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro., (© 2024. The Author(s).)

The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In mammalian embryos, DNA methylation is initialized to maximum levels in the epiblast by the de novo DNA methyltransferases DNMT3A and DNMT3B before gastrulation diversifies it across regulatory regions. Here we show that DNMT3A and DNMT3B are differentially regulated during endoderm and mesoderm bifurcation and study the implications in vivo and in meso-endoderm embryoid bodies. Loss of both Dnmt3a and Dnmt3b impairs exit from the epiblast state. More subtly, independent loss of Dnmt3a or Dnmt3b leads to small biases in mesoderm-endoderm bifurcation and transcriptional deregulation. Epigenetically, DNMT3A and DNMT3B drive distinct methylation kinetics in the epiblast, as can be predicted from their strand-specific sequence preferences. The enzymes compensate for each other in the epiblast, but can later facilitate lineage-specific methylation kinetics as their expression diverges. Single-cell analysis shows that differential activity of DNMT3A and DNMT3B combines with replication-linked methylation turnover to increase epigenetic plasticity in gastrulation. Together, these findings outline a dynamic model for the use of DNMT3A and DNMT3B sequence specificity during gastrulation., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification. Using algorithms and precise timing, we infer differentiation flows and lineage specification dynamics over the embryonic transcriptional manifold. Rapid transcriptional bifurcations characterize the commitment of early specialized node and blood cells. However, for most lineages, we observe combinatorial multi-furcation dynamics rather than hierarchical transcriptional transitions. In the mesoderm, dozens of transcription factors combinatorially regulate multifurcations, as we exemplify using time-matched chimeric embryos of Foxc1/Foxc2 mutants. Our study rejects the notion of differentiation being governed by a series of binary choices, providing an alternative quantitative model for cell fate acquisition., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Systematic numerical investigations of conformational motions in single actin molecules were performed by employing a simple elastic-network (EN) model of this protein. Similar to previous investigations for myosin, we found that G-actin essentially behaves as a strain sensor, responding by well-defined domain motions to mechanical perturbations. Several sensitive residues within the nucleotide-binding pocket (NBP) could be identified, such that the perturbation of any of them can induce characteristic flattening of actin molecules and closing of the cleft between their two mobile domains. Extending the EN model by introduction of a set of breakable links which become effective only when two domains approach one another, it was observed that G-actin can possess a metastable state corresponding to a closed conformation and that a transition to this state can be induced by appropriate perturbations in the NBP region. The ligands were roughly modeled as a single particle (ADP) or a dimer (ATP), which were placed inside the NBP and connected by elastic links to the neighbors. Our approximate analysis suggests that, when ATP is present, it stabilizes the closed conformation of actin. This may play an important role in the explanation why, in the presence of ATP, the polymerization process is highly accelerated.